水母雪莲（S.Medusa Maxim）类黄酮合成及抗逆相关基因克隆与查耳酮异构酶（CHI）基因的原核表达
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摘要
类黄酮类物质是雪莲药物的主要有效成分。植物类黄酮合成代谢途径的关键酶有苯丙氨酸裂解酶(PAL)、查耳酮合酶(CHS)、查耳酮异构酶(CHI)、黄烷酮3-羟化酶(F3H)等基因，以及一些调节基因如myb家族基因等。通过对这些基因的克隆和遗传转化，希望能够提高雪莲悬浮细胞及愈伤组织的类黄酮含量，为雪莲细胞大规模工业化发酵生产提供进一步的技术支持。
雪莲是一类生长于高寒严酷环境中的特殊植物，它具有对多种严酷逆境很强的抗性。其中与抗逆有关的基因资源于作物生产具有非常大的利用价值。通过克隆及转化这些基因，可以大大提高资源植物及作物的逆境抗性。
本实验主要克隆了多个与以上方面相关的基因cDNA及基因cDNA片段，进行了序列比较分析，并做了CHI基因的原核表达。同时对筛选文库的方法进行了改进，建立了两个文库筛选方法。重要结果如下：
1、建立了一种分部分排除筛选文库的方法。
通过对cDNA噬菌体平板划块分组，用PCR技术分部分检测排除来筛选文库。与其它筛选cDNA文库的方法相比，该方法范围可控，目标明确，能够明确地一步一步缩小目的基因的范围。而且筛选效率高、快速、简捷、省时。一般可在一周内筛选出目的基因。此方法用于同时筛选多个基因，可收到事半功倍的效果，对其它文库筛选也有借鉴意义。
2、建立了用一对引物从文库筛选多个基因的方法。
该方法利用已知序列设计引物，通过比较多个不同的基因序列，再根据这些基因之间相同或相近的部分设计引物，来同时筛选多个不同的基因。该方法主要用于从文库同时筛选克隆多个基因。
3、克隆到雪莲CHI基因(GenBank注册号：AF509335)。
CHI基因cDNA序列全长831bp，polyA信号(aataaa)在755bp-760bp之间。含有261个a、173个c、184个g、213个t，a、t总数占总碱基数的57％，有完整的阅读框，开放阅读框全长699bp。表明是该基因cDNA序列是完整的。序列间比较结果表明，与其它物种CHI基因同源性最高达85％。基因3′-端紧接着出现多个终止密码子，暗示该基因于代谢可能非常重要。该基因编码蛋白长232个氨基酸，分子量24913，预测PI为4.75。序列中的Ser和Thr含量占了接近20％。而Ala、Gly、Ile、Leu、Val四种非极性氨基酸数量占了大约37％。一个半胱氨酸(Cys)位于116位，一个组氨酸(His)位于197位。这两个氨基酸是否与CHI酶活性位点有关，需要进一步的验证。
4、克隆到雪莲F3H基因(GenBank注册号：AF509338)。
F3H基因cDNA序列全长1344bp，a、t碱基含量比c、g碱基高13.27％。嘌呤和嘧啶之间基本平衡。该序列中含有一个polyA信号(1297～1302)，尾部有polyA。该蛋白共有343个氨基酸，分子量38843道尔顿，预测PI值为5.55。与其它的物种F3H基因序列比较，同源性均在30～40％，但有一些相对保守的区域。而用于比较的其它物种已知的F3H基因之间序列比较，同源性相对要比与雪莲比较的高得多(大约在60％以上)。
蛋白质Blast比较，与茄子的加双氧酶、颠茄的天仙子胺-6-β-羟化酶、卡茄的2-酮戊
水母宰莲类故酮合成及抗逆相关基因克隆与m 基因的原佰表达——摘 要
二酸依赖的加双氧酶、天仙子的天仙子胺－6才加双氧酶、番茄的卜氨基环丙烷梭酸盐－卜氧
化酶（铁缺乏蛋白）（IDS）、拟南芥的黄烷酮 3－羟化酶样蛋白、Fe（11）／抗坏血酸氧化酶有
40～50％的同源性。与普通大麦的铁缺乏蛋白、拟南芥、水稻、白杉的乙烯形成酶、拟南芥
的花色素合酶、无色花色素加双氧酶、黄烷酮合酶有30～40％的部分同源性。蛋白质结构域
检索，与异青霉素 N－合酶有较高的结构域相似性。与 2－酮戊二酸－Fe（11）加氧酶
（20－Felloxy）的部分结构域也有很好的相似性。推测该基因是雪莲类黄酮合成代谢特有
的酶基因，它引导雪莲的特殊次生代谢物的合成。
5、克隆到两个HSP70基因。
其中全KHSP70基因cDNA（GenBank注册号：AF509336）K 2224hp，其氨基酸序列
全长647，分于量为冗US道尔顿。预测pl值为4．95。另一条HSP70基囚C＊＊A片段长
14S4bP。比较二条序列发现具有如下的特点：①氨基酸除第7位A、V A换外，其余完全相
同。②碱基序列有35个碱基不同，在这35个碱基之间，有22个是C、T之间的互换，9个
是A、G之间的互换，2个A、T间互换，且个T、G间互换，1个C、G间互换。从中可以看
出，碱基之间的变异主要集中在C、T和A、G之间，即瞟吟与瞟吟之间、啼陡和啼陡之间的
互换，在膘吟与啼陡之间的互换很少。③从翻译后的情况来看，所有的35个差异碱基，除
第一个变异碱基在三联体密码子的第二位不同，造成氨基酸A变为V，最后一个差异碱基在
开放阅读框外以外，其余的33个差异碱基都在三联体密码子的第三位不同，没有造成氨基
酸的突变。④通过与其它的 705 HSP蛋白序列比较来看，此变异较大的区域主要集中在氨基
酸序列相对较保守的部位。⑤在 3’端，全序列比得到的部分序列多出了一段 22hP的碱基
（序列为TTATTAATCTTTCTTGTCATGC），其中A、T碱基含量高达73％。
6、获得了多个调节基因片段
通过合成引物从文库扩增，?
