【作者】 汪小福；

【导师】 高智谋； 陈锦清；

【作者基本信息】 安徽农业大学 ， 微生物学， 2007， 硕士

【摘要】 抗菌肽是生物体防御系统产生的一类对外源病菌具有高效杀灭活性的小分子多肽,在植物抗病基因工程中具有重要的应用价值。Thanatin是刺肩蝽（PodisusmacuLiventris）成虫经诱导产生的一种抗菌肽,由21个氨基酸残基组成,该抗菌肽对细菌以及真菌都有很强的抗菌活性,本研究通过转基因技术将其转入模式植物拟南芥和油菜,以提高植物的抗病性尤其是对菌核病的抗性。1 Thanatin的融合植物表达载体的构建根据GenBank登录的Thanatin序列设计引物,分别合成前端含信号肽和连接肽的基因片段,即SP-Thanatin和LP-Thanatin,分别克隆到pMD 18-T载体上。通过SP-Thanatin3’端设计了BglⅡ切点,LP-Thanatin 5’设计了Bam HI切点,利用BglⅡ和Bam HI为同尾酶的特点,LP-Thanatin Bgl-Ⅱ/BamHI双酶切片断可多轮次替换SP-Thanatin上的终止密码子（TAG）构建成可剪切多拷贝抗菌肽融合植物表达载体。结果鉴定1-5拷贝的Thanatin融合植物表达载体构建成功。2转基因油菜和拟南芥的获得通过农杆菌介导和In planta技术将1-5拷贝的Thanatin融合植物表达载体分别转化油菜和拟南芥,由于转油菜的周期长和效率低,目前我们只获得5拷贝的T₀代转基因油菜,而获得了1-5拷贝的全部转基因T₀代拟南芥,对转基因油菜和拟南芥进行菌核病抗性鉴定,结果显示转基因植株中并无预期中的高抗菌核病类型。3 Thanatin的亚细胞定位和原核表达构建SP-Thanatin-GFP融合基因,利用基因枪轰击洋葱表皮。洋葱表皮细胞瞬时表达融合蛋白,在荧光显微镜下观察显示荧光蛋白特异性地定位于细胞间隙,表明信号肽能成功的将目的蛋白引导到细胞外。同时构建Thanatin的原核表达载体pGEX-Thanatin,诱导表达后经12%SDS-PAGE分析,显示在约36KD处有特异性条带,证明Thanatin能在大肠杆菌中表达且效果良好。更多还原

【Abstract】 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 BglⅡin 3’ and LP-Thanatin contained BamHⅠin 5’ BglⅡand BamHⅠhave 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.更多还原