大豆CHR2-1基因生物信息学分析与大豆遗传转化

Bioinformatics Analysis of Soybean CHR2-1 Gene and Its Genetic Transformation to Soybean

大豆异黄酮是有助于人类身体健康的一类次级代谢产物,大豆查尔酮还原酶(chalcone reductase, CHR)基因是控制大豆异黄酮主要组分大豆苷元生物合成的关键酶之一。为拓宽大豆CHR研究范围,促进异黄酮代谢机理研究,进而推进大豆品种改良,本研究利用基因工程技术对大豆CHR2-1基因进行克隆及生物信息学分析,并且构建植物表达载体pCAMBIA3301-CHR2-1,通过农杆菌介导法将目的基因整合到东农50受体大豆基因组中,PCR筛选鉴定获得大豆转化植株。结果表明:GmCHR2-1基因编码的蛋白属于非分泌型蛋白,可能在细胞质中发挥主要功能,属于非跨膜类蛋白并不在细胞中发生迁移;该蛋白存在22个潜在磷酸化位点,其中苏氨酸(Thr)4个、丝氨酸(Ser)18个;该蛋白由α-螺旋(40.63%)、延伸链(14.29%)、β-转角(4.13%)和无规则卷曲(40.95%)4个部分组成。PCR鉴定表明,转化植株基因组包含草丁膦抗性基因Bar、终止子NOS和启动子35S位点,初步确定已将GmCHR2-1转入到东农50大豆品种基因组中,并获得T;阳性植株转化苗12株。

Soybean isoflavones is a class of secondary metabolites that contribute to human health. Soybean chalcone reductase(CHR) gene is one of the key enzymes controlling the biosynthesis of daidzein, the main component of soybean isoflavones. In order to broaden the research types of soybean CHR, promote the research of isoflavone metabolism mechanism, and then promote the improvement of soybean varieties, this study used genetic engineering technology to perform gene cloning and bioinformatics analysis of GmCHR2-1.And then we constructed a recombinant plant expression vector pCAMBIA3301-CHR2-1, transformed the vector into the Dongnong 50 soybean receptor through agrobacterium-mediated method, and identified regenerated soybean transformed plants with PCR screening. The results showed that the protein encoded by the GmCHR2-1 gene is a non-secretory protein and may play a major role in the cytoplasm. The protein is a non-transmembrane protein and does not migrate in the cell. The protein has 22 potential phosphorylation sites, including four threonine(Thr), and eighteen serine(Ser) composed of 40.63% α-helix, 14.29% extended chain, 4.13% β-turn and 40.95% random curl. PCR identified that the genome of the transformed plant contained the glufosinate resistance gene Bar, the terminator NOS and the promoter 35 S site, and it was preliminarily determined that GmCHR2-1 was transferred into the genome of Dongnong 50 soybean variety, and we finally obtained 12 T;positive plants.