Flavonoids are the main components for the medical effect in Sausurrea medusa. The key Enzymes for the synthesis pathway of plant flavonoids include phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and a number of regulatory genes as some Myb family genes mediate the transcriptional activation of the flavonoids biosynthesis. We hope to gain a higher production of flavonoids through transforming these genes into suspension cells of Sausurrea medusa, and provide a solid ground on technology for further large scale produce of suspension cells.
Sausurrea medusa, which live in the high and inclement environments, have an excellent resistance character against many adversities. The genes that related to these resistance characters in Sausurrea medusa would be of great economic value on crop cultivation. We can improve the adversities resistance of resource plants and crops by transforming these genes into them.
Several complete or partial genes cDNA were cloned from cDNA library of Sausurrea medusa, and two new methods for screening cDNA library were established in this paper. All the results as the following:
1. Subsection method for cDNA library screening. With this method, cDNA phage plate was cut into several blocks, dilute each section using dilution buffer and detect them by PCR respectively. Then the target genes were obtained by screening cDNA library. Comparing with other methods, this method has many advantages such as controlled range and clear target, and also quick and simple for obtaining the target genes. It is possible to get a target gene in one week in general by this method. We can get twice the result with half the effort when we screen several genes in the same time. It is also suitable to screen other libraries.
2. Another method-creening several genes from cDNA library using one pair of primers by PCR was brought forward here. In this method, a pair of primers have been designed basing on the same or the similar parts of several sequences, and been used to screen over two genes from cDNA library by PCR at the same time. It's a good method for cloning such genes involved some similar or same partial sequences, and could be applied to screening some other libraries.
3. One chalcone isomerase gene (No: AF509335) was cloned from cDNA library. This gene involves 831bp, 57% a and t, a polyA signal, and one 31 bp polyA tail. One 699bp open reading
-5 CHI jfflM)^^ - Abstract
frame was found then. Putative protein includes 232 amino acids and 20% Ser and Thr, pi 4.15.
4. A Flavanone 3-hydroxylase (No: AF509338) complete cDNA was isolated from cDNA library, which is 1344bp length in bases sequence, a and t are higher than c and g in bases content, a polyA signal and 31bp polyA be involved in it. Putative protein includes 343 amino acids, pi 5.55. Only 30-40% conserved parts have been found in the bases sequence comparing with other genes, which is far lower than that when comparing with each other in themselves. Some similar domains to Isopenicillin N synthase and partial similar domains to 2OG-Fe(II) oxygenase superfamily were found when Search the protein conserved domains in GenBank.. Protein Blast shows that it has 40-50% identities with dioxygenase (eggplant), hyoscyamine 6-beta-hydroxylase (Atropa belladonna), 2-oxoglutarate-dependent dioxygenase (Solanum chacoense), hyoscyamine (6S)-dioxygenase (henbane), hyoscyamine 6-beta-hydroxylase-like protein (Solanum tuberosum), flavanone 3-hydroxylase (Oryza sativa), flavanone 3-beta-hydroxylase (Arabidopsis thaliana), putative Fe(II)/ascorbate oxidase (Arabidopsis thaliana), and some other enzymes. These hint that this gene may functions as completely distinct action with such F3H we have known.
5. One HSP70 gene complete cDNA (No: AF509336 ) and a partial cDNA were cloned from library. 2224bp be involved in the complete cDNA and 1484bp in the partial cDNA. Putative protein includes 647 amino acids in the complete HSP70, and its' MW is 70715 D, PI 4.95. Several charact
雪莲是一类生长于高寒严酷环境中的特殊植物，它具有对多种严酷逆境很强的抗性。其中与抗逆有关的基因资源于作物生产具有非常大的利用价值。通过克隆及转化这些基因，可以大大提高资源植物及作物的逆境抗性。
本实验主要克隆了多个与以上方面相关的基因cDNA及基因cDNA片段，进行了序列比较分析，并做了CHI基因的原核表达。同时对筛选文库的方法进行了改进，建立了两个文库筛选方法。重要结果如下：
1、建立了一种分部分排除筛选文库的方法。
