本文關(guān)鍵詞： DOP HPLC pehA pehB 克隆表達 基因敲除　出處：《華中師范大學》2016年碩士論文　論文類型：學位論文

【摘要】：鄰苯二甲酸二辛酯(Di-n-octyl ortho-phthalate,DOP)是鄰苯二甲酸的酯化衍生物,屬于鄰苯二甲酸酯類(PAEs,又名酞酸酯)。應用于涂料、食品包裝材料、潤滑劑、藥品、化肥、化妝品、農(nóng)藥、醫(yī)療器械、兒童玩具等領(lǐng)域,是一種廣泛利用的增塑劑。DOP具有生物富集作用,對人體造成神經(jīng)毒性、生殖毒性和多器官癌變等病變,嚴重威脅到人的生存與發(fā)展,已經(jīng)引起國內(nèi)外廣泛關(guān)注。美國環(huán)保局(EPA)已經(jīng)將其列入優(yōu)先控制有毒污染物,DEP、DMP與DOP也被中國政府確定為優(yōu)先控制污染物。毒理學實驗表明DOP對人體具有致突變性、致癌性、致畸形和擬／抗甲狀腺激素活性、擬／抗雌激素活性等內(nèi)分泌干擾特性；同時是一種環(huán)境激素類化合物。環(huán)境中的DOP主要通過生物降解和光解這兩種途徑,光解速度較慢、周期較長,因此生物降解是DOP降解的最主要方式。目前對DOP微生物降解的研究包括降解菌的篩選、鑒定、降解途徑及降解特性分析,對降解途徑中蛋白的主要功能的研究較少。本文以戈登氏菌屬Gordonia sp. HS-NH1為材料,克隆表達了鄰苯二甲酸酯降解過程中的關(guān)鍵酶鄰苯二甲酸酯酶(PehA)和鄰苯二甲酸單酯酶(PehB),并研究了其功能和特性。結(jié)合基因敲降技術(shù)構(gòu)建了缺失轉(zhuǎn)運蛋白ABC/MFS基因的敲除突變株,并對突變株的代謝途徑與降解特性進行了研究。主要結(jié)果如下：通過基因組掃描得知戈登氏菌HS-NH1的pehA基因全長594bp,編碼197個氨基酸,經(jīng)過blast比對發(fā)現(xiàn)其與紅球菌屬Rhodococcus sp. YK2的pehA序列(GI：21388688)同源性較高,達到98%；pehB基因全長852bp,編碼283個氨基酸,與戈登氏菌屬Gordonia sp. P8219的pehB序列(GI：85677422)具有99%的相似性。采用基因克隆技術(shù),通過設(shè)計引物、PCR擴增,成功構(gòu)建了重組表達質(zhì)粒pET-pehA和pET-pehB,并在E.coliBL21得到高效表達。SDS-PAGE電泳分析,重組酶PehA和PehB的分子量分別為27KDa和30Kda。酶活性檢測表明重組酶PehA和PehB對長鏈的PAE具有良好的水解活性。為進一步深入研究該酶的功能與結(jié)構(gòu)奠定了一定的基礎(chǔ)。利用同源重組的方法敲除了戈登氏菌(Gordonia sp.)HS-NH1的ABC和MFS轉(zhuǎn)運蛋白基因,成功構(gòu)建了ABC轉(zhuǎn)運蛋白敲除突變菌株和MFS轉(zhuǎn)運蛋白敲除突變菌株,分別命名為HS-NH1-△MFS和HS-NH1-△ABC。對突變菌株的代謝途徑、基因功能與降解特性進行了研究。研究結(jié)果表明：戈登氏菌(Gordonia sp.)HS-NH1降解DOP的最適生長條件為30℃、pH 7.0,該菌能在120小時內(nèi)DOP降解率為80%。突變株HS-NH1-△MFS在同等條件下(溫度為30℃、pH 7.0)對DOP的降解效率顯著下降,降解效率僅有20%；而突變菌株HS-NH1-△ABC不能以DOP為唯一碳源的培養(yǎng)機中生長。底物廣譜性分析表明,戈登氏菌(Gordonia sp.HS-NH1)能高效利用常見的PAE(如DOP.DEHP),及其代謝物(PCA)。突變菌株HS-NH1-△MFS能夠利用長鏈的鄰苯二甲酸酯類,如DOP、DEHP等,但利用效率較低；不能利用短鏈的PAE,如鄰苯二甲酸二甲酯(DMP)、鄰苯二甲酸二乙酯(DEP)以及鄰苯二甲酸(PA)與原兒茶酸PCA等；而HS-NH1-△ABC不能在PAE及其代謝產(chǎn)物作作為唯一碳源的培養(yǎng)基中生長。說明MFS與ABC轉(zhuǎn)運蛋白都能轉(zhuǎn)運鄰苯二甲酸,將鄰苯二甲酸運輸?shù)郊毎麅?nèi)進行進一步降解,ABC轉(zhuǎn)運蛋白作為PA主要的運輸?shù)鞍?MFS轉(zhuǎn)運蛋白作為輔助的運輸?shù)鞍住?br/>[Abstract]:The adjacent benzene two formic acid two ester (Di-n-octyl ortho-phthalate DOP) is esterified derivatives of phthalate two benzoic acid, phthalic acid esters (belonging to two PAEs, also known as phthalates). Used in coatings, food packaging materials, lubricants, chemicals, fertilizers, pesticides, cosmetics, medical equipment, toys and other fields, is a widely the use of.DOP plasticizer with bioaccumulation on human disease causing neurotoxicity, reproductive toxicity and multi organ carcinogenesis, a serious threat to human survival and development, has aroused widespread concern at home and abroad. The United States Environmental Protection Agency (EPA) has been included in the priority control of toxic pollutants, DEP, DMP and DOP are Chinese the government has identified as priority pollutants. Toxicological experiments show that DOP has mutagenicity, carcinogenicity and teratogenicity on the human body, quasi / anti thyroid hormone activity, pseudo / anti estrogenic activity and other endocrine disturbance characteristics; at the same time is A kind of environmental hormone compounds. In the environment of DOP is mainly through the two ways of biodegradation and photolysis, photodegradation is slow, long cycle, so the biodegradation is the main way of the degradation of DOP. The current research on microbial degradation of DOP degrading bacteria including screening, identification, analysis of degradation and