Study On Vectors Construction And Expression Of Antimicrobial Peptide Thanatin To Improve Plant Resistance To Sclerotiniose

Antimicrobial peptide is a small peptide with high antimicrobial activity against exterior pathogen coming form recovery system of organism.It play an important role in the project of plant disease resistance improvement.Thanatin,a 21-residue peptide,is an inducible insect peptide with a broad range of antimicrobial activity against bacteria and fungi.In this study the gene has been used to transform plants to improve disease resistance especially to Sclerotinia scolerotiorum.1 Construction of Thanatin fusion vectors for plant expressionBase on the sequence in GenBank,we construct the genes with 5' fusion signal peptide or link peptide,and then they were cloned in pMD18-Tvector.SP-Thanatin contained Bglâ…¡in 3' and LP-Thanatin contained BamHâ… in 5' Bglâ…¡and BamHâ… have the same sequence of adhansive end,so the co-adhansive end restriction and ligation strategy was used add the repeat unit one by one.PCR amplification and DNA sequencing analysis confirmed that 1-5 copies of the gene were correctly inserted into the vector.2 Transformation of oilseed rape and Arabidopsis thalianaThe fused vector of Thanatin was incorporated into Brassica napus and Arabidopsis thaliana via Agrobacterium-mediated transformation,for long period and low efficiency of oilseed rape transformation,we only gained 5-copy Transgenic oilseed rape,but get all vectors(1-5 copies of Thanatin)transgenic Arabidopsis.The experiment of Sclerotinia resistance assessment indicated that the transgenic Arabidopsis did not has favorable fastness compare with wild type as we hope.3 Thanatin sub-cellular location and prokaryotic expressionWe constructed fusion gene SP-Thanatin-GFP and introduced it into onion epidermal cell by bomb ardment.Onion epidermal cell transiently expressing fusion protein was observed under fluorescence microscope.Result showed that fusion protein was accumulated in intercelluLar space.It indicating signal-peptide can lead fused protein to accumulate in the interecelluar space.At the same time we constructed Thanatin prokaryotic expression vector,After induce expression 12%SDS-PAGE analysis showed a special protein band at 36KD.Tt meaned that fused Thanatin can expression in E.coli. In conclusion,in this study,we constructed Thanatin expression vector for plant transformation,and get transgenic Brassica napus and Arabidopsis thaliana.