农杆菌携带柞蚕抗菌肽基因转入烟草的研究

柞蚕抗菌肽具有广谱杀菌功能,对烟草等茄科作物的青枯病假单胞菌(Pseudomonassolanacearum)具有较强的杀菌效果。人工合成抗菌肽基因(122 bp)转入根癌农杆菌(Agrobnctcrium tumefaciena),感染烟草叶盘,诱导成苗。通过对卡那霉素敏感筛选,胭脂碱脱氢酶检定及抗菌肽片段探针杂交,确认抗菌肽基因已转入烟草。现正研究其在烟草中表达及抗青枯病的可能性。

TRANSFER OF SYNTHETIC ANTIBACTERIAL PEPTIDE D GENE OF ANTHERAEA PERNYI INTO TOBACCO PLANT

Some inducible antibacterial peptide (AP) were developed from the haemolymph of Chinese oak silkworm,Aniheraea pernyi pupae.The AP had broad-spectrum inhibition on bacteria such as wilt disease,Pseudomonas solanacearum of tobacco etc.The AP-D has been isolated and its amino acid sequence has been determined (36 peptides).We designed the nucleotides sequence of AP-D gene (122 bp) and synthesized by by DNA synthesizer.The M13 mp8 DNA was ligted with AP-D gene and transformed into E.soft JM103.The recombinant plasmid cut with Sai 1 and co24 plasmid containing CaMV 35S promotor cut with Xho I were ligated and transformed into Agrobacterium tuonefaciens by triple mating method.A Selected medium containing Km,Cm,Spc and Str was used to select the colonies which bad insert with AP-D gene.A.turnefaciens containing AP-D gene was used to infect the leaf dics of tobacco.After 4 weeks,we found some plantlets differentiation at the redges of leaf dics.These plants were resisted to Km,the nopaline detecton and Southern blot hybridization with AP-D gene probe showed that AP-D gene had been integrated into tobacco plant.The expression product should detected with its monoclonal antibody.We are now studying this in order to introduce the AP-D gene into to bacco plant.