PTI Detection In Protoplasts And Characterization Of CDPK Gene Family In Disease Resistance Of Hevea Brasiliensis

Natural rubber is indispensable industrial materials and strategic materials,which is important for national defense and economic development.The disease of rubber tree is one of the most important factors that lead to serious yield losses in rubber production.Therefore,finding out a suitable way to effectively improve the diseases resistance of rubber tree and reduce the losses of natural rubber is of great significance.Because of many problems on genetic transformation system in rubber tree,the research on its functional genomics and plant pathology had lagged obviously.Nowadays,the prevention and cure of diseases in rubber tree is mainly focusing on chemical control.However,the chemical control has many disadvantages,such as high cost,serious pollution,etc.With the widespread use of biotechnology,people put forward a new idea of rubber tree genetic engineering disease-resistant breeding.On this basis,we took Hevea brasiliensis 7-33-97 varieties as experimental material and established the detection system of innate immune response of rubber tree by using the transient mesophyll protoplast system,which providing the technical basis for the study of rubber tree pathology.In order to study the rubber disease-resistant mechanism and find some pivotal rubber tree disease resistance genes,we utilized the method of gene differential expression analysis based on transcriptome to detect the expression of calcium-dependent protein kinase(CDPK)gene family.Clones and functional analyzes were performed.The main results are as follows:Part.1 An efficient transient mesophyll protoplast system for investigation of the innate immunity responses in the rubber tree(Hevea brasiliensis)1.The expression of the disease-related genes after two kinds of PAMPs treatment was analysed by RT-PCR and Dual-Luciferase Reporter assay system.Results showed that the expression of HbPR1 and HbPR5 significantly enhanced after treatment with flg22.However,chitin induced the expression of HbPR5,but not HbPR1.Expression of HbMAPK3 and HbMAPK6 was not affected by flg22 or chitin at the transcriptional level.These results implied that HbPR1 and HbPR5,not HbMAPK3 and HbMAPK6,were related with PTI in rubber tree,but the signaling pathways were different.2.The phosphorylation of HbMAPKs was detected by western-blot afer treatment with.Results showed that HbMAPKs were activated by flg22 but not by chitin.This result implied that HbMAPKs may play different roles in different signaling pathways.3.After treatment with two kinds of PAMPs,the accumulatin of reactive oxygen species(ROS)in mesophyll protoplast was analysed.Results showed that both flg22 and chitin can induce ROS accumulation in rubber tree mesophyll protoplasts,which indicated that flg22 and chitin can induce the accumulation of intracellular ROS to activate rubber defense response.4.After treatment with two kinds of PAMPs,the activity of antioxidant enzymes in mesophyll protoplast was analysed.Results showed that,both flg22 and chitin can promote the activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT),which indicated thses antioxidant enzymes are involved in the innate immunity of rubber tree induced by different PAMPs.Part.2 Analysis of differentially expressed genes in the transcriptional group and screening of disease resistance-related genes in rubber trees:Differentially expressed genes profile in leaves of rubber tree was analyzed by RNA-seq after SA treatment.As a whole,among the obtained 44,477 EST,SA treatment could induce expression changes in about 30%genes,and about 800 candidate genes related to disease resistance were screened artificially,with more up-regulated genes than down-regulated genes.In addition,in order to understand the biological function of the genes,we carried out a pathway enrichment analysis based on the KEGG database.Results demonstrated that the factors associated with disease resistance of plants were divided into the following categories:1.Components regulating ROS production included up-regulated APX,CDPK,CaMCML and NOS,and down-regulated NADPH oxidase,etc.2.Components involved in the PTI signal transduction pathway included FLS2,BAK1,MEKK1,MKK1/2,MEKK4/5,WRKY35/33 and WRKY22/29,which were all up-regulated,suggesting that S A treatment activated the PTI of plants.3.Components involved in the ETI signal transduction pathway included