Bioinformatics Analysis Of NB-ARC Family Associated With Hulless Barley Leaf Stripe And Functional Validation Of Candidate Genes

Hulless barley(Hordeum vulgare L.var.nudum.Hook.f.)is a major grain crop in the Tibetan Plateau of China,also known as naked barley.Leaf stripe disease is a common fungal seed-borne disease in the barley production process.Once the leaf stripe disease occurs,the yield of hulless barley will be greatly reduced,and the quality of hulless barley will also be seriously affected.In this study,the barley reference genome information was used to screen the barley NB-ARC gene family,and the previous full-length transcriptome and transcriptome sequencing results were used to identify the lesf stripe disease-related candidate genes Hvn RPS2 and Hvn RGA3.Their expression patterns under streak stress were analyzed.The leaf stripe disease function of Hvn RPS2 gene was verified by VIGS technology,which preliminarily revealed the role of this gene in the process of hulless barley resistance to leaf stripe disease.The main findings are as follows:1.Based on the barley reference genome,298 NB-ARC family members were identified,then chromosomal mapping was performed,and gene structures,protein domains and motifs,protein evolution,and collinearity with Arabidopsis,maize,and rice were analyzed.The results showed that 298 barley NB-ARC genes were unevenly distributed on seven barley chromosomes;the gene and protein structures of the 298 barley NB-ARC gene family members were significantly different,and they were divided into 9 groups;most of the family members,The synonymous mutation of,was not subject to natural selection pressure;there was no collinear relationship between barley and Arabidopsis,and 7 pairs and 9 pairs of replication events occurred in barley,maize and rice,respectively.2.Using the results of transcriptome and full-length transcriptome sequencing of previous resistant barley variety ’Kunlun 14’ and susceptible barley variety ’Z1141’under stripe stress,7 of the 298 NB-ARC genes in barley were differentially expressed NB-ARC gene.GO analysis significantly enriched them mainly in cellular and metabolic processes;KEGG analysis found that one NB-ARC gene(gene ID:HORVU2Hr1G089020)was enriched in the pathway of interaction between plant diseases and pathogenic bacteria.The results of transcriptome analysis showed that the expression levels of Hvn RPS2 and Hvn RGA3(gene ID: HORVU2Hr1G087730)were significantly decreased and increased under leaf stripe disease stress.According to the above research results,Hvn RPS2 and Hvn RGA3 were identified as highland barley resistance genes related to leaf stripe disease.3.Hvn RPS2 and Hvn RGA3 genes were cloned from the leaves of ’Kunlun 14’ and’Z1141’ by homologous cloning technology.Sequence analysis showed that the nucleotide sequence of Hvn RPS2 and the amino acid sequence encoded by it were 100%identical in the two varieties;the amino acid sequences encoded by Hvn RGA3 were98.25% identical.q RT-PCR showed that the expression patterns of Hvn RPS2 and Hvn RGA3 genes in the resistant barley variety ’Kunlun 14’ and the susceptible barley’Z1141’ first decreased,then increased and then decreased with the extension of the streak stress time.The gene expression level of susceptible leaves was extremely significantly down-regulated,and the down-regulated value of the expression level of resistant varieties was significantly higher than that of susceptible varieties.It was speculated that Hvn RPS2 and Hvn RGA3 genes played an important negative feedback regulation in the process of highland barley resistance to leaf stripe disease.4.The anti-stripe function of Hvn RGA3 gene was preliminarily verified by VIGS technology.The results of semi-quantitative and fluorescence quantitative detection showed that after the expression of Hvn RGA3 gene was significantly reduced,the disease resistance of leaves was significantly higher than that of the control group.Play an important negative feedback role.The results of this study provided a new idea for the study of hulless barley NB-ARC genes in disease resistance breeding.