Construction of Antifreeze Protein Gene AFP Expression Vector and Transformation into Alfalfa Callus

doi:10.11733/j.issn.1007-0435.2009.06.015

›› 2009, Vol. 17 ›› Issue (6): 779-783.DOI: 10.11733/j.issn.1007-0435.2009.06.015

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Construction of Antifreeze Protein Gene AFP Expression Vector and Transformation into Alfalfa Callus

HAO Feng¹, LIU Xiao-jing¹, MAO Juan², YU Tie-feng¹, ZHANG De-gang¹

1. Pratacultural College, Gansu Agricultural University, Lanzhou, Gansu Province 730070, China;
2. College of Agronomy, Gansu Agricultural University, Lanzhou, Gansu Province 730070, China

Received:2009-04-09 Revised:2009-07-22 Online:2009-12-15 Published:2009-12-15

抗冻蛋白基因AFP表达载体构建及转化紫花苜蓿初报

郝凤¹, 刘晓静¹, 毛娟², 于铁峰¹, 张德罡¹

1. 甘肃农业大学, 草业学院, 兰州, 730070;
2. 甘肃农业大学, 农学院, 兰州, 730070

通讯作者: 刘晓静,E-mail:liuxj@gsau.edu.cn
作者简介:郝凤(1985- ),女,汉族,黑龙江鹤岗市人,硕士研究生,研究方向为草地资源与生态,E-mail: haofeng1026 @163.com
基金资助:
甘肃省农牧厅生物技术专项(GNSW-2004-07)

Abstract

Abstract: In order to enhance the freezing tolerance of alfalfa,the carrot genomic DNA was taken as template,the target gene was amplified using PCR method and connected to the PGEM-T Easy Vector vector,the target fragment was connected to the engineering plasmid PBI121 after double digestion by Xbal Ⅰ and Sac Ⅰ,then alfalfa Hetian was transformed through Agrobacterium-mediated transformation.The results show that the antifreeze protein gene AFP was successfully cloned,AFP plant expression vector was constructed,and the kanamycin-resistant callus of alfalfa was obtained.The optimal kanamycin selection concentration of alfalfa Hetian callus was 60 mg·L^-1.Based on the identification by PCR technology,AFP antifreeze protein gene was integrated into the alfalfa genome.

Key words: Antifreeze protein gene AFP, Vector construction, Genetic transformation, Alfalfa

摘要： 以胡萝卜基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上,经XbalⅠ和SacⅠ双酶切,将目的片段连接到PBI121工程质粒上,通过农杆菌介导法转化和田苜蓿,为提高紫花苜蓿的抗冻性奠定基础.结果表明,成功克隆出抗冻蛋白基因AFP,并构建成AFP植物表达载体.获得了具有卡那霉素抗性的苜蓿愈伤组织,和田苜蓿愈伤组织的最佳Kan筛选浓度为60 mg/L.经PCR技术鉴定,AFP抗冻蛋白基因已整合到苜蓿基因组中.

关键词: 抗冻蛋白基因AFP, 载体构建, 遗传转化, 紫花苜蓿

CLC Number:

Q946

HAO Feng, LIU Xiao-jing, MAO Juan, YU Tie-feng, ZHANG De-gang. Construction of Antifreeze Protein Gene AFP Expression Vector and Transformation into Alfalfa Callus[J]. , 2009, 17(6): 779-783.

郝凤, 刘晓静, 毛娟, 于铁峰, 张德罡. 抗冻蛋白基因AFP表达载体构建及转化紫花苜蓿初报[J]. , 2009, 17(6): 779-783.

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Construction of Antifreeze Protein Gene AFP Expression Vector and Transformation into Alfalfa Callus

抗冻蛋白基因AFP表达载体构建及转化紫花苜蓿初报

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