油菜EST资源中抗菌肽基因的生物信息学分析及BnLTP2的活性验证

发布时间：2018-03-30 11:49

本文选题：油菜　切入点：抗菌肽　出处：《南阳师范学院》2017年硕士论文

【摘要】：抗菌肽在植物天然免疫系统中具有重要的作用,它是一种由基因编码的小分子多肽,通常由10～50个氨基酸构成,其中大部分抗菌肽是碱性的阳离子肽,带2到7个正电荷。抗菌肽在生物天然防御体系中占有重要的地位,它在体内体外都能表现出抗菌活性。为实现高通量挖掘植物抗菌肽基因,本研究从NCBI数据库(https://www.ncbi.nlm.nih.gov/nucest)下载油菜(Brassica napus) EST序列1185911条到本地服务器,将油菜EST资源首先通过前处理和重拼接过程,获得201200个unigene,通过blastx, tblastn程序将unigene与来自于抗菌肽数据库APD2中的已知抗菌肽序列比对,利用GO, Interproscan, Clustalw等工具对这些基因进行功能注释、分类和结构预测,从中筛选获得972个包含候选抗菌肽的基因,这些候选的抗菌肽基因种类包括27类,其中很多这些同源的不同的抗菌肽基因是来自于不同的动植物和微生物,通过去重复序列后共获得605个新的抗菌肽序列,使用Phylip等软件进行进化树等分析,结果表明这些基因分属于脂转移蛋白基因(237个),防御素基因(Defesin,106个),蚊皮素(Snakin,91个),Hipposin(79个),橡胶蛋白类(Hevein,32个),硫素(Thionins15个)等6个家族,并对其中典型抗菌肽基因家族进行了二级结构分析。挑选预测到的一个植物转脂蛋白家族的抗菌肽基因BnLTP2,它的蛋白质序列由27个氨基酸组成。通过重叠延伸PCR的实验方法,把合成的BnLTP2抗菌肽基因克隆到pET30a-EDDIE-GFP表达载体上,在大肠杆菌中表达得到的包涵体融合蛋白会在体外复性,然后通过融合蛋白EDDIE的自我剪切并纯化后获得没有增加任何多余氨基酸的抗菌肽产物,经SDS-PAGE电泳发现在5 kD处有一条抗菌肽BnLTP2条带。分别以藤黄八叠球菌(Micrococo luteus)作为革兰氏阳性菌代表,大肠杆菌(Escherichia coli)作为革兰氏阴性菌代表,酵母菌(Pichia pastorisGS115)作为真菌的代表以管碟法作为检测抗菌肽BnLTP2抑菌活性的标准方法。结果表明BnLTP2对大肠杆菌、藤黄八叠球菌和毕赤酵母菌具有较强的抑菌活性。以油菜的主要致病菌核盘菌(Sclerotinia sclerotiorum)作为丝状真菌的代表,检测抗菌肽BnLTP2对丝状真菌的抗真菌活性。结果表明BnLTP2同样对核盘菌也有较强的抑菌活性。将纯化后的BnLTP2分别溶解在PBS、25%TFE.50%TFE和75%TFE溶液中,进行圆二色谱检测,从峰图中发现在194 nm处出现一个正峰,在208和222 nm处有一个负峰,这说明该抗菌肽具有典型的α螺旋结构,但是峰值不高,说明抗菌肽BnLTP2中螺旋含量较低。植物转脂蛋白LTP家族成员一般由91到95个氨基酸残基构成,它的最大特征是分子组成中含有八个高度保守的半胱氨酸,并缺乏色氨酸,在分子中有一个疏水腔,脂肪酸分子可以被这个疏水腔所容纳,由四对二硫键连接四个α螺旋构成稳固的结构。BnLTP2的蛋白序列由27个氨基酸组成,包含四个半胱氨酸,KICCPRTIDRNIYNACRLTGASMTNCA,但与LTP家族同源性最高,推测是完整LTP蛋白的部分结构。
[Abstract]:Antimicrobial peptides play an important role in plant innate immune system, which is composed of a small molecule gene encoding the polypeptide, usually consisting of 10~50 amino acids, most antimicrobial peptides are cationic peptide alkaline, with 2 to 7 positive charges. The antibacterial peptide plays an important role in the biological natural defense system. It can exhibit antibacterial activity in vivo and in vitro. Plant antibacterial peptide genes for high flux mining, this study from the NCBI database (https://www.ncbi.nlm.nih.gov/nucest) (Brassica napus) EST download rape sequence 1185911 to the local server, the first through the pretreatment of rapeseed EST resources and re stitching process, 201200 UniGene, obtained by blastx, UniGene and tblastn program from the APD2 database of antibacterial peptide known antimicrobial peptide sequences, using GO, Interproscan, Clustalw and other tools for function of these genes Notes, classification and structure prediction, screened 972 candidate genes containing antibacterial peptide in these candidate antibacterial peptide gene types include 27 categories, many of these homologous different antibacterial peptide gene from plants and microorganisms in different animals, 605 new antimicrobial peptide sequences by sequence repeat after were obtained, using Phylip analysis