甘蓝型油菜含MATH结构域基因BnMT-1干扰载体的构建及遗传转化

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。

Construction of the RNAi Vector of BnMT-1 Gene with MATH Domain from Rapeseed and its Genetic Transformation

In this paper,taking suppression subtractive hybridization library of rapeseed seed at different developmental stage as research object,we amplified a 327 bp gene fragment containing MATH domain related to lipin with designed specific primers,which was named BnMT-1.According to principles of RNA interference,the target fragment was inserted to both sides of chalcone intron of pFGC5941,nap in forwards and reverse way.The RNA interference vector PP-MT was constructed successfully by restriction endonuclease digestion and plasmid PCR identification.Furthermore,PP-MT RNAi vector was transformed into rapeseed by agrobacterium-mediated transformation.PCR assay of genomic DNA from transformed plants demonstrated that RNAi vector was transformed into rapeseed successfully.Two positive transgenic plants were obtained,which provided foundations for further functional research of BnMT-1 gene in rapeseed.