ω-醇溶蛋白基因的克隆及通过RNAi抑制小麦α-醇溶蛋白基因家族表达研究
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摘要
小麦醇溶蛋白主要赋予面粉的粘性。目前,已从普通小麦(Triticum aestivum)中克隆了大量的α-、γ-醇溶蛋白基因,但是已克隆的ω-醇溶蛋白基因数量还很少。根据小麦属ω-醇溶蛋白基因设计了一对简并引物,以体细胞杂种渐渗系Ⅱ-12及其亲本小麦Jinan 177和十倍体长穗偃麦草的gDNA为模板,扩增得到了16个ω-醇溶蛋白基因序列,长度变化为924～1146bp。其中,6个序列中因为含有编码框内提前终止密码子,因此可能是假基因。这些序列与已知的ω-醇溶蛋白基因序列有高度的相似性。通过分析ω-醇溶蛋白基因推导的氨基酸序列发现ω-醇溶蛋白由两侧短的N-端与C-端保守区及中间长重复可变区组成。系统进化分析表明这些ω-醇溶蛋白基因聚为三个亚家族：第一个包括来源于5个长穗偃麦草和1个Ⅱ-12的序列,表明至少有一个长穗偃麦草ω-醇溶蛋白基因通过体细胞杂交渗入Ⅱ-12中；第二个包括3个来自Ⅱ-12和6个来自于Jinan177的序列,它们都属于ARQ/E型ω-醇溶蛋白基因；第三个由三个来自于普通小麦的SRL型ω-醇溶蛋白基因组成。我们还发现,来自于第二亚家族的Ⅱ-12的一个ω-醇溶蛋白基因在其C端有一个Cys位点,这可能使其与面粉品质相关。所以我们预测,通过体细胞渐渗的方式使得杂种Ⅱ-12产生了新的ω-醇溶蛋白基因。
乳糜泻(coeliac disease)是患者进食麦类食品后,对其中的醇溶谷蛋白不耐受而引起的慢性小肠吸收不良综合症,而α-醇溶蛋白含有大量可导致乳糜泻疾病的毒性肽段。本论文通过对已发布的α-醇溶蛋白基因进行聚类,找出它们保守性较强的片段,根据本实验室得到的一个来自于体细胞杂种渐渗系Ⅱ-12的α-醇溶蛋白基因设计引物,经PCR扩增得到保守性片段作为干扰α-醇溶蛋白基因家族的出发序列,并构建了RNA干扰真核表达载体p3301-Ubi-AR12与p3301-Ubi-AR34。通过农杆菌介导的转化方法,分别将上述两个载体转化小麦株系,经PCR筛选后,初步得到了30株阳性株系。利用酸性聚丙烯酰胺凝胶电泳分别对这三十株阳性株系的种子进行了醇溶蛋白组成的分析检测,发现其中有六株籽粒表现出明显的α-醇溶蛋白干扰现象。为进一步研究RNA干扰是否对植株的高、低分子量谷蛋白亚基产生影响,我们通过SDS-聚丙烯酰胺凝胶电泳对上述六株产生RNA干扰效应的植株进行分析,发现它们的HMW-GS组成与对照相比没有发生改变,而LMW-GS发生了一定程度的改变。这为进一步研究RNA干扰技术在小麦谷蛋白的功能提供了理论基础。
The wheat gliadins are mainly responsible for viscosity of dough. To date, a number ofα-andγ-gliadin genes have been cloned from common wheat, but only severalω-gliadin genes have been published on GenBank. A pair of degenerate PCR primers that targeted the entire coding sequence of the Triticeae co-gliadin genes was used to amplify the DNA of a somatic hybrid introgression line and its two parents Jinan177, a bread wheat genotype(Triticum aestivum L.) and a tall wheatgrass (Agropyron elongatum, 10x). Cloning and sequencing of the resulting amplicons identified 16 distinct sequences, ranging in length from 924 to 1146 bp. Six of the sequences represented pseudogenes. The sequences shared a high degree of homology to knownω-gliadin sequences. The primary structure of the deduced polypeptides comprised a short N-terminal and C-terminal conserved domain, and a long repetitive and variable domain. A phylogenetic analysis produced three major clades:the first contained five tall wheatgrass parent sequences and one introgression line sequence, suggesting the transfer of at least one tall wheatgrassω-gliadin gene into the introgression line; the second group clustered three introgression line sequences with six Jinan 177 sequences, both of these groups were populated by ARQ/E-typeω-gliadins; the third group comprised three bread wheat SRL-typeω-gliadins. The co-gliadin of one of the introgression lines in group 2 has a cysteine residue in its C-terminal domain which may allow it to be involved in the determination of end-use quality. We present that somatic hybridisation of bread wheat could introgress wild and produce novelω-gliadin genes in the introgression lines.
