Transformation Of Anti-microbial Protein AP1 Encoded Gene And The Mediated Resistance To Bacterial Wilt Disease Of Transgenic Potato And Tobacco

APi is a plant endogenic anti-microbial protein isolated from a bacterialwilt-resistant potato variety MS42.3. Based on the nucleotide sequence of itsencoded gene api, a pair of specific primers P 1/P2 containii~ two restriction sites ofenzymes AccI and XhoI, respectively, were designed and synthesized. Using plasmidpAP2l 7(a procaryote expression vector of api gene) as template, the 1.2kb api genefragment was Obtained by PCR amplification primered by P1/P2?After digested byAccI and XhoI, the api gene fragment was ligased into a linear plasmid generatedfrom the tool vector pG 0 4A which was digested by the same restriction enzymes.After transformation and recombinants screening, an intemediate vector pAP4Acontaining the api gene's plant expression box "2E-35S- Q -api" ~s obtained. TheDNA fragment of "2E-35S- Q-api" was obtained by digestion of the plasmidpAP4A with EcoRI and HindIII and then conjuncted into a plant expression vectorpBll2l which was digested by the same restriction enzymes. After identification byPCR and restriction digestion, a plant high-efficient expression plasmid of api genewas obtained and named as pHap 1.pHapl was first transformed into Agrobacterium tumefizciens strain LBA4404by electroporation first and then idroduced into the sterile lnbe culture seedlings ofdifferent susceptible potato cuitivars `Mira', `Zhong-5-4' and `Jinguan', and tobaccocultivar NC89, respectively, by the leaf disc infectioti metlx,d. Via callus-inductionand kananiycin selection, 16, 8, 5 and 40 kanamycin resistance regenerated plants ofMira, Zhong-5-1, Jinguan and NC89 were obtained respectively. Results ofmolecular detection by PCR, Southern- and Northem.bloting indicated that the api2gene was successfully integrated into the genomes of potato and tobacco plants andtranscribed correctly.Under greenhouse conditions, two transgenic lines of `Mira' (Ts-M-2 andTs-M-5) and 8 transgenic plants of tobacco were evaluated for resistance to bacterialwilt caused by Ralstonia solanacearum. The experimental results showed that thetransgenic plants acquired a significantly increased resistance to this diseasecomparing to non-transgenic ones. The transgenic lines or plants could delay the wiltsymptom development and make disease index reduced.According to results of homology analysis on internet by Blast-n and Blast-x, itwas suggested that the api gene exhibit relatively high homology to the gene familyof Acid phosphatase. The fusion protein of api gene expressed in the Escherichiacoli and the crude protein from the transgenic plants were detected for the specificenzymatic activity of acid phosphatase. Results indicated that the acid phosphataseactivity of expression product of api gene was significantly higher than that of control ones.