Identification And Function Analysis Of MP Of Southern Rice Black-streaked Dwarf Virus

Rice black-streaked dwarf disease induced by Southern rice black-streaked dwarf virus(SRBSDV)caused increasingly serious damage in the rice and maize planting areas in south of China.The complete genomes of SRBSDVs were sequenced,the biology and molecular biology of SRBSDV were studied,and the genome structure,some virus-encoded proteins were made clear at present.While the functions of most virus-encoded proteins and pathogenic mechanism of the virus are still unclear,the molecular mechanism of MP which assists SRBSDV transport of cell to cell in plant is barely understood.Therefore,four genes of SRBSDV Anhui isolate which encoding the unstructured protein were cloned in this topic,the potential movement protein was made clear by molecular identification,and the mechanism of MP was further researched.1.Identification of SRBSDV MPFour genes of SRBSDV S5-2,S7-1,S7-2,S9-2 were cloned and constructed into pCAM2300 to obtain pCAM-S5-2-GFP,pCAM-S7-1-GFP,pCAM-S7-2-GFP and pCAM-S9-2-GFP or pBin438 to obtain pBin-S5-2,pBin-S7-1,pBin-S7-2 and pBin-S9-2,respectively.(1)Localizations of MP in N.Benthamiana leaf epidermal cells The fluorescences distributed equably in the plasmodesma before plasmolysing when infiltrated with pCAM-S5-2-GFP,pCAM-S7-1-GFP,pCAM-S7-2-GFP or pCAM-S9-2-GFP;The fluorescence produced by pCAM-S7-1-GFP could localize in the plasmodesma with distribution of discrete dot after plasmolysing,while pCAM-S5-2-GFP,pCAM-S7-2-GFP and pCAM-S9-2-GFP can not.(2)Self-movement of MP in cells The dilutions of A.tumefaciens containing pCAM-S5-2-GFP,pCAM-S7-1-GFP,pCAM-S7-2-GFP or pCAM-S9-2-GFP were inoculated on N.Benthamiana,respectively.pCAM-S7-1-GFP could move automatically in cells of N.Benthamiana,while pCAM-S5-2-GFP,pCAM-S7-2-GFP and pCAM-S9-2-GFP can not,they localized in the single cell.(3)The complementation of MP and PVX(?P25)-GFP Co-infiltrate PVX(?P25)-GFP(a PVX movement-defective vector)with pBin-S5-2,pBin-S7-1,pBin-S7-2 or pBin-S9-2,respectively.PVX(? P25)-GFP could move in cells of N.Benthamiana with the help of pBin-S7-1,while pBin-S5-2,pBin-S7-2 or pBin-S9-2can not.These works indicated S7-1 might encode movement protein of SRBSDV.2.Self-interaction of P7-1 and interaction between P7-1 and CP of SRBSDVGenes of SRBSDV S7-1 and S10 were cloned and constructed into pCV-cYFP and pCV-nYFP to obtain pCV-S7-1-cYFP,pCV-S7-1-nYFP,pCV-S10-cYFP and pCV-S10-nYFP or pGADT7 and pGBKT7 to obtain pGAD-S7-1,pGBK-S7-1,pGAD-S10 and pGBK-S10.(1)Validation of self-interaction of P7-1 and interaction between P7-1 and CP of SRBSDV by bimolecular fluorescence complementation(BIFC).Co-infiltrate pCV-S7-1-nYFP and pCV-S7-1-cYFP with pCV-S7-1-nYFP and pCV-S10-c YFP as well as pCV-S7-1-cYFP and pCV-S10-nYFP,respectively.The results show that protein encoded by S7-1 can interact with itself,appearing clear yellow fluorescence,while can not interact with CP.(2)Validation of self-interaction of P7-1 and interaction between P7-1 and CP of SRBSDV by yeast two hybrid(Y2H)Co-transform pGAD-S7-1 and pGBK-S7-1,pGBK-S7-1 and pGAD-S10,pGAD-S 7-1 as well as pGBK-S10 into yeast,respectively.The result show that,only co-transformed pGAD-S7-1 and pGBK-S7-1can grow white yeast fell,suggesting that protein encoding by SRBSDV S7-1 can interacted with itself,but can not interacted with CP.3.Prokaryotic expression and purification of SRBSDV S7-1Gene S7-1 of SRBSDV was cloned and constructed into prokaryote expression vector pET-GST.After the recombinant plasmid pET-S7-1 was transformed into Escherichia coli BL21(DE3),the recombinant protein with an approximate molecular weight of 68 kD was obtained with IPTG induction and Ni2+-NTA affinity column purification.Western blot analysis revealed that the GST monoclonal antibody could specifically bind to purified fusion protein,which indicated that the recombinant protein was acquired.The work laid a solid foundation for further research on function of S7-1 gene.