调控大豆异黄酮合成相关转录因子基因克隆与功能验证

Soybean isoflavone is one of the most important secondary metabolites with antitumor bioactivity for human health. Therefore, the key problem is that how to increase effectively the isoflavone content in soybean seeds. Currently, the effective method is to breed the cultivar with high isoflavone content by the molecular biochemical way. In this study, on the base of the soybean high-density genetic linkage map, we have mapped the quantitative trait loci (QTLs) underlying isoflavone content and analyzed their molecular biological information. The genes of key enzymes and transcription factors will be cloned based on the molecular information of reference genome comparison and GO ontology. The gene sequencing will be compared between both soybean parents to search the mutant loci. Fine mapping will be conducted using the High Resolution Melting (HRM) method based on the soybean secondary-segregation population. The closely linked molecular markers are also to be explored using the dCAPs primers according to the mutant loci. For candidate genes, the spatial and temporal expression will be analyzed by the real-time PCR and its subcellular location will be conducted using the green fluorescent protein (GFP) fusion method. The gene functions will also be clarified based on transformation methods of soybean hairy root and cotyledonary nodes using the RNA interference, overexpression, and HPLC technologies. Meanwhile, the promotor banding and target sites between the candidate transcription factors and key enzymes will be also identified by the yeast one-hybrid and gel mobility shift assays. Our objective is to illustrate the molecular network regulating the isoflavone accumulation in soybean seeds in order to facilitate the soybean molecular breeding for isoflavone content.

大豆异黄酮是一种重要抗癌生物活性物质，对人体健康具有重要作用，因此，如何有效地提高大豆种子中的异黄酮含量是亟需解决的关键问题，而采用基因调控手段培育高异黄酮含量的大豆品种，是行之有效的方法。本项目在构建的高密度遗传连锁图谱基础上，将定位到与异黄酮显著相关的QTL进行生物信息学分析，通过与参考基因组比对和GO基因注释，克隆关键酶和转录因子，并进行双亲序列比对，寻找基因突变位点；采用高分辨率溶解曲线（HRM）方法结合次级分离群体进行精细定位，针对突变位点设计dCAPs引物，开发紧密连锁分子标记。针对候选基因，采用实时定量PCR研究其时空表达，经与GFP蛋白融合分析其亚细胞定位；利用大豆发状根和子叶节转化方法，结合RNAi、过表达和HPLC技术，认证候选基因功能；采用酵母单杂交和凝胶阻滞试验验证候选转录因子与关键酶启动子的结合及其靶位点，以期解析大豆异黄酮的分子调控网络，为异黄酮分子育种服务。

大豆异黄酮是一种重要的抗癌生物活性物质，对人体健康具有重要作用，因此开展大豆异黄酮分子标记辅助育种具有重要意义。本研究首先明确了大豆种质的异黄酮地理分布规律，构建了大豆高密度遗传连锁图谱，定位了异黄酮含量相关的24个稳定QTL，精细定位主效QTL（qIF20-2），并在SSR130-SSR175区间内发现了5个与大豆异黄酮含量相关的非同义突变基因，包括1个提前终止基因和1个起始密码子丢失基因，其中提前终止基因（GmZF1）编码锌指蛋白类转录因子；其次，分析了高低异黄酮亲本GmZF1基因在发育种子中的表达量和异黄酮积累差异，过表达或RNAi抑制表达的发状根中GmZF1基因的表达量和异黄酮含量均显著高于或低于对照；利用1472份大豆自然群体的表型和基因型数据，明确了GmZF1的优势单倍型；亚细胞定位显示该基因定位在细胞核中；经转录激活分析发现该基因在高低异黄酮亲本中均具有转录激活活性，但在高异黄酮亲本中的转录激活活性更高；最后，转GmZF1基因的过表达植株或RNAi植株的叶片中的表达量和异黄酮含量均显著高于或低于对照，进一步验证了GmZF1基是异黄酮积累的正调控因子；另外，通过双荧光素酶系统也证明了GmZF1基因通过上调CHS1和IFS2基因，下调F3H基因来促进异黄酮积累，为大豆异黄酮分子设计育种提供理论支撑。