Studies On The Transgenic Tobacco And Tomato With Quorum-quenching Gene From Ralstonia Solanacearu

Aac gene could quench the quorum-sensing of Ralstonia solanacearum.Based on the nucleotide sequence of its encoded gene aac,a pair of specific primers P₁/P₂ containing two restriction sites of enzymes Xhoâ… were designed and synthesized.Using pET-aac as template, the 2388 bp aac gene fagment was obtained by PCR amplification primered by P₁/P₂.Then connect with pGEM-T easy.After digested by Xhoâ… ,the aac gene fragment was ligased into a linear plasmid generated from the tool vector pUC19-Ω4A which was digested by the same restraction enzyme.And then digested the pUC19-Ω4A-aac with EcoRâ… /Hindâ…¢,conjuncted into a plant expression vector pBI121 which was digestion by the same restraction enzymes.After identification by PCR and restriction digestion,a plant high-efficient expression plasmid of aac gene was obtained.pBI121-Ω4A-aac was first transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation first and thenintroduced into tobacco cultivar'NC89' and tomatto cultivar 'zhongshu-5'.Under Kanamycin selection pressure,57,24 resistant regenerated plants of NC89 and 'zhongshu-5' were obtained respectively.Results of molecular detection by PCR,RT-PCR, Southern,ELISA,Western indicated that the aac gene wassuccessfully integrated into the genomes of tobacco and tomato plants and transcribed correctly.Under greenhouse condition,two transgenic lines were evaluated for resistance to bacterial wilt caused by Ralstonia solanacearum.The experimental results showed that the transgenic plants acquired a significantly increased resistance to this disease comparing to non-ransgenic ones.The transgenic lines or Plants could delay the wilt symptom development and make disease index redueed.