Investigations On Stress Response Genes Of EXO70 Family In Arabidopsis

Biotic stresses(such as insect pests,diseases,etc.)and abiotic stresses(such as saline-alkaline,drought,high temperature,cold damage,etc.)are seriously affect the growth and development of plants.Thus,studying the defense mechanism of plant against the stress condition,identifying novel resistant genes,cultivating stress tolerant cultivars,would have a great significance to increase crop yield and reduce environmental pollution caused by pesticides application.Membrane trafficking in eukaryotic cells mediates the transport of molecules between membrane structures,which involved in a variety of biological processes including stress responses.During exocytosis,the specific recognition between post-Golgi vesicles and the target membranes is mediated by an evolutionary conserved multiprotein complex named the exocyst.The exocyst is comprised of 8 subunits,among which EXO70 plays a key role in exocyst assembly at target membrane.EXO70 genes in terrestrial plants expanded greatly in the genomes with their function much unknown,it is speculated that individual EXO70 members of plants are involved in different biological processes,such as stress responses.There are 23EXO70 genes in Arabidopsis genome,only 3 of them were reported to participate in stress responses so far.In this thesis,the stress responses of EXO70 genes in Arabidopsis were investigated by using reverse genetics,molecular biology and cell biology,and the regulate mechanisms had been preliminarily investigated.The main results are as followed:1.Firstly,Phylogenetic analysis was performed to reveal the evolutionary relationships among the EXO70 family of Arabidopsis.The phylogenetic analysis revealed that 23 Arabidopsis EXO70 proteins belong to a family with high homology.To study the function of individual EXO70,23 homozygous T-DNA inserted mutants were identified by genotyping,these mutant lines were used for further phenotypic analysis under different stress conditions.2.Based on public microarray data,the expression patterns of EXO70 s had been investigated by Heatmap methods,results showed that EXO70 B subfamily,EXO70 D subfamily,EXO70 E subfamily and 5 genes in EXO70 H subfamily were up-regulated to either biotic or abiotic stresses.3.The response of the EXO70 family to different stress treatments was analyzed by semi-quantitative PCR,The results showed that the expression of EXO70 A subfamily,EXO70E2,EXO70F1,EXO70G1 and EXO70H7 were up-regulated,and the expression of EXO70 B subfamily,EXO70 D subfamily and EXO70G2 were down-regulated by SA treatment,EXO70 B subfamily,EXO70 D subfamily,EXO70 E subfamily gene,EXO70F1,EXO70G1 and EXO70H7 were up-regulated by salt stress and drought stress.These results indicated that these genes might be involved in the stress response.4.The twenty-three T-DNA inserted mutants were treated by root inoculation of zoospores of Verticillium dahlia(JR2),and we found that exo70b1-2,exo70e2,exo70h2-2 and exo70h7-1 were more sensitive to JR2.The plaque diameter measurement and trypan blue staining after inoculation with zoospores of Botrytis cinerea revealed that exo70b1-1,exo70h1,exo70h2-2,exo70h3-2,exo70h7-3 were more susceptible to Botrytis cinerea.Expression of PR1 and SID2 were examined by Q-PCR(fluorescence quantitative Polymerase Chain React),results showed that the plants which inoculated with B.cinerea exhibited elevated expression levels in exo70b1 mutant,indicating that these EXO70 genes are involved in responses to B.cinerea infection through PR1 and SA pathway.5.To investigate the function of stress-responsive EXO70 genes,the localization of GFP tagged EXO70 proteins were investigated.In Pro EXO70B1::EXO70B1::GFP transgenic plants,GFP signals were present in the cytoplasm and plasma membrane,while in 35S::EXO70H7::e GFP transgenic plants,GFP signals were present in plasma membrane,cytoplasm and nucleus.These results suggested the functions of both genes are related to trafficking between PM and cytoplasms,most likely in exocytosis.6.To identify the members of EXO70 family responded to abiotic stresses,we treated the mutants with Na Cl and ABA,we found that main root growth of exo70h4 and exo70f1 were more sensitive to Na Cl and ABA treatment than that of wild type,suggesting EXO70H4 and EXO70F1 might be involved in plant response to salt stress through ABA pathway.In summary,based on our results,we proposed that different EXO70 members in plants exhibited different behaviors in the responses to biotic and abiotic stresses.These results are helpful to understand the biological significance of EXO70 family and lay a working foundation for the subsequent study of the mechanism of EXO70 involved in stress responses.