通过对cDNA噬菌体平板划块分组，用PCR技术分部分检测排除来筛选文库。与其它筛选cDNA文库的方法相比，该方法范围可控，目标明确，能够明确地一步一步缩小目的基因的范围。而且筛选效率高、快速、简捷、省时。一般可在一周内筛选出目的基因。此方法用于同时筛选多个基因，可收到事半功倍的效果，对其它文库筛选也有借鉴意义。
2、建立了用一对引物从文库筛选多个基因的方法。
该方法利用已知序列设计引物，通过比较多个不同的基因序列，再根据这些基因之间相同或相近的部分设计引物，来同时筛选多个不同的基因。该方法主要用于从文库同时筛选克隆多个基因。
3、克隆到雪莲CHI基因(GenBank注册号：AF509335)。
CHI基因cDNA序列全长831bp，polyA信号(aataaa)在755bp-760bp之间。含有261个a、173个c、184个g、213个t，a、t总数占总碱基数的57％，有完整的阅读框，开放阅读框全长699bp。表明是该基因cDNA序列是完整的。序列间比较结果表明，与其它物种CHI基因同源性最高达85％。基因3′-端紧接着出现多个终止密码子，暗示该基因于代谢可能非常重要。该基因编码蛋白长232个氨基酸，分子量24913，预测PI为4.75。序列中的Ser和Thr含量占了接近20％。而Ala、Gly、Ile、Leu、Val四种非极性氨基酸数量占了大约37％。一个半胱氨酸(Cys)位于116位，一个组氨酸(His)位于197位。这两个氨基酸是否与CHI酶活性位点有关，需要进一步的验证。
4、克隆到雪莲F3H基因(GenBank注册号：AF509338)。
F3H基因cDNA序列全长1344bp，a、t碱基含量比c、g碱基高13.27％。嘌呤和嘧啶之间基本平衡。该序列中含有一个polyA信号(1297～1302)，尾部有polyA。该蛋白共有343个氨基酸，分子量38843道尔顿，预测PI值为5.55。与其它的物种F3H基因序列比较，同源性均在30～40％，但有一些相对保守的区域。而用于比较的其它物种已知的F3H基因之间序列比较，同源性相对要比与雪莲比较的高得多(大约在60％以上)。
蛋白质Blast比较，与茄子的加双氧酶、颠茄的天仙子胺-6-β-羟化酶、卡茄的2-酮戊
水母宰莲类故酮合成及抗逆相关基因克隆与m 基因的原佰表达——摘 要
二酸依赖的加双氧酶、天仙子的天仙子胺－6才加双氧酶、番茄的卜氨基环丙烷梭酸盐－卜氧
化酶（铁缺乏蛋白）（IDS）、拟南芥的黄烷酮 3－羟化酶样蛋白、Fe（11）／抗坏血酸氧化酶有
40～50％的同源性。与普通大麦的铁缺乏蛋白、拟南芥、水稻、白杉的乙烯形成酶、拟南芥
的花色素合酶、无色花色素加双氧酶、黄烷酮合酶有30～40％的部分同源性。蛋白质结构域
检索，与异青霉素 N－合酶有较高的结构域相似性。与 2－酮戊二酸－Fe（11）加氧酶
（20－Felloxy）的部分结构域也有很好的相似性。推测该基因是雪莲类黄酮合成代谢特有
的酶基因，它引导雪莲的特殊次生代谢物的合成。
5、克隆到两个HSP70基因。
其中全KHSP70基因cDNA（GenBank注册号：AF509336）K 2224hp，其氨基酸序列
全长647，分于量为冗US道尔顿。预测pl值为4．95。另一条HSP70基囚C＊＊A片段长
14S4bP。比较二条序列发现具有如下的特点：①氨基酸除第7位A、V A换外，其余完全相
同。②碱基序列有35个碱基不同，在这35个碱基之间，有22个是C、T之间的互换，9个
是A、G之间的互换，2个A、T间互换，且个T、G间互换，1个C、G间互换。从中可以看
出，碱基之间的变异主要集中在C、T和A、G之间，即瞟吟与瞟吟之间、啼陡和啼陡之间的
互换，在膘吟与啼陡之间的互换很少。③从翻译后的情况来看，所有的35个差异碱基，除
第一个变异碱基在三联体密码子的第二位不同，造成氨基酸A变为V，最后一个差异碱基在
开放阅读框外以外，其余的33个差异碱基都在三联体密码子的第三位不同，没有造成氨基
酸的突变。④通过与其它的 705 HSP蛋白序列比较来看，此变异较大的区域主要集中在氨基
酸序列相对较保守的部位。⑤在 3’端，全序列比得到的部分序列多出了一段 22hP的碱基
（序列为TTATTAATCTTTCTTGTCATGC），其中A、T碱基含量高达73％。
6、获得了多个调节基因片段
通过合成引物从文库扩增，?
Flavonoids are the main components for the medical effect in Sausurrea medusa. The key Enzymes for the synthesis pathway of plant flavonoids include phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and a number of regulatory genes as some Myb family genes mediate the transcriptional activation of the flavonoids biosynthesis. We hope to gain a higher production of flavonoids through transforming these genes into suspension cells of Sausurrea medusa, and provide a solid ground on technology for further large scale produce of suspension cells.
Sausurrea medusa, which live in the high and inclement environments, have an excellent resistance character against many adversities. The genes that related to these resistance characters in Sausurrea medusa would be of great economic value on crop cultivation. We can improve the adversities resistance of resource plants and crops by transforming these genes into them.
Several complete or partial genes cDNA were cloned from cDNA library of Sausurrea medusa, and two new methods for screening cDNA library were established in this paper. All the results as the following:
1. Subsection method for cDNA library screening. With this method, cDNA phage plate was cut into several blocks, dilute each section using dilution buffer and detect them by PCR respectively. Then the target genes were obtained by screening cDNA library. Comparing with other methods, this method has many advantages such as controlled range and clear target, and also quick and simple for obtaining the target genes. It is possible to get a target gene in one week in general by this method. We can get twice the result with half the effort when we screen several genes in the same time. It is also suitable to screen other libraries.
2. Another method-creening several genes from cDNA library using one pair of primers by PCR was brought forward here. In this method, a pair of primers have been designed basing on the same or the similar parts of several sequences, and been used to screen over two genes from cDNA library by PCR at the same time. It's a good method for cloning such genes involved some similar or same partial sequences, and could be applied to screening some other libraries.
3. One chalcone isomerase gene (No: AF509335) was cloned from cDNA library. This gene involves 831bp, 57% a and t, a polyA signal, and one 31 bp polyA tail. One 699bp open reading
-5 CHI jfflM)^^ - Abstract
frame was found then. Putative protein includes 232 amino acids and 20% Ser and Thr, pi 4.15.
