Screening Of Antifungal Candidate Genes In Nicoitana Tomentosiformis And Identification Of Genes Resistant To Black Shank

Tobacco is one of the important economic plants in China, but the disease is an important factor intobacco production. Therefore, exploring the disease-resistant genes in tobacco is especially important.Tobacco fungus disease is one of the diseases which damage the economic benefits, the study on itshows a relative hysteresis compared to the study on tobacco virus, so the further study is in need. Inthis study,the honghuadajinyuan which is fine and widely grown is used as material, we obtainedfungus disease resistant gene from Nicotiana tomentosiformis genome Then RNAi vectors wereconstructed according to the gateway cloning technique. Using Agrobacterium-mediated method, thevectors were transformed into honghuadajinyuan and its transformation efficiency were analysed by themethods of bioinformatics. Finally, the transgenic tobacco were vaccinated against the black shank andobserved its resistance, the results as follows:(1) After the sequencing of the Nicotiana tomentosiformis genome, we have obtained a quantity ofpredicted genes through hammer software. By blasting these genes on NCBI website, twelve antifungalcandidate genes were obtained. The deduced amino acid sequences of these genes share high identitywith nine proteins which are related to the fungus diseases. Analysing the tewlve screened fungusresistance candidate genes, we found the proteins which were encoded by the candidate genes havecommon domain, and these domains are the typical resistance conserved domains of R genes.(2) We designed specific primers according to the non-conservative regions of these genes, andcloned interference clips of fungus-resistant genes using reverse transcriptase cDNA ofhonghuadajinyuan as templates. Then the RNAi vectors of these candidate genes were constructedthrough gateway cloning techniques and they have been proved to be correct by restrictionendonuclease digestion of HindⅢ and XbaI, and the sequencing result have the same conclusion.(3) We put the RNAi vectors into honghuadajinyuan via Agrobacterium-mediated transformation.Transgenic tobacco plants have been obtained through tissue culture, hygromycin screening and PCRmethod. Registered their number and transferred them to the incubator, prepared for the follow-upexperimental work.(4) After hygromycin selection and PCR analysis: The percentage of transgenic tobacco plantscarrying a single gene is64.5%, carrying two genes is25.3%, more than three is rare. The result showsthat most of the foreign gene integrated into the tobacco genome in the form of single gene. And theRT-PCR result showed that the RNAi vectors have high silencing efficiency.(5) Inoculated the transgenic tobacco plants with black shank. The result as follows: there are nosignificant changes compared to the control. Two reasons can cause such results. Frist, these genes haveunrelated to the balck shank. Second, the resisrance of black shank in honghuadajinyuan is horizontalresistance, single gene can not cause significant effects.(6) The interfere fragments of these antifungal candidate genes that have been transformed intotobacco were submitted to NCBI to blast. Two genes showed a100%identity with two R genespublished in the NCBI, we speculated they were antifungal genes in the ordinary tobacco. On the base of the experiment results, we can clone the full length DNA for the further study on the specificfunction.