The Expression Vector Construction Of Wheat Chloroplast And Transformation Analysis

Since the chloroplast DNA was proved by Ris and Plaut in 1962, genome sequence diagrams of higher plants,such as tobacco, rice, corn, spinach, alfalfa, and other Chlamydomonas chloroplast,and algae had been completed.Recently,the phenomenon of pollen drift caused considerable dispute in nuclear genetic engineering.Because chloroplast had many advantages,such as maternal genetic, site-specific integration, high-level expression,safey and stability, researchers pay more attention to chloroplast genetic engineering. At present, tobacco, potato, tomato, rapeseed and rice have been transformed successfully.But related research does not appear in wheat.This experiment was to establish wheat chloroplast transformation system by related transformation systems.It had significant effect on GM varieties of wheat and resistance breeding.The results are as follows:1) We got the sequence of rbcL gene and trnM, trnV gene from Genbank database and designed two pairs of specific primers, and then cloned them from Wheat chloroplast genome.2) we predicted 16 S rRNA specific promoter region of Wheat chloroplast genome by bioinformatics,and then cloned it from Wheat chloroplast genome by PCR.3) A site-specific integration and expression vector for wheat chloroplast genetic transformation, pRNGY, was constructed. It contained the homologous recombinant fragment 1-the rbc L, the homologous recombinant fragment 2- integral DNA fragment of trnM and trnV, the selectable marker npt II gene and the reporter gene gfp were regulated by the promoter Prrn and the terminator PsbA 3′from wheat chloroplast genome.We expected that we bombarded wheat immature embryos by gene gun, and then we could get spectinomycin-resistant callus by antibiotics of Kanamycin. We have successfully constructed the expression vector of wheat chloroplasts, but genetic transformation in the time we did not succeed in tansforming it into wheat chloroplast genome.