| Soybean mosaic virus（SMV）disease seriously harms the yield and quality of soybean[Glycine max（L.）Merr]worldwide.Due to pathogenic differentiation,SMV was currently divided into 22 strains（SC1-SC22）in China,of which SC8 was an endemic strain in soybean production areas in southern China,causing a serious threat to soybean production in China.Cultivation of soybean disease-resistant varieties is the most economical,environmentally friendly and effective method for the prevention and treatment of SMV.The study of cloning of disease resistance genes and disease resistance mechanisms can provide a theoretical basis for soybean disease resistance breeding.It is importantly significant for soybean SMV disease resistance breeding.The contents of this study are:（1）Through the RILs population of the cross combination of Kefeng No.1 and Nannong 1138-2,3863 SNPLDB markers were used to locate Rsc8 to determine the Rsc8 resistance fragment,and then select candidate genes in the resistance fragment for SMV induced expression analysis between diseased and susceptible varieties to determine the target candidate genes to be identified in this study;（2）Cloning resistance candidate genes,and then SMV-induced expression analysis between resistant and susceptible varieties,subcellular localization,and tissue-specific expression analysis among resistant variety were performed;（3）Virus induced gene silencing of Rsc8 candidate genes on Kefeng No.l and detection of SMV accumulation to verify their function;（4）Overexpression of the Rsc8 candidate genes on Nannong 1138-2 to verify their function.The main results are as follows:1.Identification of soybean mosaic virus SC8 disease resistance fragment and candidate genesBased on the phenotype and genotype datas of 419 families in the RILs combining Kefeng No.1 and Nannong 1138-2,the disease resistance fragment of SC8 was located on chromosome 2 between markers Gm0213405027 and Gm0212352061,and the genetic distance from the two markers was 5.8 cM and 1.0 cM,respectively.17 genes were selected in the disease resistance fragment,namely Gm.02g121300,Gm.02g121400,Gm.02g121500,Gm.02g121600,Gm.02g121700,Gm.02g121800,Gm.02g121900,Gm.02g122000,Gm.02g122100,Gm.02g122200,Gm.02g122300,Gm.02g122400,Gm.02g122500,Gm.02g122900,Gm.02g123700,Gm.02g124300,Gm.02g127800,then these genes were analyzed for SMV induced expression between resistant and susceptible varieties.After inoculation with SMV,the expression levels of the two genes Gm.02g121500 and Gm.02g121600 were significantly different between resistant and susceptible varieties at 2h.Kefeng No.1 was significantly higher than Nannong 1138-2,indicating that the expression of candidate genes of the disease-resistant variety Kefeng No.1 can quickly respond to SMV infection,while the other genes had little difference between resistant and susceptible varieties.Therefore,the two genes Gm.02g121500 and Gm.02g121600 of soybean mosaic virus strain SC8 resistance interval were finally selected as the target genes of this study to identify whether they were involved in the resistance of soybean to SMV.2.Cloning and expression analysis of soybean Rsc8 candidate genescDNA fragments of the complete ORFs of the genes Gm.02g121500 and Gm.02g121600 in Kefeng No.l and Nannong 1138-2 were cloned.The CDS of Gm.02g121500 was 735bp in length,encoding 244 amino acids,with an isoelectric point of 6.68 and a relative molecular mass of 28.2 kDa;The CDS of Gm.02g121600 was 732 bp in length,encoding 243 amino acids,with an isoelectric point of 8.65 and a relative molecular mass of 28.2 kDa.By sequence comparison,it was found that there was a SNP difference in Gm.02g121500 CDS sequences between the Kefeng No.1and Nannong 1138-2,and there was no difference in amino acid sequence.The Gm.02g121600 CDS sequences between the Kefeng No.1 and Nannong 1138-2 were identical.Through amino acid sequence analysis,the amino acid sequences encoded by Gm.02g121500 and Gm.02g121600 contained typical domains of MADS-box gengs.Phylogenetic analysis showed that Gm.02g121500 belonged to the SEP subfamily,Gm.02g121600 gene belonged to the AP1 subfamily,SEP and AP1 are subfamilies related to the ABCDE model,and tissue-specific expression analysis found that Gm.02g121500 and Gm.02g121600 were highest expressed in flowers,followed by bean pods,which were significantly higher than other tissues,indicating that Gm.02g121500,Gm.02g121600 may be involved in soybean growth process,especially development of flower organ.The subcellular localization results showed that Gm.02g121500 and Gm.02g121600 were expressed on the nucleus.3.Virus induced gene silencing of Rsc8 candidate genesAfter verifying that the VIGS system is available,the infection of BPMV was detected successfully after constructing the VIGS recombinant vector by inoculation on the first fully expanded primary leaves.Gm.02g121500 in Kefeng No.1 was silenced by about 75%,and Gm.02g121600 was silenced by about 80%.SMV（SC8）was inoculated on the diseased leaves containing BPMV.The qRT-PCR results showed a small increase in 21 days compared to 14 days,but SC8 accumulation did not increase significantly compared to empty vector;DAS-ELIS A results showed that,they were inoculated with SMV to detect the accumulation of SMV in the upper leaves after that VIGS-Gm.02g121500 and VIGS-Gm.02g121600 plants were silenced the corresponding genes,the test result of Kefeng No.1 was negative.The results of qRT-PCR detection of SMV accumulation were consistent,which was basically consistent with the results of DAS-ELIS A.Therefore,the results do not well prove that the target genes were involved in SMV disease resistance.This may be due to the low expression level of the target genes,which were not completely silent.Whether these two MADS-box transcription factors are involved in SMV resistance needs further verification.4.Overexpression of Rsc8 candidate genesGm.02g121500 in Nannong 1138-2 was overexpressed about 8 times.Later,SMV（SC3,SC7,SC8）was inoculated on BPMV diseased leaves,separately.The qRT-PCR results showed that,OE-Gm.02g121500 plants in Nannong 1138-2 had accumulated SMV after 14 and 21 days,but lower than empty vector.The accumulation at 21 days was increased compared to 14 days.The accumulation of SMV at 14 days was significantly lower than that of the empty vector,and the difference between the accumulation of SMV at 21 days and the empty vector was not significant,especially SC8.The DAS-ELISA results were consistent with the qRT-PCR detection results.This result did not prove that Gm.02g121500 was involved in SMV disease resistance.Gm.02g121600 was overexpressed about 200 times,and OE-Gm.02g121600 plants were inoculated with SC3,SC7,and SC8,respectively.After 14 days and 21 days,there were almost no accumulation in SC3 and SC7.Although SC8 had a small amount of accumulation,the accumulation in 21 days increased from 14 days,but its content was much less than empty vector and could be ignored.In addition,DAS-ELISA results showed that after inoculation with SC3 and SC7,the SMV test was negative and SC8 was positive,but the content was lower than the control,which was consistent with the qRT-PCR test results.This result indicated that the candidate gene Gm.02g121600 as a transcription factor may positively regulate SMV disease resistance,and not for specific strains,it is likely that this gene is involved in SMV broad-spectrum resistance. |
