不结球白菜L-半乳糖酸-1,4-内酯脱氢酶基因(GLDH)cDNA片段克隆与RNAi表达载体构建  

Cloning of GLDH gene cDNA fragment from non-heading Chinese cabbage and construction of its corresponding RNAi binary vector

作　　者：高红亮[1] 李英[1] 宋玉萍[1] 马成英[1] 侯喜林[1] 

机构地区：[1]南京农业大学园艺学院/作物遗传与种质创新国家重点实验室,江苏南京210095

出　　处：《南京农业大学学报》2008年第4期20-24,共5页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(30500343)

摘　　要：根据植物中hpRNA(hairpin RNA)原理,选择GLDH基因cDNA序列同源性较低区域设计1对携带双酶切位点的特异引物,以不结球白菜‘苏州青’叶片为材料提取总RNA,利用RT-PCR技术克隆了GLDH基因片段。构建中间克隆载体,经过3次亚克隆将测序正确的GLDH基因片段分别以正向和反向两种形式插入到间隔基因YYT的两端,经鉴定已插入到植物表达载体中,成功地构建了干扰GLDH基因表达的RNAi植物双元表达载体pRNAi-GLDH,并将表达载体转化到农杆菌菌株LBA4404中,为深入研究GLDH基因功能提供有效的工具。According to the principle of hpRNA（hairpin RNA）in the plant,a cDNA fragment encoding L-galactono-1,4-lactone dehydrogenase（GLDH）was cloned from non-heading Chinese cabbage by the method of RT-PCR on the basis of the specific primers with double enzyme sites.GLDH fragment was ligated with YYT interval region at forward and reverse orientations through three sub-cloning via mid-clone vector.The fusion gene was inserted into plant vector to construct the recombinant plasmid pRNAi-GLDH,which was then transformed into Agrobacterium LBA4404.This will provide an effective tool for the further study of GLDH gene function.

关 键 词：不结球白菜 GLDH基因 RNAi植物双元表达载体 

分 类 号：S634.3[农业科学—蔬菜学]