4. A Flavanone 3-hydroxylase (No: AF509338) complete cDNA was isolated from cDNA library, which is 1344bp length in bases sequence, a and t are higher than c and g in bases content, a polyA signal and 31bp polyA be involved in it. Putative protein includes 343 amino acids, pi 5.55. Only 30-40% conserved parts have been found in the bases sequence comparing with other genes, which is far lower than that when comparing with each other in themselves. Some similar domains to Isopenicillin N synthase and partial similar domains to 2OG-Fe(II) oxygenase superfamily were found when Search the protein conserved domains in GenBank.. Protein Blast shows that it has 40-50% identities with dioxygenase (eggplant), hyoscyamine 6-beta-hydroxylase (Atropa belladonna), 2-oxoglutarate-dependent dioxygenase (Solanum chacoense), hyoscyamine (6S)-dioxygenase (henbane), hyoscyamine 6-beta-hydroxylase-like protein (Solanum tuberosum), flavanone 3-hydroxylase (Oryza sativa), flavanone 3-beta-hydroxylase (Arabidopsis thaliana), putative Fe(II)/ascorbate oxidase (Arabidopsis thaliana), and some other enzymes. These hint that this gene may functions as completely distinct action with such F3H we have known.
5. One HSP70 gene complete cDNA (No: AF509336 ) and a partial cDNA were cloned from library. 2224bp be involved in the complete cDNA and 1484bp in the partial cDNA. Putative protein includes 647 amino acids in the complete HSP70, and its' MW is 70715 D, PI 4.95. Several charact
引文
Akhilesh K.Tyagi Plant genes and their expression Current Science,2001,80(2):161
Allan Force, Angel Amores et al Hox Cluster Organization in the Jawless Vertebrate Petromyzon marinus Journal of Experimental Zoology(MOL DEV EVOL), 2002 294:30—46
Arakaki AK, Ceccarelli EA.Eur Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones. J Biochem 2000 Oct; 267(20):6239-48
Amaravadi L, King M W. A rapid and efficient nonradioactive method for screening recombinant DNA libraries[J] Biotechniques, 1994,16(1):98～103
Bednar PA, McCaffrey C, Shan K. Introduction of unnatural amino acids into chalcone isomerase. Bioconjug Chem 1991 Jul-Aug;2(4):211-6
Benton W D, Davis R W. Screening λ gt Recombinant clones by hybridization to single plaques in situ[J] Science,1977,196:180
Bruce W,Folkerts O,Garnaat C,Crasta O,Roth B,Bowen B.Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P.Plant Cell 2000 Jan;12(1):65-80
Burbulis IE, Pelletier MK, Cain CC, Shirley BW. Are flavonoids synthesized by a multi-enzyme complex? SAAS Bull Biochem Biotechnol 1996;9:29-36
Burbulis IE, Winkel-Shirley B Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. Proc Natl Acad Sci USA 1999 Oct 26;96(22):12929-34
Cain CC, Saslowsky DE, Walker PA, Shirley BW. Expression of chalcone synthase and chaicone isomerase proteins in Arabidopsis seedlings. Plant Mol Biol 1997 Oct; 35(3):377-81
Charrier B, Coronado C, Kondorosi A, Ratet P. Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydrofiavonol-4-reductase encoding genes.Plant Mol Biol 1995 Nov;29(4):773-86
Diallinas G, Kanellis AK. A phenylalanine ammonia-lyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding. Plant Mol Biol 1994 Oct;26(1):473-9
Eilers,M., G. Schatz. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature, 1986,322:228-232。
Fang Yue-qun, Wang Chun-hui, Xie Gui-lin, Wang Bin-rui Analysis of amplification efficiency and control of false positive in nest-PCR[J] Progress in microbiology and immunology 2001,29(1):43～48
Carl W. Dieffenbach and Gabriela S. Dveksler PCR Primer: A Laboratory manual[M] Cold Spring Harbor Laboratory Press,1995
Grotewold E, Peterson T. Isolation and characterization of a maize gene encoding chalcone flavonone isomerase, Mol Gen Genet 1994 Jan;242(1):1-8
Hagen RE, Dunlap WJ, and Wender SH, Seasonal variation of naringin and certain other flavanone glycosides in juice sacs of Texas Ruby Red grapefruit. Food Science 1966,31:542-547
Jackson-Constan D, Keegstra K. Arabidopsis genes encoding components of the chloroplastic
protein import apparatus. Plant Physiol 2001 Apr;125(4):1567-76
James M. Tepperman, Tong Zhu, Hur-Song Chang et al Multiple transcription-factor genes are early targets of phytochrome A signaling PNAS, 2001.98(16):9437-9442
Jez JM, Bowman ME, Dixon RA, Noel JP. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nat Struct Biol 2000 Sep;7(9):786-91
Jez JM, Noel JP. Reaction mechanism of chalcone isomerase, pH dependence, diffusion control, and product binding differences. J Biol Chem 2002 Jan 11;277(2):1361-9
Kimura Y,Aoki T,Ayabe S. Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxy- and 6'-deoxychaleones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids. Plant Cell Physiol 2001 Oct; 42(10):1169-73
Kropat J, Oster U, Rudiger W, Beck CF. Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus. Plant J 2000 Nov;24(4):523-31
Kubasek WL, Ausubel FM, Shirley BW. A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings. Plant Mol Biol 1998 May;37(2):217-23
Liang P, Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J] Science, 1992,257(14):967～971
Li QB, Guy CL. Evidence for non-circadian light/dark-regulated expression of Hsp70s in spinach leaves. Plant Physiol 2001 Apr;125(4):1633-42
Lisitsyn Ni, Lisitsyn Na, Wigler M. Cloning the differences between two complex genomes[J] Science,1993,259(12):946～951
Manthey JA, Buslig BS, Advances in Experimental Medicine and Biology Vol.439, Flavonoids in the living system, 1999,Plenum Press New York
McKhann HI, Hirsch AM. Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcriptlevels occur in young roots and root tips. Plant Mol Biol 1994 Mar;24(5):767-77
Muir SR, Collins GJ, Robinson S, Hughes S, Bovy A, Ric De Vos CH, van Tunen AJ, Verhoeyen ME Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols.. Nat Biotechnol 2001 May;19(5):470-4
Noh B,Spalding EP. Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings. Plant Physiol 1998 Feb;116(2):503-9
Pay-Yen Chen Chen-Kuen Weng Shaw-Ching Soong Kin-Ying To Genetic Modification of Flower Color in Tobacco by Overexpression of Petunia Chalcone Synthase
Pelletier MK, Burbulis IE, Winkel-Shirley B Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings. Plant Mol Biol 1999 May;40(1):45-54
Pelletier MK, Shirley BW. Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase. Plant Physiol 1996 May; 111(1):339-45
Rouseff RL, Martin SF, Youtsey CO, Quantitative survey of narirutin, naringin, hesperidin, and neohesperidin in Citrus. J. Agric. Food. Chem. 1987.35:1027-1030
Ryan KG, Swinny EE, Winefield C, Markham KR. Flavonoids and UV photoprotection in
Arabidopsis mutants. Z Naturforsch[C]. 2001 Sep-Oct;56(9-10):745-54.
