抗冻基因CBF2表达载体构建及转化紫花苜蓿的研究

草业学报 ›› 2011, Vol. 20 ›› Issue (2): 193-200.

抗冻基因CBF₂表达载体构建及转化紫花苜蓿的研究

刘晓静^1,2*,郝凤^1,2,张德罡^1,2,毛娟³,于铁峰^1,2

1.甘肃农业大学草业学院, 甘肃兰州 730070;
2.草业生态系统教育部重点实验室中-美草地畜牧业可持续发展研究中心, 甘肃兰州 730070;
3.甘肃农业大学农学院,甘肃兰州 730070

收稿日期:2009-12-08 出版日期:2011-02-25 发布日期:2011-04-20
作者简介:刘晓静(1968-),女,甘肃酒泉人,副教授,博士。E-mail:liuxj@gsau.edu.cn
基金资助:
甘肃省农牧厅生物技术专项(GNSW-2004-07)资助。

Construction of antifreeze gene CBF₂ expression vector and transformation into alfalfa callus

LIU Xiao-jing^1,2, HAO Feng^1,2, ZHANG De-gang^1,2, MAO Juan³, YU Tie-feng^1,2

1.College of Grassland Science, Gansu Agricultural University, Lanzhou 730070, China;
2.Key Laboratory of Grassland Ecosystem of Ministry of Education, Sino-U.S.Centers for Grazing land Ecosystem Sustainability, Lanzhou 730070, China;
3.College of Agronomy,Gansu Agricultural University, Lanzhou 730070, China

Received:2009-12-08 Online:2011-02-25 Published:2011-04-20

摘要/Abstract

摘要： 本研究以拟南芥基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上构建成克隆载体T-CBF₂。用BamHⅠ和SacⅠ分别对克隆载体T-CBF₂和植物表达载体PBI121进行双酶切,获得目的片段和线性质粒。在T₄ DNA连接酶的作用下进行定向连接,构建成植物表达载体P-T-CBF₂。采用直接转化法将重组子导入根癌农杆菌EHA105。经PCR鉴定,重组质粒已成功导入根癌农杆菌中。通过农杆菌介导法转化和田苜蓿,现在已经得到转基因的苜蓿愈伤组织。

Abstract: Arabidopsis thaliana genomic DNA was selected as a template to amplify the target gene by PCR and connect it to the PGEM-T Easy Vector for construction of the T-CBF₂. The target fragment and linear plasmids were obtained from the cloning vector T-CBF₂ and from the plant expression vector PBI121 with dual digestion using BamHⅠand Sac I, respectively. The plant expression vector P-T-CBF₂ was built through directional connections using T₄ DNA ligase. PCR identification proved that the recombinant had been transferred into Agrobacterium tumefaciens and it was then introduced into Medicago sativa cv. Hetian through Agrobacterium-mediated transformation. Transfer of the target gene to alfalfa callus was successful.

中图分类号:

刘晓静,郝凤,张德罡,毛娟,于铁峰. 抗冻基因CBF₂表达载体构建及转化紫花苜蓿的研究[J]. 草业学报, 2011, 20(2): 193-200.

LIU Xiao-jing, HAO Feng, ZHANG De-gang, MAO Juan, YU Tie-feng. Construction of antifreeze gene CBF₂ expression vector and transformation into alfalfa callus[J]. Acta Prataculturae Sinica, 2011, 20(2): 193-200.

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抗冻基因CBF₂表达载体构建及转化紫花苜蓿的研究

Construction of antifreeze gene CBF₂ expression vector and transformation into alfalfa callus

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