Transcriptome Analysis And Resistance Related Gene Mining In Response To Xanthomonas Arboricola Pv.juglandis Infection In Walnut

Walnuts(Juglans regia L.)is a widely distributed economic forest tree with nutritional value and is the first of the four major nuts in the world.Bacterial black spot of walnut caused by Xanthomonas arboricola pv.juglandis(Xaj)is the most serious bacterial disease that harms walnut fruit quality.At present,the most safe and economical method is to screen and cultivate resistant varieties.However,the molecular mechanism of walnut response to Xanthomonas infection remains unclear.Therefore,in this study,the pathogenic bacteria were isolated and identified from the diseased fruits,and the leaves of the four varieties ’Xiangling’,’Qingxiang’,’Franquette’ and ’Chandler’ were artificially inoculated to identify their susceptibility by incidence and phenotype.Transcriptome sequencing analysis was conducted on two cultivars,’Qingxiang’ and ’Xiangling’,which were infected but had obvious differences,to analyze the differences in response of resistant and susceptible cultivars to pathogenic bacteria from the transcript level.By screening transcription factors related to disease resistance,cloning and functional identification of related genes were performed.This study provides reference value for further study of molecular mechanism of walnut response to Xanthomonas infection and can be used as molecular theoretical basis for screening and breeding resistant varieties.The main research results are as follows:1.Through the isolation of pathogenic bacteria of walnut fruit outer epidermal plaques,the pathogen Xaj was determined after molecular identification.The leaves of four walnut clones of ’Qingxiang’,’Xiangling’,’Franquette’ and ’Chandler’ were artificially inoculated.Based on the phenotypic changes,incidence and time of disease spots after inoculation,the order of disease resistance of the four varieties was preliminarily suggested as’ Chandler’>’Franquette’>’Qingxiang’>’Xiangling’.2.The leaves of ’Qingxiang’ and ’Xiangling’ at different periods after inoculation were selected for transcriptome sequencing analysis to compare the differences in gene expression between the disease-resistant varieties and the control group.There were 10,366 differentially expressed genes in different periods after inoculation with Xaj ’Qingxiang’.There were 6,990 differentially expressed genes in ’Xiangling’ at different periods after inoculation.The number of differentially expressed genes in disease-resistant varieties was higher than that in susceptible varieties at all stages,but all showed a trend of increasing first and then decreasing.The difference was that the number of ’Qingxiang’ was the highest after 7 days of inoculation,while the number of ’Xiangling’ was the highest after 3days of inoculation,which was consistent with the onset time.3.The differentially expressed genes of resistant cultivar ’Qingxiang’ and susceptible cultivar ’Xiangling’ were enriched by GO annotation and KEGG pathway,respectively.It was found that the two cultivars were different in terms of type and quantity.Key genes were screened out by constructing gene co-expression networks.Key genes were found in plant hormone signal transduction,MAPK signaling pathway-plant and Carotenoid biosynthesis are significantly enriched in these three pathways.Fifty-eight differentially expressed transcription factors related to black spot disease were obtained,among which AP2/ERF-ERF,MYB,b HLH and WRKY were the most.4.JrWRKY07-2,JrWRKY18 and JrWRKY47 genes obtained by transcriptome sequencing were cloned.The total length of JrWRKY07-2 was 591 bp,encoding 198 amino acids.JrWRKY18 was 753 bp in length and encoded 250 amino acids.The total length of JrWRKY47 is 939 bp,encoding 312 amino acids,all of which have one WRKYGQK characteristic sequence.Transgenic 84 K Yang was treated with salicylic acid and ababolic acid.It was found that JrWRKY07-2 and JrWRKY47 were both treated with salicylic acid and reached the highest value at 1 h,and reached the lowest value at 6 h.While JrWRKY18 was treated with salicylic acid and the expression decreased at 6 h.JrWRKY07-2 showed the most sensitive response to abscisic acid,while JrWRKY47 showed a slow response to abscisic acid with a small change trend.Although JrWRKY18 showed a late response time,it showed a significant increase and decrease trend.These results indicated that JrWRKY07-2,JrWRKY18 and JrWRKY47 genes could be involved in salicylic acid and abscisic acid signaling pathways of walnut,and thus participate in the response mechanism of walnut to pathogen infection.It lays a foundation for the follow-up study of gene function.