Cloning And Functional Analysis Of SNARE Protein Genes From Rice (Oryza Sativa L.)

Rice is the most important staple crop that is cultivated worldwide.In the many rice-growing areas there are many pathogens like fungi,bacteria or viruses,frequent salinity and extreme drought to impede rice growth and production.It has been one of main objects for the researchers to elucidate the mechanism of plant resistance or tolerance to a variety of stresses and improve the ability of crops to resistance to the stresses.This thesis aims to do functional analysis of four SNARE protein genes from rice and proteomic analysis of rice blast defense responsive plasma membrane associate proteins,and main results are given as follows.The membrane fusion in vesicle trafficking in the cells of eukaryotic organisms is mediated by soluble-N-ethyl-maleimide-sensitive fusion protein attachment protein receptor(SNARE) proteins,which are highly conserved from various species.Increasing research has demonstrated SNAREs involved in the formation of the cell plate,interacting with ion channel proteins,and gravity sensing in plants.SNARE proteins might play important roles in plant growth,development and response to abiotic and biotic stresses. Here we report the cloning and expression characterization of OsSNAP32 from rice,a novel member of SNAP25-type proteins.The OsSNAP32 comprises 283 amino acid residues with the molecular weight of 31.3kD,and contains Qb-SNARE and Qc-SNARE domains in the N- and C-terminal.By the onion epidermal cells transient expression method,it revealed that OsSNAP32 had the plasma membrane located character of SNAP25-type proteins.Semi-quantity RT-PCR assay showed the expression of OsSNAP32 was significantly activated in rice seedlings treated by H₂O₂,PEG6000,SA,ABA,low temperature and hot or inoculated by rice blast(Magnaporthe grisea) and blight (Xanthomonas campestris pv.vesicator).The T₁ OsSNAP32 over-expressing transgenic plants seemed increased resistance ability to rice blast.The results suggested that this gene may be belong to a novel gene family encoding SNAP25-type proteins involved in the rice responses to biotic and abiotic stresses.In additon,we report three novel plant SNARE(NPSN) genes isolated from rice and named OsNPSN11,OsNPSN12 and OsNPSN13.They have 80-85%nucleotide identity and similar genomic organization,with ten exons and nine introns in each gene.OsNPSN11, OsNPSN12 and OsNPSN13 are located on chromosomes 6,3 and 7,respectively.Multiple alignment of deduced amino acid sequences indicate that the OsNPSN11～13 proteins are homologous to AtNPSNs from Arabidopsis,containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region.Transient expression analysis in onion epidermal cells revealed that OsNPSN11～13 were located on the plasma membrane. The cDNA of OsNPSN11 was subcloned into pET-30a,and the recombinant protein expressed in E.coli was obtained.With the polyclonal antibody made by the purified protein, western blot result showed that OsNPSN11 was located on the plasma membrane in rice cells.Semi-quantitative RT-PCR assays showed that the expression of OsNPSN11～12 were significantly activated in rice seedlings treated with H₂O₂,but decreased under salt and PEG6000 stresses.Transformed yeast cells with OsNPSN11～13 had better growth rates than wild type or empty vector transformants when cultured in solid or liquid media containing various concentrations of H₂O₂.However the transformed yeast cells with OsNPSN11～13 were more sensitive to salt and mannitol stresses.The transgenic tobacco with OsNPSN11～13 grow better than wild type when treated with H₂O₂,while showed more sensitive to salt and mannitol treatments.These results indicate that the products of OsNPSN11～13 may be involved the signal transduction in yeast and tobacco responses to abiotic stresses.We also study about the function of OsNPSN11～13 under biotic stresses.Semi RT-PCR results showed that the expression of OsNPSN11～12 in variety Heikezijing were induced by rice blight and rice blast incubation,however,the expression of OsNPSN11～12 in variety Suyunuo were unchanged by rice blast incubation.According to the different expression pattern between the varieties Heikezijing and Suyunuo,we transformed OsNPSN11cloned from Heikezijing to Suyunuo.After identification by PCR,RT-PCR and Sourthern blot,the T₁ OsNPSN11 over-expressing transgenic rice seedlings also showed increased disease resistance ability.These results indicated the roles of OsNPSN11 in disease response of rice.Plant plasma membrane(PM) proteins play important roles in signal transduction during defense response to an attacking pathogen.By using an improved method of PM protein preparation,purification and 2-D gel analysis,we conducted PM proteomic analysis of the rice Heikezijing leaves to identify PM components involved in the early defense response to rice blast fungi(Magnaporthe grisea Hoku 1).A total of 23 regulated protein spots were observed on 2-D gels of PM fractions at 8 and 24 h after pathogen inoculation, of which were induced with different levels.23 protein spots with predicted functions in plant defense were identified by MS,including 7 MS/MS identified protein spots.All of the induced proteins can be divided in to four types:first,associate with metabolizing,such as triosephosphate isomerase(TIM),6-phosphogluconate dehydrogenase(6PGDH), glyceraldehyde-3-phosphate dehydrogenase(GAPD),enoyl-CoA hydratase(ECH),malate dehydrogenase(MDH),alcohol dehydrogenase(ADH),1,4-benzoquinone reductase(QR), quinone oxidoreductase(QR2) and formyltetrahydrofolate synthetase(FTHFS);second, associate with regulating peptide,as developmentaly regulated plasma membrane polypeptide(DREPP),reversibly glycosylated polypeptide(RGP) and 20S proteasome subunit;third,associate with detoxification enzymes,such as ascorbate peroxidase(APX) and Cu/Zn superoxide dismutase(SOD);fourth,associate with signal transduction,such as annexin and glutathione-S-transferase(GST).Our study would provide a starting point for functional research of PM proteins in the rice defense.