| Rice stripe virus(RSV)causes rice stripe disease and occurs in rice growing areas in Jiangsu,Zhejiang,and Yunnan provinces,resulting in 40-50% reduction in rice yield.RSV causes stripe symptoms in rice leaves,with severed dead heart and increasing tillering.RSV is a representative member of the genus Tenivivirus.It is a negative sense RNA virus and its genome is composed of four single-stranded RNAs.It encodes 7 non-overlapping reading frames for the virus by ambisense coding strategy.RSV RNA3 encodes p3 of a 23.9 kDa protein that is a silencing suppressor and plays an important role in viral infection.The current p3 variation and its role in plants is a research hotspot.In order to deeply discuss the variation of RSV p3 gene and obtain its antiserum,the p3 gene of RSV isolate from Sichuan Province was cloned by RT-PCR,and its evolutionary tree was constructed by MEGA 5.0.The phylogenetic tree was analyzed and cloned.On the prokaryotic expression vector pET-32 a,the recombinant plasmid pET-32a/p3 was transformed into E.coli BL21 pLysS,and after induction by IPTG,the protein was purified and the rabbit was immunized to obtain its polyclonal antibody,the polyclonal antibody of the protein has a potency of 1:2000.In this study,the diseased leaf that showed the symptoms of fringe,yellowing,and curling was collected from the rice planting area in Huidong County,Sichuan Province.The monoclonal antibody against RSV CP was used for dot-ELISA.The results showed that 9 out of 13 samples were positive.Positive samples were extracted total RNA and amplified by RT-PCR using RSV p3 gene-specific primers to obtain 636 nt fragments.Phylogenetic analysis showed that the nucleotide similarity between Sichuan isolate Huid04(KX611339)and Yunnan isolates KunM08(JQ927423)and DaL08(JQ927420)was the highest,which was 96.86% and 97.01%,respectively,and had a close genetic relationship.Antiserum was obtained after immunization of rabbits and was confirmed by Western blot of RSV p3.In order to clarify the role of p3 gene in plants,previous studies found that transgenic p3 rice has a certain resistance to Magnaporthe oryzae and Xanthomonas oryzae pv.There is less viral accumulation in the N.benthamiana and there are almost no symptoms of the virus.In order to investigate the function of p3 gene in N.benthamiana in vivo,we used Trizol method to extract the total RNA of N.benthamiana and perform transcriptome sequencing.Results showed that there were a total of 2533 differentially expressed genes in the p3 gene after N.benthamiana expression(screening conditions,using solanaceous as the reference genome with p value ? 0.05 and a multiple of difference |log2Foldchange| ? 1),of which 597 genes were up-regulated,with 1936 The genes are down regulated.These differential genes were polymerized in 43 GO functional groups and 59 KEGG pathways,the main pathways were ribosome(ko03010),photosynthesis(ko00195),photosynthesis antenna protein(ko00196)and carbon metabolism(ko01200),the each pathway has more than 80 differentially expressed genes aggregated in each pathway.Twelve differentially expressed genes were arbitrarily selected in the transcriptome sequencing results to perform RT-qPCR validation of the transcriptome results.The validation results were consistent with the results of the transcriptional sequencing.After GO and KEGG analysis of the transcriptome,among the four key pathways and differentially expressed genes with differences greater than 2,10 selected genes were obtained after screening,which releated to nuclear genes,resistance genes,photosynthesis pathways and energy metabolism pathways.In order to clarify the function of candidate differentially expressed genes in the p3 transcriptome of N.benthamiana,we used the tobacco rattle virus(TRV)mediated gene silencing(Virus-induced gene silencing,VIGS)and Potato X virus(Potato Virus X,PVX)-mediated gene overexpression techniques to verify the effect of genes on symptom formation.It was found that TRV-mediated silencing of histone-related genes(3.NbHis-2,4.NbHIS-3,5.NbHis-5)caused leaf shrinkage and malformation or chlorosis,whereas the genes of the same type had varying degrees of shrinkage or faded green.Whether overexpressing or silencing genes associated with the heat shock transcription factor(2.NbHSFP)indicates that the genes is associated with the formation of chlorotic or green mottled symptoms.After TRV-mediated silencing of energy metabolism-related genes(1.NbATP-2 and 13.NbOLA-2),there were different degrees of new leaf curls,deformities,and yellow veins,respectively.TRV-mediated silencing of the resistance gene(8.NbLRR-2)causes severe curls and deformities in new leaf.Components of the photosynthesis system I(19.NbPSB-2 and 21.NbPSB-4)were not characterized by silencing of this gene in this study.In this study,the gene related to rhodanese(23.NbRLD-2)was down-regulated at the transcriptome level.Overexpression of the gene showed green mottle.In summary,this study performed evolutionary analysis and antibody preparation of RSV Sichuan isolate p3 by using phylogenetic analysis,antibody preparation techniques,transcriptome sequencing technology,bioinformatics analysis,and RT-PCR techniques.According to the transcriptome,p3 led to the difference of endogenous genes in N.benthamiana,10 genes were selected in the major functional classes of differentially expressed genes,then the functional and phenotypic analysis of these genes were shown by mediated vector of TRV and PVX.These provide a basis for an in-depth understanding of the relationship between p3 and host interactions,virus disease,and plant anti-virus.To sum up,given some rice with rice streak disease in sichuan province,and found that RSV was harmful to rice in the east county of Sichuan province.The phylogenetic analysis of RSV Sichuan isolate p3 was found to be close to that of Yunnan.The prokaryotic expression of RSV Sichuan isolated p3 was prepared,and the polyclonal antibody of p3 protein was prepared.The transcriptome analysis of p3 transgenic N.benthamiana was carried out,and the expression of p3 gene was found to affect the gene expression of endogenous gene.The 10 differentially expressed genes were analyzed in silence and overexpression,and these genes were found to affect the normal growth of N.benthamiana. |
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