Identification Of HSP20 Family Genes In Citrus And Their Expression In Pathogen Infection Responses Citrus Canker

HSP20 genes are abundant and highly conserved in higher plants,they play vital defense roles when plants are under biotic or abiotic stress.Citrus canker,caused by Xanthomonas citri subsp.citri（Xcc）,is a bacterial disease,which severely influence citrus industry,and its pathogenicity is significantly enhanced under higher temperature and humidity environmental condition.In order to investigate the role of HSP20 genes in response to Xcc infection,we first identified genes of HSP20 family in citrus sinensis（CsHSP20）,and analyzed the CsHSP20genes biological information.Secondly,comparative analysis of the expression HSP20 family genes in the transcriptome induced by Xcc infection in disease resistant Citron C-05 and susceptible rock sweet orange,and performing q RT-PCR verification and analysis after stress treatment.Finally,cloning the HSP20 genes ORF in Citron C-05 and sweet orange.After the candidate genes were cloned,the overexpression vector was constructed,and the overexpression was performed in sweet orange leaves and Arabidopsis thaliana for functional verification.This study helpful to analyze the differences between the CsHSP20 genes and also provides data support for the later research and analysis of the gene function of the family.At the same time,which provides data support and theoretical reference for the analysis and enrichment of Citrus C-05 germplasm against citrus canker pathogen.The main results of this research are as follows:（1）Bioinformatic analysis result showed that there are 42 genes of HSP20 family in citrus sinensis,which can be divided into 11 subfamilies,they encode 135～373 amino acid and are distributed on 9 chromosomes,their protein molecular weight are from 15.17 to 41.71 k Da,isoelectric point are from 4.53 to 10.07,almost 92.9%of them don’t contain any intron or only one,and each subfamily share highly similar structures and conserved motifs,their promoter also has cis-acting elements response to hormones or stress.（2）In order to access the role of HSP20 genes in response to Xcc and abiotic stress in resistant germplasm Citron C-05 with susceptible sweet orange,we have injected Xcc into the leaves of Citron C-05 and conducted a series of heat stress with small Citron C-05 plants.The relative expression levels of HSP20 have been screened by q RT-PCR,the results showed that HSP17.9,HSP23.3,HSP18.5 and HSP18.0 were up-regulated in both Xcc and heat stress,but their relative expression level in Citron C-05 is notably different from that of sweet orange under Xcc infection,especially HSP23.3.Interestingly,the induced expression patterns are consistent with the result of the transcriptomic analysis infected by Xcc.At the same time,the amino acids encoded in the different citron species material are very conservative.（3）Through gene cloning,we were obtained the CsHSP17.9（474 bp）,CsHSP23.3（609bp）,CsHSP18.5（489 bp）and CsHSP18.0（474 bp）in sweet orange,and Cm HSP23.3（612 bp）,Cm HSP17.9（2 559 bp）and Cm HSP18.5（489 bp）in Citron C-05.These genes have been transiently overexpressed in sweet orange leaves,and Xcc quantitative analysis of Citron C-05 HSP23.3 showed that Xcc reproduction was inhibited.At the same time,the HSP23.3overexpression vector containing Citron C-05 was transformation of Arabidopsis thaliana T₂generation strain after inoculation with Pst.DC3000 showed that the Arabidopsis thaliana containing the HSP23.3 gene of Citron C-05 had milder symptoms and there is less bacterial growth than the Col＿0 type.