Subcellular Localization Of The Proteins Encoded By Rice Stripe Virus(RSV) In Spodoptera Frugiperda(Sf9)

Rice stripe virus (RSV), belonging to the genus Tenuivirus, is one of the most economically important pathogens of rice and causes severe diseases in rice fields, especially in China. And it has caused great loss to agricultural production. RSV is an multipartite RNA virus with the ambisense coded strategy, its gnome consists of four kinds of single-stranded RNA (ssRNA), RNA1, RNA2, RNA3 and RNA4. RSV encodes seven proteins, including RNA-dependent-RNA polymerase(RdRp), NS2, NSvc2, CP, NS3, SP and NSvc4.The total RNA was extracted from RSV infected rice. The strep tag gene and NS2, NSvc2-N, CP, SP, NSvc4 genes were obtained as a fusion gene with RT-PCR (reverse transcription-polymerase chain reaction) approach, then the fusion gene was inserted into pMD-18T vector at BamHⅠ/KpnⅠsites to produce pMD-NS2, pMD-NSvc2-N, pMD-CP, pMD-SP and pMD-NSvc4. After digestion with BamHⅠ/KpnⅠand sequencing, the recombinant plasmid pFB-NS2, pFB-NSvc2-N, pFB-CP, pFB-SP, pFB-NSvc4 were construdted by digesting the plasmid of pMD-NS2, pMD-NSvc2-N, pMD-CP, pMD-SP, pMD-NSvc4 and the transposing vector pFastBac1 in BamHⅠ/KpnⅠsites. Each recombinant plasmid was sequenced to ensure that the target gene is correctly inserted into the pFastBac1 vector and in right open reading frame. The recombinant plasmid was amplified in E. coli DH5α, and then re-transformed into E. coli DH10Bac compent cells to obtain the recombinant bacmid DNA, including rbpFB-NS2, rbpFB-NSvc2-N, rbpFB-CP, rbpFB-SP and rbpFB-NSvc4. The recombinant bacmid DNA were isolated and transfected into Sf9 cells, 72 hour after, collected the Sf9 cells. The expression of the target gene in Sf9 cells was confirmed by western blotting, and the subcellular localization of target proteins were observed by Immune fluorescence experiment. The green fluorescence from Sf9 cells infected by rbpFB-NS2 centralized in cell membrane. The green fluorescence from Sf9 cells infected by rbpFB-NSvc2-N centralized in cytoplasm in the form of vesicles. The green fluorescence from Sf9 cells infected by rbpFB-SP centralized in the whole cytoplasm. The green fluorescence from Sf9 cells infected by rbpFB-NSvc4 mainly centralized in and surrounded cell in the form of filamentous. The green fluorescence from Sf9 cells infected by rbpFB-CP centralized in cytoplasm in the form of irregular pointlike structure.Cell is the basic unit of life, and protein reflect the life activities directly. The cells were divided into different partitions, and the nature protein play its normal biological function only in the specific parts of cell, so the study of subcellular localization to protein is very necessary. The information about subcellular localization of protein can suggests its biological function. The subcellular localization of protein not only help us to infer the function, but also provide necessary information to other aspects of protein research, such as protein interactions.