用于植物基因表达载体构建的质粒改造及其应用

摘要

为克服目前常用于植物基因表达载体构建的质粒所具有的酶切位点有限,目的基因片段难于插入和连接,缺少植物基因表达所必须的启动子、终止子、筛选标记等功能元件的缺点,本研究构建了一个用于植物基因表达载体构建的质粒栽体pNULPGE200.该质粒载体引入了植物基因表达最常用的CaMV 35S启动子(cauliflowermosaic virus 35S promoter)和NOS终止子(nopaline synthase terminator),以及之间的多克隆酶切位点MCS(multiple cloning site).利用pNULPGE200构建植物基因表达栽体,经PCR等方法克隆得到的目的基因可以直接连接到35S启动子与NOS终止子之间,使目的基因能够在植物体内稳定表达;同时该质粒载体还具有独立表达的卡那霉素NPT Ⅱ (neomycinphosphotransferase Ⅱ)耐性基因和sGFP (synthetic green-fluorescent protein with S65T mutation)绿色荧光蛋白报告基因,可用于基因转化时的筛选.本研究以假单胞菌(Pseudomonas putida)携带质粒的二甲苯单加氧酶(xylene monooxygenase)编码基因为材料,分别利用本文质粒载体和常规的质粒栽体pBI121构建了植物表达栽体,验证了文中质粒栽体的实用性.%In order to overcome the defects that the commonly used plasmids have a limited number of restriction sites for target gene cloning, and lack expressing elements such as promoter, terminator and selection marker genes, a plasmid named pNULPGE200 for construction of plant gene expression vector was modified. The plasmid contains cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (NOS) ter-minator, and a multiple cloning site between them. The target gene amplified by PCR can be inserted directly between the 35S promoter and the NOS terminator, and can be expressed in plants stably. pNULPGE200 also contains two independent expression marker genes, encoding neomycin phosphotransferase Ⅱ(NPT Ⅱ) and synthetic green-fluorescent protein with S65T mutation (sGFP), which can be used for mutual selection in plant transformation. The practicability of the plasmid was confirmed by comparing with a conventional plas-mid pBI121 in the construction of the gene encoding xylene monooxygenase from Pseudomonas putida to create a plant gene expression vector.