本文選題：油菜　切入點(diǎn)：抗菌肽　出處：《南陽(yáng)師范學(xué)院》2017年碩士論文

【摘要】：抗菌肽在植物天然免疫系統(tǒng)中具有重要的作用,它是一種由基因編碼的小分子多肽,通常由10～50個(gè)氨基酸構(gòu)成,其中大部分抗菌肽是堿性的陽(yáng)離子肽,帶2到7個(gè)正電荷�？咕脑谏锾烊环烙w系中占有重要的地位,它在體內(nèi)體外都能表現(xiàn)出抗菌活性。為實(shí)現(xiàn)高通量挖掘植物抗菌肽基因,本研究從NCBI數(shù)據(jù)庫(kù)(https://www.ncbi.nlm.nih.gov/nucest)下載油菜(Brassica napus) EST序列1185911條到本地服務(wù)器,將油菜EST資源首先通過(guò)前處理和重拼接過(guò)程,獲得201200個(gè)unigene,通過(guò)blastx, tblastn程序?qū)nigene與來(lái)自于抗菌肽數(shù)據(jù)庫(kù)APD2中的已知抗菌肽序列比對(duì),利用GO, Interproscan, Clustalw等工具對(duì)這些基因進(jìn)行功能注釋、分類(lèi)和結(jié)構(gòu)預(yù)測(cè),從中篩選獲得972個(gè)包含候選抗菌肽的基因,這些候選的抗菌肽基因種類(lèi)包括27類(lèi),其中很多這些同源的不同的抗菌肽基因是來(lái)自于不同的動(dòng)植物和微生物,通過(guò)去重復(fù)序列后共獲得605個(gè)新的抗菌肽序列,使用Phylip等軟件進(jìn)行進(jìn)化樹(shù)等分析,結(jié)果表明這些基因分屬于脂轉(zhuǎn)移蛋白基因(237個(gè)),防御素基因(Defesin,106個(gè)),蚊皮素(Snakin,91個(gè)),Hipposin(79個(gè)),橡膠蛋白類(lèi)(Hevein,32個(gè)),硫素(Thionins15個(gè))等6個(gè)家族,并對(duì)其中典型抗菌肽基因家族進(jìn)行了二級(jí)結(jié)構(gòu)分析。挑選預(yù)測(cè)到的一個(gè)植物轉(zhuǎn)脂蛋白家族的抗菌肽基因BnLTP2,它的蛋白質(zhì)序列由27個(gè)氨基酸組成。通過(guò)重疊延伸PCR的實(shí)驗(yàn)方法,把合成的BnLTP2抗菌肽基因克隆到pET30a-EDDIE-GFP表達(dá)載體上,在大腸桿菌中表達(dá)得到的包涵體融合蛋白會(huì)在體外復(fù)性,然后通過(guò)融合蛋白EDDIE的自我剪切并純化后獲得沒(méi)有增加任何多余氨基酸的抗菌肽產(chǎn)物,經(jīng)SDS-PAGE電泳發(fā)現(xiàn)在5 kD處有一條抗菌肽BnLTP2條帶。分別以藤黃八疊球菌(Micrococo luteus)作為革蘭氏陽(yáng)性菌代表,大腸桿菌(Escherichia coli)作為革蘭氏陰性菌代表,酵母菌(Pichia pastorisGS115)作為真菌的代表以管碟法作為檢測(cè)抗菌肽BnLTP2抑菌活性的標(biāo)準(zhǔn)方法。結(jié)果表明BnLTP2對(duì)大腸桿菌、藤黃八疊球菌和畢赤酵母菌具有較強(qiáng)的抑菌活性。以油菜的主要致病菌核盤(pán)菌(Sclerotinia sclerotiorum)作為絲狀真菌的代表,檢測(cè)抗菌肽BnLTP2對(duì)絲狀真菌的抗真菌活性。結(jié)果表明BnLTP2同樣對(duì)核盤(pán)菌也有較強(qiáng)的抑菌活性。將純化后的BnLTP2分別溶解在PBS、25%TFE.50%TFE和75%TFE溶液中,進(jìn)行圓二色譜檢測(cè),從峰圖中發(fā)現(xiàn)在194 nm處出現(xiàn)一個(gè)正峰,在208和222 nm處有一個(gè)負(fù)峰,這說(shuō)明該抗菌肽具有典型的α螺旋結(jié)構(gòu),但是峰值不高,說(shuō)明抗菌肽BnLTP2中螺旋含量較低。植物轉(zhuǎn)脂蛋白LTP家族成員一般由91到95個(gè)氨基酸殘基構(gòu)成,它的最大特征是分子組成中含有八個(gè)高度保守的半胱氨酸,并缺乏色氨酸,在分子中有一個(gè)疏水腔,脂肪酸分子可以被這個(gè)疏水腔所容納,由四對(duì)二硫鍵連接四個(gè)α螺旋構(gòu)成穩(wěn)固的結(jié)構(gòu)。BnLTP2的蛋白序列由27個(gè)氨基酸組成,包含四個(gè)半胱氨酸,KICCPRTIDRNIYNACRLTGASMTNCA,但與LTP家族同源性最高,推測(cè)是完整LTP蛋白的部分結(jié)構(gòu)。
[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.

【學(xué)位授予單位】：南陽(yáng)師范學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q946

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