| Vegetable soybean(Glycine max(L.)Merr.)belong to the bean,also called edamame,it is an important legume vegetable.In its growth and development process,it is often affected by a variety of pests and diseases.Soybean Mosaic virus(Soybean Mosaic virus,SMV)disease is the most widely distributed in many diseases,one of the biggest disease impact on yield and quality,its hazard area wide,destructive,seriously affect the food with Soybean yield and quality.SMV classified as the genus potyvirus in the family potyviridae with narrow hosts,limited primarily to the leguminosea.Potyvirus is the largest plant viruse in world and responsible for severe economic losses to agriculture.The 11 proteins of Potyvirus encoded interaction with other encoded proteins and host proteins to complete the infection cycle of the virus.One of P3 protein is Potyvirues three membrane proteins,and Potyvirues polymer protein variability in the biggest,so the P3 protein and its host interaction relationship is more conducive to our research membrane-associated proteins in the function and mechanism of viral replication,thus further analysis Potyvirues replication mechanism and provide theoretical guidance for prevention and cure of diseases.The lab early isolates using SMV6067-1 sequence of P3 build bait carrier,by yeast two hybrid(Y2H)from the initial screening with ’nannong 1138-2’ cDNA library,36 host proteins were screened for interaction with soybean Mosaic virus P3 protein.In this study,two of the most potent host proteins were selected,endo-1,3-beta-glucanase and cysteine proteinase inhibitor respectively named GmGLu and GmCYS,and the length of the coding region of the two genes was cloned from the soybean sensitive variety ·nannong 1138-2’.The main results are as follows:(1)Membrane protein yeast two hybrid system was performed according to the manufacturer’s protocols this study,SMV P3 and full-length coding regions of two soybean protein(GmGLu.GmCYS)respectively constructed into the yeast expression vector pBT3-STE and pPR3-N,Co-transformation of yeast results show that the SMV P3 respectively with two candidates for soybean protein in SD/-Ade/-His/-Leu-Trp/X-α-Gal medium grew well,and blue colonies were considered as positive clones.The results show that the interaction of SMV P3 with the two candidate soybean proteins.(2)Bimolecular fluorescence complementation were performed to confirmed the interaction of the above in vivo.SMV P3 and two soybean protein(GmGLu,GmCYS)were inserted into the BiFC pEarleyGate201-YC and pEarleyGate202-YN.The fusion expression vectors were introduced into onion epiderma cells with transient expression by Agrobacterium-mediated method.The agro-infiltrated leaves were observed under a spectral confocal laser scanning microscope.The results show that the strong interactions SMV P3 with two soybean protein,emerging steady fluorescent.Interaction of P3 with soybean GmGLu occurred in the cytomembrane of Nicotiana benthamiana cells.Interaction of P3 with soybean GmCYS occurred in both the cytomembrane and cell nucleus.Subcellular localization results were similar to BiFC results,GmGLu protein was located in the cell membrane,GmCYS protein was located in the cell membrane and nucleus.The above results show that the interactions SMV P3 with two candidate soybean proteins.(3)The study analyzed genes expression of soy’bean genes using RT-PCR after soybean mosaic virus(SMV)treatment at a different time.Results showed that under the infection of SMV,the expression of soybean protein in two vegetables was up-regulated in 24hpi,48hpi,72hpi and 14dpi.GmGLu expression level rising after 24h treatment,showed the highest expression level after 72h treatment,There was a significant difference at 72h.GmCYS expression level showed the highest in 24h treatment,expression level had a downward after 48h treatment.The results indicate that the direct interactions SMV P3 with two soybean proteins(GmGLu、GmCYS),and SMV P3 might have a role in virus infection within cells. |
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