| 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。 |
| 关键词: 甘蓝 ProDH基因 基因克隆 RNA干扰 载体构建 |
| DOI：10.11841/j.issn.1007-4333.2011.03.015 |
| 投稿时间：2010-09-14 |
| 基金项目: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) |
|
| Cloning of ProDH gene in cabbage and constructionof the RNAi vector |
|
ZHANG Na, HUANG Yun-yu, FENG Jie, LIU Li-sha, YANG Peng, ZHAO Bing, GUO Yang-dong
|
| (College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China) |
| Abstract: |
| 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,BrProDH and 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. |
| Key words: cabbage ProDH gene clone RNA interference vector construction |
