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磺酸基转移酶(sulphotransferase,简称SOT)是硫代葡萄糖苷(glucosinolate,简称硫苷)核心结构合成途径中的关键酶之一。本研究采用反向遗传学方法从大豆基因组中克隆Gm SOT1基因并对其功能进行初步研究。首先采用PCR方法扩增大豆Gm SOT1基因,并将其连接到pRNAi载体上的35S(Ca MV35S)启动子后面;将35S:Gm SOT1基因片段从pRNAi载体上双酶切下来,连接到植物双元表达载体p CAMBIA2301的多克隆位点上,且命名为p CAMBIA2301-35S:Gm SOT1;采用根癌农杆菌介导的遗传转化方法转化大豆成熟胚,并考察其初步表达结果。结果表明:大豆GmSOT1基因的编码区(coding sequences,简称CDS)由1 014 bp的核苷酸组成,编码一个长度为337个氨基酸残基的蛋白质;35S:Gm SOT1被成功导入大豆胚细胞,初步观察发现Gm SOT1过量表达导致大豆胚根明显膨大弯曲。
Sulphotransferase (SOT) is one of the key enzymes in the synthetic pathway of glucosinolate (abbreviated as glucosinolate). In this study, reverse genetics was used to clone Gm SOT1 gene from soybean genome and its function was studied. Firstly, the soybean Gm SOT1 gene was amplified by PCR and ligated into the 35S (Ca MV35S) promoter on the pRNAi vector. The 35S: Gm SOT1 gene fragment was double-digested from the pRNAi vector and ligated into the plant binary The expression vector p CAMBIA2301 was cloned into pCBBIA2301 and named p CAMBIA2301-35S: Gm SOT1. The Agrobacterium tumefaciens-mediated genetic transformation method was used to transform mature embryos of soybean and the primary expression results were obtained. The results showed that the encoding sequence of soybean GmSOT1 gene consisted of 1 014 bp coding sequence and encoded a 337 amino acid residue protein. The 35S: Gm SOT1 was successfully introduced into soybean germ cells, It was observed that overexpression of Gm SOT1 led to marked bulging and bending of the soybean root.