摘要
为克服目前常用于植物基因表达载体构建的质粒由于酶切位点有限,目的基因片段难于插入,缺少植物基因表达所必须的启动子、终止子和筛选标记等功能元件,无法构建多个基因表达载体等缺点,本研究通过对大肠杆菌(Escherichia coli)质粒p UC18和双核农杆菌(Agrobacterium tumefaciens)Ti(tumer inducing)质粒p BI121进行改造,建立了一套适用于植物基因表达载体构建的质粒系统,即重组质粒p KAFCR80和p KAFCR100。p KAFCR80具有植物基因表达最常用的CAMV 35S启动子(cauliflower mosaic virus 35S promoter)和NOS终止子(Nopaline synthase terminator),并在其上游、中间和下游引入了多个克隆酶切位点,利用PCR等方法克隆得到的目的基因片段可以多种方式连接到35S启动子与NOS终止子之间;p KAFCR100具有卡那霉素NPTⅡ(neomycin phosphotransferaseⅡ)耐性基因和s GFP(synthetic green-fluorescent protein with S65T mutation)绿色荧光蛋白报告基因,以及其中间的多克隆酶切位点。p KAFCR80与p KAFCR100两个质粒的配合使用,可以方便地构建植物单个或多个基因表达载体。本研究以PCR扩增的甜叶菊葡萄糖基转移酶三个基因为材料,利用该质粒系统以单独和组合形式成功地构建了植物表达载体,验证了该系统的实用性。
In order to overcome the defects that the commonly used plasmids are limited in insufficiency of restriction site for target gene cloning, lack of expressing elements such as promoter, terminator and selection marker genes, as well as difficult in construction of multi-gene in an expression vector, we established a plasmid system suitable for construction of plant gene expression vector, which was recombinant plasmids pKAFCR80 and pKAFCR100, by modifying an Escherichia coli high express vector pUC18, and an Agrobacterium tumefociens binary vector pBI121 respectively, pKAFCR80 contained CAMV （cauliflower mosaic virus） 35S promoter and NOS （nopaline synthase） terminator, and introduced multiple cloning sites at their upstream, intermediate and downstream. The target gene fragments cloned by PCR and other methods could be connected between the 35S promoter and the NOS terminator in a variety of ways. Also, pKAFCR100 contained NPT Ⅱ （neomycin phosphotransferase Ⅱ ）, sGFP （synthetic green-fluorescent protein with S65T mutation） genes and a multiple cloning site. The combination of pKAFCR80 and pKAFCR100 two plasmids could expediently construct plant single-gene or multi-gene expression vector. In this study, three genes encoding for uridine diphosphate glycosyltransferases amplified by PCR from stevia were used as the materials. Using the plasmid system, the plant expression vector was successfully constructed in a single and combinatorial manner, and the practicability of the system was verified.
出处
《分子植物育种》
CAS
CSCD
北大核心
2018年第4期1138-1146,共9页
Molecular Plant Breeding
基金
宁夏大学引进人才科研启动基金(BQC2012001)
国家自然科学基金(31460062)共同资助
关键词
载体构建
质粒系统
多基因表达载体
新方法
甜叶菊葡萄糖基转移酶基因
Vector construction, Plasmid system, Multi-gene expression vector, Novel method, Stevia uridine diphosphate glycosyltransferase genes
