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通过RT-PCR、同源克隆和RACE等方法由甘蓝总RNA扩增得到了甘蓝脯氨酸脱氢酶基因cDNA全长(1 719 bp),其中包含了一个1 497 bp的完整开放阅读框,编码498个氨基酸,与已发表的十字花科植物ProDH基因均具有85%以上的同源性。在此基础上设计并克隆干扰片段,利用酶切连接的方法将该基因干扰片段正反向插入到载体pFGC-1008的GUS内含子两侧,经限制性内切酶酶切和测序鉴定,证明植物表达载体pFGC-gPDH已构建成功,为进一步研究该基因的功能创造了条件。
The full-length cDNA of proline dehydrogenase gene (1 719 bp) was amplified by RT-PCR, homologous cloning and RACE from total cabbage, which contained a complete 1 497 bp open reading frame (ORF) Encoding 498 amino acids, with published Crucifer ProDH gene with more than 85% homology. Based on this, the interference fragment was designed and cloned, and inserted into the GUS intron of vector pFGC-1008 in both forward and reverse directions by restriction enzyme digestion. The fragment was identified by restriction enzyme digestion and sequencing, It was proved that the plant expression vector pFGC-gPDH was constructed successfully, which provided conditions for further study on the function of this gene.