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。
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.
出处
《草地学报》
CAS
CSCD
北大核心
2009年第6期779-783,共5页
Acta Agrestia Sinica
基金
甘肃省农牧厅生物技术专项(GNSW-2004-07)
关键词
抗冻蛋白基因AFP
载体构建
遗传转化
紫花苜蓿
Antifreeze protein gene AFP
Vector construction
Genetic transformation
Alfalfa
