| Rice (Oryza sativa) is one of the world’s three major food crops and the most important food crops in China. Rice bacterial leaf blight and rice blast are two important disease threats to rice production, which are caused by bacterial Xanthomonas oryzae pv oryzae and Magnaporthe oryzae, respectively. Identification of disease-resistant rice genetic resources and improvement of rice defense are two effectively strategies to control diseases. These strategies are economic efficiency and environment-friendly.In this study, the Arabidopsis NHO1(Locus:AT1G80460) was used to retrieve its homologous genes in the rice genome and a rice homologous gene OsNHO1, cDNA of OsNHO1was isolated by RT-PCR. Then two vectors, over-expression and RNA interference, respectively, were constructed. Transgenic rice with these vector were obtained using rice cultivar TP309by Agrobacterium-mediated transformation. After propagation for3-4generations and substantial PCR verification, homozygous lines with stable expression were obtained., These lines were then tested for disease resistance against rice bacterial blight to explore OsNHO1in innate immunity. The main results were as follows:1. Full length cDNA of OsNHO1was obtained by RT-PCR and confirmed by DNA sequencing. The open reading frame (ORF) of OsNHO1is1590bp, encoding529amino acids with a conserved NBD-sugar-kinase domain.2. Expression level of OsNHO1gene with treatment of JA, SA and inoculation of rice bacterial blight strain PXO99was analysed using Real-Time PCR. Found the expression of OsNHO1genes was not identical after treated with various factors, and earlier expression after treatment SA, JA and PXO99are similar.3. By analysis the gene in Arabidopsis mutants nhol function, through the study of the bacterial count of arabidopsis different material experiment, found the rice OsNHO1gene can partly restore nhol homologous gene in arabidopsis mutants.4. By the method of enzyme digestion connection constructed rice OsNHO1expression vector and RNAi inhibit expression vector, get the length of the gene promoter gene expression vector, and use electric shock process each carrier into agrobacterium LBA4404, use the agrobacterium mediated rice genetic transformation technology will be the carrier TP309import rice, obtain transgenic plants.5. In order to do the complementary experiment in Rice, using the genome on rice, candidate of the OsNHO12Kb gene fragment upstream, PlantCARE analysis results show that the section contains TATAbox, CAATbox, CGTCA-motif, TC-rich repeats, TGA-element, GARE-motif, ABRE and other regulatory elements. The promoter sequence upstream of the OsNHO1gene amplified1400bp fragment length, and together with the OsNHO1gene to construct a plant expression vector of Gusgene, and used the nho1mutant of rice as materials,the genetic transformation. 6. After four generations of planting screening and DNA, PCR identification, get the genetic stability of genetically modified pure rice, nhol mutant M207and RNAi plant72-11,85-3.7. By using pure plant do semi-quantitative RT-PCR analysis and Real Time PCR analysis, it shows that overexpression of plant OsNHO1quantity is four times that of wild type gene expression, RNAi transgenic and nhol OsNHO1gene expression of mutant plants is0.5times that of the wild type, obtain the homozygous transgenic system conform to the requirements of the experiments. The transgenic plants are correct and expected to play the effect OsNHO1gene expression increased of overexpression plants, OsNHO1gene expression decreased of RNAi plants and nhol mutant Genetically modified pure line of selected rice pathogens PXO99, it shows that OsNHO1increased expression of plant resistance to pathogen, the disease spot15%smaller than wild typeAnd RNAi transgenic plants and decline in nhol mutant rice resistance of pathogenic bacteria,18%longer than the wild type of disease spot. In vitro inoculation with rice blast fungus Y34experiments, results showed that, OsNHO1overexpressing plants were0.5times less than the wild type plants, and RNAilesion is2times that of wild type, in the defense response of rice blast resistanceplayed a role.8. To detect the disease resistance related marker gene expression, RNAi in OsNHO1rice, nhol mutant and wild type rice TP309expression differences of the results found OsPR5and OsNHOl trend is the same in three kinds of material expression, GLU and OsNHO1instead, presumed to rice OsNHO1downstream related resistance genes can be induced by gene expression.9. Determine the OsNHO1expression, RNAi rice, nhol mutant and wild type rice TP309physiological and biochemical indexes of different material, found OsNHO1overexpression of rice leaf cuticular wax content and leaf relative content of NO increased, RNAi and nhol mutant rice in the rice decreased, illustrate OsNHO1expression and the content of wax and NO exist positive correlation. |
