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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。
Soy isoflavones are a class of secondary metabolites synthesized in the phenylalanine metabolic pathway. Phenylalanine ammonia lyase (PAL) is the key enzyme and rate-limiting enzyme in the phenylalanine metabolic pathway. Our previous study found that the relative expression of PAL gene and the content of isoflavone had a synergistic trend of decrease and decrease. PAL-3 family gene family member PAL-3 (XM_003542493) One of the main expression members with higher expression level. In this experiment, we first cloned the PAL2-3 gene in soybean; then we constructed the overexpression vector pCAMBIA3301-GmPAL2-3 and transformed the recombinant plasmid pCAMBIA3301-GmPAL2-3 into Agrobacterium tumefaciens EHA105. The transformed plants were obtained from the transformation system of soybean cotyledon. The transformants of T1 generation were subjected to PPT assay, Barr test of exogenous marker gene and fluorescent quantitative PCR assay. The content of isoflavones in the transgenic plants was determined. The results showed that the expression level of PAL2-3 gene in T 1 generation transgenic plants was 5.11-1 11.24 times of the control, and the highest content of total isoflavones (2 587.63 μg · g -1) was the control (1616.90 μg · g -1 -1)). Therefore overexpression of PAL2-3 gene in soybean can increase isoflavone content in soybean grain.