红花油体表达人透明质酸酶基因的初步研究  

Expression of human hyaluronidase gene in oil body of safflower

作　　者：杨晶[1] 李洪锐[1] 易善勇[1] 郭咏昕[1] 黄建[1] 卢震[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9期191-195,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金："十二五"国家高新技术研究与发展计划项目(2011AA100606);中小企业创新基金项目(13C26212201223);长春市重大科技攻关项目(2014077);吉林省教育厅项目(201458/2015190)

摘　　要：【目的】获得转人源透明质酸酶基因PH20(hPH20)红花,为hPH20药用蛋白的生产奠定基础。【方法】人工合成hPH20基因,并在两端引入NcoⅠ/HindⅢ酶切位点。将合成的hPH20基因与pUC57载体连接,构建克隆载体pUC57-hPH20,对其进行双酶切鉴定。用NcoⅠ/HindⅢ酶切pUC57-hPH20获得hPH20基因,将其插入到植物油体高效表达载体pOBT上,构建重组质粒pOBT-hPH20,进行PCR和双酶切鉴定。采用冻融法将重组质粒pOBT-hPH20转入根瘤农杆菌EHA105中,通过农杆菌介导法转化红花,对转hPH20基因红花植株进行PCR检测。【结果】成功构建了hPH20基因的植物油体表达载体pOBT-hPH20,获得了3株PCR检测呈阳性的转hPH20基因红花植株。【结论】建立了完善的红花再生体系,获得了转hPH20基因的红花植株。[Objective] The transgenic safflower was obtained to express human hyaluronidase gene （hPH20） and lay foundation for development of hPH20 products. [Method] The hPH20 gene was synthe- sized and Nco Ⅰ/Hind Ⅲ enzyme sites were introduced at both ends. Then the hPH20 gene was inserted into plant expression vector pOBT by Nco Ⅰ/Hind Ⅲ. The recombinant plasmid was named pOBT-hPH20 and was transferred into Agrobacterium tumefaciens EHA105. The hPH20 gene was introduced into saf- flower via Agrobacterium-mediated method and positive transgenic plants were determined by PCR analy- sis. [Result] The recombinant plasmid pOBT-hPH20 was successfully constructed. PCR confirmed that hPH20 gene was integrated into the genome of safflower plant and three transgenic plants were obtained. [Conclusion] The safflower regeneration system was constructed successfully and hPH20 gene was suc- cessfully transformed into safflower plants.

关 键 词：红花 透明质酸酶hPH20 油体 

分 类 号：Q786[生物学—分子生物学]