Proteomics Analysis Of Rice And Maize Chloroplast And Construction Of Maize Chloroplast Transformation System

Chloroplast is an organell which has extranuclear genetic material. There are only 80 to 100 proteins encoded by chloroplast genome, and the other proteins related to chloroplast metabolism encoded by nuclear genome.As a complex memebrane system, it is so difficult to extract protein from chloroplast that it has been a challenge to analyze entire chloroplast proteome,restricted to arabidopsis and so on ,not thorough research in rice and maize.With the development of mass spectrometry (MS), protemics is becoming a technique of high throughput and high sensitivity, which makes it possible to analyze entire chloroplast proteome. So we researched chloroplast proteome of maize and rice using this technique combined with traditional technology. First, global chloroplast was purified from maize and rice. The soluble total proteins with high quality were extracted by optimized method, and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique.By orbitrap analysis, 620 chloroplast proteins were obtained in maize, of which 368 proteins located in chloroplast, 160 proteins in cytoplasm, 56 proteins in mitochondrion and 17 new proteins located in chloroplast blasting with PPDB. While, 231 chloroplast proteins were obtained in rice, of which 117 proteins located in chloroplast, 93 proteins in cytoplast, and 26 proteins in mitochondrion blasting with PPDB.Those proteins located in cytoplast and mitochondrion may be existed in chloroplast. After blasting with PPDB, most of the proteins in PPDB are predicted proteins, but they are identified as expressed proteins by MS analysis. The chloroplast proteome were analyzed in maize and rice to obtain more proteins and understand the protein composition of chloroplast, which provides enough evidences for the study of photosynthetic systems, all kinds of enzymes and metabolism of chloroplast.With high express efficiency and environment safety, chloroplast transformation is a technique which uses the principle of homologous recombination to recombinate target DNA into chloroplast genome, making the target DNA expressed in chloroplast. It has been used in chlamydomonas, tobacco, arabidopsis and rice successfully.In this study, we constructed transformation vectors of maize chloroplast and transformed maize callus by using gene gun method, hoping to construct maize chloroplast transformation system.Firstly, we cloned 16SrRNA promoter and terminator rbcL3'of maize chloroplast. The promoter was optimized by bring in the SD sequence of rbcL and nucleotide sequence related to translation, which can enhance the promoter translation.Using proper primer, we cloned three pairs of fragments, trnC, ropB,atpB, rbcL and trnL, ndhD,which were used as homologous fragment in homologous recombination of target DNA and chloroplast genome. Futhermore, we choosed optimized EPSPSA15N gene and hygromycin resistance as target gene and selection marker respectively, constructing transformation vectors of maize chloroplast,pCB-PTN,pBR-PTN, pBR-PTN-pht,pBR-PTN-pht.We transformed maize callus by gen gun method, and now we are screening resistance callus, wishing to obtain regeneration plant of maize and construct transformation system of maize chloroplast .