degradation characteristics, research the function of protein degradation pathway in less. Based on Gordon's genus Gordonia sp. HS-NH1 as material, the cloning and expression of phthalic acid ester in the degradation process of the two key enzymes of phthalic acid esterase (PehA) and two adjacent benzene two formic acid (PehB) and monoesterase, its function and characteristic of the combination. Knockdown of ABC/MFS gene deletion transporter knockout mutant, and the metabolism and degradation characteristics of mutant strains were studied. The main results are as follows: that Gordon's bacterium HS-NH1 by genome scanning The full-length 594bp of pehA gene, encoding 197 amino acids, found after comparison of blast pehA sequence and Rhodococcus YK2 Rhodococcus sp. (GI:21388688) high homology reached 98%; the full-length 852bp pehB gene, encoding 283 amino acid sequences, pehB and Gordon's Gordonia sp. P8219 sp. (GI:85677422) with 99% similarity by using gene cloning technique, we designed a pair of primers, PCR amplification, the recombinant expression plasmid pET-pehA and pET-pehB, and expressed in E.coliBL21.SDS-PAGE electrophoresis, molecular recombination enzyme PehA and PehB were detected for 27KDa and 30Kda. enzyme activity showed that the recombinant enzyme PehA and PehB on the long chain PAE with hydrolysis activity good. Laid a foundation for further study of the structure and function of the enzyme. Using the method of homologous recombination in bacteria on Gordon's (Gordonia sp.) HS-NH1 ABC and MFS Transporter gene, construct ABC transporter knockout mutant and MFS transporter knockout mutant strains, which were named as HS-NH1- MFS and HS-NH1- ABC. of delta delta pathway mutants, gene function and degradation characteristics were studied. The results show that: Gordon's bacteria (Gordonia sp.) the optimum growth conditions the degradation of HS-NH1 DOP is 30 DEG C, pH 7, the bacteria in 120 hours DOP degradation rate of 80%. mutant HS-NH1- MFS under the same conditions (temperature of 30 DEG C, pH 7) the degradation rate of DOP was significantly decreased, the degradation efficiency of only 20%; growth machine and mutant HS-NH1- ABC cannot with DOP as the sole carbon source. The substrate spectrum analysis showed that Gordon's bacteria (Gordonia sp.HS-NH1) can effectively use the common PAE (such as DOP.DEHP), and its metabolite (PCA). The mutant strain HS-NH1- Delta MFS can use two formic acid phthalate esters of long chain Class, such as DOP, DEHP and so on, but the low utilization efficiency; cannot use the short chain PAE, such as the adjacent benzene two formic acid two methyl ester (DMP), adjacent benzene two formic acid ethyl ester two (DEP) and phthalic acid (two PA) and protocatechuic acid PCA; and HS-NH1- ABC not as delta growth medium is the sole carbon source in PAE and its metabolites. The results showed that MFS and ABC transporters can transport two phthalic acid, phthalic acid two will be transported into cells for further degradation, ABC transporter protein PA as a major transport protein of MFS transporters as auxiliary transport protein.

【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：X172;Q78

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1 李景龍;Dordonia sp.菌屬peh基因簇亞克隆表達及轉(zhuǎn)運蛋白分析[D];華中師范大學;2016年

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