黄花梨及其绿皮芽变果皮发育特性和差异表达基因的克隆与功能分析
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摘要
果皮色泽是梨果重要的农艺性状和商品外观品质之一,也是消费者在购买前先得到的感官感受之一。培育不同果皮色泽的梨果以满足消费者多元化的需求是梨育种的一个长期目标,梨的果皮色泽研究正逐渐成为梨研究的热点。研究褐皮和绿皮梨果皮发育和果皮色泽形成期间的差异表达基因,可以深入理解梨褐皮和绿皮果实的形成机制,推进褐皮和绿皮梨分子育种理论形成和育种工作开展。褐皮梨及其绿皮芽变为梨的果皮色泽研究提供了理想的研究材料。
本文首先对黄花梨(果皮为褐色)的绿皮芽变——绿皮黄花进行了生物学特性的研究,其次对比研究了黄花梨和绿皮黄花梨成熟果实果皮结构、表皮膜结构,不同发育时期果点的发育、果皮发育和表皮膜发育,绿皮黄花梨成熟果实不同颜色果锈果皮与黄花梨褐色果皮的特性,然后以确定的黄花梨果皮色泽关键发育期时的两种梨果皮为材料,提取了高分子量RNA,用差异显示PCR (DDRT-PCR)和5'RACE等技术克隆了在黄花梨和绿皮黄花梨果皮中差异表达的梨防御素基因PpyDFN1全长和苯丙氨酸解氨酶基因片段,并对PpyDFN1进行了序列分析和亚细胞定位研究；接着对黄花梨和绿皮黄花梨不同发育时期果皮中参与苯丙烷次生代谢的苯丙氨酸解氨酶基因PpyPAL1、肉桂酸-4-羟化酶基因PpyC4H和肉桂酰CoA氧化还原酶基因PpyCCR表达水平进行了分析,以评估果皮发育期间两种梨果皮中的次生代谢水平差异。最后对PpyDFN1在黄花梨和绿皮黄花梨中的表达特点、转基因烟草中的功能和体外抑菌功能进行了研究。主要研究结果如下。
1.绿皮黄花为黄花梨稳定的绿皮芽变,其果皮为绿色,成熟期较黄花梨提前7-10天,果实可溶性总糖、总可溶性固形物和可滴定酸高于黄花梨,果实硬度低于黄花梨,其它植物学特性和结果习性都与黄花梨接近。
2.黄花梨与绿皮黄花梨的果点发育明显不同,黄花梨果点形成早而集中,果点发育快,果点大小较同期绿皮黄花梨果点大,而绿皮黄花梨果点形成晚,果点发育慢；黄花梨果面蜡质在花后10周以前较多,花后10周以后果面蜡质不断减少,而绿皮黄花梨果面蜡质从观察开始的花后4周到果实成熟一直存在；黄花梨角质膜花后10周前基本完整,从10周时从果点周围出现裂口到果实成熟时完全龟裂,而绿皮黄花梨果皮角质膜从幼果到果实成熟都保持完整,很少破裂；开花后10周以前,黄花梨与绿皮黄花梨表皮膜都是完整和无色透明的,开花后10周以后,黄花梨角质膜逐渐破裂,直至完全龟裂,裸露的表皮细胞木栓化,表皮膜呈现黄褐色,而绿皮黄花梨表皮膜从幼果到成熟都相对完整,无色透明,其表皮细胞很少木栓化；黄花和绿皮黄花梨果皮结构从外到内依次为角质膜、表皮细胞层和亚表皮细胞层,成熟黄花梨果皮表面粗糙,角质膜不完整,其下或裸露的表皮细胞木栓化,不能被间苯三酚染色,颜色为黄褐色,而成熟绿皮黄花梨果皮表面光滑,角质膜完整,其下表皮细胞为绿色或无色,没有木栓化。成熟黄花梨果实的表皮膜为黄褐色,是果皮颜色的决定者,而绿皮黄花梨果实表皮膜无色透明,不决定果皮色泽。
3.绿皮黄花梨的深锈果皮与黄花梨的黄褐色果皮本质上是一样的,而浅锈果皮和深锈果皮的区别主要在于角质膜的受损程度、表皮细胞的木栓化程度和木栓化表皮细胞层的完整性。
4.用黄花梨和绿皮黄花梨果皮为材料,建立了一种从果皮中分离高分子量RNA(HMW RNA)和低分子量RNA(LMW RNA)方法。用果皮中所提取的高分子量RNA合成的cDNA作为DDRT-PCR模板,克隆差异片段,结合5'RACE及5’同源简并引物扩增,克隆了黄花梨和绿皮黄花梨果皮中差异表达基因——梨防御素基因PpyDFN1(GenBank登录号为HM044853)和苯丙氨酸解氨酶基因PpyPAL1。实时荧光定量PCR进一步确定了两个基因在8周的黄花梨和绿皮黄花梨果皮中表达量存在差异,其中PpyDFN1在黄花梨果皮中的表达量为绿皮黄花梨果皮中表达量的6.7倍,苯丙氨酸解氨酶基因PpyPAL1在黄花梨果皮中的表达量为绿皮黄花梨果皮中的2.2倍。
黄花梨防御素基因PpyDFN1的全长cDNA序列为567bp,包括72bp的5’非翻译区,258bp的3’非翻译区和237bp的编码区。在5’非翻译区含有一个(TC)7简单重复序列。编码区编码一条长度为78个氨基酸残基组成的多肽链,该多肽链的分子量为8.65KDa,等电点为9.36。BLASTP序列分析表明,该多肽链为防御素的前体序列。信号肽预测分析表明,多肽链的前31个氨基酸序列为防御素的信号肽,余下47个氨基酸残基组成的序列为成熟的防御素。梨防御素三维结构的同源比对建模表明,防御素PpyDFN1呈现出典型的由一个α-螺旋,三个反向平行的p-折叠组成的βαββ防御素结构。基因组序列克隆和分析表明,PpyDFN1的基因组序列在黄花和绿皮黄花中没有差异,都含有一个内含子和2个外显子。构建的亚细胞定位载体用于轰击洋葱表皮细胞结果表明,PpyDFN1定位在细胞壁上。
5.黄花梨和绿皮黄花梨花后6周到10周的果皮中PpyPAL1、PpyC4H和PpyCCR表达量较高,其中在黄花梨果皮中ppyPAL1和PpyCCR表达量显著高于绿皮黄花梨果皮中的表达量。上述基因高表达量发生时期与果点形成和扩大期吻合,一定程度上反映了此时期果皮中旺盛的次生代谢活动。而黄花梨果皮中PpyPAL1和PpyCCR较高的表达量,反映了黄花梨果皮较绿皮黄花梨果皮中有着更为旺盛的苯丙烷类物资代谢活动。
6.梨防御素PpyDFN1在根、叶片、花梗、萼片、花瓣、雌蕊和柱头、雄蕊、种子、果肉和果皮中基础性表达。PpyDFN1基因的表达表现出果点发育时期依赖性,在果点形成和扩大期表达量较高,在果点形成后期表达量较低；PpyDFN1在黄花梨果皮中的表达量总体上较绿皮黄花梨高。机械损伤、乙烯、水杨酸(SA)、茉莉酸甲酯(MeJA)和H202都能促进PpyDFN1的表达,而ABA不能。
7.表达PpyDFN1的烟草叶片提高了对灰霉菌的抗性；转PpyDFN1基因植株节间缩短,高度降低,开花延迟,花器官较短,花梗较短,花梗较粗,花筒较粗等；表达PpyDFN1基因的T1代转基因幼苗的根系明显短于非转基因对照。用镍离子树脂从转基因植株中纯化了PpyDFN1,纯化的蛋白洗脱液可以抑制农杆菌的生长。
Fruit color is one of the important agronomic traits and appearance quality of goods for pear, and is also consumers'first sense before their purchasing decisions. Breeding pear cultivars bearing fruits with different fruit color to meet consumers'diversified needs is a long-term goal for pear breeding, and now works on pear fruit color is becoming a research focus. Study on fruit skin development of russet and green pears, and differentially expressed genes during fruit color formation in pear could help deeply understand the formation mechanism of pear russet fruit and green fruit, promote russet and green pear molecular breeding theory formation and breeding work. Russet pear and its green bud sports supply the ideas materials for pear fruit color research.
In this article, biological characteristics was firstly studied on Huanghua pear (russet fruit) green skin bud sport——Green Huanghua. Secondly, studies on structure of fruit peel of ripe fruits and epidermal structure for Huanghua and Green Huanghua pear, fruit dot development, fruit skin development and epidermis development, the characters of Green Huanghua ripe fruit peels with different level of fruit rust and Huanghua pear russet peel were performed. Thirdly, high molecular weight RNA was isolated from the two kinds of pear peels sampled at the key stage of fruit skin development, and differentially expressed genes——the full length of pear defensin gene PpyDFNl and fragment of phenylalanine ammonia-lyase gene PpyPAL1in Huanghua and Green Huanghua pear peels were cloned by differential display reverse transcription polymerase chain reaction (DDRT-PCR) and rapid amplification of cDNA5'end (5'RACE). The sequence of PpyDFN1was further analy sized and subcellular location of PpyDFN1was studied. Next, the expression level of genes involved in phenylpropanoid secondary metabolites including phenylalanine ammonia-lyase gene PpyPAL1, cinnamic acid-4-hydroxylase gene PpyC4H and cinnamoyl-CoA oxidoreductase gene PpyCCR was determined in peels of Huanghua and Green Huanghua pear at different developmental stages to evaluate the level of secondary metabolism. Finally, the expression analysis of PpyDFNl in Huanghua and Green Huanghua pear, functional analysis in transgenic tobacco plants and bacterial inhibition in vitro were studied. The results were showed as follows.
1. Green Huanghua is a stable green skin bud sport of Huanghua pear. Its fruit skin is green, maturiy is7to10days ahead of Huanghua pear, and the total soluble sugar, total soluble solids and titratable acidity is higher than that of Huanghua pear fruit, but the fruit hardness is lower than Huanghua pear. Besides that, other botanical characteristics and fruiting habit are close to Huanghua pear.
2. Huanghua and Green Huanghua pear shared different fruit dot development pattern, in which the fruit dot on Huanghua fruit formed earlier and more intensively, developed faster and had the larger size than Green Huanghua fruit dot at the same developmental stage, whereas fruit dot on Green Huanghua fruit formed later, developed slower; Huanghua pear fruit surface wax before10weeks after flowering (WAF) was more, but continually decreased from10WAF, whereas Green Huanghua pear fruit surface wax was constantly present on the fruit from the observed fruit of4WAF to ripe fruit; the cuticle of Huanghua fruit was basically intact before10WAF, but the cuticle at the edge of fruit dot began to crack at10WAF, and the whole fruit cuticle completely cracked at the maturity, whereas Green Huanghua fruit cuticle from the young fruit to fruit ripening remained intact and rarely rupture; Before10WAF, the fruit epidermal membrane of Huanghua and Green Huanghua pear were both intact, colorless and transparent, but after10WAF, the fruit cuticle of Huanghua pear gradually cracked and finally completely cracked into pieces, exposed epidermal cells suberized and the epidermal membrane showed russet, whereas the epidermal membrane of Green Huanghua pear was almost intact, colorless, transparent and suberization seldom occurred for epidermal cells; Ripe pear peel structure followed by the cuticle, the epidermal cell layer and sub-epidermal cell layer from outside to inside. For ripe Huanghua pear, its fruit surface was rough, cuticle completely cracked, the epidermal cells exposed or underneath the cuticle suberized, showing russet, and could not be stained by phloroglucinol solution, whereas for ripe Green Huanghua pear, its fruit surface was smooth, cuticle was intact and epidermal cells underneath it was green or colorless, without suberization. The epidermal membrane of ripe Huanghua pear fruit was russet, and determined the fruit color, whereas the epidermal membrane of ripe Green Huanghua pear fruit was coloeless and transparent, did not determine the fruit color.
3. Green Huanghua pear peel with heavy rust was the same as Huanghua pear peel in substance, and the differences of fruit peel with light rust and heavy rust was the degree of cuticle injury, the suberized degree of epidermal cells, and the integrality of the suberized epidermal cell layer.
4. A method to isolate high molecular weight RNA (HMW RNA) and low molecular weight RNA (LMW RNA) from pear peels was developed using the Huanghua and Green Huanghua pear peel as materials. Using the cDNA synthesized from the HMW RNA of fruit peels as template, DDRT-PCR was carried out to clone differential expressed gene fragments. Pear defensin gene PpyDFN1(accession number HM044853) and fragment of phenylalanine ammonia-lyase gene PpyPAL1, as differentially expressed genes were cloned by5'RACE and5'homologous degenerate primers amplification based on the differentially expressed fragments. The two genes'differential expression in Huanghua and Green Huanghua pear peels of8WAF by quantitative real-time PCR (qRT-PCR). The expression level of PpyDFN1and PpyPAL1in Huanaghua pear peel was6.7times and2.2times higher than that in Green Huanghua pear, respectively.
The full length cDNA of Huanghua pear defensin gene PpyDFN1was567bp, containing5'untranslated region (UTR) with a length of72bp,3'UTR with a length of258bp, and coding region with a length of237bp. The5'UTR contained a (TC)7simple repeat sequence. The coding region encoded a polypeptide composed of78amino acid residues with molecular weight (MW)8.65kDa and isoelectric point (PI)9.36. BLASTP analysis indicated the polypeptide was precursor of defensin PpyDFN1. Signal prediction indicated that the first31amino acid residues was the signal peptide of PpyDFN1, and the left47amino acid residues was the mature defensin. Comparative homology modeling of PpyDFN1indicated that, PpyDFN1had a typical defensin structure consisting of a triple-stranded antiparallel (3-sheet and an α-helix, which was organized in a βαββ Clone and analysis of the genome sequence of PpyDFN1indicated that the genome sequence of PpyDFN1in Huanghua and Green Huanghua showed no difference and they both contained one intron and two exons. Results of bombardment on onion epidermal cells using the constructed vector for PpyDFN1subcellular location showed that defensin PpyDFN1located on the cell wall.
5. PpyPAL1, PpyC4H and PpyCCR showed higher expression level in peels of Huanghua and Green Huanghua pear from6WAF to10WAF, and the expression level of PpyPALl and PpyCCR in Huanghua pear peel was apparently higher than that in Green Huanghua pear. The period of above genes'higher expression according with the stage of fruit dot forming and enlargement, to some extent reflected the strong secondary metabolic activity in the peel at fruit dot forming and enlargement stage. Whereas the higher expression level of PpyPALl and PpyCCR in Huanghua pear peels, reflected the stronger phenylpropanoid secondary metabolic activity in Huanghua pear peel than that in Green Huanghua pear peel.
