转角质细胞生长因子-2基因红花研究  

Construction of safflower with keratinocyte growth factor-2 gene(KGF2)

作　　者：杨晶[1] 王兰[1] 黄建[1] 陈玉斌 郭咏昕[1] 易善勇[1] 官丽莉[1] 杜林娜[1] 李海燕[1] 李校堃[1] 姜潮[1] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118

出　　处：《西北农林科技大学学报（自然科学版）》2015年第2期210-216,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家高新技术研究与发展计划项目(2011AA100606);吉林省中小企业创新基金项目(13C26212201223);吉林省教育厅项目(201458);教育部博士点基金青年教师基金项目(20122223120002);长春市重大科技攻关项目(2014077)

摘　　要：【目的】构建转角质细胞生长因子-2基因(KGF2)红花,为KGF2药用蛋白的生产奠定了基础。【方法】利用融合PCR方法扩增人源KGF2基因与拟南芥油体蛋白Oleosin基因的融合基因,将融合基因克隆至表达载体pOP上,构建了重组质粒pOP-OL-KGF2,采用冻融法将质粒pOP-OL-KGF2转入根癌农杆菌EHA105中,通过农杆菌介导法转化红花,采用PCR检测筛选转基因红花阳性植株,SDS-PAGE检测Oleosin-KGF2融合蛋白的表达情况。【结果】成功构建了植物特异表达载体pOP-OL-KGF2,转染后共获得了12株转基因红花植株,其中2株鉴定为阳性,阳性率为17%,对转化植株进行DNA水平检测和蛋白水平检测,可知KGF2基因已经整合进红花基因组中,且有所表达。【结论】成功获得转KGF2基因的红花株系。【Objective】Safflower with keratinocyte growth factor gene(KGF2)was constructed to improve KGF2 protein production.【Method】Molecular biology method was used to amplify KGF2 with Oleosinfusion gene.The fusion gene was cloned into the expression vector pOP to construct recombinant plasmid pOP-OL-KGF2 and the freeze-thaw method was used to transfer plasmid pOP-OL-KGF2 into Agrobacterium tumefaciens EHA105.It was transformed into safflower cotyledons by Agrobacterium-mediated.Positive transgenic plants were determined by PCR analysis.【Result】Plant binary expression vector pOP-OL-KGF2 was successfully constructed.A total of 12 positive safflower plants were obtained after transfection with a positive rate of 17%.From the DNA and protein level,KGF2 gene was integrated into the safflower genome and target protein was expressed.【Conclusion】Safflower lines with KGF2 gene were obtained successfully.

关 键 词：角质细胞生长因子-2 红花 遗传转化 

分 类 号：Q943.2[生物学—植物学]