Adzuki Bean DIR Gene Family Identification And Functional Verification Of VaDIR33 Gene

Adzuki bean rust caused by Uromyces vignae occurs commonly in all adzuki bean growing areas in Heilongjiang Province.In severe cases,the disease can lead to premature leaf senescence and early abscission,which is a serious threat to adzuki bean production.Selection and planting disease-resistant varieties are one of the most economical and effective methods to control crop rust.However research on rust resistance genes and resistance mechanisms of adzuki bean has been poorly reported,limiting the cultivation and utilization of disease-resistant varieties of adzuki bean.In the early stage,i TRAQ technology was used to analyze the differently expressed proteins of adzuki bean rust-resistant variety “QH1” at different times after inoculation,and it was found that the abundance of several Dirigent(DIR)proteins in adzuki bean increased significantly after inoculation,suggesting that DIR proteins play an important role in rust resistance of adzuki bean.In order to further explore the function of DIR gene and analyze the molecular mechanism of rust resistance,this study started with the whole genome of adzuki bean DIR gene family,and clarfied the number,structure,chromosome localization and other characteristics of DIR gene family members.At the same time,q RT-PCR was used to determine the expression pattern of DIR family members in response to U.vignae infection in different resistant and susceptible adzuki bean varieties.On this basis,the relationship between DIR gene expression and changes in lignin and lignan content was analyzed,then selected the DIR gene that was significantly up-regulated and closely related to changes in lignin and lignan content during infection.The Tobacco transient expression system was used to confirm its role in the production of lignin and lignan in plants.This laid the groundwork for further research into how the DIR gene controls the production of lignin and lignan and helps plants resist disease.Based on the above research,the following research results were obtained:1.A total of 33 DIR genes were screened from adzuki bean genome using bioinformatics method,and chromosomal localization showed that 29 DIR genes were located on 8 different chromosomes,and the remaining 4 members were not located on chromosomes.Phylogenetic analysis showed that Va DIRs include four subfamilies,DIR-a(n=6),DIR-b/d(n=17),DIR-e(n=6)and DIR-f(n=4).The presence of hormones and pathogen response elements in all Va DIRs promoter regions was discovered through analysis of cis-acting elements in member promoter regions,implying that Va DIRs may play an important role in the response to biological stress of adzuki bean.2.The expression patterns of Va DIRs were analyzed during the response to rust infection in resistant and suscepitible cultivars by using q RT-PCR technology.The results showed that 23 Va DIRs showed different levels of up-regulation after inoculation with the rust infection,among which 9 Va DIRs were significantly up-regulated in the early stage of rust infection(120 h～192 h).,and 11 Va DIRs were significantly up-regulated in the late stage of rust infection(120h～192h).The expression levels of Va DIR33 were significantly higher than that of the susceptible variety during the whole infection process,indicating that different members of DIR gene family play an important regulatory role at differnet stages of adzuki bean response to U.vignae infection.3.In order to clarify the influence of Va DIRs on lignin content during the infection process of U.vignae,this study analyzed the changes of lignin content in different resistant and susceptible adzuki bean varieties at different times after inoculation.The results showed that among the disease-resistant varieties.The growth rate of lignin content increased by 23.90% and 23.98% at120 h and 192 h after inoculation,respectively,compared with the control,susceptible variety increased only by 11.64% and 18.85%.In disease-resistant varieties,Va DIR33 had a significant positive correlation with the growth rate of lignin content with a correlation coefficient of r=0.650(P =0.004).4.In order to clarify the influence of Va DIRs on lignan content during the infection process of U.vignae,this study analyzed the changes of lignan content in different resistant and susceptible adzuki bean varieties at different times after inoculation.The results showed that among the resistant varieties,The growth rate of lignan content increased by 23.10% and 20.57% at 120 h and 192 h after inoculation,respectively,compared with the control,susceptible variety increased onlu by 10.65% and 16.36%.Moreover,Va DIR33 was significantly positively correlated with the growth rate of lignan content in resistant variety with a correlation coefficient of r=0.637(P=0.004).5.In order to verify the role of DIR gene in disease resistance,Va DIR33 gene was cloned,and transiently expressed in tobacco leaves by agrobacterium tumefaciens mediated method.Subcellular localization of Va DIR33 protein was observed and its effects on lignin and lignan metabolism in tobacco were analyzed.The subcellular localization results showed that Va DIR33 protein was localized in the cytoplasm.The contents of lignin and lignan in tobacco leaves transiently expressing Va DIR33 gene increased by 0.06 mg/g,0.07 mg/g and 0.06 mg/g after 2,4and 6 d of transient expression,respectively.The content of lignan increased by 0.11 mg/g and0.22 mg/g after 4 and 6 d of transient expression,respectively.These results indicated that Va DIR33 is involved in the synthesis of lignin and lignan in plants.