6. PpyDFNl gene was constitutively expressed in roots, leaf, stalk, sepal, petal, stigma and style, stamen, seed, flesh, and fruit peel. The expression level of PpyDFNl was depended on fruit dot developmental stages and PpyDFNl had a higher expression level during fruit dot forming and enlargement stage, whereas had a lower expression level in the later stage for fruit dot development; PpyDFNl showed a higher expression level in Huanghua pear peel than that in Green Huanghua pear. Wounding, ethylene, SA, MeJA, and H2O2strongly induced PpyDFNl expression, but ABA did not.
7. Transgenic plants constitutively expressing the pear defensin PpyDFNl exhibited enhanced resistance to B. cinerea infection. Transgenic tobacco plants expressing PpyDFNl had shorter internodes, lower plant height, delayed flowering time, shorter flower organs, shorter stalks, thicker stalks and thicker flower tube. Root of T1generation seedlings harboring the PpyDFNl gene was significantly shorter than that of seedlings from wild type seeds. PpyDFN1was isolated from the transgenic tobacco leaves using the Nickel-ion resin and the eluate containing PpyDFNl could inhibit the growth of Agrobacterium tumefaciens.
本文首先对黄花梨(果皮为褐色)的绿皮芽变——绿皮黄花进行了生物学特性的研究,其次对比研究了黄花梨和绿皮黄花梨成熟果实果皮结构、表皮膜结构,不同发育时期果点的发育、果皮发育和表皮膜发育,绿皮黄花梨成熟果实不同颜色果锈果皮与黄花梨褐色果皮的特性,然后以确定的黄花梨果皮色泽关键发育期时的两种梨果皮为材料,提取了高分子量RNA,用差异显示PCR (DDRT-PCR)和5'RACE等技术克隆了在黄花梨和绿皮黄花梨果皮中差异表达的梨防御素基因PpyDFN1全长和苯丙氨酸解氨酶基因片段,并对PpyDFN1进行了序列分析和亚细胞定位研究；接着对黄花梨和绿皮黄花梨不同发育时期果皮中参与苯丙烷次生代谢的苯丙氨酸解氨酶基因PpyPAL1、肉桂酸-4-羟化酶基因PpyC4H和肉桂酰CoA氧化还原酶基因PpyCCR表达水平进行了分析,以评估果皮发育期间两种梨果皮中的次生代谢水平差异。最后对PpyDFN1在黄花梨和绿皮黄花梨中的表达特点、转基因烟草中的功能和体外抑菌功能进行了研究。主要研究结果如下。
1.绿皮黄花为黄花梨稳定的绿皮芽变,其果皮为绿色,成熟期较黄花梨提前7-10天,果实可溶性总糖、总可溶性固形物和可滴定酸高于黄花梨,果实硬度低于黄花梨,其它植物学特性和结果习性都与黄花梨接近。
2.黄花梨与绿皮黄花梨的果点发育明显不同,黄花梨果点形成早而集中,果点发育快,果点大小较同期绿皮黄花梨果点大,而绿皮黄花梨果点形成晚,果点发育慢；黄花梨果面蜡质在花后10周以前较多,花后10周以后果面蜡质不断减少,而绿皮黄花梨果面蜡质从观察开始的花后4周到果实成熟一直存在；黄花梨角质膜花后10周前基本完整,从10周时从果点周围出现裂口到果实成熟时完全龟裂,而绿皮黄花梨果皮角质膜从幼果到果实成熟都保持完整,很少破裂；开花后10周以前,黄花梨与绿皮黄花梨表皮膜都是完整和无色透明的,开花后10周以后,黄花梨角质膜逐渐破裂,直至完全龟裂,裸露的表皮细胞木栓化,表皮膜呈现黄褐色,而绿皮黄花梨表皮膜从幼果到成熟都相对完整,无色透明,其表皮细胞很少木栓化；黄花和绿皮黄花梨果皮结构从外到内依次为角质膜、表皮细胞层和亚表皮细胞层,成熟黄花梨果皮表面粗糙,角质膜不完整,其下或裸露的表皮细胞木栓化,不能被间苯三酚染色,颜色为黄褐色,而成熟绿皮黄花梨果皮表面光滑,角质膜完整,其下表皮细胞为绿色或无色,没有木栓化。成熟黄花梨果实的表皮膜为黄褐色,是果皮颜色的决定者,而绿皮黄花梨果实表皮膜无色透明,不决定果皮色泽。
3.绿皮黄花梨的深锈果皮与黄花梨的黄褐色果皮本质上是一样的,而浅锈果皮和深锈果皮的区别主要在于角质膜的受损程度、表皮细胞的木栓化程度和木栓化表皮细胞层的完整性。
4.用黄花梨和绿皮黄花梨果皮为材料,建立了一种从果皮中分离高分子量RNA(HMW RNA)和低分子量RNA(LMW RNA)方法。用果皮中所提取的高分子量RNA合成的cDNA作为DDRT-PCR模板,克隆差异片段,结合5'RACE及5’同源简并引物扩增,克隆了黄花梨和绿皮黄花梨果皮中差异表达基因——梨防御素基因PpyDFN1(GenBank登录号为HM044853)和苯丙氨酸解氨酶基因PpyPAL1。实时荧光定量PCR进一步确定了两个基因在8周的黄花梨和绿皮黄花梨果皮中表达量存在差异,其中PpyDFN1在黄花梨果皮中的表达量为绿皮黄花梨果皮中表达量的6.7倍,苯丙氨酸解氨酶基因PpyPAL1在黄花梨果皮中的表达量为绿皮黄花梨果皮中的2.2倍。
黄花梨防御素基因PpyDFN1的全长cDNA序列为567bp,包括72bp的5’非翻译区,258bp的3’非翻译区和237bp的编码区。在5’非翻译区含有一个(TC)7简单重复序列。编码区编码一条长度为78个氨基酸残基组成的多肽链,该多肽链的分子量为8.65KDa,等电点为9.36。BLASTP序列分析表明,该多肽链为防御素的前体序列。信号肽预测分析表明,多肽链的前31个氨基酸序列为防御素的信号肽,余下47个氨基酸残基组成的序列为成熟的防御素。梨防御素三维结构的同源比对建模表明,防御素PpyDFN1呈现出典型的由一个α-螺旋,三个反向平行的p-折叠组成的βαββ防御素结构。基因组序列克隆和分析表明,PpyDFN1的基因组序列在黄花和绿皮黄花中没有差异,都含有一个内含子和2个外显子。构建的亚细胞定位载体用于轰击洋葱表皮细胞结果表明,PpyDFN1定位在细胞壁上。
5.黄花梨和绿皮黄花梨花后6周到10周的果皮中PpyPAL1、PpyC4H和PpyCCR表达量较高,其中在黄花梨果皮中ppyPAL1和PpyCCR表达量显著高于绿皮黄花梨果皮中的表达量。上述基因高表达量发生时期与果点形成和扩大期吻合,一定程度上反映了此时期果皮中旺盛的次生代谢活动。而黄花梨果皮中PpyPAL1和PpyCCR较高的表达量,反映了黄花梨果皮较绿皮黄花梨果皮中有着更为旺盛的苯丙烷类物资代谢活动。
6.梨防御素PpyDFN1在根、叶片、花梗、萼片、花瓣、雌蕊和柱头、雄蕊、种子、果肉和果皮中基础性表达。PpyDFN1基因的表达表现出果点发育时期依赖性,在果点形成和扩大期表达量较高,在果点形成后期表达量较低；PpyDFN1在黄花梨果皮中的表达量总体上较绿皮黄花梨高。机械损伤、乙烯、水杨酸(SA)、茉莉酸甲酯(MeJA)和H202都能促进PpyDFN1的表达,而ABA不能。
7.表达PpyDFN1的烟草叶片提高了对灰霉菌的抗性；转PpyDFN1基因植株节间缩短,高度降低,开花延迟,花器官较短,花梗较短,花梗较粗,花筒较粗等；表达PpyDFN1基因的T1代转基因幼苗的根系明显短于非转基因对照。用镍离子树脂从转基因植株中纯化了PpyDFN1,纯化的蛋白洗脱液可以抑制农杆菌的生长。
Fruit color is one of the important agronomic traits and appearance quality of goods for pear, and is also consumers'first sense before their purchasing decisions. Breeding pear cultivars bearing fruits with different fruit color to meet consumers'diversified needs is a long-term goal for pear breeding, and now works on pear fruit color is becoming a research focus. Study on fruit skin development of russet and green pears, and differentially expressed genes during fruit color formation in pear could help deeply understand the formation mechanism of pear russet fruit and green fruit, promote russet and green pear molecular breeding theory formation and breeding work. Russet pear and its green bud sports supply the ideas materials for pear fruit color research.
In this article, biological characteristics was firstly studied on Huanghua pear (russet fruit) green skin bud sport——Green Huanghua. Secondly, studies on structure of fruit peel of ripe fruits and epidermal structure for Huanghua and Green Huanghua pear, fruit dot development, fruit skin development and epidermis development, the characters of Green Huanghua ripe fruit peels with different level of fruit rust and Huanghua pear russet peel were performed. Thirdly, high molecular weight RNA was isolated from the two kinds of pear peels sampled at the key stage of fruit skin development, and differentially expressed genes——the full length of pear defensin gene PpyDFNl and fragment of phenylalanine ammonia-lyase gene PpyPAL1in Huanghua and Green Huanghua pear peels were cloned by differential display reverse transcription polymerase chain reaction (DDRT-PCR) and rapid amplification of cDNA5'end (5'RACE). The sequence of PpyDFN1was further analy sized and subcellular location of PpyDFN1was studied. Next, the expression level of genes involved in phenylpropanoid secondary metabolites including phenylalanine ammonia-lyase gene PpyPAL1, cinnamic acid-4-hydroxylase gene PpyC4H and cinnamoyl-CoA oxidoreductase gene PpyCCR was determined in peels of Huanghua and Green Huanghua pear at different developmental stages to evaluate the level of secondary metabolism. Finally, the expression analysis of PpyDFNl in Huanghua and Green Huanghua pear, functional analysis in transgenic tobacco plants and bacterial inhibition in vitro were studied. The results were showed as follows.
1. Green Huanghua is a stable green skin bud sport of Huanghua pear. Its fruit skin is green, maturiy is7to10days ahead of Huanghua pear, and the total soluble sugar, total soluble solids and titratable acidity is higher than that of Huanghua pear fruit, but the fruit hardness is lower than Huanghua pear. Besides that, other botanical characteristics and fruiting habit are close to Huanghua pear.
2. Huanghua and Green Huanghua pear shared different fruit dot development pattern, in which the fruit dot on Huanghua fruit formed earlier and more intensively, developed faster and had the larger size than Green Huanghua fruit dot at the same developmental stage, whereas fruit dot on Green Huanghua fruit formed later, developed slower; Huanghua pear fruit surface wax before10weeks after flowering (WAF) was more, but continually decreased from10WAF, whereas Green Huanghua pear fruit surface wax was constantly present on the fruit from the observed fruit of4WAF to ripe fruit; the cuticle of Huanghua fruit was basically intact before10WAF, but the cuticle at the edge of fruit dot began to crack at10WAF, and the whole fruit cuticle completely cracked at the maturity, whereas Green Huanghua fruit cuticle from the young fruit to fruit ripening remained intact and rarely rupture; Before10WAF, the fruit epidermal membrane of Huanghua and Green Huanghua pear were both intact, colorless and transparent, but after10WAF, the fruit cuticle of Huanghua pear gradually cracked and finally completely cracked into pieces, exposed epidermal cells suberized and the epidermal membrane showed russet, whereas the epidermal membrane of Green Huanghua pear was almost intact, colorless, transparent and suberization seldom occurred for epidermal cells; Ripe pear peel structure followed by the cuticle, the epidermal cell layer and sub-epidermal cell layer from outside to inside. For ripe Huanghua pear, its fruit surface was rough, cuticle completely cracked, the epidermal cells exposed or underneath the cuticle suberized, showing russet, and could not be stained by phloroglucinol solution, whereas for ripe Green Huanghua pear, its fruit surface was smooth, cuticle was intact and epidermal cells underneath it was green or colorless, without suberization. The epidermal membrane of ripe Huanghua pear fruit was russet, and determined the fruit color, whereas the epidermal membrane of ripe Green Huanghua pear fruit was coloeless and transparent, did not determine the fruit color.
3. Green Huanghua pear peel with heavy rust was the same as Huanghua pear peel in substance, and the differences of fruit peel with light rust and heavy rust was the degree of cuticle injury, the suberized degree of epidermal cells, and the integrality of the suberized epidermal cell layer.
4. A method to isolate high molecular weight RNA (HMW RNA) and low molecular weight RNA (LMW RNA) from pear peels was developed using the Huanghua and Green Huanghua pear peel as materials. Using the cDNA synthesized from the HMW RNA of fruit peels as template, DDRT-PCR was carried out to clone differential expressed gene fragments. Pear defensin gene PpyDFN1(accession number HM044853) and fragment of phenylalanine ammonia-lyase gene PpyPAL1, as differentially expressed genes were cloned by5'RACE and5'homologous degenerate primers amplification based on the differentially expressed fragments. The two genes'differential expression in Huanghua and Green Huanghua pear peels of8WAF by quantitative real-time PCR (qRT-PCR). The expression level of PpyDFN1and PpyPAL1in Huanaghua pear peel was6.7times and2.2times higher than that in Green Huanghua pear, respectively.
The full length cDNA of Huanghua pear defensin gene PpyDFN1was567bp, containing5'untranslated region (UTR) with a length of72bp,3'UTR with a length of258bp, and coding region with a length of237bp. The5'UTR contained a (TC)7simple repeat sequence. The coding region encoded a polypeptide composed of78amino acid residues with molecular weight (MW)8.65kDa and isoelectric point (PI)9.36. BLASTP analysis indicated the polypeptide was precursor of defensin PpyDFN1. Signal prediction indicated that the first31amino acid residues was the signal peptide of PpyDFN1, and the left47amino acid residues was the mature defensin. Comparative homology modeling of PpyDFN1indicated that, PpyDFN1had a typical defensin structure consisting of a triple-stranded antiparallel (3-sheet and an α-helix, which was organized in a βαββ Clone and analysis of the genome sequence of PpyDFN1indicated that the genome sequence of PpyDFN1in Huanghua and Green Huanghua showed no difference and they both contained one intron and two exons. Results of bombardment on onion epidermal cells using the constructed vector for PpyDFN1subcellular location showed that defensin PpyDFN1located on the cell wall.
