【作者】 张隽；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2011， 博士

【摘要】 拟除虫菊酯(以下简称菊酯)是人工合成的一类高效、广谱、神经致毒性杀虫剂,被广泛应有于农业病虫害的防治和家庭卫生害虫的杀灭。作为高毒、难降解的有机磷和有机氯农药替代品,菊酯类除草剂在世界范围内得到广泛应用,目前菊酯类杀虫剂占我国农业杀虫剂使用面积的三分之一以上。相对有机磷和有机氯农药,菊酯类杀虫剂对哺乳动物低毒,环境危害较低。但最近的研究表明该类杀虫剂对哺乳动物的神经、生殖、免疫、内分泌和心血管系统等仍有明显的毒副作用；此外,菊酯类杀虫剂对蜜蜂、鱼类和水生无脊椎动物等非靶标性生物高毒,比如对鱼的急性毒性浓度一般在μg/L级。菊酯类杀虫剂在环境中的残留和降解越来越成为关注的焦点。以微生物降解为基础的生物修复是一种环境友好型污染物消除技术。本研究拟从拟除虫菊酯杀虫剂降解菌的分离筛选入手,同时研究其降解途径,通过易错PCR技术对菊酯水解酶进行体外定向进化,获得比酶活大幅提高的突变酶,解决了常规菌株难以降解联苯菊酯的问题。一、氯氰菊酯杀虫剂降解菌的分离和鉴定从生产菊酯杀虫剂的废水处理系统中分离到三株新菌株,分别命名为LQY-7T、LQY-18T和BA-3T。利用多相分类学技术对这三株菌的分类学地位进行了研究。菌株LQY-7T革兰氏染色阴性,菌体杆状,无鞭毛,无芽孢；菌落颜色为黄色,圆形并向上凸起,边缘光滑,直径2-4mm,并且粘着于平板上；细胞内无非扩散性黄色素。好氧,化能异养型。该菌株的呼吸醌为MK-6和MK-7；主要的脂肪酸为iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH, anteiso-C15:0和iso-C15:0 3-OH; DNAG+C含量为34 mol%,将菌株LQY-7鉴定为Flavobacterium属的一个新种,命名为Flavobacterium haoranii sp. nov.。LQY-18T革兰氏染色反应呈阴性,无鞭毛,无芽孢,在TSA琼脂培养基上能够形成的黄色、表面凸起、光滑湿润,有边缘规则的菌落；主要的呼吸醌为MK-7；主要的脂肪酸为summed feature 3 (C16:1ω6c and/or C6:1cω7c), iso-C15:0和iso-C17:0 3-OH);包含磷脂酰乙醇胺,鞘脂,未知氨基磷脂,未知氨基脂,未知脂；DNA G+C含量为40.3 mol%。通过以上多相分类结果和16S rRNA基因序列相似性分析结果,将LQY-18鉴定为鞘氨醇杆菌属的一个新种,命名为Sphingobacterium wenxiniae sp. nov.。菌株BA-3T革兰氏染色反应呈阴性,无芽孢,杆状,无鞭毛,在MLB固体培养基上茵落呈灰白色、圆形、边缘规整、突起、不透明、质地均匀,严格好氧；主要的脂肪酸是C16：0、C14：02 -OH和Summed feature 8 (C18:1ω6c and/or C18:1ω7c);所含极性酯主要为乙醇酸磷酯、磷脂酰甘油、双磷脂酰甘油和神经鞘糖脂类和鞘糖脂；主要的醌是Q-10,主要的多胺是亚精胺；G+C含量为63.8 mol%。因此根据表型及多相分类特征、16S rRNA系统发育和DNA-DNA杂交分析,将菌株BA-3鉴定为鞘脂菌属的一个新种,命名为江苏鞘酯菌Sphingobium jiangsuense sp. nov.。二、菊酯水解酶PytH的定向进化根据已报道的菊酯水解酶基因序列设计引物,通过PCR从降解菌株JZ-2中克隆到了菊酯水解酶pytH基因,pytH结构基因共有840个碱基,编码280个氨基酸序列。将pytH基因连接到表达质粒载体pET29a上,在E.coli BL21实现了基因的高效表达。通过易错PCR对野生型pytH基因进行随机突变,获得了1个联苯菊酯水解活力增强的突变体LB37。和野生型PytH相比,突变体LB37有2个氨基酸位点发生改变(Y19F,H127R),其水解联苯菊酯的动力学参数kcat和kcat/Km比原始PytH分别提高6和70倍。通过定点突变技术,将野生型PytH的这两个氨基酸突变为相应的突变体LB37氨基酸,获得两个突变体M19(Y19F)和M127(H127R),酶动力学参数的测定结果表明,突变体M19的kcat/Km比原始PytH提高了5.5倍,M127的kcat/Km比原始PytH提高了14倍。三、菌株JZ-2对3-苯氧基苯甲酸的代谢途径研究菌株JZ-2以3-PBA为唯一碳源生长时,能够在24小时内完全降解100 mg·L-13-PBA；利用MS/MS技术检测了该菌株降解3-PBA的代谢产物,并检测了相关降解酶的酶活,结果表明3-PBA在3-苯氧基苯甲酸1,2双加氧酶的作用下断裂醚键生成邻苯二酚和间羟基苯甲酸,邻苯二酚经1,2-双加氧酶作用开环降解,而间羟基苯甲酸不能进一步降解。这是一条不同于国内外已有报道的3-PBA微生物降解代谢新途径。用MEGA分析软件将已报道的邻苯二酚1,2-双加氧酶基因进行了同源性比对,选择高保守区域设计兼并引物,以菌株JZ-2基因组DNA为模板成功扩增出约400bp的特异性条带,通过在GeneBank上进行核酸和蛋白质序列比对,发现该片段与咔唑降解菌Sphingomonas sp. KA1 plasmid pCAR3邻苯二酚1,2-双加氧酶(GeneBank Aeeession No. AB270530)同源性为98%,将catA基因的一个片段克隆至自杀性质粒pJQ200SK,转化至E. coli DH5αλpir中获得同源重组载体pJQ-catA,通过三亲接合转入JZ-2中,通过同源重组单交换将pJQ200SK插入catA基因获得突变子JZ-2-△catA.JZ-2-△catA失去了降解邻苯二酚的功能,并且由于邻苯二酚的反馈抑制使3-PBA降解速率下降,24h降解率约为30%,并且突变株不能在含有3-PBA的无机盐平板上生长,进一步证明了3-PBA是经过邻苯二酚的代谢过程被完全降解。采用以sacB基因为反向筛选标记的同源重组载体,将3-HBA降解基因mbhDHIM整合到Sphingobium faniae JZ-2T的染色体上,构建了遗传稳定、不含外源抗性基因的可以完全矿化3-PBA的工程菌株JZ-2-mbhDHIM。四、降解菌剂对菊酯污染的植株修复技术应用应用菊酯农药降解菌剂进行了浙江丽水茶叶的菊酯污染修复研究,通过试验发现降解菌的降解效果与菌剂用量成正相关,降解菌剂对不同类菊酯的降解效果有显著差异,适量的降解菌剂对甲氰菊酯、氯氟氰菊酯有较好的降解效果,对溴氰菊酯降解效果较差。降解菌剂对甲氰菊酯、氯氟氰菊酯3d后降解率达60%以上,7d后降解率达70%以上,而对溴氰菊酯3d后降解率只有32.26%,7d后降解率56.21%,降解菌的使用时间以药后1d傍晚处理效果最好。更多还原

