Identification And Preliminarily Functional Verification Of SMV Resistant Gene In Soybean Based On Transcriptome Analysis

Soybean [Glycine Max(L.)Merr.] is an important food and oil crop in China.Soybean Mosaic Virus(SMV)belongs to Potyvirus,a global viral disease that poses a serious threat to the yield and quality of soybean.Based on their different symptoms on 10 soybean differentials,SMV was classified into 22 strains(SC1-SC22)in China,of which the SC18 strain was widely distributed in the southern and Northeastern China,posing a threat to soybean production.At present,chemical control cannot effectively prevent SMV.The most economical and effective SMV control methods are to excavate resistant genes,breed resistant cultivars,and study resistant mechanisms.The main contents of this study were as follows:(1)To study the transcriptional levels of resistant cultivar Kefeng No.1 and susceptible cultivar Nannong 1138-2 after inoculation with SC18,identify the differentially expressed genes,and analyze the defense response of resistant cultivar to SC18 and the regulatory networks that may be related to disease resistance through enrichment analysis of differentially expressed genes.(2)The candidate genes for resistance were screened,and the reliability of the sequencing results was tested by q RT-PCR.Meanwhile,the expression analysis of the screened candidate genes was conducted to determine the target genes to be verified in this study.(3)The target gene was cloned in the resistant and susceptible cultivars,and its sequence was analyzed.The VIGS verification of the target gene was carried out,and the accumulation of SC18 was detected to preliminarily verify its function.The main research results are as follows:1.Transcriptome analysis of resistant and susceptible soybean cultivars inoculation with SC18After RNA-Seq of resistant cultivar Kefeng No.1 and susceptible cultivar Nannong1138-2,9227,7357,and 11014 differentially expressed genes(DEGs)were identified in resistant cultivar compared with control 0 hpi,and meanwhile 9995,6938,and 12512 DEGs were identified in susceptible cultivar at 6 hpi,48 hpi and 5 dpi.Enrichment analysis based on DEGs found that after SC18 inoculation,biosynthesis of secondary metabolites,plants-pathogen interaction,plant hormone signal transduction,isoflavones metabolic,phenylpropanoid biosynthesis and MAPK signal transduction in resistant cultivar and susceptible cultivar significantly enriched,these pathways were involved soybeans to SMV defense.In addition,we also found that the four genes encoding EDS1(enhanced disease susceptibility1)Glyma.06G187300,Glyma.06G187200,Glyma.06G187400,and Glyma.04G177700 in the plant-pathogen interaction pathway were down-regulated in susceptible cultivar and no differential expression in resistant cultivar at 6 hpi.EDS1 is indispensable in the SA defense pathway.This result indicates that the early related defense pathways of susceptible cultivar are inhibited.The analysis of plant hormone signal transduction pathway showed that compared with 0 hpi,ethylene pathway related genes of susceptible cultivars were down-regulated at48 hpi or 5 dpi,while all ethylene pathway genes of resistant cultivars were up-regulated.In the jasmonic acid pathway,compared with 0 hpi,JAZ and MYC2 were inhibited in the susceptible cultivar at 48 hpi,while the resistant cultivar JAZ was inhibited,but some genes related to MYC2 were up-regulated.At 5 dpi,JAZ was still inhibited,and some genes related to MYC2 were up-regulated in susceptible cultivar,while the genes related to JAZ and MYC2 were up-regulated in resistant cultivar.NPR1 plays an important role in SA-responsive ISR and is a major regulator of plant SAR.Compared with 0 hpi,NPR1 was down-regulated in susceptible cultivar at 48 hpi and up-regulated at 5 dpi,while up-regulated in resistant cultivar all the time.These results reflected the different defenses of susceptible cultivar Nannong 1138-2 and resistant cultivar Kefeng No.1 against SC18 infection and the differences in transcription level.The resistant cultivar had a faster defense against SMV infection.2.Screening and qRT-PCR verification of disease resistance related genesSeven candidate genes that may be related to disease resistance were screened by Venn diagram,gene expression and gene function analysis.These genes were verified by q RT-PCR.The expression trend of q RT-PCR and RNA-Seq for 70% of the genes was no difference,and the correlation coefficients between them reached were more than 0.9,showing significant correlation.These results indicate that RNA-Seq was reliable.The expression level of Glyma.12G183400 was first decreased and then increased in susceptible cultivar,and first increased and then decreased in resistant cultivar.Compared with control0 hpi,the expression level of Glyma.12G183400 was up-regulated at all time points in resistant cultivar and down-regulated at all time points in susceptible cultivar.Therefore,Glyma.12G183400 was selected as the target gene in this study.3.Using VIGS to verify the function of Glyma.12G183400The coding sequences(CDS)of Glyma.12G183400 was 1473 bp in length,encoding491 amino acids,and CDS in Nannong 1138-2 and Kefeng No.1 were cloned separately.By Blast sequence alignment analysis,it was found that the CDS sequences of Nannong1138-2 and Kefeng No.1 had one base causing the difference in amino acid sequences,which were translated into arginine and lysine in Nannong 1138-2 and Kefeng No.1,respectively.After silencing Glyma.12G183400,the silencing efficiency was 76% in the susceptible cultivar and 60% in the resistant cultivar.15 days after inoculation of soybean mosaic virus isolate SC18 in BPMV-infected leaves,q RT-PCR was performed to detect virus accumulation in plants of susceptible and resistant cultivars,and the results showed that the virus accumulation in susceptible cultivar was 1.9 times higher than empty-vector plants,and the difference was significant.However,the difference in virus accumulation was not significant in silent and empty-vector plants of Kefeng No.1,The results of DAS-ELISA is consistent with that of q RT-PCR.These results suggest that silencing of Glyma.12G183400 increased virus accumulation in susceptible cultivar and Glyma.12G183400 plays an important role in soybean defense against SMV.