5. PpyPAL1, PpyC4H and PpyCCR showed higher expression level in peels of Huanghua and Green Huanghua pear from6WAF to10WAF, and the expression level of PpyPALl and PpyCCR in Huanghua pear peel was apparently higher than that in Green Huanghua pear. The period of above genes'higher expression according with the stage of fruit dot forming and enlargement, to some extent reflected the strong secondary metabolic activity in the peel at fruit dot forming and enlargement stage. Whereas the higher expression level of PpyPALl and PpyCCR in Huanghua pear peels, reflected the stronger phenylpropanoid secondary metabolic activity in Huanghua pear peel than that in Green Huanghua pear peel.
6. PpyDFNl gene was constitutively expressed in roots, leaf, stalk, sepal, petal, stigma and style, stamen, seed, flesh, and fruit peel. The expression level of PpyDFNl was depended on fruit dot developmental stages and PpyDFNl had a higher expression level during fruit dot forming and enlargement stage, whereas had a lower expression level in the later stage for fruit dot development; PpyDFNl showed a higher expression level in Huanghua pear peel than that in Green Huanghua pear. Wounding, ethylene, SA, MeJA, and H2O2strongly induced PpyDFNl expression, but ABA did not.
7. Transgenic plants constitutively expressing the pear defensin PpyDFNl exhibited enhanced resistance to B. cinerea infection. Transgenic tobacco plants expressing PpyDFNl had shorter internodes, lower plant height, delayed flowering time, shorter flower organs, shorter stalks, thicker stalks and thicker flower tube. Root of T1generation seedlings harboring the PpyDFNl gene was significantly shorter than that of seedlings from wild type seeds. PpyDFN1was isolated from the transgenic tobacco leaves using the Nickel-ion resin and the eluate containing PpyDFNl could inhibit the growth of Agrobacterium tumefaciens.
引文
Allen A, Snyder AK, Preuss M, Nielsen EE, Shah DM, Smith TJ.2008. Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth. Planta,227:331-339.
Almeida MS, Cabral KMS, Kurtenbach E, Almeida FCL, Valente AP.2002. Solution structure of Pisum sativum defensin 1 by high resolution NMR:plant defensins, identical backbone with different mechanisms of action. J. Mol. Biol.,315:749-757.
Aluru M, Curry J, O'Connell MA.1999. Nucleotide sequence of a defensin or y-thionin-like gene (accession no. AF128239) from habanera chili. Plant Physiol.,120:633.
Amarante C, Banks NH, Ganesh S.2001. Relationship between character of skin cover of coated pears and permeance to water vapour and gases. Postharvest Biol. Tec.,21:291-301.
Anuradha TS, Divya K, Jami SK, Kirti PB.2008. Trangenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep.,27:1777-1786.
Arulsekar S, Parfolt DE, Keste DE.] 986. Comparison of isozyme variability in peach and almond cultivars. J. Hered.,77:272-274.
Azuma A, Kobayashi S, Goto-Yamamoto N, Shiraishi M, Mitani N, Yakushiji H, Koshita Y.2009. Color recovery in berries of grape (Vitis vinifera L.)'Benitaka', a bud sport of'Italia', is caused by a novel allele at the VvmybAl locus. Plant Sci.,176:470-478.
Benbouza H, Jacquemin JM, Baudoin JP, Mergeai G.2006. Optimization of a reliable, fast, cheap and sensitive silver staining method to detect SSR markers in polyacrylamide gels. Biotechnol. Agron. Soc. Environ,10(2):77-81.
Bendtsen JD, Nielsen H, von Heijne G, Brunak S.2004. Improved prediction of signal peptides:SignalP 3.0. J. Mol. Biol.,340:783-795.
Bernards MA, Fleming WD, Llewellyn DB, Priefer R, Yang X, Sabatino A, Plourde GL.1999. Biochemical characterization of the suberization-associated anionic peroxidase of potato. Plant Physiol.,121:135-146.
Bernards MA, Summerhurst DK, Razem FA.2004. Oxidases, peroxidases and hydrogen peroxide:the suberin connection. Phytochem Rev.,3:113-126.
Bernards MA.2002. Demystifying suberin. Can. J. Bot,80:227-240.
Bozkurt O, Unver T, Akkaya MS.2007. Genes associated with resistance to wheat yellow rust disease identified by differential display analysis. Physiol. Mol. Plant P.,71:251-259.
Broekaert WF, Cammue BPA, De Bolle MFC, Thevissen K, De Samblanx G, Osborn RW.1997. Antimicrobial peptides from plants. Crit. Rev. Plant Sci.,16:297-323.
Broekaert WF, Terras FRG, Cammue BPA, Osborn RW.1995. Plant defensins:novel antimicrobial peptides as components of the host defense system. Plant Physiol.,108:1353-1358.
Bruix M, Jimenez MA, Santoro J, Gonzalez C, Colilla FJ, Mendez E, Rico M.1993. Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins. Biochem.,32:715-724.
Carra A, Gambino G., Schubert A.2007. A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues. Anal Biochem.,360:318-320.
Carreno J, Martinez A, Almela L, Fernandez-Lopez JA.1995. Proposal of an index for the objective evaluation of the colour of red table grapes. Food Res. Int.,28(4):373-377.
Carvalho AdeO, Gomes VM.2009. Plant defensins—prospects for the biological functions and biotechnological properties. Peptides,30:1007-20.
Chang, S, Puryear J, Cairney J.1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep.,11:113-116.
Cheng GW, Breen PJ.1991. Activity of Pheyla lanine ammonia lyase and concent ration of anthocyanins and phenolics in developing strawberry fruit. J. Amer. Soc. Hortic. Sci.,116 (5):865-869.
Chevreau E, Leuliette S, Gallet M.1997. Inheritance and linkage of isozyme loci in pear (Pyrus communis L.). Theor. Appl. Genet.,94(3,4):498-506.
Chevreau E, Manganaris AG, Gallet M.1999. Isozyme segregation in five apple progenies and potential use for map construction. Theor. Appl. Genet.,98(2):329-336.
Chiang CC, Hadwiger LA.1991. The Fusarium solani-induced expression of a pea gene family encoding high cysteine content proteins. Mol. Plant Microbe Interact.,4:324-331.
Choi MS, Kim YH, Park HM, Seo BY, Jung JK, Kim ST, Kim MC, Shin DB, Yun HT, Choi IS, Kim CK, Lee JY.2009. Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against Brown planthopper (Nilaparvata lugens) in transgenic rices. Mol. Cells,28:131-137.
Currie AJ, Noiton DA, Lawes GS, et al.1997. Preliminary results of differentiating apple sports by pollen ultrastructures. Euphytica,98:155-161.
de Beer A, Vivier MA.2008. Vv-AMP1, a ripening induced peptide from Vitis vinefera shows strong antifungal activity. BMC Plant Biol.,8:75.
de Godoy A, Cordenunsi BR, Lajolo FM, do Nascimento JRO.2010. Differential display and suppression subtractive hybridization analysis of the pulp of tipening banana. Sci. Hortic.,124: 51-56.
De Lano WL.2002. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA, USA., http://www.pymol.org
de Zelicourt A, Letousey P, Thoiron S, Campion C, Simoneau P, Elmorjani K, Marion D, Simier P, Delavault P.2007. Ha-DEF1, a sun flower defensin, induces cell death in Orobanche parasitic plants. Planta,226:591-600.
Deng ZN, Gentile A, Nicolosi E, Domina F, Vardi A, Tribulato E.1995. Identification of in vivo and in vitro lemon mutants by RAPD markers. J. Hortic. Sci.,70(1):117-125.
Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD.1996. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A.,93:6025-6030.
Do HM, Lee SC, Jung HW, Sohn KH, Hwang BK.2004. Differential expression and in situ localization of a pepper defensin (CADEF1) gene in response to pathogen infection, abiotic elicitors and environmental stresses in Capsicum annuum. Plant Sci.,166:1297-1305.
Doughty J, Dixon S, Hiscock SJ, Willis AC, Parkin IAP, Dickinson HG.1998. PCP-A1, a defensin-like brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression. Plant Cell,10:1333-1347.
du Preez MG, Rees DJG.2008. Investigating the molecular mechanism of anthocyanin production in 'Bon Rouge'Pyrus communis L. Acta. Horticul.,800:383-390.
Dussi MC, Sugar D, Wrolstad RE.1995. Characterizing and quantifying anthocyanins in red pears and the effect of light quality on fruit color. J. Amer. Soc. Hortic. Sci.,120(5):785-789.
Fabi JP, Lajolo FM, do Nascimento JRO.2009. Cloning and characterization of transcripts differentially expressed in the pulp of ripening papaya. Sci. Hortic.,121:159-165.
Fant F, Vranken W, Broekaet W, Borremans F.1998. Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR. J. Mol. Biol.,279:257-270.
Fedorova M, van de Mortel J, Matsumoto PA, Cho J, Town CD, Vandenbosch KA, Gantt JS, Vance CP. 2002. Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula. Plant Physiol.,130:519-537.
Ferrer A, Remon S, Negueruela A I, Oria R.2005. Changes during the ripening of the very late season Spanish peach cultivar Calanda feasibility of using CIELAB coordinates as maturity indices. Sci. Hortic.,105:435-446.
Finkina El, Shramova El, Tagaev AA, Ovchinnikova TV.2008. A novel defensin from the lentil Lens culinaris seeds. Biochem. Bioph. Res. Co.,371:860-865.
Fischer TC, Gosch C, Pfeiffer J, Halbwirth H, Halle C, Stich K, Forkmann G.2007. Flavonoid genes of pear (Pyrus communis). Trees,21:521-529.
Gamble J, Jaeger SR, Roger Harker F.2006. Preferences in pear appearance and response to novelty among Australian and New Zealand consumers. Postharvest Biol. Tec.,41:38-47.
Gang DR, Wang J, Dudareva N, Nam KH, Simon JE, Lewinsohn E, Pichersky E.2001. An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol.,125:539-555.
Gao AG, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang J, Rommens C.M. 2000. Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat. Biotechnol.,18:1307-1310.
Garcia-Olmedo F, Molina A, Alamillo JM, Rodriguez-Palenzuela P.1998. Plant defense peptides. Biopolymers,47:479-491.
Graca J, Santos S.2007. Suberin:a biopolyester of plants'skin. Macromol Biosci.,7:128-135.
Graca J.2010. Hydroxycinnamates in suberin formation. Phytochem Rev.,9:85-91.
Graham MA, Silverstein KA, Cannon SB, VandenBosch KA.2004. Computational identification and characterization of novel genes from legumes. Plant Physiol.,135:1179-1197.
Graham MA, Silverstein KAT, VandenBosch KA.2008. Defensin-like genes:genomic perspectives on a diverse superfamily in plants. Crop. Sci.,48:s3-s11.
Granger AR, Clarke GR, Jackson JF.1993. Sweet cherry cultivar identification by leaf isozyme polymorphism. Theor. Appl. Genet.,86(4):458-464.
Gu Q, Kawata EE, Morse MJ, Wu HM, Cheung AY.1992. A flower-specific cDNA encoding a novel thionin in tobacco. Mol. Gen. Genet.,234:89-96.
Guo S, Zheng Y, Joung J, Liu S, Zhang Z, Crasta OR, Sobral BW, Xu Y, Huang S, Fei Z.2010. Transcriptome sequencing and comparative analysis of cucumber flowers with different sex types. BMC Genomics,11:384-396.
Hanks JN, Snyder AK, Graham MA, Shah RK, Blaylock LA, Harrison MJ, Shah DM.2005. Defensin gene family in Medicago trvncatula:structure, expression and induction by signal molecules. Plant Mol. Biol.,58:385-399.
Hauptmann M, Pleschberger H, Mai C, Follrich J, Hansmann C.2010. The potential of color measurements with the CIEDE2000 equation in wood science. Eur. J. Wood Prod., DOI 10.1007/s00107-011-0575-6
He FM, Zhu YP, Zhang YZ.2008. Identification and characterization of differentially expressed genes involved in pharmacological activeties of roots of Panax notoginseng during plant growth. Plant Cell Rep.,275:923-930.
Huang GJ, Lai HC, Chang YS, Sheu MJ, Lu TL, Huang SS, Lin YH.2008. Antimicrobial, dehydroascorbate reductase, and monodehydroascorbate reductase activities of defensin from sweet potato [Ipomoea batatas (L.) Lam.'Tainong 57'] storage roots. J. Agric. Food Chem.,56:2989-95.
Iandolino AB, Goes da Silva F, Lim H, Choi H, Williams LE, Cook DR.2004. High-quality RNA, cDNA, and derived EST libraries from Grapevine (Vitis vinifera L.). Plant Mol. Biol. Rep.,22: 269-278.
Janisiewicz WJ, Pereira IB, Almeida MS, Roberts DP, Wisniewski M, Kurtenbach E.,2008. Improved biocontrol of fruit decay fungi with Pichia pastoris recombinant strains expressing Psd 1 antifungal peptide. Postharvest Biol. Tech.,47:218-225.
Jha S, Chattoo BB.2010. Expression of a plant defensin in rice confers resistance to fungal phytopathogens. Transgenic Res.,19:373-384.
Kant P, Liu WZ, Pauls P.2009. PDC1, a corn defensin peptide expressed in Escherichia coli and Pichia pastoris inhibits growth of Fusarium graminearum. Peptides,30:1593-1599.
Kanzaki H, Nirasawa S, Saitoh H, Ito M, Nishihara M, Terauchi R, Nakamura I.2002. Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor. Appl. Genet.,105:809-814.
Karim K, Hatem CM, Mokhtar T, Mbarek BN.2011. Identification of salt stress-induced transcripts in barley leaves by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Afr. J. Biotechnol.,10(83):19356-19364.
Karunanandaa B, Singh A, Kao T-H.1994. Characterization of a predominantly pistil-expressed gene encoding a thionin-like protein of Petunia inflata. Plant Mol. Biol.,26:459-464.
Kato M, Kamo T, Wang R, Nishikawa F, Hyodo H, Ikoma Y, Sugiura M, Yano M.2002. Wound-induced ethylene synthesis in stem tissue of harvested broccoli and its effect on senescence and ethylene synthesis in broccoli florets. Postharvest Biol. Tec.,24:69-78.
Kobayashi S, Ishimaru M, Ding CK, Yakushiji H, Goto N.2001. Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Plant Sci.,160:543-550.
