甘蓝型油菜pep基因片段的克隆和种子特异性反义表达载体的构建  

Cloning of a Sequence Encoding PEP from Bassica napus and Constructing of Antisense Seed-Specific Pep Expression Vector

作　　者：张志刚[1] 白德朗[1] 陈烈臣[2] 熊兴华[3] 李栒[3] 官春云[3] 

机构地区：[1]国家杂交水稻工程技术研究中心,湖南长沙410125 [2]湖南农业大学期刊社,湖南长沙410128 [3]作物基因工程湖南省重点实验室,湖南长沙410128

出　　处：《湖南农业大学学报（自然科学版）》2005年第3期249-252,共4页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家"863"计划资助项目(国家生字1997-185)

摘　　要：丙酮酸羧化酶(PEP)是控制油菜蛋白质/油脂含量比例的一个关键酶,抑制种子中pepc基因的表达,使其底物(丙酮酸)更多地朝生成油脂的方向流动,对提高种子的含油量,增加油菜的经济价值具有重要的意义.利用PCR技术从甘蓝型油菜湘油15号基因组中扩增了PEP基因片段,并将其克隆到pGEM-TEasy载体上进行测序.测序结果表明:扩增片段长576bp,与Yannai报道的PEP基因相应区域的同源性为95%.用扩增引物上设计的BamHI和SacI两位点酶将PEP基因片段切下,反向插入到pBI121.N质粒的Napin启动子之后,构建了种子特异性反义PEP表达载体,并通过农杆菌介导法将反义PEP基因转化到湘油15号中.Phosphoenolpyruvate carboxylase (PEP) plays an important role in the control of the ratio of the protein/lipid content in rape (Brassica napus).To modify the expression of pepc gene in rape by co-suppression may facilitate the flow of phosphoenolpyruvate into lipid at times of high lipid accumulation and enhance rape commercial value.A PEP gene fragment was amplified from Xiangyou15 is genomic DNA by polymerase chain reaction and subsequently ligated into pGE M-T Easy vector,the DNA sequence analysis indicated that the ligated fragment was 576 bp in size and shared 95% homology with the corresponding sequence of the reported PEP gene.The 576 bp fragment of the recombinant pGEM-T Easy vector was digested by BamHI/SacI(the new restriction sites were introduced by PCR amplification primers ) and ligated into the corresponding sites of the pBI121.N in the antisense orientation.The antisense PEP of the transgenegenic plasmid was testified under the control of the napin promoter.

关 键 词：甘蓝型油菜 PCR 反义PEP 种子特异性表达载体 

分 类 号：Q785[生物学—分子生物学] S565.4[农业科学—作物学]