Adzuki Bean WRKY Gene Family Identification And Expression Analysis In Response To Uromyce Vignae Infection

Vigna angularis is a traditional grain crop in China.Because of its short growth period and barren tolerance,it plays an important role in the adjustment of agricultural planting structure.However,adzuki bean rust caused by Uromyces vignae can lead to dry leaves and early deciduous leaves of adzuki bean,which seriously affects the yield and quality of adzuki bean.In the early stage of this study,the results of transcriptomic analysis showed that the expression of several WRKY transcription factors was significantly up-regulated in rust resistant adzuki bean varieties 24 hours after inoculation.The results showed that the expression of transcription factors in rust resistant adzuki beans was significantly up-regulated 24 hours after vaccination.WRKY transcription factors are widely involved in plant disease resistance process,regulate plant disease resistance response and the establishment of signal transduction pathway,so it is speculated that adzuki bean WRKY gene plays an important role in rust resistance.In order to further clarify the function of adzuki bean WRKY transcription factor in rust resistance,the whole genome data of adzuki bean was used to identify the members of WRKY transcription factor in adzuki bean genome by means of biological information method,and the chromosome location,evolution and sequence characteristics of WRKY family members were systematically analyzed in this study.At the same time,the expression of adzuki bean WRKY transcription factor in different resistant varieties was analyzed by real-time fluorescence quantitative PCR?qRT-PCR?at different stages of rust infection.The full length of VaWRKY33 gene was cloned with adzuki bean genomic DNA as reference,and the role of VaWRKY33 in rust resistance and its relationship with ethylene resistance signal pathway were preliminarily clarified.The main findings are as follows:1.Using bioinformatics method,it was found that there were 90 WRKY transcription factors in adzuki bean genome,which were inhomogeneously divided into 11 chromosomes of adzuki bean.The highest distribution was 12 on chromosome 3,at least 1 on chromosome 8,and18 of them were not located on chromosome.The analysis of the characteristics of the WRKY protein of adzuki bean shows that the WRKY domain is highly conserved and contains the WRKYGQK 7 peptide domain and the zinc finger structure.The zinc finger structure types are CX₄CX_22-23HXH,CX_4-5CX₂₃HXH and CX₇CX_23–24HXC,respectively.The adzuki bean WRKY gene family can be divided into Group?,Group II and Group III types,with 17,61 and 12members,respectively.Group II can be further subdivided into two subgroup:II-a?6?,II-b?16?,II-c?21?,II-d?7?and II-e?11?.The adzuki bean WRKY domain evolution is highly conserved,which contains 8 conserved elements.The results of this study indicate that the WRKY domain of adzuki bean is relatively highly conserved.2.Based on the preliminary RNA-Seq sequencing data of the research group,the expression pattern of adzuki bean WRKY gene was clustered.It was found that the WRKY transcription factor could be grouped into 6 groups at 48 h,and 8 WRKY transcription factors were not expressed in the transcript group data.According to the relationship between the WRKY transcription factor and the transcription factor of adzuki bean WRKY,19 adzuki bean WRKY genes were selected for qRT-PCR.The candidate WRKY transcription factors could be divided into three categories according to the relative expression level of the inoculated treatment and the control.The first group consisted of five WRKY members,which were up-regulated at 12 and 24h after inoculation in the disease-resistant variety and up-regulated at 192 h after inoculation in the susceptible variety.The second group had 5 genes,which was up-regulated at 48 and 120 h after the inoculation in the disease-resistant variety,and there was no significant difference in the level of the expression of the inoculated and non-inoculated control in the susceptible variety.And the 9 members of the third group were significantly up-regulated in the disease-resistant variety at 120 h after the inoculation,and the expression was up-regulated at 192h after the inoculation in the susceptible variety.The results showed that adzuki bean VaWRKY gene played an important role in the response of adzuki bean to rust infection.In addition,it was found that VaWRKY33 was up-regulated in all stages of rust infection in resistant varieties,suggesting that the gene played a positive regulatory effect in the process of rust resistance in adzuki beans.3.In order to further analyze the role of VaWRKY33 in rust resistance of adzuki bean,the full length of adzuki bean VaWRKY33 gene was cloned with genomic mRNA sequence as reference and adzuki bean genomic DNA as template.Sequencing results showed that the full length of adzuki bean VaWRKY33 gene 2442 bp,was separated by four introns,and the length of four introns was 95 bp,518 bp,112 bp and 97 bp,respectively.In order to clarify the relationship between VaWRKY33 involvement in rust resistance and ethylene signaling pathway in adzuki beans,the effect of exogenous 1-aminocyclopropanecarboxylic acid?ACC?treatment on the expression of VaWRKY33 was analyzed.The results showed that compared with water treatment,the expression of VaWRKY33 increased significantly at 48 h after ACC treatment,and reached the peak value of 57.02 times as much as that of the control at 60 h after treatment,and then began to decrease,but it was still significantly higher than that of the control.After 2 days of ACC treatment and 0 hours after inoculated with rust,the expression of VaWRKY33 was significantly up-regulated,which was 285.29 times higher than that of the control treatment,and reached the peak value 3 days after 24 h(ACC treatment,indicating that the expression of adzuki bean VaWRKY33 gene was induced by ACC treatment,indicating that VaWRKY33 gene may be involved in the resistance of adzuki bean to rust by affecting ethylene signaling pathway.