铜绿假单胞菌GF31氯氰菊酯降解酶的基因克隆及原核表达

发布时间：2018-01-14 16:34

本文关键词：铜绿假单胞菌GF31氯氰菊酯降解酶的基因克隆及原核表达　出处：《广西大学》2017年硕士论文　论文类型：学位论文

【摘要】：拟除虫菊酯是从天然除虫菊素经历两代发展而成的一类高效、广谱性杀虫剂;由于其大量使用给生态环境和人类健康带来危害而亟待治理;微生物降解是目前比较有效降解拟除虫菊酯的途径。近些年来国内外关于拟除虫菊酯降解的研究大多集中在降解微生物的筛选、降解特性和降解途径的研究等方面;关于降解酶基因的克隆、表达、分子改造和降解机理的研究还不够深入。本研究以实验室保存的一株具有拟除虫菊酯降解能力的铜绿假单胞菌PseudomonasaeruginosaGF31为研究对象,根据前期纯化获得的氯氰菊酯降解酶的氨基酸序列,通过NCBI数据库搜索同源序列,确定以同源性最高的PAO1假定的氨肽酶序列为模板设计引物,从GF31中克隆氯氰菊酯降解酶基因,命名为APs。通过SMART数据库分析APs基因编码的氨基酸序列,按照该蛋白的不同结构片段进行克隆,分别是ORF(全长基因)、SgI(去信号肽和前导肽的成熟蛋白)、PA(保留Pfam及Peptidase结构域)、Pep(Peptidase),基因片段大小分别为:1611bp、1503bp、1014bp和645bp。通过与载体pET28a/pET30a/pET32a构建表达质粒,转化大肠杆菌BL21(DE3)、Rosseta(DE3)、Rosseta-gami成功构建了基因工程菌。对构建的工程菌表达氯氰菊酯降解酶的情况进行了探索,发现该酶在BL21(DE3)、Rosseta(DE3)中以包涵体形式表达,在Rosseta-gami中以可溶形式融合表达;通过镍柱亲和层析对氯氰菊酯降解酶进行了纯化和酶活测定,结果显示SgI片段编码的蛋白24h对氯氰菊酯的降解率为12.7%。该酶为蛋白酶其中的氨肽酶,经过测定,对底物L-亮氨酸-对硝基苯胺(Leu-pNA)的比活力为1255.2U/mg。为了研究目的基因的表达水平,制备了目的基因APs和内参基因16SrRNA的质粒标准品,按10倍梯度稀释测定并绘制标准曲线,得到的标准曲线R~20.99,扩增效率=90-110%,成功建立了相对实时荧光定量PCR方法,可用来检测铜绿假单胞菌氯氰菊酯降解酶基因的表达水平。初步以此方法测定了工程菌相比于野生菌GF31中氯氰菊酯降解酶基因的表达水平变化。
[Abstract]:Pyrethroids are from natural pyrethrum through two generations of development and become a kind of high efficient, broad-spectrum insecticide; due to its extensive use to bring harm to the ecological environment and human health and urgent treatment; microbial degradation is the more effective way of degradation of pyrethroids. In recent years research on pyrethroid degradation at home and abroad are mostly concentrated in the screening of microbial degradation, degradation characteristics and the degradation pathway of; cloning and expression, on the degrading enzyme gene, molecular transformation and degradation mechanism is not deep enough. In this study, the laboratory was preserved with pyrethroid degrading Pseudomonas aeruginosa PseudomonasaeruginosaGF31 research the object, according to the amino acid sequence of purified cypermethrin degrading enzyme obtained by the early NCBI database searching homologous sequences, to determine the highest homology PAO1 false Aminopeptidase sequence as template primers were designed to clone the Cypermethrin Degrading Enzyme Gene from GF31, named APs. by the amino acid sequence of APs gene encoding the SMART database analysis, was cloned according to the different structural fragments of the protein, which are ORF (gene), SgI (signal peptide and mature protein to leading peptide the PA (Pfam), retained and Peptidase domain), Pep (Peptidase), gene fragment respectively: 1611bp, 1503bp, 1014bp and 645bp. through the construction of pET28a/pET30a/pET32a vector plasmid was transformed into E.coli BL21 (DE3), Rosseta (DE3), Rosseta-gami gene has been successfully constructed. The construction of engineering strain engineering bacteria the expression of Cypermethrin Degrading Enzyme was studied, found that the enzyme in BL21 (DE3), Rosseta (DE3) in the form of inclusion body expression in Rosseta-gami in the soluble form of fusion expression; through Ni NTA affinity chromatography of Cypermethrin Ester degrading enzymes were purified and enzyme activity determination, the results showed that SgI fragment encoding the 24h protein, the degradation rate of Cypermethrin was 12.7%. the enzyme for aminopeptidase, the protease after determination of substrate L- l-leucine-p-nitroanilide (Leu-pNA) specific activity was in order to study the expression level of 1255.2U/mg. gene standard plasmid, APs gene and 16SrRNA gene were prepared by 10 fold dilution were determined and plotted the standard curve, the standard curve obtained by R~20.99, the amplification efficiency =90-110%, successfully established a relatively real-time fluorescent quantitative PCR method can be used to detect the expression of Pseudomonas aeruginosa of Cypermethrin Degrading Enzyme Gene. Changes in expression level compared to the wild strain GF31 Cypermethrin Degrading enzyme gene engineering bacteria were determined preliminarily by the method.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172;X592

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