致敏蛋白α-亚基缺失型大豆近等基因系的创制及相关基因的图位克隆与功能验证

The α-subunit of 7S globulin, the components of soybean storage protein as well as the main allergens, is one of the important factors affecting the nutritional and processing quality of soybean. Near-isogenic lines (NILs) are one effective type of plant genetic resources for gene-mapping technique, which are developed through repeated backcrossing and extensive selection for a trait of interest. By comparing such NILs containing the presence or absence of the target region, a large number of genes controlling the desirable traits have been isolated through advanced molecular technologies,because these pairs of NILs were useful and valuable for determining the target gene in many crops. In the present study, using a Japanese variety "riB" with (α′+α)-subunits deficiency of 7S globulin (β-conglycinin) as the donor parent, and soybean variety Dongnong47 with high oil content, wildly planted in Heilongjiang Province of China, as the recurrent parent, the NILs (BC4F5) with respect to α-subunit-null gene will be developed through four cycles of backcrossing and advanced five selfing generation. Marker-assistant selection (MAS) were applied for screening of individuals with the genomic regions of a recurrent parent Dongnong47 in BC2F2 and BC3F3 generation, individuals with the highest recurrent parent genome (RPG) content were selected using for backcross so as to accelerate the development of NILS for α-subunit-null trait. Theses NILs (BC4F3:4) will be confirmed by SDS-PAGE analysis and background MAS (lines showing greater than 99% RPG content will be selected), as well as phenotype traits evaluation. Then with NIL gene-mapping approach, we are going to: (1) study the effect of α-subunit-null loci on yield traits, nutritional quality and proceeding quality in soybean.(2)detect the polymorphic molecular markers closely related to the trait of α-subunit-null for the α- null/normal gene pool by bulked segregation analysis (BSA) in BC2F2 population, with SSR markers evenly distributed on soybean genome, so that preliminary map α-subunit-null gene. (3) develop a large mapping population with heterozygous BC5F2 plants. Based on the results obtained in the initial screen for α- subunit gene allelic diversity, if co-segregated SSR markers were not identified in candidate region, we are going to develop microsatellite markers, insertion deletion polymorphism markers or cleaved amplified polymorphic sequence markers so as to complete fine mapping the α-subunit-null gene on chromosome.These works will provide particularly powerful genetic materials for further isolating, map-based gene cloning and its function study of α-subunit-null gene and get greater efficiency for cultivation of allergic subunit deficiency soybean.

7S球蛋白α亚基是大豆种子贮藏蛋白的重要组分之一，α缺失可以在去除致敏蛋白的同时提高大豆蛋白的营养价值、改善其加工品质。近等基因系（NIL）是理论上可以最大限度降低遗传背景差异、使单基因效应得以充分表达的高级作图群体。目前，国内外尚没有利用α缺失型NIL对α缺失相关基因的遗传效应及缺失机理进行研究的报道。本研究以日B为供体亲本，东农47为轮回亲本；采用冬季南繁加代、春季SDS-PAGE法筛选α缺失个体，夏季回交的方法加快世代进程，通过准确的近等性评价，育成α缺失型NILα-DN47。利用α-DN47分析α缺失相关基因的遗传效应，构建F2作图大群体，完成相关基因的精细定位，通过基因组序列信息图位克隆法克隆α缺失相关基因，并对该基因功能进行验证，为分子标记辅助选育α缺失型大豆新品种提供永久性遗传学材料平台，为明晰α缺失相关基因的遗传效应、了解其基因功能，揭示缺失特性的分子调控机制提供理论依据。

本研究首先通过常规杂交结合四轮回交，同时利用分子标记辅助遗传背景选择法，完成全基因组遗传背景扫描，创制了仅在第20号染色体存在目的基因差别的、致敏蛋白α-亚基缺失特性稳定遗传、主要农业性状与轮回亲本东农47基本相同、遗传背景回复率大于99%的理想的致敏蛋白α-亚基缺失型近等基因系（NIL）群体。利用NIL群体，完成了致敏蛋白α-亚基缺失基因的遗传效应分析，致敏蛋白α-亚基的缺失，可以在有效除去致敏蛋白的同时，全面改良大豆蛋白的氨基酸品质、提高必须氨基酸含量及氨基酸总量。游离氨基酸含量增加、特别是游离精氨酸含量的增加，是α-亚基缺失基因主要的遗传效应。致敏蛋白‘α-亚基缺失基因’是大豆蛋白质品质改良优良的等位变异。通过图位克隆的方法，将α-亚基缺失相关基因精细定位于大豆第20号染色体SSR标记BARCSOYSSR_20_987和BARCSOYSSR_20_996之间，α-亚基缺失基因cgy-2与BARCSOYSSR_20_987和BARCSOYSSR_20_996两引物间的距离均为0.5cM。在精细定位确认的 122.6 kb区域内，NCBI(http://www.ncbi.nlm.nib.gov)网站共注释了14个完整的基因。综合功能预测分析及转录差异比较的结果，我们首先完成了其中八个关键候选基因GLYMA20G28530、GLYMA20G28540、GLYMA20G28571、GLYMA20G28670、GLYMA20G28631、GLYMA20G28545、GLYMA20G28550及GLYMA20G28610的克隆，进行了以上八个基因的过表达及RNAi干扰载体的大豆遗传转化工作，获得了部分基因的T2代转基因阳性植株。qRT-PCR分析的结果表明，在种子发育的过程中，以上八个基因在致敏蛋白α-亚基缺失型突变体与其轮回亲本东农47之间，均显著差异表达。完成了GLYMA20G28571、GLYMA20G28670的亚细胞定位分析，其中Glyma20g28571定位在大的细胞器、Glyma20g28670基因被定位在细胞核里。