本文選題：MCs + 降解基因�。� 參考：《武漢理工大學(xué)》2015年碩士論文

【摘要】：微囊藻毒素(Microcytins,MCs)是一類環(huán)狀的七肽,具有強烈的毒性。微生物降解被認為是MCs在水體中去除的主要途徑。目前已經(jīng)分離出60余株MCs降解菌,它們大部分具有相同的MCs降解機理。已有研究表明,至少有3種蛋白水解酶參與了該過程,編碼這3個酶的基因mlrA、mlrB、mlrC和編碼轉(zhuǎn)運蛋白的mlrD基因聚集在一個5.8 kb的基因簇中。盡管在多株MCs降解菌中檢測到了mlrA基因,但是目前只獲得了4株MCs降解菌的mlrA基因全序列。并且,多數(shù)MCs降解菌未進行mlrB、mlrC和mlrD基因檢測,獲得的基因全序列更是有限。由于缺乏MCs降解基因的全序列信息,所以目前對該基因的來源及進化過程尚不清楚。本研究以此前從滇池沉積物中分離得到一株高效的MCs降解菌Sphingopyxis sp.X20為材料,通過克隆方法得到mlr基因簇全序列,對mlrA,mlrB,mlrC,mlrD四個基因及其編碼的酶進行了生物信息學(xué)分析,并對關(guān)鍵基因mlrA進行異源表達驗證。本文主要研究內(nèi)容及結(jié)果如下:(1)經(jīng)PCR擴增獲得目的片段,測序拼接后得到mlrA基因全長是1011bp。編碼336個氨基酸,含有163個疏水性氨基酸,58個親水性氨基酸,具有6處跨膜區(qū),其信號肽位于1-26處區(qū)域。X20菌mlrA基因與Sphingopyxis sp.C-1 mlrA基因相似性達到99%,系統(tǒng)發(fā)育分析結(jié)果表明,X20菌mlrA基因與Sphingopyxis sp.C-1的mlrA基因一起形成一個進化分支。mlrA基因異源表達產(chǎn)物對MCs具有降解活性,可以將MCLR降解成線性MCLR,證明所得序列正確。(2)通過PCR擴增方法得到mlrD基因全序列。mlrD基因全長為1272 bp,編碼423個氨基酸,MlrD酶的分子量為44.849 kDa,含有221個疏水性氨基酸,80個親水性氨基酸,具有10處跨膜區(qū),其信號肽位于1-38處區(qū)域,不存在信號肽。X20菌mlr D基因與Sphingopyxis sp.C-1 mlrD基因序列相似度達到99%。(3)通過采用步移PCR方法得到mlrC全序列。mlrC基因的全長為1587 bp,編碼528個氨基酸,含有200個疏水性氨基酸和97個親水性氨基酸。在整個氨基酸結(jié)構(gòu)中,MlrC酶疏水性不高,沒有跨膜區(qū),也沒有氨基酸信號肽。系統(tǒng)發(fā)育分析結(jié)果表明,X20菌mlrC基因與Sphingopyxis sp.C-1 mlrC基因同源性較高。(4)利用步移PCR技術(shù)未能擴增出mlrB基因全序列,故選擇構(gòu)建基因組文庫的方法獲得mlr B基因全序列。mlrB基因全長1581 bp,有526個氨基酸,其中含有193個疏水性氨基酸,113個親水性氨基酸。MlrB酶的分子量為57.6 kDa,在MlrB酶結(jié)構(gòu)中不存在跨膜區(qū)和信號肽。系統(tǒng)發(fā)育分析結(jié)果表明,X20菌mlrB基因與Sphingopyxis sp.C-1菌mlrB基因親緣性很高。
[Abstract]:Microcystins (microcystins MCs) are a class of cyclic heptapeptide with strong toxicity. Microbial degradation is considered to be the main way to remove MCs from water. At present, more than 60 strains of MCs have been isolated, most of which have the same mechanism of MCs degradation. It has been shown that at least three proteolytic enzymes are involved in this process, and the genes that encode these three enzymes, mlrAhlBrC, and MLRD, which encode transporter, are clustered in a 5.8 kb gene cluster. Although the mlrA gene was detected in several strains of MCS-degrading bacteria, only 4 strains of MCS-degrading bacteria were found to have the full mlrA gene sequence. Moreover, most of the MCs degrading bacteria were not detected by the ml rC and mlrD genes, and the complete sequence of the genes was even limited. Due to the lack of full sequence information of MCs degradation gene, the origin and evolution of MCs degradation gene are unclear. In this study, a highly efficient strain of Sphingopyxis sp.X20 was isolated from sediment of Dianchi Lake, and the complete sequence of mlr gene cluster was obtained by cloning method. The bioinformatics analysis of four genes and their encoding enzymes were carried out. The key gene mlrA was verified by heterologous expression. The main contents and results of this study are as follows: (1) the target fragment was amplified by PCR, and the mlrA gene was sequenced to be 1011bpp. Encoding 336 amino acids, containing 163 hydrophobic amino acids, 58 hydrophilic amino acids, with 6 transmembrane regions, Its signal peptide was located in 1-26 regions. The similarity between the mlrA gene and Sphingopyxis sp.C-1 mlrA gene was 99%. Phylogenetic analysis showed that the X20 mlrA gene and the mlrA gene of Sphingopyxis sp.C-1 formed an evolutionary branch, and the heterologous expression product of Sphingopyxis sp.C-1 could degrade MCs. MCLR can be degraded into linear MCLR. (2) the complete sequence of mlrD gene is 1272 BP, encoding 423 amino acids, the molecular weight of MlrD enzyme is 44.849 kDa. it contains 221 hydrophobic amino acids and 80 hydrophilic amino acids. There are 10 transmembrane regions and the signal peptide is located in 1-38 regions. There is no similarity between the signal peptide. X20 mlr D gene and Sphingopyxis sp.C-1 mlrD gene sequence. (3) the full length of mlrC complete sequence .mlrC gene is 1587 BP, encoding 528 amino acids by stepwise PCR, and the sequence of Sphingopyxis sp.c-1 mlrD gene is similar to that of Sphingopyxis sp.mlrD gene. It contains 200 hydrophobic amino acids and 97 hydrophilic amino acids. In the whole amino acid structure, the hydrophobicity of MlrC is not high, there is no transmembrane region, and there is no amino acid signal peptide. Phylogenetic analysis showed that the sequence of mlrC gene of X20 strain and Sphingopyxis sp.C-1 gene were highly homologous. (4) the full sequence of mlrB gene could not be amplified by step PCR. Therefore, the whole sequence of mlr B gene .mlrB gene was obtained by using the method of constructing genomic library, with a total length of 1581 BP and 526 amino acids. There were 193 hydrophobic amino acids and 113 hydrophilic amino acids. The molecular weight of MlrB enzyme was 57.6 kDa. There was no transmembrane region and signal peptide in the structure of MlrB enzyme. Phylogenetic analysis showed that the relationship between the MLrB gene of X20 strain and that of Sphingopyxis sp.C-1 strain was very close to that of Sphingopyxis sp. C-1 strain.
【學(xué)位授予單位】：武漢理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X172;Q78