Saslowsky D, Winkel-Shirley B. Localization of flavonoid enzymes in Arabidopsis roots. Plant J 2001 Jul;27(1):37-48
Satyanarayana T, Gowda S, Mawassi M, Albiach-Marti MR, Ayllon MA, Robertson C, Garnsey SM, Dawson WO. Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly. Virology 2000 Dec 5;278(1):253-65
Searles L L, Jokerst R S, Binghham P M, Voelker, R. A., and Greenleaf, A. L Molecular cloning of sequences from a drosophila RNA polymerase Ⅱ locus by P element transposon tagging[J] Cell, 1982,31:585
Slightom JL, Custom polymerase-chain-reaction engineering of a plant expression vector. Gene 1991,100:251-255
Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C. Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Mol Biol 1994 Mar;24(5):743-55
Sun XT, Li B, Zhou GM, Tang WQ, Bai J, Sun DY, Zhou RG. Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70. Plant Cell Physiol 2000 Jun;41(6):804-10
Sung DY, Vierling E, Guy CL. Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiol 2001 Jun;126(2):789-800
Song W Y, Wang G L, Chen L L, Kim H S, Pi L Y, Holsten T, Gardner J, Wang B, Zhai W X, Zhu L H, Fanquet C, Ronald P C A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21[J] Science, 1995,270:1804
Takumi T,Lodish HF. Rapid cDNA cloning by PCR screening. Bio Techniques, 1994,17:443-444
Terai Y, Fujii I, Byun SH, Nakajima O, Hakamatsuka T, Ebizuka Y, Sankawa U. Cloning of chalcone-flavanone isomerase cDNA from Pueraria lobata and its overexpression in Escherichia coli. Protein Expr Purif 1996 Sep;8(2):183-90
Ufuk Koca, Diane Luth, Mark Berhow, and Gloria A. Moore Manipulation of the Flavonoid Biosynthetic Pathway in Citrus to Decrease Bitter Taste and/or to Increase The Flavonoid Content
Voisine C, Schilke B, Ohlson M, Beinert H, Marszalek J, Craig EA. Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Y fh1. Mol Cell Biol 2000 May;20(10):3677-84
Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA Cell Biol 1991,10(3):233-8
Wood AJ, Davies E. A cDNA encoding chalcone isomerase from aged pea epicotyls Plant Physiol 1994 Apr;104(4):1465-6
Yang Xiao-Ming, Xie Ling, Hu Zhi-Yuan, Qiu Zhao-Hua, Wu Chu-Tse, He Fu-Chu. A Method of Screening an Arrayed cDNA Library by PCR[J] Chinese Biochemical Journal 1997,13(5):505～508
Zheng Fei-yun, Jin Jian-zhong et al. The PCR Prime7rs Design of Beer Spoilage Lactic Acid Bacteria Liquor Making Mar.,2002,29(2)
陈发菊，扬映根，赵德修，等．我国雪莲植物的种类、生境分布及化学成分的研究进展[J]．植物学通报，1999，16(5)：561～566．
陈金瑞，王叶富，邱林刚等，云南植物研究，1989．11(3)：271～275
戴茹娟，李宁 利用PCR法从文库中快速克隆人促红细胞生成素基因 农业生物技术学报．1996，4(1)．76-80
方悦群，王春晖，谢贵林，王秉瑞 套式PCR检测体系扩增效率的分析及对假阳性的控制[J] 微生物学免疫学进展，2001，29(1)：43～48
方溪，叶伟胜，温志永等，1983．天津医学院学报，1：41
国家环境保护局，中国科学院植物研究所．中国珍稀濒危保护植物名录．(第一册)．北京：科学出版社，1987
韩书亮，大苞雪莲四种成分抗癌作用研究〔J〕癌变·畸变·突变，1995，7：80-83
何新，李观海，陈汉瑜，西北药学学报，1990．5(3)：17～19
胡迎春 张开泰 PCR快速筛选质粒cDNA文库方法的建立 中华放射医学与防护杂志．1999，19(5)．310-312
李观海，药学通报，1979．14(2)：86
李观海，刘发，赵荣春，雪莲对大鼠实验性关节急性炎症的作用〔J〕药学学报，1980．15：368～370
李君山，蔡少青，中国药学杂志，1998．33(8)：449～452
李瑜，贾忠建，杜枚等，高等学校化学学报，1985．6(5)：417～420
李瑜，贾忠建，朱子清，席等学校化学学报，1989．10(9)：909～912
连文琰，肖培根．雪莲花原植物的调查整理[J]．中药材，1985，6：19．
梁文生，柴进，雪莲通脉丸治疗偏瘫82例报告〔J〕江苏中医，1996，17(1)：14
林秀珍，王国祥．雪莲多糖对离体大鼠子宫的作用[J]．药学学报，1986，21(3)：220～222．
刘力生，肖显华，张龙弟等，兰州大学学报，(自然科学版)，1985．21(4)：80～83
刘学志，孙明德，吴欣，天津医学院学报，1983．2：38～43
刘勇民，沙吾提·伊克木．维吾尔药志(上册)．乌鲁木齐：新疆人民出版社，1985，385～389
鲁国东，王宗华，郑学勤，谢联辉 cDNA文库和PCR技术相结合的方法克隆目的基因 农业生物技术学报 1998．6(3)，257-262)
青藏高原综合考察队 西藏植物志：第四卷[Z] 北京：科学出版社，1995 878
