Research Of Resistance Against Rice Stripe Virus And Rice Black Stripe Dwarf Virus Based On MiRNA And SiRNA Strategies

Rice is one of the most important food crops in China, which accounts for 30% of the total sown area of grain, and the yield is close to 44% of the total grain output. The rice quality is good and the value is high, it is the main commodity grain of our country. In addition, it can be used as raw material for fermentation industry. Rice disease is one of the main bottlenecks to limit the yield and quality of rice. Rice stripe disease and rice black streak dwarf disease are two of the most serious rice diseases, causing great damage to rice production. When the disease is serious, the diseased plant dies, and eventually leads to a significant reduction in rice yield. Even plants survived to maturity, also showed growth severely hampered. These two viruses are all transmitted by Laodelphax striatellus. Under field conditions, RSV and RBSDV always infect plants simultaneously, and a synergistic effect of viruses may cause more severe symptoms In recent years, rice stripe disease and rice black streak dwarf disease is popular worldwide, causing serious damage to rice production.Sequence-specific gene silencing is a key mechanism for the regulation of endogenous gene expression and defense against exogenous genomic invaders in plants. It is mediated by exogenous or endogenous double-stranded RNAs(dsRNAs), which are recognized by the endonuclease Dicer and digested into 21-25 nt short double-stranded RNAs. Small interfering RNAs(siRNAs) and microRNAs(miRNAs) are the two principal types of small RNAs(sRNAs) involved in RNA silencing. RNAi technique is a common strategy to cultivate anti-viral transgenic plant. Great progress has been made in the use of artificial microRNA mediated virus resistance strategy in the cultivation of transgenic plants, and it is considered as a kind of strategy with great application value. Producing both RSV and RBSDV resistant transgenic plants through dimeric amiRNA approach precursor that encode efficient amiRNAs in rice plants have not been reported.At present, the control of plant virus disease by RNA silencing has been paid attention and widely used. With the deeping of research, the use of RNA silencing to prevent and control plant virus disease is not only limited to the cultivation of highly resistant transgenic plants. The research that dsRNA synthesized in vitro and fed to the insect vector, lead to the loss of the virus transmission ability of the insect vector has been carried out. This method is mainly acting for blocking the transmission of plant viruses by insects.In this study, Rice stripe virus and Ricee black-streaked dwarf virus as experimental materials, the transgenic plants against RSV and RBSDV infection simultaneously were obtained through dimeric artificial microRNA precursor strategy. It is a great significance for improving the yield and quality of rice, protecting the sustainable development of rice production. In addition, Laodelphax striatellus and RSV as experimental materials, the functions of the proteins which may be involved in the transmission of insect vector were verified by RNA silencing technique. This provides an important basis for using the RNA silencing technology to control the risk of virus disease in the future. The main results and conclusions presented in this thesis are as follows:(1) Generation of transgenic rice that simultaneously resistant to RSV and RBSDVThree amiRNAs targeting the middle segment, 3′ end and 3′-UTR region of the RSV CP gene were selected using the miRNA Target Finder program WMD and were named amiR-RSVM, amiR-RSV3 and amiR-RSVU. Similarly, amiR-RBSDVM, amiR-RBSDV3 and amiR-RBSDVU targeting the RBSDV CP gene were screened. The natural miR528/miR528* sequences were replaced by viral amiRNA/amiRNA* sequences using oligonucleotide-directed mutagenesis, and six pre-amiRNAs were obtained. The pre-amiRNAs fragments were inserted into the vector pM1300 b to obtain plant expression vectors with chimeric amiRNA precursors.The recombinant binary plant expression vectors pamiRNAs were separately introduced to the Agrobacterium tumefaciens strain EHA105 using the frost thawing method. Then a transient expression assay was performed to test whether the amiRNA expression vectors efficiently produced amiRNAs. These results suggested that the dimeric amiRNA precursor could be effectively recognized and processed into functional amiRNAs that could effectively cleave the target RNA in plants.The rice calli(cv. Lindao 10) that included amiRNA structures were subjected to Agrobacterium tumefaciens-mediated transformation. We generated transgenic plants from transformed calli, and 35, 37, and 48 plants containing pamiR-M, pami R-3 and pamiR-U were obtained, respectively, through PPT tolerance selection using leaf painting assays and PCR detection. Three plant expression vectors with modified dimeric amiRNA precursors could be effectively detected and used to generate mature amiRNAs in transgenic plants.Statistical analysis of the resistance results showed that the virus-infected transgenic groups exhibited varying degrees of viral resistance. The ratios of transgenic lines resistant to RSV were 29.52%, 44.14%, 54.17% and the percentages of resistance against RBSDV were 26.66%, 36.04%, 45.83% in transgenic plants containing pamiR-M, pamiR-3 and pamiR-U, respectively.The Southern blot assay demonstrated that the exogenous genes were integrated into the rice genome with 1-7 copy numbers and at different locations in the genome of the transgenic plants. A northern blot assay indicated that the RNA silencing induced by the original amiRNAs could be bilaterally extended by the siRNA pathway. The ami RNAs, together with the secondary siRNAs, mediated the degradation of viral RNAs. A genetic stability assay showed that transgenes and amiRNA-mediated virus resistance could be stably inherited in the transgenic plants.(2) RNAi mediated gene silencing in Laodelphax striatellusThe NCuP gene has been reported in Laodelphax striatellus as candidate gene, primers were designed to clone the NCuP by RT-PCR. The qRT-PCR assay showed that NCuP in the whole development process of Laodelphax striatellus were expressed, and the expression levels of different developmental stages were different. At the age of 1 in nymphs, the expression of NCuP was highest. In addition, the expression level of NCuP in viruliferous vector insects was significantly higher than that of non viruliferous vector insects.Synthesized the dsRNAs of NCuP genes and detected the expression of target gene and virus gene in insect by qRT-PCR after nymphs were fed on an artificial diet including the dsRNA. The results showed that the expression level of NCuP in Laodelphax striatellus was reduced significantly, and the expression level of RSV CP was reduced simultaneously. Detection of the infection and transmission rate in SBPH after feeding on dsNCuP indicated that the efficiency of infection(28.89%) and transmission(23.89%) were significantly reduced when the NCuP silenced by RNAi.