Spingopyxis sp. X20菌mlr基因簇的克隆及其特性研究

发布时间：2018-06-30 21:02

  本文选题：MCs + 降解基因　； 参考：《武汉理工大学》2015年硕士论文

【摘要】：微囊藻毒素(Microcytins,MCs)是一类环状的七肽,具有强烈的毒性。微生物降解被认为是MCs在水体中去除的主要途径。目前已经分离出60余株MCs降解菌,它们大部分具有相同的MCs降解机理。已有研究表明,至少有3种蛋白水解酶参与了该过程,编码这3个酶的基因mlrA、mlrB、mlrC和编码转运蛋白的mlrD基因聚集在一个5.8 kb的基因簇中。尽管在多株MCs降解菌中检测到了mlrA基因,但是目前只获得了4株MCs降解菌的mlrA基因全序列。并且,多数MCs降解菌未进行mlrB、mlrC和mlrD基因检测,获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,所以目前对该基因的来源及进化过程尚不清楚。本研究以此前从滇池沉积物中分离得到一株高效的MCs降解菌Sphingopyxis sp.X20为材料,通过克隆方法得到mlr基因簇全序列,对mlrA,mlrB,mlrC,mlrD四个基因及其编码的酶进行了生物信息学分析,并对关键基因mlrA进行异源表达验证。本文主要研究内容及结果如下:(1)经PCR扩增获得目的片段,测序拼接后得到mlrA基因全长是1011bp。编码336个氨基酸,含有163个疏水性氨基酸,58个亲水性氨基酸,具有6处跨膜区,其信号肽位于1-26处区域。X20菌mlrA基因与Sphingopyxis sp.C-1 mlrA基因相似性达到99%,系统发育分析结果表明,X20菌mlrA基因与Sphingopyxis sp.C-1的mlrA基因一起形成一个进化分支。mlrA基因异源表达产物对MCs具有降解活性,可以将MCLR降解成线性MCLR,证明所得序列正确。(2)通过PCR扩增方法得到mlrD基因全序列。mlrD基因全长为1272 bp,编码423个氨基酸,MlrD酶的分子量为44.849 kDa,含有221个疏水性氨基酸,80个亲水性氨基酸,具有10处跨膜区,其信号肽位于1-38处区域,不存在信号肽。X20菌mlr D基因与Sphingopyxis sp.C-1 mlrD基因序列相似度达到99%。(3)通过采用步移PCR方法得到mlrC全序列。mlrC基因的全长为1587 bp,编码528个氨基酸,含有200个疏水性氨基酸和97个亲水性氨基酸。在整个氨基酸结构中,MlrC酶疏水性不高,没有跨膜区,也没有氨基酸信号肽。系统发育分析结果表明,X20菌mlrC基因与Sphingopyxis sp.C-1 mlrC基因同源性较高。(4)利用步移PCR技术未能扩增出mlrB基因全序列,故选择构建基因组文库的方法获得mlr B基因全序列。mlrB基因全长1581 bp,有526个氨基酸,其中含有193个疏水性氨基酸,113个亲水性氨基酸。MlrB酶的分子量为57.6 kDa,在MlrB酶结构中不存在跨膜区和信号肽。系统发育分析结果表明,X20菌mlrB基因与Sphingopyxis sp.C-1菌mlrB基因亲缘性很高。
[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.
【学位授予单位】：武汉理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;Q78

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