Kobayashi S, Yamamoto N, Hirochika H.2004. Retrotransposon induced mutations in grape skin color. Science,304:982.
Koike M, Okamoto T, Tsuda S, Imai R.2002. A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation. Biochem. Bioph. Res. Co.,298:46-53.
Kolattukudy PE.1980. Biopolyester membranes of plants:cutin and suberin. Science,208:990-1000.
Kumar S, Tamura K, Nei M.2004. MEGA3:Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform.,5:150-163.
Lang P, Zhang CK, Eel RC, Dane F, Dozier WA.2005. Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display. Gene,359:111-118.
Lay FT, Brugliera F, Anderson MA.2003. Isolation and properties of floral defensins from Ornamental tobacco and Petunia. Plant Physiol.,131:1283-1293.
Lay FT, Schirra HJ, Scanlon MJ, Anderson MA, Craik DJ.2003. The three-dimensional solution structure of NaD1, a new floral defensin from Nicotiana alata and its application to a homology model of the crop defense protein alfAFP. J. Mol. Biol.,325:175-188.
Liang P, Meade J D, Pardee A B.2007. A protocol for differential display of mRNA expression using either fluorescent or radioactive labeling. Nat. Protoc.,2(3):457-470.
Lievens S, Goormachtig S, Holsters M.2001. A critical evaluation of differential display as a tool to identify genes involved in legume nodulation:looking back and looking forward. Nucleic Acids Res.,29(17):3459-3468.
Liu YZ, Liu Q, Xiong JJ, Deng XX.2007. Difference of a citrus late-ripening mutant (Citrus sinensis) from its parental line in sugar and acid metabolism at the fruit ripening stage. Science in China Series C:Life Sciences,50(4):511-517.
Lu XP, Liu YZ, An JC, Hu HJ, Peng SA.2011. Isolation of a cinnamoyl CoA reductase gene involved in formation of stone cells in pear (Pyrus pyrifolia). Acta. Physiol. Plant,33:585-591.
Ma DZ, Wang HX, Ng TB.2009. A peptide with potent antifungal and antiproliferative activities from Nepalese large red beans. Peptides,30:2089-2094.
Maitra N, Cushman JC.1998. Characterization of a drought-induced soybean cDNA encoding a plant defensin (Accession No. U12150) (PGR 98-213). Plant Physiol.,118:1536.
Matsuoka M, Sanada Y.1991. Expression of photosynthetic genes from the C4 plant, maize, in tobacco. Mol. Gen. Genet.,225:411-419.
Mayer B, Houlne G, Pozueta-Romero J, Shanta ML, Shantz R.1996. Fruit-specific expression of a defensin-type gene family in bell pepper. Plant Phsysiol.,112:615-655.
Melotto M, Underwood W, He SY.2008. Role of stomata in plant innate immunity and foliar bacterial disease. Annu. Rev. Phytopathol.,46:101-122.
Melotto M, Underwood W, Koczan J, Nomura K, He SY.2006. Plant stomata function in innate immunity against bacteria invasion. Cell,126:969-980.
Mendgen K, Hahn M, Deising H.1996. Morphogenesis and mechanisms of penetration by plant pathogenic fungi. Annu. Rev. Phytopathol.,34:367-386.
Mergaert P, Nikovics K, Kelemen Z, Maunoury N, Vaubert D, Kondorosi A, Kondorosi E.2003. A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol.,132:161-173.
Meyer B, Houlne G, Pozueta-Romero J, Schantz ML, Schantz R.1996. Fruit-specific expression of a defensin-type gene family in bell pepper. Plant Physiol.,112:615-622.
Ming D, Hellekant G.1994. Brazzein, a new high potency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS. Lett.,355:106-108.
Mirouze M, Sels J, Richard O, Czernic P, Loubet S, Jacquier A, Francois IEJA, Cammue BPA, Lebrun M, Berthomieu P, Marques L.2006. A putative novel role for plant defensins:a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance. Plant J.,47:329-42.
Moreno M, Segura A, Garcia-Olmedo F.1994. Pseudothionin-Stl, a potato peptide active against potato pathogens. Eur. J. Biochem.,223:135-139.
Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant,15:473-497.
Nielsen M, Lundegaard C, Lund O, Petersen TN.2010. CPHmodels-3.0—remote homology modeling using structure-guided sequence profiles. Nucleic Acids Res.,38:w576-w581.
Oberschmidt O, Grundler FMW, Kleine M.2003. Identification of a putative cation transporter gene from sugar beet (Beta vulgaris L.) by DDRT-PCR closely linked to the beet cyst nematode resistance gene HsI (pro-1). Plant Sci.,165(4):777-784.
Odintsova TI, Egorov TA, Musolyamov AK, Odintsova MS, Pukhalskv VA, Grishin EV.2007. Seed defensins from T. kiharae and related species:genome localization of defensin-encoding genes. Biochime.,89:605-612.
Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D, Kawano N, Sakakibara T, Namiki S, Itoh K, Otsuka K, Matsuzaki M, Nozaki H, Kuoroiwa T, Nakano A, Kanaoka MM, Dresselhaus T, Sasaki N, Higashiyama T.2009. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature, 458:357-361.
Oltean L, Teischinger A, Hansmann C.2008. Wood surface discolouration due to simulated indoor sunlight exposure. Holz. Roh. Werkst,66:51-56.
Parashina EV, Serdobinskii LA, Kalle EG, Lavorova NV, Vetisov VA, Lunin VG, Naroditskii BS.2000. Genetic engineering of oilseed rape ans tomato by expression of a radish defensin gene. Russ. J. Plant Physiol.,47:417-423.
Park HC, Kang YH, Chun HJ, Koo JC, Cheong YH, Kim CY, Kim MC, Chung WS, Kim JC, Yoo JH, Koo YD, Koo SC, Lim CO, Lee SY, Cho MJ.2002. Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage. Plant Mol. Biol.,50:59-69.
Passardi F, Penel C, Dunand C.2004. Performing the paradoxical:how plant peroxidases modify the cell wall. Trends Plant Sci.,9:534540.
Pelegrini PB, Franco OL.2005. Plant y-thionins:novel insights on the mechanism of action of a multi-functional class of defense proteins. Int. J. Biochem. Cell B.,37:2239-2253.
Penninckx I A, Thomma BP, Buchala A, Metraux JP, Broekaert WF.1998. Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell,10:2103-2113.
Pollard M, Beisson F, Li Y, Ohlrogge JB.2008. Building lipid barriers:biosynthesis of cutin and suberin. Trends Plant Sci.,13:236-246.
Ponsuksili S, Wimmers K, Schellander K.2001. Application of differential display RT-PCR to identify porcine liver ESTs. Gene,280:75-85.
Ribeiro SFF, Carvalho AO, Da Cunha M, Rodrigues R, Cruz LP, Melo VMM, Vasconcelos IM, Melo EJT, Gomes VM.2007. Isolation and characterization of novel peptides from chilli pepper seeds: Antimicrobial activities against pathogenic yeasts. Toxicon,50:600-611.
Schafleitner R, Wilhelm E.2002. Isolation of wound-responsive genes from chestnut (Castanea sativa) microstems by mRNA display and their differential expression upon wounding and infection with the chestnut blight fungus (Chryphonectriaparasitica). Physiol. Mol. Plant Pathol.,61:339-348.
Scott KD, Alblett EM, Lee LS, Henry RJ.2000. AFLP markers distinguishing an early mutant of Flame Seedless grape. Euphytica,113:245-249.
Segura A, Monero M, Molina A, Garcia-Olmedo F.1998. Novel defensin subfamily from spinach (Spinacia oleracea). FEBS Lett.,435:159-162.
Silverstein KAT, Graham MA, Paape TD, VandenBosch KA.2005. Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant Physiol.,138:600-610.
Soler M, Serra O, Molinas M, Huguet G, Fluch S, Figueras M.2007. A genomic approach to suberin biosynthesis and cork differentiation. Plant Physiol.,144:419-431.
Song X, Zhou Z, Wang J, Wu F, Gong W.2004. Purification, characterization and preliminary crystallographic studies of a novel plant defensin from Pachyrrhizus erosus seeds. Acta. Cryst., D60: 1121-1124.
Stiekema WJ, Heidekamp F, Dirkse WQ Van Beckum J, De Haan P, Ten Bosch C, Louwerse JD.1988. Molecular cloning and analysis of four potato tuber mRNAs. Plant Mol. Biol.,11:255-269.
Stotz HU, Spence B, Wang Y.2009. A defensin from tomato with dual function in defense and development. Plant Mol. Biol.,71:131-143.
Swathi Anuradha T, Divya K, Jami SK, Kirti PB.2008. Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep.,27:1777-1786.
Tani E, Polidoros AN, Flemetakis E, Stedel C, Kalloniati C, Demetriou K, Katinakis P, Tsaftaris AS. 2009. Characterization and expression analysis of AGAMOUS-like, SEEDSTICK-like, and SEPALLATA-like MADS-box genes in peach (Prunus persica) fruit. Plant Physiol. Bioch.,47: 690-700.
Tavares LS, Santos Mde O, Viccini LF, Moreira JS, Miller RNG, Franco OL.2008. Biotechnological potential of antimicrobial peptides from flowers. Peptides,29:1842-1851.
Terras FRG, Eggermont K, Kovaleva V, Raikhel NV, Osborn RW, Kester A, Rees SB, Torrekens S, Van Leuven F, Vanderleyden J et al.1995. Small cysteine-rich antifungal proteins from radish:their role in host defense. Plant Cell,7:573-588.
Thomma BPHJ, Broekaert WF.1998. Tissue-specific expression of plant defensin genes PDF2.1 and PDF2.2 in Arabidopsis thaliana. Plant Physiol. Biochem.,36:533-537.
Thomma BPHJ, Camme BPA, Thevissen K.2002. Plant defensins. Planta,216:193-202.
Thompson JD, Higgins DG, Gibson TJ.1994. CLUSTALW:improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res.,22:4673-4680.
Torres AM, Bergi BO.1980. Fruit and leaf isozymes as genetics in avocado. J. Amer. Soc. Hort. Sci.,105: 614-619.
Torres GAM, Pflieger S, Corre-Menguy F, Mazubert C, Hartmann C, Lelandais-Briere C.2006. Identification of novel drought-related mRNAs in common bean roots by differential display RT-PCR. Plant Sci.,171:300-307.
Urdangarin MC, Norero NS, Broekaert WF, de la Canal L.2000. A defensin gene expressed in sunflower inflorescence. Plant Physiol. Biochem.,238(3):253-258.
van den Heuvel KJPT, Hulzink JMR, Barendse GWM, Wullems GJ.2001. The expression of tgas118, encoding a defensin in Lycopersicon esculentum is regulated by gibberellin. J. Exp. Bot.,52: 1427-1436.
van Kan JA.2006. Licensed to kill:the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci.,11: 247-253.
Veeden NJ, Land RC.1985. Identification of apple cultivars by isozyme phenotypes. J. Amer. Soc. Hort. Sci.,110:509-515.
Velculescu VE, Zhang L, Vogelstein B, Kinzler KW.1995. Serial analysis of gene expression. Science, 270:484-487.
Vijayan S, Guruprasad L, Kirti PB.2008. Prokaryotic expression of a constitutively expressed Tephrosia villosa defensin and its potent antifungal activity. Appl. Microbiol. Biotechnol.,80:1023-32.
Viljoen MM, Hoysamer M.1995. Biochemical and regulatory aspects of anthocuanin synthesis in apple and pears. Journal of the Southern African Society for Horticultural Sciences,5(1):1-6.
Vinterhalter DV, James DJ.1986. The use of peroxidase polymorphism in the identification of Mailing and Mailing Merton apple rootstock. J. Hort. Sci.,61:147-152.
Vuylsteke M, Peleman JD, van Eiik MJT.2007. AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis. Nat. Protoc.,2(6):1399-1413.
Walker AR, Lee E, Robinson SP.2006. Two new grape cultivars, bud sports of Cabernet Saurignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus. Plant Mol. Biol.,62:623-635.
Wan CY, Wilkins TA.1994. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L). Anal Biochem.,223(1):7-12.
Wang X, Tang C, Zhang G, Li Y, Wang C, Liu B, Qu Z, Zhao J, Han Q, Huang L, Chen X, Kang Z.2009. cDNA-AFLP analysis reveals differential gene expression in compatible interaction of wheat challenged with Puccinia striiformis f. sp. Tritici. BMC Genomics,10:289 doi:10.1186/1471-2164-10-289.
Wang YP, Nowak G, Culley D, Hadwiger LA, Fristensky B.1999. Constitutive expression of pea defense gene DRR206 confers resistance to blackleg (Leptosphaeria maculans) disease in transgenic canola (Brassica napus). Mol. Plant Microbe Interact.,12:410-418.
Welsh J,McClelland M.1990 Fingerprinting geuomes using PCR with arbitrary primers. Nucleic. Acids Res.,18 (24):7213-7218.
Wijaya R, Neumann GM, Condron R, Hughes AB, Poly GM.2000. Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin. Plant Sci.,159:243-55.
Williams JG, Kubeilk AR, Livak KJ, et al.1990. DNA polymorphisms amplified by arbit rary primers are useful as genetic markers. Nucleic. Acids Res.,18 (22):6531-6535.
Willingdon R.1913. Inheritance of the russet skin in the pear. Science,37(943):156.
Wisniewski ME, Bassett CL, Artlip TS, Webb RP, Janisiewicz WJ, Norelli JL, Goldway M, Droby S. 2003. Characterization of a defensin in bark and fruit tissues of peach and antimicrobial activity of a recombinant defensin in the yeast, Pichia pastor is. Physiol. Plant.,119:563-72.
Wong JH, Ng TB.2005. Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase. Peptides,26:1120-6.
Wong JH, Zhang XQ, Wang HX, Ng TB.2006. A mitogenic defensin from white cloud beans (Phaseolus vulgaris). Peptides,27:2075-81.
Xiao H, Jiang N, Schaffner E, Stockinger EJ, van der Knaap E.2008. A retransposon-mediated gene duplication underlies morphplogical variation of tomato fruit. Science,319:1527-1530.
Xu Q, Yu KQ, Zhu AD, Ye JL, Liu Q, Zhang JC, Deng XX.2009. Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange (Citus sinensis) red-flesh mutant. BMC Genomics,10:540.
