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  本文關(guān)鍵詞：銅綠假單胞菌GF31氯氰菊酯降解酶的基因克隆及原核表達　出處：《廣西大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 銅綠假單胞菌GF31 氯氰菊酯降解酶 基因克隆和表達熒光定量PCR

【摘要】：擬除蟲菊酯是從天然除蟲菊素經(jīng)歷兩代發(fā)展而成的一類高效、廣譜性殺蟲劑;由于其大量使用給生態(tài)環(huán)境和人類健康帶來危害而亟待治理;微生物降解是目前比較有效降解擬除蟲菊酯的途徑。近些年來國內(nèi)外關(guān)于擬除蟲菊酯降解的研究大多集中在降解微生物的篩選、降解特性和降解途徑的研究等方面;關(guān)于降解酶基因的克隆、表達、分子改造和降解機理的研究還不夠深入。本研究以實驗室保存的一株具有擬除蟲菊酯降解能力的銅綠假單胞菌PseudomonasaeruginosaGF31為研究對象,根據(jù)前期純化獲得的氯氰菊酯降解酶的氨基酸序列,通過NCBI數(shù)據(jù)庫搜索同源序列,確定以同源性最高的PAO1假定的氨肽酶序列為模板設(shè)計引物,從GF31中克隆氯氰菊酯降解酶基因,命名為APs。通過SMART數(shù)據(jù)庫分析APs基因編碼的氨基酸序列,按照該蛋白的不同結(jié)構(gòu)片段進行克隆,分別是ORF(全長基因)、SgI(去信號肽和前導(dǎo)肽的成熟蛋白)、PA(保留Pfam及Peptidase結(jié)構(gòu)域)、Pep(Peptidase),基因片段大小分別為:1611bp、1503bp、1014bp和645bp。通過與載體pET28a/pET30a/pET32a構(gòu)建表達質(zhì)粒,轉(zhuǎn)化大腸桿菌BL21(DE3)、Rosseta(DE3)、Rosseta-gami成功構(gòu)建了基因工程菌。對構(gòu)建的工程菌表達氯氰菊酯降解酶的情況進行了探索,發(fā)現(xiàn)該酶在BL21(DE3)、Rosseta(DE3)中以包涵體形式表達,在Rosseta-gami中以可溶形式融合表達;通過鎳柱親和層析對氯氰菊酯降解酶進行了純化和酶活測定,結(jié)果顯示SgI片段編碼的蛋白24h對氯氰菊酯的降解率為12.7%。該酶為蛋白酶其中的氨肽酶,經(jīng)過測定,對底物L(fēng)-亮氨酸-對硝基苯胺(Leu-pNA)的比活力為1255.2U/mg。為了研究目的基因的表達水平,制備了目的基因APs和內(nèi)參基因16SrRNA的質(zhì)粒標(biāo)準(zhǔn)品,按10倍梯度稀釋測定并繪制標(biāo)準(zhǔn)曲線,得到的標(biāo)準(zhǔn)曲線R~20.99,擴增效率=90-110%,成功建立了相對實時熒光定量PCR方法,可用來檢測銅綠假單胞菌氯氰菊酯降解酶基因的表達水平。初步以此方法測定了工程菌相比于野生菌GF31中氯氰菊酯降解酶基因的表達水平變化。
[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.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X172;X592

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本文編號：1424412