甘蓝脯氨酸脱氢酶基因克隆与RNAi表达载体构建  

Cloning of ProDH gene in cabbage and construction of the RNAi vector

作　　者：张娜[1] 黄韫宇[1] 冯洁[1] 刘莉莎[1] 杨鹏[1] 赵冰[1] 郭仰东[1] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193

出　　处：《中国农业大学学报》2011年第3期87-94,共8页Journal of China Agricultural University

基　　金：国家"973"计划项目(2009CB119000);国家大学生创新性实验计划(20081001903);中央高校基本科研业务费专项资金资助(2009-2-06)

摘　　要：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。RNA interference（RNAi） targeting ProDH suppresses the degradation of proline,and may increase the resistance to drought and salt stress.In this assay,full length cDNA of ProDH was cloned with RT-PCR and RACE methods from total RNA of cabbage（Brassica oleracea L.var.capitata L.）.The sequence analysis indicated that it contained 1 719 nucleotides coding for 498 amino acid residues.The ProDH gene and its deduced amino acid sequence showed a high degree of sequence homology with the AtERD5,BnProDH,BrProDHand AsProDH.The cDNA fragment was chosen to insert into plant vector pFGC-1008 at forward and reverse orientations to construct the recombinant RNAi vector.The RNA interference vector pFGC-gPDH was constructed successfully by restricting endonuclease digestion and sequencing,which would provide a construct for further functional study for drought resistance of plant.

关 键 词：甘蓝 ProDH基因 基因克隆 RNA干扰 载体构建 

分 类 号：S635[农业科学—蔬菜学] S188[农业科学—园艺学]