Dordonia sp.菌属peh基因簇亚克隆表达及转运蛋白分析

发布时间：2018-02-15 21:55

本文关键词： DOP HPLC pehA pehB 克隆表达基因敲除　出处：《华中师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：邻苯二甲酸二辛酯(Di-n-octyl ortho-phthalate,DOP)是邻苯二甲酸的酯化衍生物,属于邻苯二甲酸酯类(PAEs,又名酞酸酯)。应用于涂料、食品包装材料、润滑剂、药品、化肥、化妆品、农药、医疗器械、儿童玩具等领域,是一种广泛利用的增塑剂。DOP具有生物富集作用,对人体造成神经毒性、生殖毒性和多器官癌变等病变,严重威胁到人的生存与发展,已经引起国内外广泛关注。美国环保局(EPA)已经将其列入优先控制有毒污染物,DEP、DMP与DOP也被中国政府确定为优先控制污染物。毒理学实验表明DOP对人体具有致突变性、致癌性、致畸形和拟／抗甲状腺激素活性、拟／抗雌激素活性等内分泌干扰特性；同时是一种环境激素类化合物。环境中的DOP主要通过生物降解和光解这两种途径,光解速度较慢、周期较长,因此生物降解是DOP降解的最主要方式。目前对DOP微生物降解的研究包括降解菌的筛选、鉴定、降解途径及降解特性分析,对降解途径中蛋白的主要功能的研究较少。本文以戈登氏菌属Gordonia sp. HS-NH1为材料,克隆表达了邻苯二甲酸酯降解过程中的关键酶邻苯二甲酸酯酶(PehA)和邻苯二甲酸单酯酶(PehB),并研究了其功能和特性。结合基因敲降技术构建了缺失转运蛋白ABC/MFS基因的敲除突变株,并对突变株的代谢途径与降解特性进行了研究。主要结果如下：通过基因组扫描得知戈登氏菌HS-NH1的pehA基因全长594bp,编码197个氨基酸,经过blast比对发现其与红球菌属Rhodococcus sp. YK2的pehA序列(GI：21388688)同源性较高,达到98%；pehB基因全长852bp,编码283个氨基酸,与戈登氏菌属Gordonia sp. P8219的pehB序列(GI：85677422)具有99%的相似性。采用基因克隆技术,通过设计引物、PCR扩增,成功构建了重组表达质粒pET-pehA和pET-pehB,并在E.coliBL21得到高效表达。SDS-PAGE电泳分析,重组酶PehA和PehB的分子量分别为27KDa和30Kda。酶活性检测表明重组酶PehA和PehB对长链的PAE具有良好的水解活性。为进一步深入研究该酶的功能与结构奠定了一定的基础。利用同源重组的方法敲除了戈登氏菌(Gordonia sp.)HS-NH1的ABC和MFS转运蛋白基因,成功构建了ABC转运蛋白敲除突变菌株和MFS转运蛋白敲除突变菌株,分别命名为HS-NH1-△MFS和HS-NH1-△ABC。对突变菌株的代谢途径、基因功能与降解特性进行了研究。研究结果表明：戈登氏菌(Gordonia sp.)HS-NH1降解DOP的最适生长条件为30℃、pH 7.0,该菌能在120小时内DOP降解率为80%。突变株HS-NH1-△MFS在同等条件下(温度为30℃、pH 7.0)对DOP的降解效率显著下降,降解效率仅有20%；而突变菌株HS-NH1-△ABC不能以DOP为唯一碳源的培养机中生长。底物广谱性分析表明,戈登氏菌(Gordonia sp.HS-NH1)能高效利用常见的PAE(如DOP.DEHP),及其代谢物(PCA)。突变菌株HS-NH1-△MFS能够利用长链的邻苯二甲酸酯类,如DOP、DEHP等,但利用效率较低；不能利用短链的PAE,如邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)以及邻苯二甲酸(PA)与原儿茶酸PCA等；而HS-NH1-△ABC不能在PAE及其代谢产物作作为唯一碳源的培养基中生长。说明MFS与ABC转运蛋白都能转运邻苯二甲酸,将邻苯二甲酸运输到细胞内进行进一步降解,ABC转运蛋白作为PA主要的运输蛋白,MFS转运蛋白作为辅助的运输蛋白。
[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.

【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X172;Q78

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