邱林刚，连敏，马忠武，等 雪兔子的成分研究[J] 植物学报，1989，31(5)：398～401
全国中草药汇编编写组，全国中草药汇编，人民卫生出版社，1997，第758～761页，第889～890页
沈观冕．新疆凤毛菊属分类的初步研究．新疆植物学研究文集．北京：科学出版社，1991，102～115
孙敬三，桂耀林，植物细胞工程实验技术〔M〕北京：科学出版社，1995，403～408
谭敦炎，朱建雯等．高山红景天的繁殖生态学研究．Ⅰ生态因子与植物学、物候学特性分析．新疆农业大学学报，1997，20(3)：1～4
瓦·古巴诺娃，刘杰龙，石玉瑚．新疆雪莲的组织培养[J]．新疆农业科学，1990(5)：221～222
王本富，路杰，关家彦，天山雪莲注射液提取工艺的考察〔J〕 中国药学杂志，1996，31(5)：299～301
王惠康，林章代，何侃等，新疆雪莲化学成分的研究．药学学报，1986，21(9)：680～682
王志超．新疆天山东段的现代冰川．新疆冰川积雪研究论文集．新疆维吾尔自治区科委出版，1964，1～9
吴征镒，西藏植物志(吴征镒主编)，北京：科学出版社，1985，Vol．4：758～762：865～
881
徐军望 朱祯等 快速cDNA文库筛选和淀偻合成酶基因的分离与鉴定 高技术通讯．2001，11(8)．1～6
杨晓明，谢玲，胡志远，邱兆华，吴祖泽，贺福初 PCR介导的cDNA文库矩阵排列筛选方法[J] 生物化学杂志，1997，13(5)：505～508
余建华，程珍，郑尚珍，等 雪兔子中木脂素成分的研究[J] 中草药，1997，23(6)：283～285
余建华，郑尚珍，沈序维，中国中药杂志，1991．16(6)：356～358
赵德修，赵丽丽．雪莲花的研究进展[J]．中草药，1996，27(6)：372～374．
郑飞云，金建中等 啤酒污染乳酸菌PCR引物的设计 酿酒 2001，29(2)
中国科学院植物研究所主编．中国高等植物图鉴(第4册)．北京：科学出版社，1983．617～618
Allan Force, Angel Amores et al Hox Cluster Organization in the Jawless Vertebrate Petromyzon marinus Journal of Experimental Zoology(MOL DEV EVOL), 2002 294:30—46
Arakaki AK, Ceccarelli EA.Eur Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones. J Biochem 2000 Oct; 267(20):6239-48
Amaravadi L, King M W. A rapid and efficient nonradioactive method for screening recombinant DNA libraries[J] Biotechniques, 1994,16(1):98～103
Bednar PA, McCaffrey C, Shan K. Introduction of unnatural amino acids into chalcone isomerase. Bioconjug Chem 1991 Jul-Aug;2(4):211-6
Benton W D, Davis R W. Screening λ gt Recombinant clones by hybridization to single plaques in situ[J] Science,1977,196:180
Bruce W,Folkerts O,Garnaat C,Crasta O,Roth B,Bowen B.Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P.Plant Cell 2000 Jan;12(1):65-80
Burbulis IE, Pelletier MK, Cain CC, Shirley BW. Are flavonoids synthesized by a multi-enzyme complex? SAAS Bull Biochem Biotechnol 1996;9:29-36
Burbulis IE, Winkel-Shirley B Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. Proc Natl Acad Sci USA 1999 Oct 26;96(22):12929-34
Cain CC, Saslowsky DE, Walker PA, Shirley BW. Expression of chalcone synthase and chaicone isomerase proteins in Arabidopsis seedlings. Plant Mol Biol 1997 Oct; 35(3):377-81
Charrier B, Coronado C, Kondorosi A, Ratet P. Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydrofiavonol-4-reductase encoding genes.Plant Mol Biol 1995 Nov;29(4):773-86
Diallinas G, Kanellis AK. A phenylalanine ammonia-lyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding. Plant Mol Biol 1994 Oct;26(1):473-9
Eilers,M., G. Schatz. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature, 1986,322:228-232。
Fang Yue-qun, Wang Chun-hui, Xie Gui-lin, Wang Bin-rui Analysis of amplification efficiency and control of false positive in nest-PCR[J] Progress in microbiology and immunology 2001,29(1):43～48
Carl W. Dieffenbach and Gabriela S. Dveksler PCR Primer: A Laboratory manual[M] Cold Spring Harbor Laboratory Press,1995
Grotewold E, Peterson T. Isolation and characterization of a maize gene encoding chalcone flavonone isomerase, Mol Gen Genet 1994 Jan;242(1):1-8
Hagen RE, Dunlap WJ, and Wender SH, Seasonal variation of naringin and certain other flavanone glycosides in juice sacs of Texas Ruby Red grapefruit. Food Science 1966,31:542-547
Jackson-Constan D, Keegstra K. Arabidopsis genes encoding components of the chloroplastic
protein import apparatus. Plant Physiol 2001 Apr;125(4):1567-76
