棉花<em>GbCBF2</em>基因的克隆、互补表达载体的构建及遗传转化

doi:10.7668/hbnxb.2016.01.007

华北农学报 ›› 2016, Vol. 31 ›› Issue (1): 40-45. doi: 10.7668/hbnxb.2016.01.007

所属专题： 棉花；生物技术；

棉花GbCBF2基因的克隆、互补表达载体的构建及遗传转化

孔丽颖, 李翔, 李才运, 刘晓东, 李月

新疆农业大学农学院, 农业生物技术重点实验室, 新疆乌鲁木齐 830052

收稿日期:2015-12-01 出版日期:2016-02-28
通讯作者: 李月(1984-),女,河南许昌人,讲师,博士,主要从事棉花分子生物学研究。
作者简介:孔丽颖(1993-),女,新疆呼图壁人,主要从事生物技术研究。
基金资助:
国家自然科学基金项目(31470289);新疆农业大学校前期课题(XJAU201311);新疆农业大学大学生创新项目

Cloning,Complementary Expression Vector Construction and Transformation of GbCBF2 Gene from Cotton

KONG Liying, LI Xiang, LI Caiyun, LIU Xiaodong, LI Yue

College of Agronomy, Xinjiang Agricultural University, Key Laboratory of Agricultural Biological Technology, Urumqi 830052, China

Received:2015-12-01 Published:2016-02-28

摘要/Abstract

摘要： 为研究棉花CBF2基因在植物抗逆中的作用,构建了pCAMBIA1300-AtCBF2P-GbCBF2-AtCBF2T植物互补表达载体,并对拟南芥cbf2突变体进行遗传转化。以新海16 cDNA为材料,采用PCR的方法克隆4个棉花CBF2基因,同时克隆拟南芥CBF2基因启动子AtCBF2P和终止子AtCBF2T,并将这些基因与pBluescript SK载体连接,构建pBluescript SK-AtCBF2P-GbCBF2-AtCBF2T中间过渡载体,用Sac Ⅰ和BamH Ⅰ双酶切中间载体,获得AtCBF2P-GbCBF2-AtCBF2T核心片段,将此片段插入到pCAMBIA1300表达载体中,构建pCAMBIA1300-AtCBF2P-GbCBF2-AtCBF2T植物互补表达载体。以拟南芥cbf2突变体为材料,采用农杆菌介导的方法进行遗传转化,获得转棉花CBF2基因群体。获得转基因株系后,将为筛选出棉花的CBF2抗冻负调控基因及棉花抗逆分子育种奠定基础。

关键词: 棉花, CBF2 基因, 互补表达载体, 遗传转化

Abstract: In order to study the function of cotton CBF2 gene in plant resistance,the complementary expression vector pCAMBIA1300-AtCBF2P-GbCBF2-AtCBF2T was constructed,and transformed to Arabidopsis cbf2 mutant by genetic engineering.The four CBF2 genes were cloned from cDNA of the cotton Xinhai 16 and Arabidopsis CBF2 gene promoter AtCBF2P and termination AtCBF2T were also cloned by PCR method.Firstly,these genes were connected with pBluescript SK,and obtained the intermediate transition vector pBluescript SK-AtCBF2P-GbCBF2-AtCBF2T,then the AtCBF2P-GbCBF2-AtCBF2T fragment was obtained by cutting the intermediate transition vector with Sac Ⅰ and BamH Ⅰ,after the fragment inserted into pCAMBIA1300,the complementary expression vector pCAMBIA1300-AtCBF2P-GbCBF2-AtCBF2T was constructed.The cotton CBF2 gene was transformated into Arabidopsis cbf2 mutant by Agrobacterium mediated method and regenerative seedlings were obtained.These results will lay the foundation for screening cotton negative regulator CBF2 gene and stress-resistance molecular breeding.

Key words: Cotton, CBF2 gene, Complementary expression vector, Transformation

中图分类号:

Q78
S562.03

孔丽颖, 李翔, 李才运, 刘晓东, 李月. 棉花GbCBF2基因的克隆、互补表达载体的构建及遗传转化[J]. 华北农学报, 2016, 31(1): 40-45. doi: 10.7668/hbnxb.2016.01.007.

KONG Liying, LI Xiang, LI Caiyun, LIU Xiaodong, LI Yue. Cloning,Complementary Expression Vector Construction and Transformation of GbCBF2 Gene from Cotton[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(1): 40-45. doi: 10.7668/hbnxb.2016.01.007.

http://www.hbnxb.net/CN/Y2016/V31/I1/40

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