毛尖紫萼藓GpPOD基因克隆及其表达载体的构建  

Cloning and expression vector construction of GpPOD gene from Grimmia pilifera

作　　者：宋璐[1] 沙伟[1] 张梅娟[1] 刘博[1] 安洪雪[1] 

机构地区：[1]齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔161006

出　　处：《齐齐哈尔大学学报（自然科学版）》2013年第4期5-9,共5页Journal of Qiqihar University(Natural Science Edition)

基　　金：国家自然科学基金资助(31070180;31270254)

摘　　要：本实验室已成功构建毛尖紫萼藓干旱胁迫cDNA文库。从中选取与抗旱相关的过氧化物酶基因,命名为GpPOD。通过RACE技术克隆获得序列全长。采用RT-PCR方法克隆GpPOD基因,连接到pMD-18T simple载体上,构建克隆载体pMD18T-GpPOD,将其插入表达载体PRI101-AN中,构建表达载体PRI101-GpPOD,然后将重组质粒导入根癌农杆菌EHA105中。重组质粒pMD18T-POD、PRI101-POD的DNA测序结果与原序列相似性高达99%,证明GpPOD基因成功连接到pMD-18T simple载体上,并已克隆到表达载体PRI101-AN中;农杆菌转化后的PCR电泳图谱在约581 bp左右得到清晰条带,证明重组质粒PRI101-GpPOD已成功转入农杆菌中。本实验为后续研究GpPOD基因的遗传转化奠定了良好基础。cDNA library was succeed constructed with Grimmia pilifera drought stress in the laboratory. A peroxidase gene was selected to drought resistance and named GpPOD. The full-length sequence was obtained by using rapid amplification of eDNA ends（RACE）.The GpPOD gene was cloned by using RT-PCR method and connected it with pMD-18T simple Vetcor to construct cloning vector pMD18T-GpPOD. It was inserted into expression vector PRI101-AN for constructing expression vector PRIIO1-GpPOD, and recombinant plasmid was carried into Agrobacterium tumefaciens EHA105. The DNA sequencing result of recombinant plasmid pMD18T-GpPOD, PRI 101-GpPOD had 99% similarity with the original sequence, which indicated that GpPOD gene was connected to clone vector pMD18-T successfully and had been cloned into expression vector PRI101-AN; There were bands appeared at about 581 bp in electrophoregram of PCR of Agrobacterium transformer, indicating that PRI 101-GpPOD had been introduced into Agrobacterium tumefaciens.The experimental provided an essential basis for genetic transformation of Grimmia pilifera GpPOD gene.

关 键 词：毛尖紫萼藓 GpPOD 克隆载体 表达载体 根癌农杆菌EHA105 

分 类 号：Q343.1[生物学—遗传学]