Coeliac disease is a chronic enteropathy caused by intolerance to gluten proteins. There are many toxic peptides that can result in celiac disease in a-gliadins. In order to inhibit/decrease the expression of a-gliadin gene family, conserved fragments of all a-gliadin genes released were found by sequence aligned and one sequence from 11-12 possessed conserved fragments was selected to design the RNAi trigger sequences. Then they were used to construct the RNA interference eukaryotic expression vectors p3301-Ubi-AR12 and p3301-Ubi-AR34. As followed, both of them were transformed into the wheat strain respectively by using Agrobacterium-mediated transformation method. Thirty positive transgenic plants were obtained by PCR screening. Seeds of six plants presented apparently silencing of a-gliadins examined by acid-PAGE and band scan analysis compared with control. In order to investigate whether the composition and expression patterns of HMW-GS and LMW-GS were influenced by RNAi, SDS-PAGE profile was examined to analyse these positive lines in which present a-gliadins silencing. It was shown that the composition and expression patterns of LMW-GS were changed but HMW-GS not. It will provide useful information in further functional study of wheat gluten by RNAi.
乳糜泻(coeliac disease)是患者进食麦类食品后,对其中的醇溶谷蛋白不耐受而引起的慢性小肠吸收不良综合症,而α-醇溶蛋白含有大量可导致乳糜泻疾病的毒性肽段。本论文通过对已发布的α-醇溶蛋白基因进行聚类,找出它们保守性较强的片段,根据本实验室得到的一个来自于体细胞杂种渐渗系Ⅱ-12的α-醇溶蛋白基因设计引物,经PCR扩增得到保守性片段作为干扰α-醇溶蛋白基因家族的出发序列,并构建了RNA干扰真核表达载体p3301-Ubi-AR12与p3301-Ubi-AR34。通过农杆菌介导的转化方法,分别将上述两个载体转化小麦株系,经PCR筛选后,初步得到了30株阳性株系。利用酸性聚丙烯酰胺凝胶电泳分别对这三十株阳性株系的种子进行了醇溶蛋白组成的分析检测,发现其中有六株籽粒表现出明显的α-醇溶蛋白干扰现象。为进一步研究RNA干扰是否对植株的高、低分子量谷蛋白亚基产生影响,我们通过SDS-聚丙烯酰胺凝胶电泳对上述六株产生RNA干扰效应的植株进行分析,发现它们的HMW-GS组成与对照相比没有发生改变,而LMW-GS发生了一定程度的改变。这为进一步研究RNA干扰技术在小麦谷蛋白的功能提供了理论基础。
The wheat gliadins are mainly responsible for viscosity of dough. To date, a number ofα-andγ-gliadin genes have been cloned from common wheat, but only severalω-gliadin genes have been published on GenBank. A pair of degenerate PCR primers that targeted the entire coding sequence of the Triticeae co-gliadin genes was used to amplify the DNA of a somatic hybrid introgression line and its two parents Jinan177, a bread wheat genotype(Triticum aestivum L.) and a tall wheatgrass (Agropyron elongatum, 10x). Cloning and sequencing of the resulting amplicons identified 16 distinct sequences, ranging in length from 924 to 1146 bp. Six of the sequences represented pseudogenes. The sequences shared a high degree of homology to knownω-gliadin sequences. The primary structure of the deduced polypeptides comprised a short N-terminal and C-terminal conserved domain, and a long repetitive and variable domain. A phylogenetic analysis produced three major clades:the first contained five tall wheatgrass parent sequences and one introgression line sequence, suggesting the transfer of at least one tall wheatgrassω-gliadin gene into the introgression line; the second group clustered three introgression line sequences with six Jinan 177 sequences, both of these groups were populated by ARQ/E-typeω-gliadins; the third group comprised three bread wheat SRL-typeω-gliadins. The co-gliadin of one of the introgression lines in group 2 has a cysteine residue in its C-terminal domain which may allow it to be involved in the determination of end-use quality. We present that somatic hybridisation of bread wheat could introgress wild and produce novelω-gliadin genes in the introgression lines.
