Molecular Mechanism Of The Transcription Factor CsBZIP40 Enhancing Resistance To Citrus Bacterial Canker Disease

Citrus canker is a quarantine bacterial disease caused by Xanthomonas citri subsp.citri（Xcc）,which poses a great threat to the healthy development of citrus industry.Cultivating resistant varieties is the fundamental way to solve the threat of canker.Because molecular breeding is accurate and efficient,it has been developed rapidly in recent years.The overexpression of CsBZIP40 gene in citrus improved the resistance to canker,indicating that CsBZIP40 is an important transcription factor in response to canker infection.Transcription factors in the process of gene expression regulation will interact with other regulatory elements to affect the expression of downstream genes.To clarify the regulatory molecular mechanism of CsBZIP40 can explain the principle of improving the resistance of citrus to canker disease after transgenic,analyze the downstream genes to improve the resistance,and preliminarily analyze the regulatory mechanism of CsBZIP40.It can provide theoretical basis and resistant gene resources for further research of disease resistant molecular breeding,and improve the efficiency of molecular breeding.1.Construction and resistance evaluation of CsBZIP40 overexpressing plants.Construction of GFP-CsBZIP40 overexpression vector,transformation of citrus test-tube seedling epicotyls by Agrobacterium-mediated method,tissue culture and screening to obtain GFP-CsBZIP40 expressing transgenic citrus plants with overexpression,and evaluation of transgenic plants for ulcer resistance Plants with improved resistance to ulcer disease were obtained.Based on this material,the regulatory mechanism of CsBZIP40 transcription factor was analyzed.2.Identification of CsBZIP40 interacting protein.To screen CsBZIP40 interacting proteins,first construct a GST-CsBZIP40 fusion bait protein expression vector,express and purify the bait protein,and incubate with overexpressing CsBZIP40 transgenic citrus total protein.SDS-PAGE shows that the bait protein can specifically interact with citrus Some proteins in the protein are combined,and these protein profiles are analyzed to obtain 6 proteins that are closely related to the improvement of resistance to ulcer disease by transgenic citrus.3.CsBZIP40 regulates downstream gene identification.To analyze the differential expression of transgenic citrus genes after overexpression of CsBZIP40,this study performed transcriptome analysis on transgenic citrus plants.Transgenic citrus has 599differentially expressed genes compared with wild type,up-regulated expression,and combined analysis of biological stress response pathways,preliminary determination of some genes in plant hormone signals,cell wall related genes,PR protein genes,ros clearance related genes and catalase genes Significant differences in expression after transgenes,these differentially expressed genes may play an important role in citrus resistance.4.Identification of CsBZIP40 target genes.In this study,chromatin immunoprecipitation combined with second-generation sequencing（CHIP-seq）was used to find the target site of CsBZIP40 in the whole genome of citrus.The results showed that CsBZIP40 had a total of 634 target sites,of which 241 sites were located in the promoter region of the gene.Target gene KEGG analysis revealed that six genes were closely related to plant resistance.They are Four target genes of CsBZIP40 were screened by analyzing the data of CHIP-seq and RNA-seq.They were peroxidase gene（orange1.1t02040）,pathogenesis related gene transcription activator ERF（orange 1.1t00851）,superoxide dismutase（cs8g15520）,ethylene response transcription factor（s6g18930）.Further clarified the regulatory mechanism of CsBZIP40.5.According to the results of CHIP-seq and RNA-seq data,analysis of biochemical indicators related to the overexpression of CsBZIP40 transgenic citrus,the results showed that the jasmonic acid content in the leaves of transgenic citrus overexpression CsBZIP40increased significantly,and the salicylic acid content also increased slightly.Increasing the content of both plays an important role in activating the citrus disease resistance pathway;overexpression of CsBZIP40 reduces SOD,POD,and GST activities.Changes in these antioxidant enzyme activities may be related to increased resistance to transgenic citrus;The content of ROS,H₂O₂,and O-in overexpression of CsBZIP40 transgenic citrus leaves increased.Increasing these contents can promote the hypersensitivity reaction of transgenic citrus,prevent the development of ulcer disease in transgenic plants,and improve the disease resistance of transgenic citrus.To sum up,this paper analyzes the regulatory molecular mechanisms of transgenic citrus over-expressing CsBZIP40 in response to ulcerative bacteria from the differences in proteins,genes,and biochemical indicators,and lays the theory for precise digging of anti-ulcerative genes and molecular breeding of ulcer-resistant citrus basis.