Expression Analysis Of Related Disease Resistance Genes In The Interaction Between Citron C-05 And Xathomonas Citri Subsp.Citri

Citrus canker is a quarantine disease caused by Xathomonas citri subsp.citri.Infected plants show persistent skin damage,leaf and fruit drop,and tree decline.At present,there is no effective treatment for completely control.Selecting citrus cultivars with stronger resistance to replace susceptible ones is the substential effective method.After years of screening at the National Center for Citrus Improvement（Changsha）,Citron C-05（Citrus medica L.）was verified to be a disease-resistant genotype which showed allergic,necrotic and disease-resistant response to th canker pathogen.This thesis aims at the search for disease resistance related genes and analysis of the expression profiles of the PRR genes,so as to better understand the resistant mechanism of citron C-05,which may provide the theoretical basis of disease resistance molecular breeding.In this study,based on the transcriptome sequencing data of resistant citron C-05 and susceptible variety Bingtang sweet orange [C.sinensis（L.）Osbeck] before and after inoculation with the pathogen,a number of candidate PRR genes related to citrus canker resistance were found out,and their expression patterns were verified by quantitative PCR.Combined with the results of gene cloning and sequencing,the sequences and structures of EFR1 and BAK1-1,two genes regulated by peptic ulcer induction,were analyzed.Then the EFR1 and BAK1-1 gene sequences and their promoters were cloned,and their amino acid sequences and promoter sequences were analyzed by bioinformatics to analyze the differences between protein structure and function,as well as the differences between the key structure and cis-acting elements of the promoter sequence.The vector was constructed and the transient expression of these two genes was used to analyze the function of candidate genes and their promoters.In addition,yeast mono-hybrid was used to screen transcription factors that interact with promoters.The main conclusions are as follows:1.A series of PRR genes and BKA1 family genes were screened from the transcriptome through the sweet orange genome retrieval and comparison with the reported PRR gene homologous proteins.Transcriptomic data analysis and quantitative PCR verification showed that EFR family genes EFR1（Cs4g12920）and BAK1 family genes BAK1-1（Cs5g11310）were specifically up-regulated by ulcerative bacteria in Citron C-05,but not significantly up-regulated in sugar orange.2.In the process of responding to the invasion of ulcer pathogen,the candidate genes EFR1 and BAK1-1 related to disease resistance were not significantly up-regulated in susceptible germplasm Bingtang orange,wild citron,Nanchuan citron and Shatian pomelo,but were significantly upregulated in resistant germplasm citron C-05,American citron and bergamot,and the trend was consistent with the results of transcriptional group.3.The amino acid sequences of EFR1 and BAK1-1 in resequenced citrus germplasm were analyzed,and the results showed that there were differences in amino acid sequences among different germplasms.But there is no obvious correlation between resistance and resistance.The results of gene function verification by transient expression showed that both EFR1 and BAK1-1 could inhibit the growth of ulcer pathogen in susceptible germplasm Bingtang orange to some extent.4.Most of the cisacting elements,such as salicylic acid cis-acting element,methyl jasmonate response element,gibberellin response cis-acting element,W-box and WUN-box related to plant defense gene activation,are common to the promoters of EFR1 and BAK1-1 genes in Bingtang Orange and Citrate C-05.The results of promoter activity verification showed that both PCm-EFR1 、and PCs-EFR1 could be induced and activated by ulcer bacteria.The promoter PCs-BAK1-1of Bingtang orange could not be induced by xcc,while the promoter P_Cm-BAK1-11 of citron C-05 could be activated by xcc.It was found that the key active site of P_Cm-BAK1-11 was between P-1212 and P-1459.The active site of P_Cm-BAK1-1 is between P-0 and P-429.5.The ulceration-induced library cDNA of Citron C-05 was screened,and the transcription factors interacting with BAK1-1 promoter were identified as Cm RAP2-13,and the transcription factors interacting with EFR1 promoter were Cmb HLH66,Cm ZAT6.Transcription factors Cmb HLH66,Cm ZAT6,and Cm RAP2-13 were specifically upregulated by ulcerative bacteria in Citron C-05,and the transcription factors Cmb HLH66 and Cm ZAT6 could interact with the citron C-05 promoter EFR1 fragment,but not with the sweeet orange promoter EFR1 fragment.Transcription factor Cm RAP2-13 interacted with the BAK1-1 fragment of citron C-05 promoter,but not with the EFR1 fragment of sweet orange promoter.