|
Rice gall dwarf disease caused by the rice gall dwarf virus (Rice gall dwarf virus, RGDV), distributed in the rice growing regions of Southeast Asia and East Asia, and the local popular, has a certain economic importance. Using the yeast two-hybrid system and bimolecular fluorescence complementary technologies for rice gall dwarf virus protein self-interactions, and its interaction with the rice protein research. First construct a P2, P3, P7, P8, PN9, Pn10 Pn11, PN12 yeast expression vector and self-activation effect detection; rice gall dwarf virus RNA as a template, was amplified by RT-PCR get RGDV P2, P3 , P7, P8, Pn9, Pn10, Pn11, Pn12 the gene sequence are respectively connected to the yeast expression vector pGAD T7 PGBK are T7 build positive recombinants the pGAD - S2 the pGAD-S3 the pGAD-S7, pGAD-S8, pGAD- S9, pGAD-S11, pGAD-S12 and PGBK-S2 the PGBK-S3 the PGBK-S7, PGBK-S8, PGBK-S9, PGBK-S10, PGBK-S11, PGBK-S12. Positive recombinants were transformed into yeast AH109 and coated on the type of the auxotrophic selection medium plate detected RGDV P2, P3, P7, P8, PN9 Pn10 Pn11, PN12 protein having a self-activating activity. The results showed that the positive recombinant plasmid does not have any self-activation, and RGDV PN10 protein in yeast cells with transcriptional activation. Secondly, the RGDV P3 protein interaction studies using the yeast two-hybrid system. The the positive recombinants the pGAD-S3 and pGBK-S3 of transformed yeast AH109 and coated on the type of auxotrophy selection medium plate detected RGDV P3 protein interaction. The results show that the, RGDV P3 protein interaction. With bimolecular fluorescence complementary technology naturally in plant cells under physiological conditions to further verify the RGDV P3 protein interaction situation. The positive yeast expression plasmid pGAD - S3 as a template amplification S3 genes, the recombinant the bimolecular fluorescence complementary expression carrier pSPYCE-HA-S3 and pSPYNE-Myc-S3. And the reorganization of the HA tag pSPYCE-HA-S3 and with c-Myc tag pSPYNE-Myc-S3 of bimolecular fluorescence expression vectors were transformed into Agrobacterium GV1301 deal with Agrobacterium after mixing injection tobacco. Tobacco leaf tissue was cut at the injection site on the slide, with confocal laser scanning microscope (Leica TCS STED, Germany) observed under yellow fluorescent expression in tobacco epidermal cells and save pictures. The bimolecular fluorescence complementary test results with the yeast two-hybrid test results are consistent: RGDV P3 protein interaction. Furthermore, complementary technologies, using the yeast two-hybrid screening cDNA library of rice to RGDV P2 protein as bait. Bait plasmid RGDV pGBK-S2 and rice cDNA library plasmids were transformed into yeast AH109 transformation products coated with different auxotrophic selection medium, positive clones were screened possible interaction with the bait protein P2 colonies. Using cDNA insert fragment size of the PCR positive clones, respectively containing different size insert yeast plasmid was transformed into E. coli DH5a find homologous sequences by sequencing and Blast. According to the homologous sequence corresponding to the annotation information, analysis of the positive clones were screened by the inserted gene fragment size, the integrity of the gene sequence and the reading frame correctness. The results show that the P2 protein RGDV as bait, we screened a rice cDNA library eventually won two kinds of positive clones rice PC1, PC3 protein. According to the notes of the corresponding homologous genes in the database, combined with literature data on the interaction of protein, initially speculated that the protein interactions that may arise in RGDV virus infection, pathogenesis and host response process role. Finally, in order to further verify of RGDV P2 protein rice PC1, PC3 protein interaction between the extract after the rice leaf total RNA was amplified by RT-PCR were obtained rice PC1, PC3 full-length ORF gene sequences, respectively connected to the yeast expression vector pGADT7, pGAD-PC1 and pGAD-PC3, build positive recombinants. The bait plasmid PGBK-S2 with recombinants the pGAD - PC1 and pGAD-PC3 co-transformed yeast the phenotypes of the AH109, ??transformation products coated in different auxotrophic selection medium, the interaction between the detection RGDV P2 protein and rice PC1 PC3 protein case. The results showed that rice PC1 and PC3 proteins could with RGDV P2 protein interaction occurs. With bimolecular fluorescence complementary technology naturally in plant cells under physiological conditions P2 protein and rice PC1 RGDV the PC3 protein interaction between further validation. Positive yeast expression plasmid PGBK-S2 the pGAD-PC1, and pGAD of-PC3 as a template amplification, S2, PC1 and PC3 gene, bimolecular fluorescence of the recombinant expression vector pSPYCE-HA-P2, pSPYNE-Myc-PC1 and pSPYNE-Myc-PC3. And bimolecular fluorescence of the three recombinant expression vectors were transformed into Agrobacterium GV1301 deal with Agrobacterium pSPYCE-HA-P2 pSPYNE-Myc-PC1 and pSPYCE-HA-P2 pSPYNE-Myc-PC3 combined injection tobacco. Tobacco leaf tissue was cut at the injection site on the slide, with confocal laser scanning microscope (Leica TCS STED, Germany) observed under yellow fluorescent expression in tobacco epidermal cells and save pictures. The results showed that rice PC3 protein can occur with RGDV P2 protein interaction in yeast two-hybrid test results; rice PC1 protein can not with RGDV P2 protein interaction occurs, is inconsistent with the yeast two-hybrid test results. In summary, this paper has constructed the RGDV P2, P3, P7, P8, Pn9, Pn10, Pn11, Pn12 the yeast two-hybrid vectors and self-activation effect detection; the P3 protein by yeast two-hybrid and bimolecular fluorescence complementary technologies RGDV own mutual for; using the yeast two-hybrid complementary technologies, RGDV P2 protein bait, we screened a cDNA library of rice, two kinds of positive clones; and by yeast two-hybrid and bimolecular fluorescence complementary technologies the verification RGDV P2 protein and rice PC1, PC3 protein The interaction between. The test results will be possible for us to further understand RGDV of protein function, the pathogenesis of RGDV, host anti RGDV susceptible mechanism to provide the basis.
|