James M. Tepperman, Tong Zhu, Hur-Song Chang et al Multiple transcription-factor genes are early targets of phytochrome A signaling PNAS, 2001.98(16):9437-9442
Jez JM, Bowman ME, Dixon RA, Noel JP. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nat Struct Biol 2000 Sep;7(9):786-91
Jez JM, Noel JP. Reaction mechanism of chalcone isomerase, pH dependence, diffusion control, and product binding differences. J Biol Chem 2002 Jan 11;277(2):1361-9
Kimura Y,Aoki T,Ayabe S. Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxy- and 6'-deoxychaleones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids. Plant Cell Physiol 2001 Oct; 42(10):1169-73
Kropat J, Oster U, Rudiger W, Beck CF. Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus. Plant J 2000 Nov;24(4):523-31
Kubasek WL, Ausubel FM, Shirley BW. A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings. Plant Mol Biol 1998 May;37(2):217-23
Liang P, Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J] Science, 1992,257(14):967～971
Li QB, Guy CL. Evidence for non-circadian light/dark-regulated expression of Hsp70s in spinach leaves. Plant Physiol 2001 Apr;125(4):1633-42
Lisitsyn Ni, Lisitsyn Na, Wigler M. Cloning the differences between two complex genomes[J] Science,1993,259(12):946～951
Manthey JA, Buslig BS, Advances in Experimental Medicine and Biology Vol.439, Flavonoids in the living system, 1999,Plenum Press New York
McKhann HI, Hirsch AM. Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcriptlevels occur in young roots and root tips. Plant Mol Biol 1994 Mar;24(5):767-77
Muir SR, Collins GJ, Robinson S, Hughes S, Bovy A, Ric De Vos CH, van Tunen AJ, Verhoeyen ME Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols.. Nat Biotechnol 2001 May;19(5):470-4
Noh B,Spalding EP. Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings. Plant Physiol 1998 Feb;116(2):503-9
Pay-Yen Chen Chen-Kuen Weng Shaw-Ching Soong Kin-Ying To Genetic Modification of Flower Color in Tobacco by Overexpression of Petunia Chalcone Synthase
Pelletier MK, Burbulis IE, Winkel-Shirley B Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings. Plant Mol Biol 1999 May;40(1):45-54
Pelletier MK, Shirley BW. Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase. Plant Physiol 1996 May; 111(1):339-45
Rouseff RL, Martin SF, Youtsey CO, Quantitative survey of narirutin, naringin, hesperidin, and neohesperidin in Citrus. J. Agric. Food. Chem. 1987.35:1027-1030
Ryan KG, Swinny EE, Winefield C, Markham KR. Flavonoids and UV photoprotection in
Arabidopsis mutants. Z Naturforsch[C]. 2001 Sep-Oct;56(9-10):745-54.
Saslowsky D, Winkel-Shirley B. Localization of flavonoid enzymes in Arabidopsis roots. Plant J 2001 Jul;27(1):37-48
Satyanarayana T, Gowda S, Mawassi M, Albiach-Marti MR, Ayllon MA, Robertson C, Garnsey SM, Dawson WO. Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly. Virology 2000 Dec 5;278(1):253-65
Searles L L, Jokerst R S, Binghham P M, Voelker, R. A., and Greenleaf, A. L Molecular cloning of sequences from a drosophila RNA polymerase Ⅱ locus by P element transposon tagging[J] Cell, 1982,31:585
Slightom JL, Custom polymerase-chain-reaction engineering of a plant expression vector. Gene 1991,100:251-255
Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C. Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Mol Biol 1994 Mar;24(5):743-55
Sun XT, Li B, Zhou GM, Tang WQ, Bai J, Sun DY, Zhou RG. Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70. Plant Cell Physiol 2000 Jun;41(6):804-10