Ye W, Qin Y, Ye Z, da Silva JAT, Zhang L, Wu X, Lin S, Hu G.2009. Seedless mechanism of a new mandarin cultivar'Wuzishatangju'(Citrus reticulata Blanco). Plant Sci.,177:19-27.
Ye XY, Ng TB.2001. Peptides from pinto bean and red bean with sequence homology to cowpea 10-kda protein precursor exhibit antifungal, mitogenic, and HIV-1 reverse transcriptase-inhibitory activities. Biochem. Bioph. Res. Co.,285:424-429.
Yin F, Pajak A, Chapman R, Sharpe A, Huang S, Marsolais F.2011. Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins BMC Genomics,12:268. doi: 10.1186/1471-2164-12-268.
Zabeau M, Vos P.1993. Selective restriction fragment amplication:A general method for DNA fingerprinting. European Patent Application., No.0534858 A1.
Zelicourt A, Letousey P, Thoiron S, Campion C, Simoneau P, Elmorjani K, Marion D, Simier P, Delavault P.2007. Ha-DEF1, a sun flower defensin, induces cell death in Orobanche parasitic plants. Planta,226:591-600.
曹玉芬,刘凤之,高源,姜立杰,王昆,马智勇,张开春.2007.梨栽培品种SSR鉴定及遗传多样性.园艺学报,34(2)：305-310.
曹玉芬,刘凤之,胡红菊,张冰冰.2006.梨种质资源描述规范和数据标准.北京：中国农业出版社.
曹玉芬.2001.我国的红皮梨种质资源.中国种业,1：26-27.
曾柏全,甘霖,熊兴耀,邓秀新,邓子牛.2006.冰糖橙优良芽变的AFLP分析.江西农业大学学报,28(2)：222-225.
曾继吾,彭成绩,易干军,杜观兴,张绍平,冯春添,霍合强,钟云,周碧容,黄永红.2006.晚熟柑橘新品种‘明柳甜橘’.园艺学报,33(5)：1164.
陈大明,张上隆,金勇丰.1997.一种本本果树基因组DNA提取方法研究.浙江农业大学学报,23(6)：621-624.
陈健初,董绍华,叶兴乾,苏平.1997.芹菜汁叶绿素及色泽稳定性的研究.浙江农业大学学报,23(3)：301-304.
陈婷,王日葵,周炼,陆智明,刘涛.2010.柑橘贮藏期间色差指数变化规律的研究.农产品加工学,3：4-7.
陈希历,陈苏.1989.巴梨优良红色芽变系.山西果树.1：44-45.
丁武,魏益民.2002.色彩色差计在肉品新鲜度检验中的应用.肉类研究,4：47-48.
樊丽,方成泉,王斐,王志刚,董星光,林盛华.2008.DNA分子标记在梨育种上的应用概况.中国果树,3：49-53.
樊庆忠.2010.桃芽变中早熟新品种凤红的选育.中国果树,6：3-5.
房经贵,章镇,周兰华,陈崇顺,王三红.2001.RAPD标记鉴定果树芽变的可行性分析.果树学报,18(3)：182-185.
冯少菲.2006.黄金梨果皮发育、锈斑形成及套袋对其影响的研究.保定：河北农业大学.15.
冯月秀,李从玺,王琨.2001.梨早熟梨-六月酥.西北园艺,(5)：35.
高正辉,潘海发,徐义流,张昂,束冰.2010.‘砀山酥梨’芽变‘97-05-9’的RAPD分析.中国农学通报,26(10)：48-53.
苟永平.1997.介绍一质优的冬果梨芽变.北京农业,10：28.
郭景芬.1990.大南果梨芽变优系通过审定.中国农业科技通讯,6(2)：48.
贺立红,张进标,宾金华.2006.苯丙氨酸解氨酶的研究进展.食品科技,7：31-34.
衡伟,贾兵,叶振风,李晓峰,李雪,朱立武.2011.砀山酥梨褐皮芽变品系锈酥果皮结构分析.中国果树,3：20-22.
洪柳,邓秀新.2005.应用MSAP技术对脐橙品种进行DNA甲基化分析.中国农业科学,38：2301-2307.
胡宁三,蔡盛华,陆修闽,黄新忠.2007.柑橘类新品种——红肉蜜柚.中国农技推广,23(10)：16.
焦言英,赵桂敏,陈继忠.1999.南果梨芽变新品种红南果梨.中国果树,3：22.
金勇丰,张耀洲,陈大明,张上隆.1998.桃早熟芽变品种‘大观一号’的RAPD分析及其特异片断克隆.果树科学,15(2)：103-106.
赖国亮,吴金桃,兰永辉.1999.测色技术在炒青绿茶品质评价中的应用研究.福建茶叶,2：19-20.
黎茵,张以顺,周玉亮,陈琛,黄上志.2010.莲种子防御素基因序列分析及表达载体构建.园艺学报,37(12)：175-182.
李俊才,李宝江,于丹,刘成.2010.2个西洋梨红色芽变品种与原品种果实发育期果皮色素变化比较研究.中国果树,1：27-30.
李俊才,隋洪涛,王家珍,刘成,许英武,闫忠业,孙丽梅,蔡忠民,沙守峰,伊凯.2006.梨芽变新品种——尖把王梨的选育.果树学报,23(6)：912-913.
李俊才,王斌,高庆福,王家珍,蔡忠民,张景娥.2011.红色梨新品种‘南红梨’.园艺学报,38(9)： 1821-1822.
李敏,代洪义,李培环,王然,林振海.1983.在梨杨山酥梨等果点的形成.落叶果树,3：3-5.
李润唐,黄茂松,张春年.黄花梨芽变新品系——大香西黄梨(暂定名).2001.落叶果树,1：22.
李涛,解娟,罗雪峰,李纯凡.2010.凯特杏芽变新品种金沙杏的选育.中国南方果树,39(4)：55-56.
李晓芳,礼茂福,韩振海,许雪峰,李天忠.2008.‘鸭梨’芽变‘闫庄梨’自交亲和分子机制的初步研究.园艺学报,31(1)：13-18.
李忠,杨建平,陈远富,秦勇,唐太平,钟天才,钟天华.2010.早熟、优质梨新品系‘普威早熟1号’的选育.中国园艺文摘,6：144-145.
廖明安,张志鹏,任雅君,邓国涛,程帅,李健,吕秀兰,王永清,汪志辉,周廷国.2010.优质高产梨新品种‘川花梨’.园艺学报,37(6)：1017-1018.
廖振坤,张秋明,刘卫国,丁伟平,王春梅.2006.利用AFLP鉴定柑橘变异.果树学报,23(3)：486-488.
林真二.(吴耕民译).1981.梨.北京：农业出版社.293-318.
刘成明,梅曼彤.2002.利用RAPD分析鉴别荔枝的焦核突变体.园艺学报,29(1)：57-59.
刘平,彭士琪,郭振怀.2002.果树主要经济性状的遗传动态.河北林果研究,17(2)：267-274.
刘庆华,周启河,王奎玲,刘庆芳,吕志宁.1992.莱阳往梨果实斑痕的解剖学研究.莱阳农学院学报,9(2)：104-108.
刘中成,赵锦,刘孟军.2004. mRNA差异显示技术评述.分子植物育种,2(6)：895-900.
罗安才,李纯凡,黄仁湖,向可术,李道高.2003.奉节脐橙芽变株系的AFLP分析.农业生物技术科学,19(6)：20-24.
马克元,傅玉瑚,程福厚,苏艳英,王海星,苏春季.1990.鸭梨果点发育的解剖观察.山西果树,04：12-14.
念红忠,银立新,张晓霞,张素芳,李学林,杨瑞林.2002.华夫人葡萄芽变新品种——华变.中国南方果树,31(3)：73.
聂继云,李静,杨振峰,张红军,李明强.2006.冷冻法测定梨的石细胞含量.果树学报,23(1)：133-135.
宁允叶,熊庆娥,曾伟光,曾光荣.2003.红阳猕猴桃全红芽变系的RAPD分析.园艺学报,30(5)：511-513.
宁允叶,熊庆娥,曾伟光.2005.‘红阳’猕猴桃全红型芽变(86-3)的果实品质及花粉形态研究.园艺学报,32(3)：486-488.
蒲富慎.1979.梨的一些性状的遗传.遗传,01：25-28.
曲柏宏.2003.延边苹果梨分类地位及其授粉受精特性的研究.沈阳：沈阳农业大学3-8,23.
任秋萍,张复君,吕福堂,王贵余,朱法作,邢柱东.2007.梨浓红色新品种奥冠红梨的选育.中国 果树,6：12-13.
任志宏,雷应果,张国德.1998.大果型芽变新品种——伏红.现代农业,5：29.
申连长,马晓娣,王彦敏,傅玉瑚.2005.鸭梨芽变新品系——光鸭梨.河北果树,3：19.
沈德绪,林伯年.1985.园艺植物遗传学.北京：农业出版社.249-250.
沈德绪.1986.果树育种学.上海：上海科学技术出版社.
沈德绪.1992.果树育种学.北京：农业出版社.55-56.
盛炳成.2008.黄河故道地区果树综论.北京：中国农业出版社.267.
石荫坪,李雅志,王强生.1986.果树突变育种.上海：上海科技出版社.43-109,255-293.
束怀瑞.1999.苹果学.北京：中国农业出版社.186-196.
宋伟,王彩虹,田义轲,田伟,殷豪.2010.梨果实褐皮性状的SSR标记.园艺学报,37(8)：1325-1328.
宋忠伍.2010.‘红甜核杏’芽变的选育.北方果树,1：47-48.
陶能国.2006.甜橙(Citrus sinensis Osbeck)红肉突变体类胡萝卜素合成相关基因的克隆与特性分析.武汉：华中农业大学博士学位论文.64-67.
陶世蓉,辛华,初庆刚,曹玉芬.1999.窝梨果实结构及发育的研究.西北植物学报,19(1)：123-126.
滕元文,沈玉英,周先章.2005.砂梨果皮锈斑成因及解决对策.中国南方果树,34(3)：52-56.
王家政,王家喜,王常君.1993.红巴梨引种研究初报.落叶果树.2：39-40.
王心燕,肖璇,乔爱民,孙敏.2006.'石硖’龙眼芽变系双引物RAPD鉴定的研究.园艺学报,33(1)：134-136.
王新卫,刘金义,孙兴民,崔海荣,王燕,乔玉山,章镇.2010.中国梨生产、贸易与国际竞争力分析.中国农学通报,26(21)：202-206.
王宇霖,White A, Brewer L, Cranwel D.1997.红皮梨育种研究报告.果树科学,14(2)：71-76.
王志,闫忠业.2010.富士系苹果的发展概况.农业科技通讯,1：164-166.
魏立刚.2011.晚熟大果型梨——奥林斤秋梨.农村百事通,21：25.
魏志东,米建仓,晁晓庆.2000.桃芽变新品种——礼泉沙红桃.西北园艺,1：31-32.
吴丹,叶兴乾,陈健初.2007.杨梅汁色差分析.酿酒,34(6)：94-96.
吴桂余,吴桂法,吴建华韦军.2000.梨新品种‘大果白酥’.园艺学报,27(6)：460.
吴厚玖,陈荣柱,李育农.1984.金冠苹果果锈发生时期与绣花过程的研究.园艺学报,11(1)：51-54.
吴华清,张绍铃,吴巨友,王迎涛,吴俊.2007.‘金坠梨’自交亲和性突变机制的初步研究.园艺学报,34(2)：295-300.
吴忠华,张洪平,韩寄桥,唐道禄.1994.香梨芽变新品种新梨2号.新疆农业科学,4：175.
武瑞俊,王耀胜.2011.红地球葡萄四倍体芽变新品种红太阳的选育.山西果树,3：4.
肖璇,孙敏,王心燕,乔爱民,江波.2005.‘石砍’龙眼大果型桂花味芽变系的RAPD分析鉴定.园艺学报,32(4)：684-687.
徐凌飞,张福民.2009.早熟红色梨优良品系——早酥红.山西果树,4：44.
徐象华,斯金平,谢建秋,蓝云龙,曾燕如,江伟林,叶文伟.2006.柑橘新品种无子欧柑的选育.果树学报,23(5)：781-782.
徐晓梅,杨署光.2009.苯丙氨酸解氨酶研究进展.安徽农业科学,37(31)：15115-15119,15122.
许方.1992.梨树生物学.北京：科学出版社,150-158.
许宽勇.2009.苹果钙反向转运体基因的克隆、表达特性及其功能分析研究.南京：南京农业大学.
宣继萍.2001.富士苹果芽变的细胞学与分子生物学鉴定研究.南京：南京农业大学26-28.
鄢新民,李学营,王献革,郝婕,冯建忠.2011.苹果芽变及芽变选种回顾.河北农业科学,15(05)：75-77.
阎振立,张顺妮,张全军.2006.果树的芽变选种.果农之友,1：38-39.
杨岐生,林卿,李奕,谢毅.1999.用差异显示法从人胎脑基因文库分离一个编码序列.中国生物化学与分子生物学报,15(6)：876-880.
杨有龙.1989.美国的苹果芽变选种概况.落叶果树,4：40-41.
姚红伟,张立冬,孙金阳,刘霄霞.2010.DNA分子标记技术概述.河北渔业,7：42-46.
伊凯,闫忠业,刘志,王冬梅,杨峰,张景娥.2006.苹果芽变选种鉴定及应用研究.果树学报,23(5)：745-749.
尹永胜,王强生.1996.巴梨四倍体芽变品种的选育.中国农学通报,12(5)：22-23.
尹永胜,徐增凯,宋福正,李瑞芝,王强生.1999.巴梨芽变新品种鲁梨1号.中国果树,3：23.
俞德浚,张鹏.1979.我国梨品种的一个新系统.园艺学报,6(1)：27-32.
张东,滕元文.2011.红梨资源及其果实着色机制研究进展.果树学报,28(3)：485-492.
张凯斌,刘凤君,代汉萍,吴宇红,高兆银.2003.花盖梨芽变新品系——花盖王.中国果树,1：12-13.
张士鸿.2010.砀山酥梨果实PAL酶学特性的研究及PAL基因克隆.合肥：安徽农业大学.
张小军,徐凌飞,吴中营.2009.梨极早熟芽变品种‘六月酥’的AFLP分析.西北农林科技大学学报,37(12)：139-145,138.
张志刚,李栒,官春云,白德朗,武小金.2006.改良提取水稻胚乳和拟南芥花柱中DNA和RNA的SDS法.植物生理学通讯,42(3)：493-495.
张智涛,张立彬,于凤鸣,关骋遥.2011.大果水晶梨与其褐色突变体多酚氧化酶及过氧化物酶同工酶差异(简报).河北科技师范学院学报,25(2)：31-33.