RPM1,RPS2,PBS1,RPS5,MIN7,JAZ,etc.Part.3 Cloning and expression analysis of CDPK gene family of rubber tree:1.In this study,31 HbCDPK genes were electronically aligned according to the information of rubber tree transcriptome sequencing,which was named as HbCDPK1-31 respectively.However,in the actual cloning process,only 28 HbCDPK genes were colned successfully,while HbCDPK19,HbCDPK21 and HbCDPK30 were not be cloned.2.The 28 HbCDPK genes were analyzed by evolutionary tree analysis.It is found that HbCDPK gene family can be divided into four Subgroups.We grouped HbCDPK5,9,10,15,17,20,22,27,28,31 into Subgroup Ⅰ;HbCDPKl,6,8,11,12,14,16,23 into Subgroup Ⅱ;HbCDPK2,3,4;7,13,18,24,26,and 29 into Subgroup Ⅲ,while only HbCDPK25 into Subgroup Ⅳ.3.qPCR was used to monitor the expression of HbCDPK in rubber leaves after inoculated with different pathogens.Results showed that after inoculated with Colletotrichum gloeosporioides,HbCDPKl,4,8,13,11,15,18,20,29 were up-regulated,while HbCDPK6,13,24 were down-regulated.The expression of the remaining genes did not have significant changes.However,after inoculated with Odium heveae Steinm,HbCDPK2,3,6,7,10,14,22,23,26 were up-regulated,while HbCDPK18 and 29 were down-regulated.The expression of the remaining genes was not significantly changed.4.qPCR was used to monitor the expression of HbCDPK in rubber leaves after stimulated by different hormones(SA,JA,ET,ABA,GA).Results showed that after treated with SA,HbCDPKl,2,7,9,15,20,23,26,28,31 were up-regulated,while HbCDPK4,14,24,29 were down-regulated.The expression of remaining genes had no notebly changes.After treated with JA,HbCDPK1,6,8,12,15,18,23,27,29 were up-regulated,and the HbCDPK2,10,17,25 were down-regulated,while the expression of the remaining genes did not change significantly.After treated with ET,HbCDPK24 was up-regulated,while HbCDPK25 was up-regulated at 6h but down-regulated soon afterwards.After treated with ABA,HbCDPK3,5,8,12,16,18,20,22 were up-regulated,and HbCDPK14,25,31 were down-regulated,while the expression of the remaining genes did not change significantly.After treated with GA,HbCDPK10,14,17,20,27 were up-regulated,and HbCDPK3,24 were down-regulated,while the expression of the remaining genes did not have significant changes.These results indicated that different members of CDPK gene family in Hevea brasiliensis may play different roles in different signal transduction pathways,but there is a cross and overlap of signal transduction among different members.5.Subcellular localization of the HbCDPKs was detected by confocal laser scanning microscope.The results showed that HbCDPKl,6,8,9,12,16,18,23,25,27 were located on the cell membrane,while HbCDPK2,3,4,5,7,10,11,13,14,15,17,20,22,24,26,28,29,31 were located in the cytoplasm or organelles.6.We dectected the accumulation of reactive oxygen species(ROS)in the protoplasts of rubber tree after HbCDPK overexpression.The results showed that HbCDPK3、8、9、10、15、18、20、23、26、29 can accumulate ROS obviously in rubber tree mesophyll protoplasts.Part.4 Functional analysis of HbCDPKIS:1.HbCDPK15 encodes a 561 amino acids peptide,which molecular weight is 62.8kDa and the isoelectric point is 5.01.HbCDPK15 contain a special S_TKc protein kinase domain and EF-hand calcium combined domain which were similar to other CDPK families members.The homology of HbCDPK15 and AtCPK6 was found to be the highest by homology comparison.2.HbCDPK15 transgenic Arabidopsis was obtained to investigate the biological function of HbCDPK15.Results showed that the bacterial plaque diameter of 35s:HbCDPK15/cpk6 was much smaller than that of cpk6 mutant after inoculated with Botrytis cinerea,indicating that HbCDPK15 can improve plant resistance to Botrytis cinerea.Meanwhile,the seed germination of 35s:HbCDPKl5/cpk6 was remarkably higher than that of cpk6 mutant after treated with 150 mM NaCl,which indicated that HbCDPK15 may participate in the process of seed germination under salt stress,but not in the growth of plants.There was no significant difference in seed germination and seedling growth of the three types of Arabidopsis under ABA stress,indicating that HbCDPK15 may have no effect on plant resistance to ABA,which was consistent with the expression pattern of HbCDPK15 under ABA treatment.3.The results of Co-IP showed that HbCDPK15 can interact with HbRboh D,which suggested that HbCDPK15 may play a key role in the defense-induced oxidative burst by activating HbRboh D through direct phorylation.