Coeliac disease is a chronic enteropathy caused by intolerance to gluten proteins. There are many toxic peptides that can result in celiac disease in a-gliadins. In order to inhibit/decrease the expression of a-gliadin gene family, conserved fragments of all a-gliadin genes released were found by sequence aligned and one sequence from 11-12 possessed conserved fragments was selected to design the RNAi trigger sequences. Then they were used to construct the RNA interference eukaryotic expression vectors p3301-Ubi-AR12 and p3301-Ubi-AR34. As followed, both of them were transformed into the wheat strain respectively by using Agrobacterium-mediated transformation method. Thirty positive transgenic plants were obtained by PCR screening. Seeds of six plants presented apparently silencing of a-gliadins examined by acid-PAGE and band scan analysis compared with control. In order to investigate whether the composition and expression patterns of HMW-GS and LMW-GS were influenced by RNAi, SDS-PAGE profile was examined to analyse these positive lines in which present a-gliadins silencing. It was shown that the composition and expression patterns of LMW-GS were changed but HMW-GS not. It will provide useful information in further functional study of wheat gluten by RNAi.
引文
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98. Zhao TJ, Zhao SY, Chen HM, Zhao QZ, Hu ZM, Hou BK, Xia GM (2006) Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculums to the basal portion of wheat seedling. Plant Cell Reports 25,1199-1204
99.高梅,张国权,魏益民(2007)小麦醇溶蛋白A-PAGE电泳鉴定技术的分析。西北农林科技大学学报：自然科学版35(9)：53-57
100.耿洪伟,张庆祝,何中虎,夏兰芹,陈新民,王德森,曲延英(2006)转外源puroindoline基因小麦的获得与检测。中国农业科学39(9)：1751-1755
101.侯义龙(2010)RNA干扰及其在植物上的应用研究进展与展望。信阳师范学院学报23(2)：316-320
102.柳觐,相微微,张怀刚(2009)RNA干扰技术在小麦中的应用研究进展。麦类作物学报29(4)：725-72
103.施农农,何光源,李克秀,Jones HD, Shewry PR(2005)基因枪法获得优质HMW亚基基因表达的转基因小麦。中国农业科学38(5)：874-881
104.汪越胜,柯涛,李三和,覃建兵,刘勇,何光源(2005)转基因小麦后代HMW-GS组成的变异研究。作物学报31(4)：529-531
105.张晓东,梁荣奇,陈绪清,杨凤萍,张立全(2003)优质HMW谷蛋白亚基转基因小麦的获得及其遗传稳定性和品质性状分析。科学通报48(5)：474-479
106.张晓科,李耀科,魏益民(2002)小麦贮藏蛋白特性及其遗传转化。麦类作物学报22(2)：78-82
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97. Xue ZY, Zhi DY, Xue GP, Zhang H, Zhao YX, Xia GM (2004a) Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L) expressing a vacuolar Na+/H+antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na+. Plant Sci 167:849-859
98. Zhao TJ, Zhao SY, Chen HM, Zhao QZ, Hu ZM, Hou BK, Xia GM (2006) Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculums to the basal portion of wheat seedling. Plant Cell Reports 25,1199-1204
99.高梅,张国权,魏益民(2007)小麦醇溶蛋白A-PAGE电泳鉴定技术的分析。西北农林科技大学学报：自然科学版35(9)：53-57
100.耿洪伟,张庆祝,何中虎,夏兰芹,陈新民,王德森,曲延英(2006)转外源puroindoline基因小麦的获得与检测。中国农业科学39(9)：1751-1755
101.侯义龙(2010)RNA干扰及其在植物上的应用研究进展与展望。信阳师范学院学报23(2)：316-320
102.柳觐,相微微,张怀刚(2009)RNA干扰技术在小麦中的应用研究进展。麦类作物学报29(4)：725-72
103.施农农,何光源,李克秀,Jones HD, Shewry PR(2005)基因枪法获得优质HMW亚基基因表达的转基因小麦。中国农业科学38(5)：874-881
104.汪越胜,柯涛,李三和,覃建兵,刘勇,何光源(2005)转基因小麦后代HMW-GS组成的变异研究。作物学报31(4)：529-531
105.张晓东,梁荣奇,陈绪清,杨凤萍,张立全(2003)优质HMW谷蛋白亚基转基因小麦的获得及其遗传稳定性和品质性状分析。科学通报48(5)：474-479
106.张晓科,李耀科,魏益民(2002)小麦贮藏蛋白特性及其遗传转化。麦类作物学报22(2)：78-82
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