大豆PAL2-3基因的克隆及转化研究

大豆异黄酮是苯丙氨酸代谢途径中合成的一类次级代谢产物,在苯丙氨酸代谢途径中,苯丙氨酸解氨酶(PAL)是关键酶和限速酶。本课题组前期研究发现PAL基因的相对表达量与大豆异黄酮含量具有明显的协同增减趋势,并且在PAL基因家族成员时空表达模式分析中发现,PAL2-3(XM_003542493)是PAL基因家族中相对表达量较高的主要表达成员之一。本试验首先通过克隆大豆中PAL2-3基因;然后构建pCAMBIA3301-GmPAL2-3植物过表达载体,将构建好的pCAMBIA3301-GmPAL2-3表达载体重组质粒转化到根癌农杆菌EHA105中;采用农杆菌介导的大豆子叶节转化体系获得转化植株,并对T1代转化植株进行PPT检测,外源标记基因Bar检测以及荧光定量PCR检测,对转基因阳性植株进行大豆籽粒异黄酮含量的测定。结果表明T_1代转基因植株中PAL2-3基因的表达量是对照的5.11~11.24倍,总异黄酮含量最高的(2 587.63μg·g~(-1))是对照(1 616.90μg·g~(-1))的1.6倍。因此在大豆中过量表达PAL2-3基因可以提高大豆籽粒中异黄酮含量。

Cloning and Transformation of the PAL2-3 Gene in Soybean (Glycine max L.)

Soybean isoflavones are a class of secondary metabolites synthesized in the phenylalanine metabolic pathway.Phenylalanine ammonia-lyase(PAL) is a key enzyme and rate-limiting enzyme in the phenylalanine metabolic pathway.In the preliminary study,we found that the relative expression of PAL gene and soybean isoflavone content had a significant synergistic trend,and it was found that PAL2-3 (XM_003542493) was one of the major expression members with high relative expression in PAL gene family.In this study,we firstly cloned the PAL2-3 gene in soybean,then constructed the pCAMBIA3301-GmPAL2-3 plant overexpression vector and transformed itinto Agrobacterium tumefaciens EHA105.The transgenic plants were obtained by Agrobacterium tumefaciens-mediated transformation system,and the T1 transgenic plants were determined by PPT,exogenous marker gene Bar and quantitative PCR.The determination of isoflavones in soybean seeds was carried out on the identified transgenic plants.The results showed that the expression level of PAL2-3 gene in T1 transgenic plants was 5.11-11.24times of the control,and the highest of total isoflavones (2 587.63 pg·g-1) was 1.6 times of the control (1 616.90 pg·g-1).Therefore,overexpression of PAL2-3 gene in soybean could increase the content of isoflavones in soybean seeds.