小麦Glu-B1位点高分子量麦谷蛋白亚基的功能分析及其调控机理

The type and quantity of high molecular weight glutenin subunits (HMW-GS) from wheat flour are directly bound up with the gluten strength, which mainly influences the processing quality of wheat. It has been reported that the gluten strength has something to do with the 1Bx7 overexpression subunit from Glu-B1 locus, and it's the one increased copy of 1Bx7 in the wheat material with 1Bx7 overexpression subunit that cause its overexpression. So we can infer that by increasing the copies of 1Bx7 in varieties which are widely popularized in production using transgene technology, the wheat quality will be further improved. In our research group, the 1Bx7 subunit from high-quality wheat material derived from Australia will be transfered into Zhengmai 366 by transgene method to obtain the higher-quality new material; To analyze the influence of 1Bx7+1By8 subunits on wheat quality, we will be the first at home and abroad to utilize hpRNAi and amiRNA technologies in turn to respectively and simultaneously silence the expression of 1Bx7+1By8 subunits from Glu-B1 locus in Zhengmai 366; We plan to apply promoter stepwise deletion, yeast one-hybrid, and electrophoresis mobility shift assay to identify the key factors which regulate the subunit expression in Glu-B1 locus. The result of this project will not only demonstrate the molecular mechanism of HMW-GS expression, but also supply with new thinking in improving wheat quality by transgene method.

小麦面粉中高分子量麦谷蛋白亚基（HMW-GS）的类型和数量直接关系到面筋强度，进而影响小麦的加工品质。研究发现，面筋强度与Glu-B1位点1Bx7超量表达亚基有关，而它的表达增强是由于在含有该亚基的小麦材料中1Bx7增加了1个拷贝。据此，我们推测利用转基因技术在目前生产上大面积推广的品种中增加1Bx7的拷贝数，可以进一步提高小麦品质。本课题采用转基因方法将源于澳大利亚优质小麦材料中的1Bx7亚基导入到郑麦366中，期望获得更加优质的新材料；在国内外首次采用hpRNAi结合amiRNA技术依次分别和同时沉默郑麦366中Glu-B1位点1Bx7+1By8亚基的表达，以分析它们对小麦最终加工品质的影响；拟采用启动子分段缺失、酵母单杂交和凝胶迁移阻滞实验来分离调控Glu-B1位点亚基表达的关键因子。本项目研究结果不仅可以阐明HMW-GS表达的分子机理，还为利用转基技术改良小麦品质提供新思路。

小麦是世界范围内种植面积最广的粮食作物，它是人类主要的能量、蛋白和食用纤维的来源。小麦含有独特的面筋成分，能被加工成面包、面条、饼干等各式各样的食品来满足人们消费需求。小麦籽粒中高分子量麦谷蛋白亚基（HMW-GS）的类型和数量是决定小麦良好加工品质的关键因素。本研究利用转基因、酵母单杂等技术深入的解析了超表达1Bx7亚基对小麦面筋品质影响及其表达调控机制。利用1Dx5亚基启动子，融合1Bx7 基因读码框序列，构建了p1Dx5::1Bx7超表达载体，将该载体转入郑麦366中，获得高代转基因株系，品质分析结果表明单独增加1Bx7亚基的表达量，并不能显著提升小麦的面筋强度。定量PCR结合启动子融合GUS试验结果表明1Bx7基因在小麦籽粒中特异表达且在20天籽粒中表达量最高。生物信息学方法分析发现1Bx7基因上游启动子序列含有种子表达必要元件Skn-1模体，通过构建Skn-AbAi载体和酵母文库，筛选到1个Dof转录因子参与1Bx7基因的表达调控。此外，利用转录组测序技术分离到7个醇溶蛋白基因在弱筋材料郑麦004中强表达，通过RACE技术克隆到TaWG04基因的全长，Real-time PCR分析发现该基因在强筋品种中表达受到抑制。利用甲基化特异性PCR技术，发现在郑麦366等强筋材料中TaWG04启动子区在-758和-778bp因发生甲基化修饰导致该基因的表达受到抑制，结果初步表明DNA甲基化对小麦面筋品质具有正向调控作用。本研究结果不仅对阐明小麦面筋品质形成的分子机理具有重要科学意义，也为优质小麦品质选育提供理论和技术支撑。