Gene Cloning And Expression Analysis Of Canker-related Transcription Factor CsBZIP40 In Citrus

Xanthomonas citri subsp.citri,Xcc is the pathogenic bacteria of citrus canker disease which is one of the most disease causing serious yield losses in citrus-planting regions all over the world.The phenomenon has been affected the economic development,including fruits with scar or flower fallen down,so it is necessary to breed new species to improve the canker resistance.The goal of this experiment was finding the Xcc-associated genes by analyzing the transcriptome data which had been inoculated Xcc in Newhall Navel Orange(Citrus sinensis Osbeck)and Calamondin(Citrus madurensis)and the bioinformatics data about the BZIP family.The Canker-related transitional factor CsBZIP40 was obtained from transcriptome data which associated bioinformatics and related literatures.CsBZIP40 plays a role in citrus canker by setting expression patterns and validating the function of this gene.The research based on the public genome databases,the BZIP gene family was expertly and comprehensively annotated and named based on the chromosomal localization of all the members of BZIPs;the motifs of the BZIPs were analyzed by MEME online tool;the phylogenetic tree of BZIPs in Citrus and Arabidopsis thaliana was constructed using software Mega 7.0 based on which the category of BZIP family was obtained.A set of researches had been found that 47 BZIP genes.There are fewer gene duplication events detected from BZIP family of citrus compared with other plants,such as Arabidopsis,grapevine and so on.That is why citrus has a smaller BZIP family size.The CsBZIP40 obtained from transcriptome data which associated bioinformatics data and related literatures.It is closely related to AT1g08320 in Arabidopsis thaliana based on the evolutionary analysis.In citrus,the BZIPs have been annotated which can be divided into 10 different sub-families.CsBZIP40 belongs to sub-family D,which is always take part in the pathogen resistance in plants.The gene and its promoter was cloned.The full-length of CsBZIP40 is 5756 bp with a 1530-bp open reading frame which codes a protein containing 509 amino acids.The gene promoter contains multiple cis-elements involved in plant adversity or hormone response,such as Box-W1,HSE,ERE and so on.CsBZIP40 includes two nuclear localization signals and then the subcellular localization was confirmed by GFP fusion experiments in onion.Subcellular localization results confirm the prediction of protein localization in nucleus.It was also detected by q-RT-PCR.expression profiles induced by Xcc,Salicylic acid(SA),Jasmonic Acid Methyl Ester(MeJA),Ethylene(ET)and mechanical damage of CsBZIP40 were checked with real-time fluorescent quantitative PCR.Based on the qPCR data,the exogenous salicylic acid can induce the incresse expression of CsBZIP40 in Calamondin and present decrease in Newhall Navel Orange,in contrast,Jasmonic acid methyl ester can present a increase espression in Newhall Navel Orange and induce decrease in Calamondin.Xcc attack can significantly increase the expression level of CsBZIP40 in Calamondin but no difference in Newhall Navel Orange.The Xcc-resistance of citrus is related to the CsBZIP40 expression which be regulated the concentration of SA and MeJA in Calamondin from the patterns of the induction of exogenous hormone and Xcc.Finally,the over-expressed and RNAi vector was constructed and transformed citrus to confirm the relationship between CsBZIP40 and Xcc infection.Five over-expressed transgenic plants and eleven RNAi transgenic plants were obtained after detection of PCR.There were decrease phenomenon in the lesion area and disease index of over-expressed transgenic plants by inoculating Xcc,however,the RNAi transgenic plants presented increase results.All above,the CsBZIP40 become an important transcription factor which is closely associated with the resistance of citrus canker.This gene should be a potential candidate in the molecular breeding to improve the canker resistance of citrus.