植物双元表达载体pCRI1210的构建及其功能验证

Construction and Function Analysis of a Plant Binary Expression Vector pCRI1210

pBI121是转基因研究中的常用载体,但是其多克隆位点相对较少,所携带的GUS基因检测较为复杂。为了增加其多克隆位点,增强其表达效率,简化其检测方法,本研究在pBI121载体的基础上,在其骨架中插入含有CaMV35S启动子、棉花β-tubulin基因内含子和CaMV 35S polyA终止子的干涉表达框,该表达框含有GFP报告基因。通过验证,所插入的干涉表达框能够正常表达插入的外源基因,且GFP基因可以正常表达。该载体被命名为pCRI1210,它是一种双元植物表达载体,既可以用来当作表达载体,又可以用作干涉载体。本研究为转基因研究提供了一种非常实用的工具。

Vector is an indispensable tool in the process of transgenic research. Researchers usually connect foreign target genes with vectors and import vectors into target plants（ animals） by technical means to observe the role played by target genes. Therefore,many factors,such as the success of vector selection,the efficiency of vector transformation and expression,the difficulty of vector detection and the level of metabolic burden,determine the success or failure of the test to a certain extent. Therefore,the use of a simple,easy to transform,high expression,easy to detect and small metabolic burden is very important. Based on this,the vector pCRI1210 was successfully constructed by inserting an interference frame and replacing reporter gene on the basis of the commonly used vector pBI121. pBI121 was commonly used in the study of genetically modified（ gm）,but its multiple cloning site was relatively small,and GUS gene detection was more complicated. In order to increase its multiple cloning loci,enhance its expression efficiency,and simplify the detection method,a plant binary expression vector,named pCRI1210,was constructed on the basis of pBI121 vector,which contained CaMV 35 S promoter,cotton β-tubulin gene introns,CaMV 35S poly A terminator and interference expression box with GFP as reporter gene. Functional analysis in tobacco showed that the interference express cassette could normally expressed exogenous gene and GFP gene. The vector had multiple cleavage sites in a TUB intron 5＇and 3＇,so that target gene could be positive and reverse inserted and hairpin structure of dsRNA was builded. In addition,the vector carries multiple cloning sites at both 5＇ and 3＇ of the CaMV 35S promoter,which could easily exchange 35S promoter to study other promoter functions. pCRI1210 could be used as expression vector or interference vector. Through validation in tobacco,skin cells of the onion and the healing of the cotton embryo,it would be a very practical tool for transgenic research.