Construction Of Chloroplast Site-specific Integration Expression Vector In Wheat

The genetic materials are found in nucleus, chloroplast and mitochondria in plant cells. In the 1970s, the birth of genetic engineering technology created a new era that human controlled life process in vitro arbitrarily. Among them, nuclear genetic transformation technology was very mature, and was now very widely applied in molecular biology research and successfully used in transformation of economic crops. However, with research in-depth, many disadvantages in nuclear genetic transformation were found, such as the low rate of genetic, generations of genetic instability and negative impaction on the ecological environment. Deepening on chloroplast genomics, people realize that it is possible to transfer exogenous gene into chloroplast genome. In addition, this technique cannot be replaced by the nuclear transformation. Presently, the chloroplast genetic modification technology is gradually perfected which will become a new hotspot in the field of plant gene engineering.In recent years, the research in chloroplast transformation has made a lot of progress. It is successfully used in tobacco, potatoes, tomatoes, rape and soybean. Chloroplast has been wildly paid more attention based on its advantage. Although the chloroplast genetic engineering has many unique and attractive advantages, it startes relatively late and the operation technology is not mature in wheat compared with nuclear technology. Therefore this study was conducted and obtained some results as follows:1) Wheat chloroplasts site-specific integration expression vector were successfully constructed using DNA homologous recombination. rbcL and trnM, trnV were choosen as the homologous segment in the thesis. Firstly, primers were designed according to the sequence of wheat chloroplast genome, and two segments in 1500bp and 1000 bp from wheat chloroplast genome were cloned using the method of PCR. Sequence analysis showed that the cloned DNAs were the homologous segments we selected.2) The reporter gene GFP was cloned form pEGFPN1 with length of 800 bp. RbcL and trnM, trnV segments were ligased into pBluescript2-KS-plus vector after digested using restriction enzymes. Firstly, GFP was inserted into PTP4 vector with tobacco chloroplast cell-specific promoter and termination, and then the expressing box was inserted into two homologous segments after double enzyme digesteing. Thus the wheat chloroplast site-specific integration expression vector was successfully constructed. This specific vector will be integrated into the position between rbcL and trnM, trnV genes in wheat chloroplast genome and express highly efficient. It provides an expression vector for transforming functional genes into wheat chloroplast genome in future.Using the unique advantages of chloroplast genetic transformation system in this experiment, efficient and rapid system of wheat chloroplast transformation was established, which has important significance for the future research on wheat transformation.