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本研究利用RNA干扰的方法抑制紫苏ω-3脂肪酸脱氢酶基因FAD3的表达,以期得到低ALA含量的转基因植株。根据紫苏FAD3基因(Gen Bank登录号为KC990786.1)序列设计含有不同酶切位点的特异性扩增引物,通过扩增获得长度为258 bp的RNA干扰片段,构建以Ca MV35S启动子驱动表达的紫苏FAD3基因RNA干扰表达载体p FGC5941M-FAD3I,将该载体转入根癌农杆菌GV2301后,采用农杆菌介导法进行紫苏的遗传转化,经除草剂抗性筛选和PCR检测获得1株阳性转基因植株。利用荧光定量PCR技术对野生型和转基因阳性苗的FAD3基因进行了表达分析,结果显示,阳性植株中FAD3基因的表达受到显著抑制,FAD3基因表达量降低了80%,说明此研究构建的FAD3基因RNA干扰载体对该基因表达沉默是有效的,为FAD3基因在不饱和脂肪酸代谢过程中的功能研究提供了理论依据。
In this study, the RNA interference method was used to inhibit the expression of FAD3, a perilla wax-omega-3 fatty acid dehydrogenase gene, in order to obtain transgenic plants with low ALA content. According to the sequence of FAD3 gene (GenBank accession number KC990786.1), a specific amplification primer containing different restriction sites was designed and a 258 bp RNA interference fragment was obtained by amplification. The CaMV35S promoter The expression of perilla FAD3 gene RNA interference expression vector pFGC5941M-FAD3I, the vector was transformed into Agrobacterium tumefaciens GV2301, Agrobacterium-mediated genetic transformation of perilla, after herbicide resistance screening and PCR detection A positive transgenic plants. The expression of FAD3 gene in wild-type and transgenic-positive seedlings was analyzed by fluorescence quantitative PCR. The results showed that the expression of FAD3 gene in the positive plants was significantly inhibited and the expression of FAD3 gene was reduced by 80%. This indicated that the FAD3 gene The RNA interference vector is effective for silencing the gene expression, which provides a theoretical basis for the study of the function of FAD3 gene in the process of unsaturated fatty acid metabolism.