【參考文獻】

相關(guān)期刊論文 前10條

1 游狄杰;陳曉國;向薈圯;歐陽潦;楊冰;;微囊藻毒素降解菌Paucibacter sp.CH菌的分離鑒定及其降解特性[J];環(huán)境科學(xué);2014年01期

2 蘇雅玲;鄧一榮;;富營養(yǎng)化湖泊中微囊藻毒素及其控制去除技術(shù)[J];環(huán)境科學(xué)與技術(shù);2013年06期

3 崔亞青;俞魯;高雪蓮;馬田田;楊興明;沈其榮;;一株高效MC-RR降解菌的分離鑒定及其降解特性[J];環(huán)境工程學(xué)報;2013年02期

4 劉凱英;薛罡;程起躍;石為民;葉文婷;;微囊藻毒素-RR高效降解菌的分離鑒定及降解特性[J];環(huán)境科學(xué)與技術(shù);2012年04期

5 吳涓;鐘升;王光云;李玉成;;一株降解微囊藻毒素菌種的鑒定及其活性研究[J];中國環(huán)境科學(xué);2011年01期

6 劉曉文;李現(xiàn)堯;史全良;;一株微囊藻毒素降解菌的分離與鑒定[J];環(huán)境工程學(xué)報;2010年09期

7 姚玉玲;蔣文菁;屈秋;但德忠;;地表水和飲用水中微囊藻毒素檢測方法的研究進展[J];現(xiàn)代科學(xué)儀器;2008年05期

8 陳娟;陳灝;;鑭負載改性TiO_2太陽光催化降解微囊藻毒素的研究[J];環(huán)境工程學(xué)報;2008年07期

9 劉海燕;宦海琳;汪育文;李建宏;周耀明;;微囊藻毒素降解菌S3的分子鑒定及其降解毒素的研究[J];環(huán)境科學(xué)學(xué)報;2007年07期

10 劉博;蘇喬;湯敏謙;袁曉東;安利佳;;應(yīng)用于染色體步移的PCR擴增技術(shù)的研究進展[J];遺傳;2006年05期

相關(guān)碩士學(xué)位論文 前1條

1 王萬群;武夷菌素產(chǎn)生菌CK-15細菌人工染色體（BAC）文庫構(gòu)建[D];中國農(nóng)業(yè)科學(xué)院;2008年

，

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