Different Chimeric-gene Hairpin Structures Affect Virus Resistance Mediated By RNA Silencing In Transgenic Tobacco And Production Of Transgenic Rice Against RBSDV And RSV

RNA silencing is an immune system widely existing in the organism, which could protect the integrity of the biological genome by preventing the exogenous nucleic acid invasive foreign gene, transposable element and virus such. RNA-mediated virus resistance (RMVR) is often considered as an important manifestation of RNA silencing. The expression of virus-specific dsRNA has complete or partial homology to the gene of invasive virus. During the RNA degradation process which is induced by post-transcriptional gene silencing, the RNA silencing system recognizes complementary invasive viral RNAs and triggers the degradation of RNA from both transgene and the invasive virus. RMVR has been widely used in antiviral research for its advantages, such as a high degree of resistance (almost immune); sustaining resistance; biological safety and so on. In the process of RNA silencing, effectiveness of siRNA could be affected by the structure of the target construction. In this study, coat protein genes (CP) of Potato virus Y (PVY), Cucumber Mosaic virus (CMV), Rice black-streaked dwarf virus(RBSDV) and Rice stripe virus (RSV), suppressor genes of PVY and CMV (PVY HC-pro and CMV2b) were served as the target sequences. We designed different chimeric-gene hairpin RNA (hpRNA) constructs based on the target sequences and generated transgenic plants. We aim at systematically disserting if different chimeric-gene hpRNA constructs could affect RMVR, and producing new breed of rice which could be resistant to both RBSDV and RSV. The main results and conclusions presented in this study are as follows:1. Different target genes and chimeric-gene hairpin structures affect virus resistance mediated by RNA silencing in transgenic tobaccoThe target sequences were amplified by PCR using the corresponding primer pairs. The amplified fragments were cloned into the pUC19, pBSK, and pEASY-T cloning vectors. The reverse chimeric gene structures were constructed using these cloning vectors and were then inserted into the plant expression vector, pROKⅡ. Plant expression vectors harbouring a single-hairpin of the dual-virus chimeric-gene or a double-hairpin of the dual-virus genes pDCPSH, pHC2bSH, pDCPDH and pHC2bDH were obtained, and the vectors were introduced into tobacco NC89. Through kanamycin selection and PCR detection,106,111,104and108plants from pDCPSH, pDCPDH, pHC2bSH and pHC2bDH were obtained, respectively. The transformed plants grew normally, and there was no significant difference between the wild type and transgenic plants.The expression level of the target genes was measured by qRT-PCR. The qRT-PCR result showed that there was no significant difference in target gene expression between transgenic plants which were transformed with single-hairpin structure or double-hairpin structure vectors using the same target genes. A northern blot was performed to examine the accumulation of siRNA in the transgenic plants before inoculation. We found that the accumulation of siRNA in transgenic plants transformed with the vector of double-hairpin structure vector was significantly greater than in those transformed with the single-hairpin structure using the same target genes.We manually inoculated the To generation transgenic tobacco plants and non-transgenic plants (wild type NC89) with a CMV and PVYN mixture. After three weeks, both resistant and susceptible plants were observed in the transgenic plants. The percentage of resistant plants in the pDCPSH, pDCPDH, pHC2bSH and pHC2bDH transgenic plants were47.08%,63.25%,25.78%and37.98%, respectively. These results showed that the expression vector pDCPDH provided the highest resistance efficiency in the transgenic plants, which is three times more than that of the expression vector pHC2bSH, suggesting that the resistance ratio is affected by differences and target genes in the structures.Northern blot analysis revealed that the trangene have been expressed in level of RNA, and the accumulation level of resistant plants was lower than that of the susceptible ones. There was an inverse correlation between the resistance and the amount of RNA accumulation in the transgenic plants. The results proved this resistance was RNA-mediated. siRNAs were present in all selected resistant and susceptible transgenic plants; there was no obvious correlation between the expression level of sequence-specific siRNAs and virus resistance. The result of southern blot demonstrated that the exogenous genes were integrated into the tobacco genome. Approximately fifty transgenic tobacco plants of T1generation containing each vector were grown in the green house and were inoculated with the CMV and PVYN mixture. After three weeks, there were no symptoms of viral diseases on most of the plants, and the percentage of resistance plants reached95%or greater. This result indicated that the resistance was stably inherited by the T1generation.2. Cultivation of viral-resistant rice against both Rice Black-Streaked Dwarf Virus and Rice Stripe VirusRice stripe virus (RSV) and rice black streaked dwarf virus (RBSDV) are two serious viral diseases in rice production. Cultivating the viral resistant rice variety against both RBSDV and RSV is an effective means of reducing production losses caused by viral disease. We constructed the transient expression vectors p1300-RBSDV3’, p1300-RBSDV-M, p1300-RBSDV5’, p1300-RSV3’, p1300-RSV-M and p1300-RSV5’, which were respectively targeted the3’end, middle, and5’ end of RBSDV and RSV. The6transient expression vectors were transferred into N. benthamiana. In transient assay, Northern blot analysis revealed siRNAs were detected in all infiltrated leaves, and the accumulation of viral mRNA was significantly reduced by the middle sequences. Therefore,the intermediate sequences of RBSDV and RSV CP gene were chosen as target sequences. With the help of cloning vectors pUC19and pBSK, the plant expression vectors, pl300SH (single hairpin of chimeric-gene) and p1300DH(double hairpin of chimeric-gene), were constructed.Transgenic lines of rice CV. Lindao10were generated through Agrobacterium-mediated transformation. After herbicide-PPT selection and PCR detection, we got To transgenic plants transformed p1300SH and p1300DH,45and30, respectively.The self-fertilized seeds from To generation transgenic plants of transformed p1300SH and p1300DH were planted to obtain T1generation lines. In every transgenic line,50PPT-resistant plants were selected and inoculated with both RBSDV and RSV using viruliferous vector insects. Wild types (cv. Lindao10) were used as control. The results of virue resistance showed that eight lines of p1300SH and eight lines of p1300DH exhibited resistant phenotype, which had a susceptible score below6%, far lower than the susceptible ratio of wild types. The susceptible ratios of other lines of the two vectors were all above12%, exhibiting moderately resistant or susceptible phenotype. In these resistance assays, the ratios of resistant transgenic lines were17.78%and26.67%in p1300SH and p1300DH, respectively. The result verifies that the ratio of Ti transgenic resistant lines containing double-hairpin is higher than that contained the single-hairpin.Foreign genes had been integrated into the rice genome by Southern blot. Northern blot analysis revealed that the trangene have been expressed in level of RNA, and the accumulation level of resistant plants was lower than that of the susceptible ones. There was an inverse correlation between the resistance and the amount of RNA accumulation in the transgenic plants. The results proved this resistance was RNA-mediated. siRNAs were present in all selected resistant and susceptible transgenic plants. No obvious correlation was observed between the expression level of sequence-specific siRNAs and virus resistance.We selected T2self-fertilized progeny of resistant transgenic plants which were proved to have one or two copy from lines SH1, SH15, SH27, DH12, DH14, DH19and DH23to study the segregation pattern and stable inheritance. The results showed that transgene and RNA-mediated virus resistance can be stably inherited until T2, and two homozygous lines, DH14and DH19, were obtained.