Sung DY, Vierling E, Guy CL. Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiol 2001 Jun;126(2):789-800
Song W Y, Wang G L, Chen L L, Kim H S, Pi L Y, Holsten T, Gardner J, Wang B, Zhai W X, Zhu L H, Fanquet C, Ronald P C A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21[J] Science, 1995,270:1804
Takumi T,Lodish HF. Rapid cDNA cloning by PCR screening. Bio Techniques, 1994,17:443-444
Terai Y, Fujii I, Byun SH, Nakajima O, Hakamatsuka T, Ebizuka Y, Sankawa U. Cloning of chalcone-flavanone isomerase cDNA from Pueraria lobata and its overexpression in Escherichia coli. Protein Expr Purif 1996 Sep;8(2):183-90
Ufuk Koca, Diane Luth, Mark Berhow, and Gloria A. Moore Manipulation of the Flavonoid Biosynthetic Pathway in Citrus to Decrease Bitter Taste and/or to Increase The Flavonoid Content
Voisine C, Schilke B, Ohlson M, Beinert H, Marszalek J, Craig EA. Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Y fh1. Mol Cell Biol 2000 May;20(10):3677-84
Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA Cell Biol 1991,10(3):233-8
Wood AJ, Davies E. A cDNA encoding chalcone isomerase from aged pea epicotyls Plant Physiol 1994 Apr;104(4):1465-6
Yang Xiao-Ming, Xie Ling, Hu Zhi-Yuan, Qiu Zhao-Hua, Wu Chu-Tse, He Fu-Chu. A Method of Screening an Arrayed cDNA Library by PCR[J] Chinese Biochemical Journal 1997,13(5):505～508
Zheng Fei-yun, Jin Jian-zhong et al. The PCR Prime7rs Design of Beer Spoilage Lactic Acid Bacteria Liquor Making Mar.,2002,29(2)
陈发菊，扬映根，赵德修，等．我国雪莲植物的种类、生境分布及化学成分的研究进展[J]．植物学通报，1999，16(5)：561～566．
陈金瑞，王叶富，邱林刚等，云南植物研究，1989．11(3)：271～275
戴茹娟，李宁 利用PCR法从文库中快速克隆人促红细胞生成素基因 农业生物技术学报．1996，4(1)．76-80
方悦群，王春晖，谢贵林，王秉瑞 套式PCR检测体系扩增效率的分析及对假阳性的控制[J] 微生物学免疫学进展，2001，29(1)：43～48
方溪，叶伟胜，温志永等，1983．天津医学院学报，1：41
国家环境保护局，中国科学院植物研究所．中国珍稀濒危保护植物名录．(第一册)．北京：科学出版社，1987
韩书亮，大苞雪莲四种成分抗癌作用研究〔J〕癌变·畸变·突变，1995，7：80-83
何新，李观海，陈汉瑜，西北药学学报，1990．5(3)：17～19
胡迎春 张开泰 PCR快速筛选质粒cDNA文库方法的建立 中华放射医学与防护杂志．1999，19(5)．310-312
李观海，药学通报，1979．14(2)：86
李观海，刘发，赵荣春，雪莲对大鼠实验性关节急性炎症的作用〔J〕药学学报，1980．15：368～370
李君山，蔡少青，中国药学杂志，1998．33(8)：449～452
李瑜，贾忠建，杜枚等，高等学校化学学报，1985．6(5)：417～420
李瑜，贾忠建，朱子清，席等学校化学学报，1989．10(9)：909～912
连文琰，肖培根．雪莲花原植物的调查整理[J]．中药材，1985，6：19．
梁文生，柴进，雪莲通脉丸治疗偏瘫82例报告〔J〕江苏中医，1996，17(1)：14
林秀珍，王国祥．雪莲多糖对离体大鼠子宫的作用[J]．药学学报，1986，21(3)：220～222．
刘力生，肖显华，张龙弟等，兰州大学学报，(自然科学版)，1985．21(4)：80～83
刘学志，孙明德，吴欣，天津医学院学报，1983．2：38～43
刘勇民，沙吾提·伊克木．维吾尔药志(上册)．乌鲁木齐：新疆人民出版社，1985，385～389
鲁国东，王宗华，郑学勤，谢联辉 cDNA文库和PCR技术相结合的方法克隆目的基因 农业生物技术学报 1998．6(3)，257-262)
青藏高原综合考察队 西藏植物志：第四卷[Z] 北京：科学出版社，1995 878
邱林刚，连敏，马忠武，等 雪兔子的成分研究[J] 植物学报，1989，31(5)：398～401
全国中草药汇编编写组，全国中草药汇编，人民卫生出版社，1997，第758～761页，第889～890页
沈观冕．新疆凤毛菊属分类的初步研究．新疆植物学研究文集．北京：科学出版社，1991，102～115
孙敬三，桂耀林，植物细胞工程实验技术〔M〕北京：科学出版社，1995，403～408
谭敦炎，朱建雯等．高山红景天的繁殖生态学研究．Ⅰ生态因子与植物学、物候学特性分析．新疆农业大学学报，1997，20(3)：1～4
瓦·古巴诺娃，刘杰龙，石玉瑚．新疆雪莲的组织培养[J]．新疆农业科学，1990(5)：221～222
王本富，路杰，关家彦，天山雪莲注射液提取工艺的考察〔J〕 中国药学杂志，1996，31(5)：299～301
王惠康，林章代，何侃等，新疆雪莲化学成分的研究．药学学报，1986，21(9)：680～682
王志超．新疆天山东段的现代冰川．新疆冰川积雪研究论文集．新疆维吾尔自治区科委出版，1964，1～9
吴征镒，西藏植物志(吴征镒主编)，北京：科学出版社，1985，Vol．4：758～762：865～
881
徐军望 朱祯等 快速cDNA文库筛选和淀偻合成酶基因的分离与鉴定 高技术通讯．2001，11(8)．1～6
杨晓明，谢玲，胡志远，邱兆华，吴祖泽，贺福初 PCR介导的cDNA文库矩阵排列筛选方法[J] 生物化学杂志，1997，13(5)：505～508
余建华，程珍，郑尚珍，等 雪兔子中木脂素成分的研究[J] 中草药，1997，23(6)：283～285
余建华，郑尚珍，沈序维，中国中药杂志，1991．16(6)：356～358
赵德修，赵丽丽．雪莲花的研究进展[J]．中草药，1996，27(6)：372～374．
郑飞云，金建中等 啤酒污染乳酸菌PCR引物的设计 酿酒 2001，29(2)
中国科学院植物研究所主编．中国高等植物图鉴(第4册)．北京：科学出版社，1983．617～618
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