software for phylogenetic tree, the results show that these genes belong to lipid transfer protein gene (237), defensin gene (Defesin, 106), mosquito endothelin (Snakin, 91), Hipposin (79), rubber proteins (Hevein, 32). Sulfur (Thionins15) and other 6 families, and one of the typical antimicrobial peptide gene family was analyzed. Two level structure of a plant selected to predict lipid transfer protein family of antibacterial peptide gene BnLTP2 and its protein sequence consists of 27 amino acids. The experiment by overlap extension PCR Methods to synthesis of antibacterial peptide BnLTP2 gene was cloned into pET30a-EDDIE-GFP expression vector. The expression of protein refolding in vitro by inclusion bodies in E.coli, and then through the EDDIE fusion protein was purified and self splicing did not increase any additional amino acid antimicrobial peptide products, by SDS-PAGE electrophoresis was found in 5 at kD a strip of antibacterial peptide BnLTP2. By eight fold (Micrococo luteus Garcinia aureus) as gram positive bacteria, Escherichia coli (Escherichia coli) as the representative of gram negative bacteria, yeast (Pichia pastorisGS115) is a standard method for fungi on behalf of the tube plate method as the detection of antibacterial activity of antibacterial peptide BnLTP2. The results showed that BnLTP2 of Escherichia coli, Staphylococcus and Garcinia eight fold Pichia pastoris has strong antimicrobial activity. The main pathogen Sclerotinia rapeseed (Sclerotinia sclerotiorum) As a representative of filamentous fungi, antifungal activity against filamentous fungi. The results show that the detection of antibacterial peptide BnLTP2 BnLTP2 on Sclerotinia sclerotiorum has strong antibacterial activity. The purified BnLTP2 were dissolved in PBS, 25%TFE.50%TFE and 75%TFE in the solution, for round two from the peak of chromatography, found a positive the peak at 194 NM, with a negative peak at 208 and 222 nm, which indicated that the antibacterial peptide has the typical structure of the alpha helix, but the peak is not high, showed that the antimicrobial peptide BnLTP2 in helical content is low. The plant lipid transfer protein LTP family members by 91 to 95 amino acids, the largest it is characterized molecular composition containing eight highly conserved cysteine, and the lack of tryptophan, a hydrophobic cavity in the molecule, fatty acid molecules can be accommodated by the hydrophobic cavity, a stable structure consisting of four two disulfide linked four alpha helix. The protein sequence of BnLTP2 is composed of 27 amino acids, including four cysteine, KICCPRTIDRNIYNACRLTGASMTNCA, but the highest homology with LTP family. It is speculated that the whole LTP protein is partially constructed.

【学位授予单位】：南阳师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946

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