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本研究以甘蓝型油菜湘油15种子不同发育时期抑制差减文库为研究对象,设计特异性引物扩增得到一个与油脂代谢相关,含MATH结构域的基因片段,长度为327bp,命名为BnMT-1。根据RNA干扰的基本原理,将目的片段正反向插入到表达载体pFGC5941.nap查尔酮内含子两侧,经限制性内切酶酶切和质粒PCR扩增检测,证明已成功构建了干扰载体PP-MT。采用根癌农杆菌法将PP-MT干扰载体转入甘蓝型油菜中,对转化植株基因组DNA的PCR检测表明,干扰载体已转入到甘蓝型油菜中,共获得了两株转基因植株。这为进一步研究BnMT-1基因在甘蓝型油菜中的功能打下了基础。
In this study, the suppression subtractive library of 15 seeds of Brassica napus L. at different developmental stages was selected as the research object. A specific primer was designed to amplify a fragment of MATH-containing gene which was related to the lipid metabolism. The length of the gene was 327 bp, named BnMT- 1. According to the basic principle of RNA interference, the target fragment was inserted into both sides of the expression vector pFGC5941.nap chalcone introns according to the principle of restriction endonuclease digestion and PCR amplification of plasmid to prove that the interference was successfully established Carrier PP-MT. The Agrobacterium tumefaciens method was used to transfer the PP-MT interference vector into Brassica napus. PCR analysis of the genomic DNA of the transformed plants showed that the interference vector was transferred into Brassica napus and two transgenic plants were obtained. This laid the foundation for further study on the function of BnMT-1 in Brassica napus.