章镇,俞宏,高志红,葛绍义,陶建敏,蔡斌华.2000.梨新品种‘大果黄花’.园艺学报,27(1)：74.
赵广,乐文全,路娟,张绍铃,李俊才,吴俊.2011.梨果皮色泽变异品种(系)的AFLP与SRAP 标记鉴定.果树学报,28(4)：568-574.
赵权,王军.2010.ABA和6-BA对山葡萄果实着色及相关品质的影响.江苏农业科学,2：189-190.
郑玉龙,姜春玲,冯玉龙.2005.植物的气孔发生.植物生理学通讯,41(6)：847-850.
周蓓蓓,朱海军,生静雅,刘广勤.2011.DNA分子标记在果树种质资源遗传多样性研究中的应用.江西农业学报,23(9)：47-50.
朱立武,王艳芳,贾兵,张水明,黄永丰,杨振江.2009.安徽砀山酥梨自然保护区梨种质资源AFLP分析.果树学报,26(2)：145-150.
Almeida MS, Cabral KMS, Kurtenbach E, Almeida FCL, Valente AP.2002. Solution structure of Pisum sativum defensin 1 by high resolution NMR:plant defensins, identical backbone with different mechanisms of action. J. Mol. Biol.,315:749-757.
Aluru M, Curry J, O'Connell MA.1999. Nucleotide sequence of a defensin or y-thionin-like gene (accession no. AF128239) from habanera chili. Plant Physiol.,120:633.
Amarante C, Banks NH, Ganesh S.2001. Relationship between character of skin cover of coated pears and permeance to water vapour and gases. Postharvest Biol. Tec.,21:291-301.
Anuradha TS, Divya K, Jami SK, Kirti PB.2008. Trangenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep.,27:1777-1786.
Arulsekar S, Parfolt DE, Keste DE.] 986. Comparison of isozyme variability in peach and almond cultivars. J. Hered.,77:272-274.
Azuma A, Kobayashi S, Goto-Yamamoto N, Shiraishi M, Mitani N, Yakushiji H, Koshita Y.2009. Color recovery in berries of grape (Vitis vinifera L.)'Benitaka', a bud sport of'Italia', is caused by a novel allele at the VvmybAl locus. Plant Sci.,176:470-478.
Benbouza H, Jacquemin JM, Baudoin JP, Mergeai G.2006. Optimization of a reliable, fast, cheap and sensitive silver staining method to detect SSR markers in polyacrylamide gels. Biotechnol. Agron. Soc. Environ,10(2):77-81.
Bendtsen JD, Nielsen H, von Heijne G, Brunak S.2004. Improved prediction of signal peptides:SignalP 3.0. J. Mol. Biol.,340:783-795.
Bernards MA, Fleming WD, Llewellyn DB, Priefer R, Yang X, Sabatino A, Plourde GL.1999. Biochemical characterization of the suberization-associated anionic peroxidase of potato. Plant Physiol.,121:135-146.
Bernards MA, Summerhurst DK, Razem FA.2004. Oxidases, peroxidases and hydrogen peroxide:the suberin connection. Phytochem Rev.,3:113-126.
Bernards MA.2002. Demystifying suberin. Can. J. Bot,80:227-240.
Bozkurt O, Unver T, Akkaya MS.2007. Genes associated with resistance to wheat yellow rust disease identified by differential display analysis. Physiol. Mol. Plant P.,71:251-259.
Broekaert WF, Cammue BPA, De Bolle MFC, Thevissen K, De Samblanx G, Osborn RW.1997. Antimicrobial peptides from plants. Crit. Rev. Plant Sci.,16:297-323.
Broekaert WF, Terras FRG, Cammue BPA, Osborn RW.1995. Plant defensins:novel antimicrobial peptides as components of the host defense system. Plant Physiol.,108:1353-1358.
Bruix M, Jimenez MA, Santoro J, Gonzalez C, Colilla FJ, Mendez E, Rico M.1993. Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins. Biochem.,32:715-724.
Carra A, Gambino G., Schubert A.2007. A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues. Anal Biochem.,360:318-320.
Carreno J, Martinez A, Almela L, Fernandez-Lopez JA.1995. Proposal of an index for the objective evaluation of the colour of red table grapes. Food Res. Int.,28(4):373-377.
Carvalho AdeO, Gomes VM.2009. Plant defensins—prospects for the biological functions and biotechnological properties. Peptides,30:1007-20.
Chang, S, Puryear J, Cairney J.1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep.,11:113-116.
Cheng GW, Breen PJ.1991. Activity of Pheyla lanine ammonia lyase and concent ration of anthocyanins and phenolics in developing strawberry fruit. J. Amer. Soc. Hortic. Sci.,116 (5):865-869.
Chevreau E, Leuliette S, Gallet M.1997. Inheritance and linkage of isozyme loci in pear (Pyrus communis L.). Theor. Appl. Genet.,94(3,4):498-506.
Chevreau E, Manganaris AG, Gallet M.1999. Isozyme segregation in five apple progenies and potential use for map construction. Theor. Appl. Genet.,98(2):329-336.
Chiang CC, Hadwiger LA.1991. The Fusarium solani-induced expression of a pea gene family encoding high cysteine content proteins. Mol. Plant Microbe Interact.,4:324-331.
Choi MS, Kim YH, Park HM, Seo BY, Jung JK, Kim ST, Kim MC, Shin DB, Yun HT, Choi IS, Kim CK, Lee JY.2009. Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against Brown planthopper (Nilaparvata lugens) in transgenic rices. Mol. Cells,28:131-137.
Currie AJ, Noiton DA, Lawes GS, et al.1997. Preliminary results of differentiating apple sports by pollen ultrastructures. Euphytica,98:155-161.
de Beer A, Vivier MA.2008. Vv-AMP1, a ripening induced peptide from Vitis vinefera shows strong antifungal activity. BMC Plant Biol.,8:75.
de Godoy A, Cordenunsi BR, Lajolo FM, do Nascimento JRO.2010. Differential display and suppression subtractive hybridization analysis of the pulp of tipening banana. Sci. Hortic.,124: 51-56.
De Lano WL.2002. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA, USA., http://www.pymol.org
de Zelicourt A, Letousey P, Thoiron S, Campion C, Simoneau P, Elmorjani K, Marion D, Simier P, Delavault P.2007. Ha-DEF1, a sun flower defensin, induces cell death in Orobanche parasitic plants. Planta,226:591-600.
Deng ZN, Gentile A, Nicolosi E, Domina F, Vardi A, Tribulato E.1995. Identification of in vivo and in vitro lemon mutants by RAPD markers. J. Hortic. Sci.,70(1):117-125.
Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD.1996. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A.,93:6025-6030.
Do HM, Lee SC, Jung HW, Sohn KH, Hwang BK.2004. Differential expression and in situ localization of a pepper defensin (CADEF1) gene in response to pathogen infection, abiotic elicitors and environmental stresses in Capsicum annuum. Plant Sci.,166:1297-1305.
Doughty J, Dixon S, Hiscock SJ, Willis AC, Parkin IAP, Dickinson HG.1998. PCP-A1, a defensin-like brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression. Plant Cell,10:1333-1347.
du Preez MG, Rees DJG.2008. Investigating the molecular mechanism of anthocyanin production in 'Bon Rouge'Pyrus communis L. Acta. Horticul.,800:383-390.
Dussi MC, Sugar D, Wrolstad RE.1995. Characterizing and quantifying anthocyanins in red pears and the effect of light quality on fruit color. J. Amer. Soc. Hortic. Sci.,120(5):785-789.
Fabi JP, Lajolo FM, do Nascimento JRO.2009. Cloning and characterization of transcripts differentially expressed in the pulp of ripening papaya. Sci. Hortic.,121:159-165.
Fant F, Vranken W, Broekaet W, Borremans F.1998. Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR. J. Mol. Biol.,279:257-270.
Fedorova M, van de Mortel J, Matsumoto PA, Cho J, Town CD, Vandenbosch KA, Gantt JS, Vance CP. 2002. Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula. Plant Physiol.,130:519-537.
Ferrer A, Remon S, Negueruela A I, Oria R.2005. Changes during the ripening of the very late season Spanish peach cultivar Calanda feasibility of using CIELAB coordinates as maturity indices. Sci. Hortic.,105:435-446.
Finkina El, Shramova El, Tagaev AA, Ovchinnikova TV.2008. A novel defensin from the lentil Lens culinaris seeds. Biochem. Bioph. Res. Co.,371:860-865.
Fischer TC, Gosch C, Pfeiffer J, Halbwirth H, Halle C, Stich K, Forkmann G.2007. Flavonoid genes of pear (Pyrus communis). Trees,21:521-529.
Gamble J, Jaeger SR, Roger Harker F.2006. Preferences in pear appearance and response to novelty among Australian and New Zealand consumers. Postharvest Biol. Tec.,41:38-47.
Gang DR, Wang J, Dudareva N, Nam KH, Simon JE, Lewinsohn E, Pichersky E.2001. An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol.,125:539-555.
Gao AG, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang J, Rommens C.M. 2000. Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat. Biotechnol.,18:1307-1310.
Garcia-Olmedo F, Molina A, Alamillo JM, Rodriguez-Palenzuela P.1998. Plant defense peptides. Biopolymers,47:479-491.
Graca J, Santos S.2007. Suberin:a biopolyester of plants'skin. Macromol Biosci.,7:128-135.
Graca J.2010. Hydroxycinnamates in suberin formation. Phytochem Rev.,9:85-91.
Graham MA, Silverstein KA, Cannon SB, VandenBosch KA.2004. Computational identification and characterization of novel genes from legumes. Plant Physiol.,135:1179-1197.
Graham MA, Silverstein KAT, VandenBosch KA.2008. Defensin-like genes:genomic perspectives on a diverse superfamily in plants. Crop. Sci.,48:s3-s11.
Granger AR, Clarke GR, Jackson JF.1993. Sweet cherry cultivar identification by leaf isozyme polymorphism. Theor. Appl. Genet.,86(4):458-464.
Gu Q, Kawata EE, Morse MJ, Wu HM, Cheung AY.1992. A flower-specific cDNA encoding a novel thionin in tobacco. Mol. Gen. Genet.,234:89-96.
Guo S, Zheng Y, Joung J, Liu S, Zhang Z, Crasta OR, Sobral BW, Xu Y, Huang S, Fei Z.2010. Transcriptome sequencing and comparative analysis of cucumber flowers with different sex types. BMC Genomics,11:384-396.
Hanks JN, Snyder AK, Graham MA, Shah RK, Blaylock LA, Harrison MJ, Shah DM.2005. Defensin gene family in Medicago trvncatula:structure, expression and induction by signal molecules. Plant Mol. Biol.,58:385-399.
Hauptmann M, Pleschberger H, Mai C, Follrich J, Hansmann C.2010. The potential of color measurements with the CIEDE2000 equation in wood science. Eur. J. Wood Prod., DOI 10.1007/s00107-011-0575-6
He FM, Zhu YP, Zhang YZ.2008. Identification and characterization of differentially expressed genes involved in pharmacological activeties of roots of Panax notoginseng during plant growth. Plant Cell Rep.,275:923-930.
Huang GJ, Lai HC, Chang YS, Sheu MJ, Lu TL, Huang SS, Lin YH.2008. Antimicrobial, dehydroascorbate reductase, and monodehydroascorbate reductase activities of defensin from sweet potato [Ipomoea batatas (L.) Lam.'Tainong 57'] storage roots. J. Agric. Food Chem.,56:2989-95.
Iandolino AB, Goes da Silva F, Lim H, Choi H, Williams LE, Cook DR.2004. High-quality RNA, cDNA, and derived EST libraries from Grapevine (Vitis vinifera L.). Plant Mol. Biol. Rep.,22: 269-278.
Janisiewicz WJ, Pereira IB, Almeida MS, Roberts DP, Wisniewski M, Kurtenbach E.,2008. Improved biocontrol of fruit decay fungi with Pichia pastoris recombinant strains expressing Psd 1 antifungal peptide. Postharvest Biol. Tech.,47:218-225.
Jha S, Chattoo BB.2010. Expression of a plant defensin in rice confers resistance to fungal phytopathogens. Transgenic Res.,19:373-384.
Kant P, Liu WZ, Pauls P.2009. PDC1, a corn defensin peptide expressed in Escherichia coli and Pichia pastoris inhibits growth of Fusarium graminearum. Peptides,30:1593-1599.
Kanzaki H, Nirasawa S, Saitoh H, Ito M, Nishihara M, Terauchi R, Nakamura I.2002. Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor. Appl. Genet.,105:809-814.
Karim K, Hatem CM, Mokhtar T, Mbarek BN.2011. Identification of salt stress-induced transcripts in barley leaves by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Afr. J. Biotechnol.,10(83):19356-19364.
Karunanandaa B, Singh A, Kao T-H.1994. Characterization of a predominantly pistil-expressed gene encoding a thionin-like protein of Petunia inflata. Plant Mol. Biol.,26:459-464.
Kato M, Kamo T, Wang R, Nishikawa F, Hyodo H, Ikoma Y, Sugiura M, Yano M.2002. Wound-induced ethylene synthesis in stem tissue of harvested broccoli and its effect on senescence and ethylene synthesis in broccoli florets. Postharvest Biol. Tec.,24:69-78.
Kobayashi S, Ishimaru M, Ding CK, Yakushiji H, Goto N.2001. Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Plant Sci.,160:543-550.
Kobayashi S, Yamamoto N, Hirochika H.2004. Retrotransposon induced mutations in grape skin color. Science,304:982.
Koike M, Okamoto T, Tsuda S, Imai R.2002. A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation. Biochem. Bioph. Res. Co.,298:46-53.
Kolattukudy PE.1980. Biopolyester membranes of plants:cutin and suberin. Science,208:990-1000.
Kumar S, Tamura K, Nei M.2004. MEGA3:Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform.,5:150-163.
Lang P, Zhang CK, Eel RC, Dane F, Dozier WA.2005. Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display. Gene,359:111-118.
Lay FT, Brugliera F, Anderson MA.2003. Isolation and properties of floral defensins from Ornamental tobacco and Petunia. Plant Physiol.,131:1283-1293.
Lay FT, Schirra HJ, Scanlon MJ, Anderson MA, Craik DJ.2003. The three-dimensional solution structure of NaD1, a new floral defensin from Nicotiana alata and its application to a homology model of the crop defense protein alfAFP. J. Mol. Biol.,325:175-188.
