Production Of Rice Stripe Virus Resistant And Marker-free Transgenic Rice And Production Of Polyclonal Antibody Of Rice Black Streaked Dwarf Virus Coat Protein

Rice stripe disease and Rice black-streaked dwarf disease are the most widespread andimportant threats to rice in our country. The pathogen of rice stripe disease is Rice stripe virus(RSV). The susceptible rice plant exhibited typical symptoms including withered inner leaves,chlorotic stripes, mottled leaves, subsequently premature wiltting and annually causes aserious negative effect on crop production grain yield. Rice black-streaked dwarf disease,with the pathogen of Rice black-streaked dwarf virus (RBSDV), infects rice plants and leadsto the appearance of pronounced stunting and also darkening of the foliage, dark-coloredfoliage, and more tillers than normal plants at a very early stage of growth. Plants that areinfected survive until harvest although they are severely stunted. These two viruses are alltransmitted by Laodelphax striatellus. Since recent years, these two disease have broken outin several areas and results in considerable decrease of rice production.Previous studies have shed light on the mechanism of RNA-mediated virus resistance(RMVR), making it available as a potential strategy with broad applications and practicalsignificance in anti-virus genetic engineering. Selection marker genes that encode antibioticand herbicide resistance are widely used to identify the rare transformation of crop plants, Inthe recent decade, the use of antibiotic resistance and herbicide resistance genes has arousedwide public concern and has negative effect on large-scale planting of transgenic cropsbecause of the potential risk to ecology and human. Twin T-DNAs system is considered aconvenient and common strategy for eliminating marker genes after their use. In addition,rapid, sensitive and effective method is desired in detecting and identifying virus in the field.However, RBSDV existed in infected rice plants at a low concentration and instable state andis difficult to be abstracted and purified. Prokaryotic expression and preparation of polyclonalantibody of RBSDV CP provides effective method for detecting virus in the field. In the present study, we constructed twin T-DNA vectors that target the400nucleotidesof RSV CP and SP, respectively. Through Agrobacterium-mediated transformation, weobtained selectable marker-free transgenic rice with strong RSV resistance to developmarker-free and RSV-resistant rice varieties. In addition, the nucleotide sequence of RBSDVS10was obtained by using RT-PCR. RBSDV CP gene was prokaryotic expressed,subsequently, we obtained the sensitive and effective anti-RBSDV CP polyclonal antibody byusing rabbits, providing an approaches for a large number of viral detection in field. The mainresults and conclusions presented in this thesis are as follows:(1) Production of RSV resistant and marker-free Transgenic RiceThe modified pCAMBIA-1300that harboured two separate T-DNAs was utilized toconstructed the vectors that expressed the inverted-repeat (IR) structures targeting the400ntof rice stripe vrius (RSV) coat protein (CP) gene.Transgenic rice lines were obtained through Agrobacterium-mediated transformation.Seven independent events that harbored both the hpt marker gene and the target gene (RSVCP) were obtained in the primary transformants.Two lines of pDTRSVCP that harbored the homozygous target gene but without the hptgene exhibited high resistance (with the susceptible ratio of less than5%) against RSV.Molecular analysis of the resistant transgenic plants confirmed the stable integration andexpression of the target genes. Lower levels of transgenes transcripts and specific smallinterfering RNAs were observed in the resistant transgenic plants, suggesting that the virusresistance was induced by RNAi.(2) Preparation of Polyclonal Antibody of Rice Black Streaked Dwarf Virus S10The nucleotide sequence of the RBSDV S10which encodes coat protein (CP) wascloned from RBSDV Shandong isolate using RT-PCR. The RBSDV CP gene was subclonedinto the prokaryotic expression vector PET-30a(+). Subsequently the resulted vector wastransformed into Escherichia. coli BL21(DE3) and the target protein carrying a His-tag wassuccessfully induced and expressed.The expressed protein was purified by using Ni-NAT resin column. Anti-RBSDV CPpolyclonal antibody was produced using rabbits. Using the antibody, ELISA and Western blotcould be performed, providing a sensitive and effective approaches for detection of RBSDV.