Construction Of Recombinant Plant Expression Plasmids Containing SRBSDV 12 Genes And Phenotype Analysis Of Transgenic Rice

Southern rice black-streaked dwarf virus(SRBSDV) is a novel and tentative species belonging to Fijivirus, Reoviradae. The rice infected by SRBSDV shows dwarfism, dark green leaves, folded near the leaf base, anatropous fibril in the internodes and tumors in the stem with high tillering position and browning roots. Previous researches found SRBSDV genome contained 10 double strand RNA, which were respectively named S1~S10 according to the length of the gene. S5, S7 and S9 respectively encode two proteins, and others only encode one protein, respectively. The function of the genes were still remained unknown, especially the interaction between the virus protein and the rice host still needs further exploration. In order to classify the function and pathogenesis of genes of SRBSDV, this study constructed 12 plant expression plasmids of SRBSDV genes, respectively, via agrobacterium-mediated method to obtain the constitutive expression of SRBSDV Rd RP and CP of rice. Phenotype observation were conducted in T1 generation transgenic rice and the relative expression of CP gene in rice were dected by real-time fluorescent quantitative PCR. The results of this study are following:1. Constructed recombinant plant expression plasmids of 12 SRBSDV genes, and obtained the engineering agrobacterium. The 12 encoded genes of the virus, S1, S3, S4, S5-1, S5-2, S6, S7-1, S7-2, S8, S9-1, S9-2 and S10, were amplified by RT-PCR, the total RNA of SRBSDV infected rice leaves as template. The segments were connected to the plant expression plasmids p Cambia1302 after sequencing. The recombinant plasmids were respectively named p C1302-P1, p C1302-P3, p C1302-P4, p C1302-P51, p C1302-P52, p C1302-P6, p C1302-P71, p C1302-S72, p C1302-P8, p C1302-P91, p C1302-P92 and p C1302-P10. Electric shock method was used to transfer the recombinant plasmids mentioned above into agrobacterium EHA105.2. Obtained 4 strains of Rd RP transgenic rice plants and 6 strains of CP transgenic rice plants. Used the japonica variety “Nipponbare” embryonic callus as vectors, the agrobacterium carried the p C1302-P1 and p C1302-P10 was conducted to infect the vectors material. Then the callus were screened twice in the medium containing hygromycin. After predifferentiation, differentiation, rooting and seedling step-drilling, we obtained 4 strains of Rd RP transgenic rice plants and 6 strains of CP transgenic rice plants. And the exogenous gene CP was detected by Southern blot, confirming the strains single copy transformant plants.3. Phenotype analysis of T1 generation Rd RP transgenic rice plants. There is no obvious difference between the transgenic plants and the non-transgenic plants in the seedling stage. During the tillering stage, some transgenic plants showed albinistic. The lobus cardiacus of the plants were normal, but other leaves turned white in different degrees, and the plants became diminished.4. Molecular detection and phenotype analysis of T1 generation CP transgenic rice plants. T1 generation plants were detected by PCR, showing that the exogenous gene followed the law of segregation. There is no obvious difference between the transgenic plants and the non-transgenic plants in the seedling stage. During the tillering stage, some transgenic plants became significantly diminished, dwarfism and tillers decreased.5. Established RT-q PCR detection method of Relative expression of CP of the virus. RT-q PCR analyzed the CP relative expression in the seedling stage, tillering stage and heading stage of each strain. Results indicated that CP could be transcribed in the three stages.These results have provided a basic to further exploring the pathogenesis of Rd RP and CP of SRBSDV during the infection in rice.