Liang P, Meade J D, Pardee A B.2007. A protocol for differential display of mRNA expression using either fluorescent or radioactive labeling. Nat. Protoc.,2(3):457-470.
Lievens S, Goormachtig S, Holsters M.2001. A critical evaluation of differential display as a tool to identify genes involved in legume nodulation:looking back and looking forward. Nucleic Acids Res.,29(17):3459-3468.
Liu YZ, Liu Q, Xiong JJ, Deng XX.2007. Difference of a citrus late-ripening mutant (Citrus sinensis) from its parental line in sugar and acid metabolism at the fruit ripening stage. Science in China Series C:Life Sciences,50(4):511-517.
Lu XP, Liu YZ, An JC, Hu HJ, Peng SA.2011. Isolation of a cinnamoyl CoA reductase gene involved in formation of stone cells in pear (Pyrus pyrifolia). Acta. Physiol. Plant,33:585-591.
Ma DZ, Wang HX, Ng TB.2009. A peptide with potent antifungal and antiproliferative activities from Nepalese large red beans. Peptides,30:2089-2094.
Maitra N, Cushman JC.1998. Characterization of a drought-induced soybean cDNA encoding a plant defensin (Accession No. U12150) (PGR 98-213). Plant Physiol.,118:1536.
Matsuoka M, Sanada Y.1991. Expression of photosynthetic genes from the C4 plant, maize, in tobacco. Mol. Gen. Genet.,225:411-419.
Mayer B, Houlne G, Pozueta-Romero J, Shanta ML, Shantz R.1996. Fruit-specific expression of a defensin-type gene family in bell pepper. Plant Phsysiol.,112:615-655.
Melotto M, Underwood W, He SY.2008. Role of stomata in plant innate immunity and foliar bacterial disease. Annu. Rev. Phytopathol.,46:101-122.
Melotto M, Underwood W, Koczan J, Nomura K, He SY.2006. Plant stomata function in innate immunity against bacteria invasion. Cell,126:969-980.
Mendgen K, Hahn M, Deising H.1996. Morphogenesis and mechanisms of penetration by plant pathogenic fungi. Annu. Rev. Phytopathol.,34:367-386.
Mergaert P, Nikovics K, Kelemen Z, Maunoury N, Vaubert D, Kondorosi A, Kondorosi E.2003. A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol.,132:161-173.
Meyer B, Houlne G, Pozueta-Romero J, Schantz ML, Schantz R.1996. Fruit-specific expression of a defensin-type gene family in bell pepper. Plant Physiol.,112:615-622.
Ming D, Hellekant G.1994. Brazzein, a new high potency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS. Lett.,355:106-108.
Mirouze M, Sels J, Richard O, Czernic P, Loubet S, Jacquier A, Francois IEJA, Cammue BPA, Lebrun M, Berthomieu P, Marques L.2006. A putative novel role for plant defensins:a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance. Plant J.,47:329-42.
Moreno M, Segura A, Garcia-Olmedo F.1994. Pseudothionin-Stl, a potato peptide active against potato pathogens. Eur. J. Biochem.,223:135-139.
Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant,15:473-497.
Nielsen M, Lundegaard C, Lund O, Petersen TN.2010. CPHmodels-3.0—remote homology modeling using structure-guided sequence profiles. Nucleic Acids Res.,38:w576-w581.
Oberschmidt O, Grundler FMW, Kleine M.2003. Identification of a putative cation transporter gene from sugar beet (Beta vulgaris L.) by DDRT-PCR closely linked to the beet cyst nematode resistance gene HsI (pro-1). Plant Sci.,165(4):777-784.
Odintsova TI, Egorov TA, Musolyamov AK, Odintsova MS, Pukhalskv VA, Grishin EV.2007. Seed defensins from T. kiharae and related species:genome localization of defensin-encoding genes. Biochime.,89:605-612.
Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D, Kawano N, Sakakibara T, Namiki S, Itoh K, Otsuka K, Matsuzaki M, Nozaki H, Kuoroiwa T, Nakano A, Kanaoka MM, Dresselhaus T, Sasaki N, Higashiyama T.2009. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature, 458:357-361.
Oltean L, Teischinger A, Hansmann C.2008. Wood surface discolouration due to simulated indoor sunlight exposure. Holz. Roh. Werkst,66:51-56.
Parashina EV, Serdobinskii LA, Kalle EG, Lavorova NV, Vetisov VA, Lunin VG, Naroditskii BS.2000. Genetic engineering of oilseed rape ans tomato by expression of a radish defensin gene. Russ. J. Plant Physiol.,47:417-423.
Park HC, Kang YH, Chun HJ, Koo JC, Cheong YH, Kim CY, Kim MC, Chung WS, Kim JC, Yoo JH, Koo YD, Koo SC, Lim CO, Lee SY, Cho MJ.2002. Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage. Plant Mol. Biol.,50:59-69.
Passardi F, Penel C, Dunand C.2004. Performing the paradoxical:how plant peroxidases modify the cell wall. Trends Plant Sci.,9:534540.
Pelegrini PB, Franco OL.2005. Plant y-thionins:novel insights on the mechanism of action of a multi-functional class of defense proteins. Int. J. Biochem. Cell B.,37:2239-2253.
Penninckx I A, Thomma BP, Buchala A, Metraux JP, Broekaert WF.1998. Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell,10:2103-2113.
Pollard M, Beisson F, Li Y, Ohlrogge JB.2008. Building lipid barriers:biosynthesis of cutin and suberin. Trends Plant Sci.,13:236-246.
Ponsuksili S, Wimmers K, Schellander K.2001. Application of differential display RT-PCR to identify porcine liver ESTs. Gene,280:75-85.
Ribeiro SFF, Carvalho AO, Da Cunha M, Rodrigues R, Cruz LP, Melo VMM, Vasconcelos IM, Melo EJT, Gomes VM.2007. Isolation and characterization of novel peptides from chilli pepper seeds: Antimicrobial activities against pathogenic yeasts. Toxicon,50:600-611.
Schafleitner R, Wilhelm E.2002. Isolation of wound-responsive genes from chestnut (Castanea sativa) microstems by mRNA display and their differential expression upon wounding and infection with the chestnut blight fungus (Chryphonectriaparasitica). Physiol. Mol. Plant Pathol.,61:339-348.
Scott KD, Alblett EM, Lee LS, Henry RJ.2000. AFLP markers distinguishing an early mutant of Flame Seedless grape. Euphytica,113:245-249.
Segura A, Monero M, Molina A, Garcia-Olmedo F.1998. Novel defensin subfamily from spinach (Spinacia oleracea). FEBS Lett.,435:159-162.
Silverstein KAT, Graham MA, Paape TD, VandenBosch KA.2005. Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant Physiol.,138:600-610.
Soler M, Serra O, Molinas M, Huguet G, Fluch S, Figueras M.2007. A genomic approach to suberin biosynthesis and cork differentiation. Plant Physiol.,144:419-431.
Song X, Zhou Z, Wang J, Wu F, Gong W.2004. Purification, characterization and preliminary crystallographic studies of a novel plant defensin from Pachyrrhizus erosus seeds. Acta. Cryst., D60: 1121-1124.
Stiekema WJ, Heidekamp F, Dirkse WQ Van Beckum J, De Haan P, Ten Bosch C, Louwerse JD.1988. Molecular cloning and analysis of four potato tuber mRNAs. Plant Mol. Biol.,11:255-269.
Stotz HU, Spence B, Wang Y.2009. A defensin from tomato with dual function in defense and development. Plant Mol. Biol.,71:131-143.
Swathi Anuradha T, Divya K, Jami SK, Kirti PB.2008. Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep.,27:1777-1786.
Tani E, Polidoros AN, Flemetakis E, Stedel C, Kalloniati C, Demetriou K, Katinakis P, Tsaftaris AS. 2009. Characterization and expression analysis of AGAMOUS-like, SEEDSTICK-like, and SEPALLATA-like MADS-box genes in peach (Prunus persica) fruit. Plant Physiol. Bioch.,47: 690-700.
Tavares LS, Santos Mde O, Viccini LF, Moreira JS, Miller RNG, Franco OL.2008. Biotechnological potential of antimicrobial peptides from flowers. Peptides,29:1842-1851.
Terras FRG, Eggermont K, Kovaleva V, Raikhel NV, Osborn RW, Kester A, Rees SB, Torrekens S, Van Leuven F, Vanderleyden J et al.1995. Small cysteine-rich antifungal proteins from radish:their role in host defense. Plant Cell,7:573-588.
Thomma BPHJ, Broekaert WF.1998. Tissue-specific expression of plant defensin genes PDF2.1 and PDF2.2 in Arabidopsis thaliana. Plant Physiol. Biochem.,36:533-537.
Thomma BPHJ, Camme BPA, Thevissen K.2002. Plant defensins. Planta,216:193-202.
Thompson JD, Higgins DG, Gibson TJ.1994. CLUSTALW:improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res.,22:4673-4680.
Torres AM, Bergi BO.1980. Fruit and leaf isozymes as genetics in avocado. J. Amer. Soc. Hort. Sci.,105: 614-619.
Torres GAM, Pflieger S, Corre-Menguy F, Mazubert C, Hartmann C, Lelandais-Briere C.2006. Identification of novel drought-related mRNAs in common bean roots by differential display RT-PCR. Plant Sci.,171:300-307.
Urdangarin MC, Norero NS, Broekaert WF, de la Canal L.2000. A defensin gene expressed in sunflower inflorescence. Plant Physiol. Biochem.,238(3):253-258.
van den Heuvel KJPT, Hulzink JMR, Barendse GWM, Wullems GJ.2001. The expression of tgas118, encoding a defensin in Lycopersicon esculentum is regulated by gibberellin. J. Exp. Bot.,52: 1427-1436.
van Kan JA.2006. Licensed to kill:the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci.,11: 247-253.
Veeden NJ, Land RC.1985. Identification of apple cultivars by isozyme phenotypes. J. Amer. Soc. Hort. Sci.,110:509-515.
Velculescu VE, Zhang L, Vogelstein B, Kinzler KW.1995. Serial analysis of gene expression. Science, 270:484-487.
Vijayan S, Guruprasad L, Kirti PB.2008. Prokaryotic expression of a constitutively expressed Tephrosia villosa defensin and its potent antifungal activity. Appl. Microbiol. Biotechnol.,80:1023-32.
Viljoen MM, Hoysamer M.1995. Biochemical and regulatory aspects of anthocuanin synthesis in apple and pears. Journal of the Southern African Society for Horticultural Sciences,5(1):1-6.
Vinterhalter DV, James DJ.1986. The use of peroxidase polymorphism in the identification of Mailing and Mailing Merton apple rootstock. J. Hort. Sci.,61:147-152.
Vuylsteke M, Peleman JD, van Eiik MJT.2007. AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis. Nat. Protoc.,2(6):1399-1413.
Walker AR, Lee E, Robinson SP.2006. Two new grape cultivars, bud sports of Cabernet Saurignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus. Plant Mol. Biol.,62:623-635.
Wan CY, Wilkins TA.1994. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L). Anal Biochem.,223(1):7-12.
Wang X, Tang C, Zhang G, Li Y, Wang C, Liu B, Qu Z, Zhao J, Han Q, Huang L, Chen X, Kang Z.2009. cDNA-AFLP analysis reveals differential gene expression in compatible interaction of wheat challenged with Puccinia striiformis f. sp. Tritici. BMC Genomics,10:289 doi:10.1186/1471-2164-10-289.
Wang YP, Nowak G, Culley D, Hadwiger LA, Fristensky B.1999. Constitutive expression of pea defense gene DRR206 confers resistance to blackleg (Leptosphaeria maculans) disease in transgenic canola (Brassica napus). Mol. Plant Microbe Interact.,12:410-418.
Welsh J,McClelland M.1990 Fingerprinting geuomes using PCR with arbitrary primers. Nucleic. Acids Res.,18 (24):7213-7218.
Wijaya R, Neumann GM, Condron R, Hughes AB, Poly GM.2000. Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin. Plant Sci.,159:243-55.
Williams JG, Kubeilk AR, Livak KJ, et al.1990. DNA polymorphisms amplified by arbit rary primers are useful as genetic markers. Nucleic. Acids Res.,18 (22):6531-6535.
Willingdon R.1913. Inheritance of the russet skin in the pear. Science,37(943):156.
Wisniewski ME, Bassett CL, Artlip TS, Webb RP, Janisiewicz WJ, Norelli JL, Goldway M, Droby S. 2003. Characterization of a defensin in bark and fruit tissues of peach and antimicrobial activity of a recombinant defensin in the yeast, Pichia pastor is. Physiol. Plant.,119:563-72.
Wong JH, Ng TB.2005. Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase. Peptides,26:1120-6.
Wong JH, Zhang XQ, Wang HX, Ng TB.2006. A mitogenic defensin from white cloud beans (Phaseolus vulgaris). Peptides,27:2075-81.
Xiao H, Jiang N, Schaffner E, Stockinger EJ, van der Knaap E.2008. A retransposon-mediated gene duplication underlies morphplogical variation of tomato fruit. Science,319:1527-1530.
Xu Q, Yu KQ, Zhu AD, Ye JL, Liu Q, Zhang JC, Deng XX.2009. Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange (Citus sinensis) red-flesh mutant. BMC Genomics,10:540.
Ye W, Qin Y, Ye Z, da Silva JAT, Zhang L, Wu X, Lin S, Hu G.2009. Seedless mechanism of a new mandarin cultivar'Wuzishatangju'(Citrus reticulata Blanco). Plant Sci.,177:19-27.
Ye XY, Ng TB.2001. Peptides from pinto bean and red bean with sequence homology to cowpea 10-kda protein precursor exhibit antifungal, mitogenic, and HIV-1 reverse transcriptase-inhibitory activities. Biochem. Bioph. Res. Co.,285:424-429.
Yin F, Pajak A, Chapman R, Sharpe A, Huang S, Marsolais F.2011. Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins BMC Genomics,12:268. doi: 10.1186/1471-2164-12-268.
Zabeau M, Vos P.1993. Selective restriction fragment amplication:A general method for DNA fingerprinting. European Patent Application., No.0534858 A1.
Zelicourt A, Letousey P, Thoiron S, Campion C, Simoneau P, Elmorjani K, Marion D, Simier P, Delavault P.2007. Ha-DEF1, a sun flower defensin, induces cell death in Orobanche parasitic plants. Planta,226:591-600.
曹玉芬,刘凤之,高源,姜立杰,王昆,马智勇,张开春.2007.梨栽培品种SSR鉴定及遗传多样性.园艺学报,34(2)：305-310.
曹玉芬,刘凤之,胡红菊,张冰冰.2006.梨种质资源描述规范和数据标准.北京：中国农业出版社.
曹玉芬.2001.我国的红皮梨种质资源.中国种业,1：26-27.
曾柏全,甘霖,熊兴耀,邓秀新,邓子牛.2006.冰糖橙优良芽变的AFLP分析.江西农业大学学报,28(2)：222-225.
曾继吾,彭成绩,易干军,杜观兴,张绍平,冯春添,霍合强,钟云,周碧容,黄永红.2006.晚熟柑橘新品种‘明柳甜橘’.园艺学报,33(5)：1164.
陈大明,张上隆,金勇丰.1997.一种本本果树基因组DNA提取方法研究.浙江农业大学学报,23(6)：621-624.
陈健初,董绍华,叶兴乾,苏平.1997.芹菜汁叶绿素及色泽稳定性的研究.浙江农业大学学报,23(3)：301-304.
陈婷,王日葵,周炼,陆智明,刘涛.2010.柑橘贮藏期间色差指数变化规律的研究.农产品加工学,3：4-7.
陈希历,陈苏.1989.巴梨优良红色芽变系.山西果树.1：44-45.
丁武,魏益民.2002.色彩色差计在肉品新鲜度检验中的应用.肉类研究,4：47-48.
樊丽,方成泉,王斐,王志刚,董星光,林盛华.2008.DNA分子标记在梨育种上的应用概况.中国果树,3：49-53.
樊庆忠.2010.桃芽变中早熟新品种凤红的选育.中国果树,6：3-5.
房经贵,章镇,周兰华,陈崇顺,王三红.2001.RAPD标记鉴定果树芽变的可行性分析.果树学报,18(3)：182-185.
冯少菲.2006.黄金梨果皮发育、锈斑形成及套袋对其影响的研究.保定：河北农业大学.15.
冯月秀,李从玺,王琨.2001.梨早熟梨-六月酥.西北园艺,(5)：35.
高正辉,潘海发,徐义流,张昂,束冰.2010.‘砀山酥梨’芽变‘97-05-9’的RAPD分析.中国农学通报,26(10)：48-53.
苟永平.1997.介绍一质优的冬果梨芽变.北京农业,10：28.
郭景芬.1990.大南果梨芽变优系通过审定.中国农业科技通讯,6(2)：48.
贺立红,张进标,宾金华.2006.苯丙氨酸解氨酶的研究进展.食品科技,7：31-34.
衡伟,贾兵,叶振风,李晓峰,李雪,朱立武.2011.砀山酥梨褐皮芽变品系锈酥果皮结构分析.中国果树,3：20-22.
洪柳,邓秀新.2005.应用MSAP技术对脐橙品种进行DNA甲基化分析.中国农业科学,38：2301-2307.
胡宁三,蔡盛华,陆修闽,黄新忠.2007.柑橘类新品种——红肉蜜柚.中国农技推广,23(10)：16.
焦言英,赵桂敏,陈继忠.1999.南果梨芽变新品种红南果梨.中国果树,3：22.
金勇丰,张耀洲,陈大明,张上隆.1998.桃早熟芽变品种‘大观一号’的RAPD分析及其特异片断克隆.果树科学,15(2)：103-106.
赖国亮,吴金桃,兰永辉.1999.测色技术在炒青绿茶品质评价中的应用研究.福建茶叶,2：19-20.
黎茵,张以顺,周玉亮,陈琛,黄上志.2010.莲种子防御素基因序列分析及表达载体构建.园艺学报,37(12)：175-182.
李俊才,李宝江,于丹,刘成.2010.2个西洋梨红色芽变品种与原品种果实发育期果皮色素变化比较研究.中国果树,1：27-30.
李俊才,隋洪涛,王家珍,刘成,许英武,闫忠业,孙丽梅,蔡忠民,沙守峰,伊凯.2006.梨芽变新品种——尖把王梨的选育.果树学报,23(6)：912-913.
李俊才,王斌,高庆福,王家珍,蔡忠民,张景娥.2011.红色梨新品种‘南红梨’.园艺学报,38(9)： 1821-1822.
李敏,代洪义,李培环,王然,林振海.1983.在梨杨山酥梨等果点的形成.落叶果树,3：3-5.
李润唐,黄茂松,张春年.黄花梨芽变新品系——大香西黄梨(暂定名).2001.落叶果树,1：22.
李涛,解娟,罗雪峰,李纯凡.2010.凯特杏芽变新品种金沙杏的选育.中国南方果树,39(4)：55-56.
李晓芳,礼茂福,韩振海,许雪峰,李天忠.2008.‘鸭梨’芽变‘闫庄梨’自交亲和分子机制的初步研究.园艺学报,31(1)：13-18.
李忠,杨建平,陈远富,秦勇,唐太平,钟天才,钟天华.2010.早熟、优质梨新品系‘普威早熟1号’的选育.中国园艺文摘,6：144-145.
廖明安,张志鹏,任雅君,邓国涛,程帅,李健,吕秀兰,王永清,汪志辉,周廷国.2010.优质高产梨新品种‘川花梨’.园艺学报,37(6)：1017-1018.
廖振坤,张秋明,刘卫国,丁伟平,王春梅.2006.利用AFLP鉴定柑橘变异.果树学报,23(3)：486-488.
林真二.(吴耕民译).1981.梨.北京：农业出版社.293-318.
刘成明,梅曼彤.2002.利用RAPD分析鉴别荔枝的焦核突变体.园艺学报,29(1)：57-59.
刘平,彭士琪,郭振怀.2002.果树主要经济性状的遗传动态.河北林果研究,17(2)：267-274.
刘庆华,周启河,王奎玲,刘庆芳,吕志宁.1992.莱阳往梨果实斑痕的解剖学研究.莱阳农学院学报,9(2)：104-108.
刘中成,赵锦,刘孟军.2004. mRNA差异显示技术评述.分子植物育种,2(6)：895-900.
罗安才,李纯凡,黄仁湖,向可术,李道高.2003.奉节脐橙芽变株系的AFLP分析.农业生物技术科学,19(6)：20-24.
马克元,傅玉瑚,程福厚,苏艳英,王海星,苏春季.1990.鸭梨果点发育的解剖观察.山西果树,04：12-14.
念红忠,银立新,张晓霞,张素芳,李学林,杨瑞林.2002.华夫人葡萄芽变新品种——华变.中国南方果树,31(3)：73.
聂继云,李静,杨振峰,张红军,李明强.2006.冷冻法测定梨的石细胞含量.果树学报,23(1)：133-135.
宁允叶,熊庆娥,曾伟光,曾光荣.2003.红阳猕猴桃全红芽变系的RAPD分析.园艺学报,30(5)：511-513.
宁允叶,熊庆娥,曾伟光.2005.‘红阳’猕猴桃全红型芽变(86-3)的果实品质及花粉形态研究.园艺学报,32(3)：486-488.
蒲富慎.1979.梨的一些性状的遗传.遗传,01：25-28.
曲柏宏.2003.延边苹果梨分类地位及其授粉受精特性的研究.沈阳：沈阳农业大学3-8,23.
任秋萍,张复君,吕福堂,王贵余,朱法作,邢柱东.2007.梨浓红色新品种奥冠红梨的选育.中国 果树,6：12-13.
任志宏,雷应果,张国德.1998.大果型芽变新品种——伏红.现代农业,5：29.
申连长,马晓娣,王彦敏,傅玉瑚.2005.鸭梨芽变新品系——光鸭梨.河北果树,3：19.
沈德绪,林伯年.1985.园艺植物遗传学.北京：农业出版社.249-250.
沈德绪.1986.果树育种学.上海：上海科学技术出版社.
沈德绪.1992.果树育种学.北京：农业出版社.55-56.
盛炳成.2008.黄河故道地区果树综论.北京：中国农业出版社.267.
石荫坪,李雅志,王强生.1986.果树突变育种.上海：上海科技出版社.43-109,255-293.
束怀瑞.1999.苹果学.北京：中国农业出版社.186-196.
宋伟,王彩虹,田义轲,田伟,殷豪.2010.梨果实褐皮性状的SSR标记.园艺学报,37(8)：1325-1328.
宋忠伍.2010.‘红甜核杏’芽变的选育.北方果树,1：47-48.
陶能国.2006.甜橙(Citrus sinensis Osbeck)红肉突变体类胡萝卜素合成相关基因的克隆与特性分析.武汉：华中农业大学博士学位论文.64-67.
陶世蓉,辛华,初庆刚,曹玉芬.1999.窝梨果实结构及发育的研究.西北植物学报,19(1)：123-126.
滕元文,沈玉英,周先章.2005.砂梨果皮锈斑成因及解决对策.中国南方果树,34(3)：52-56.
王家政,王家喜,王常君.1993.红巴梨引种研究初报.落叶果树.2：39-40.
王心燕,肖璇,乔爱民,孙敏.2006.'石硖’龙眼芽变系双引物RAPD鉴定的研究.园艺学报,33(1)：134-136.
王新卫,刘金义,孙兴民,崔海荣,王燕,乔玉山,章镇.2010.中国梨生产、贸易与国际竞争力分析.中国农学通报,26(21)：202-206.
王宇霖,White A, Brewer L, Cranwel D.1997.红皮梨育种研究报告.果树科学,14(2)：71-76.
王志,闫忠业.2010.富士系苹果的发展概况.农业科技通讯,1：164-166.
魏立刚.2011.晚熟大果型梨——奥林斤秋梨.农村百事通,21：25.
魏志东,米建仓,晁晓庆.2000.桃芽变新品种——礼泉沙红桃.西北园艺,1：31-32.
吴丹,叶兴乾,陈健初.2007.杨梅汁色差分析.酿酒,34(6)：94-96.
吴桂余,吴桂法,吴建华韦军.2000.梨新品种‘大果白酥’.园艺学报,27(6)：460.
吴厚玖,陈荣柱,李育农.1984.金冠苹果果锈发生时期与绣花过程的研究.园艺学报,11(1)：51-54.
吴华清,张绍铃,吴巨友,王迎涛,吴俊.2007.‘金坠梨’自交亲和性突变机制的初步研究.园艺学报,34(2)：295-300.
吴忠华,张洪平,韩寄桥,唐道禄.1994.香梨芽变新品种新梨2号.新疆农业科学,4：175.
武瑞俊,王耀胜.2011.红地球葡萄四倍体芽变新品种红太阳的选育.山西果树,3：4.
肖璇,孙敏,王心燕,乔爱民,江波.2005.‘石砍’龙眼大果型桂花味芽变系的RAPD分析鉴定.园艺学报,32(4)：684-687.
徐凌飞,张福民.2009.早熟红色梨优良品系——早酥红.山西果树,4：44.
徐象华,斯金平,谢建秋,蓝云龙,曾燕如,江伟林,叶文伟.2006.柑橘新品种无子欧柑的选育.果树学报,23(5)：781-782.
徐晓梅,杨署光.2009.苯丙氨酸解氨酶研究进展.安徽农业科学,37(31)：15115-15119,15122.
许方.1992.梨树生物学.北京：科学出版社,150-158.
许宽勇.2009.苹果钙反向转运体基因的克隆、表达特性及其功能分析研究.南京：南京农业大学.
宣继萍.2001.富士苹果芽变的细胞学与分子生物学鉴定研究.南京：南京农业大学26-28.
鄢新民,李学营,王献革,郝婕,冯建忠.2011.苹果芽变及芽变选种回顾.河北农业科学,15(05)：75-77.
阎振立,张顺妮,张全军.2006.果树的芽变选种.果农之友,1：38-39.
杨岐生,林卿,李奕,谢毅.1999.用差异显示法从人胎脑基因文库分离一个编码序列.中国生物化学与分子生物学报,15(6)：876-880.
杨有龙.1989.美国的苹果芽变选种概况.落叶果树,4：40-41.
姚红伟,张立冬,孙金阳,刘霄霞.2010.DNA分子标记技术概述.河北渔业,7：42-46.
伊凯,闫忠业,刘志,王冬梅,杨峰,张景娥.2006.苹果芽变选种鉴定及应用研究.果树学报,23(5)：745-749.
尹永胜,王强生.1996.巴梨四倍体芽变品种的选育.中国农学通报,12(5)：22-23.
尹永胜,徐增凯,宋福正,李瑞芝,王强生.1999.巴梨芽变新品种鲁梨1号.中国果树,3：23.
俞德浚,张鹏.1979.我国梨品种的一个新系统.园艺学报,6(1)：27-32.
张东,滕元文.2011.红梨资源及其果实着色机制研究进展.果树学报,28(3)：485-492.
张凯斌,刘凤君,代汉萍,吴宇红,高兆银.2003.花盖梨芽变新品系——花盖王.中国果树,1：12-13.
张士鸿.2010.砀山酥梨果实PAL酶学特性的研究及PAL基因克隆.合肥：安徽农业大学.
张小军,徐凌飞,吴中营.2009.梨极早熟芽变品种‘六月酥’的AFLP分析.西北农林科技大学学报,37(12)：139-145,138.
张志刚,李栒,官春云,白德朗,武小金.2006.改良提取水稻胚乳和拟南芥花柱中DNA和RNA的SDS法.植物生理学通讯,42(3)：493-495.
张智涛,张立彬,于凤鸣,关骋遥.2011.大果水晶梨与其褐色突变体多酚氧化酶及过氧化物酶同工酶差异(简报).河北科技师范学院学报,25(2)：31-33.
章镇,俞宏,高志红,葛绍义,陶建敏,蔡斌华.2000.梨新品种‘大果黄花’.园艺学报,27(1)：74.
赵广,乐文全,路娟,张绍铃,李俊才,吴俊.2011.梨果皮色泽变异品种(系)的AFLP与SRAP 标记鉴定.果树学报,28(4)：568-574.
赵权,王军.2010.ABA和6-BA对山葡萄果实着色及相关品质的影响.江苏农业科学,2：189-190.
郑玉龙,姜春玲,冯玉龙.2005.植物的气孔发生.植物生理学通讯,41(6)：847-850.
周蓓蓓,朱海军,生静雅,刘广勤.2011.DNA分子标记在果树种质资源遗传多样性研究中的应用.江西农业学报,23(9)：47-50.
朱立武,王艳芳,贾兵,张水明,黄永丰,杨振江.2009.安徽砀山酥梨自然保护区梨种质资源AFLP分析.果树学报,26(2)：145-150.
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