【Abstract】 Synthetical pyrethroids (SPs), which are synthetic version of an extract from the chrysanthemum, are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic and environmentally persistent organochlorine and organophosphorus pesticides. Currently, SPs annual sales are estimated to reach above 1.5 billion dollars and rank the second in global insecticide sales. In China, SPs account for 1/3 of total pesticides application area.Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate and organochlorine insecticides, exposure to pyrethroid pesticides might cause damage to reproductive, immune, endocrine and cardiovascular systems. Especially, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, with LC50 values less than 0.5μg/L and factor of safety lower than 0.063 to fish.Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment. Bioremediation based on the degradation ability of microorganisms was an environment friendly method in the treatment of pollutants.The aim of this study was to isolate pyrethroid-degrading strains, elucidate the degradation pathway, clone the degradation-related genes, which would be helpful to research the gene function and establish foundation of construct the genetic engineered strain by enhanced degrading capacity.Three novel bacteria strains designated LQY-7T, LQY-18Tand BA-3Twere isolated from activated sludge in a synthetic pyrethroid-manufacturing wastewater treatment facility in Yangnong Chemical Group Co., LTD., Jiangsu Province, China by using a classic enrichment method. Strains LQY-7T, LQY-18Tand BA-3T showed obviously distincted morphological, chemotaconomic and phylogentic characteristics from their cloest neighbors. Based on the polyphasic taxonomic results, the isolated strains LQY-7T, LQY-18T and BA-3Twere assigned to the members of the genus Flavobacterium, Sphingobacterium and Sphingobium.LQY-7T cells are Gram-negative rods,0.3-0.7μm in width and 1.3-2.0μm in length, non-spore-forming, and motile by gliding. Congo red is not absorbed and flexirubin-type pigments are not produced. Colonies on TSA are yellow, glistening, sticky, convex and circular with entire margins. The major fatty acids (>5%) are iso-C15:0(39.5%), iso-C15:1 G (26.3%), iso-C17:03-OH (7.9%), anteiso-C15:0(6.3%) and iso-Ci1:0 3-OH (5.6%). The major isoprenoid quinone is MK-7 (87%) and MK-6 (13%), the DNA G+C content is 34 mol%. on the basis of the phylogenetic evidence and fatty acids together with the phenotypic characteristics, strain LQY-7T should be classified as representing a novel species of the genus Flavobacterium, for which the name Flavobacterium haoranii sp. nov. is proposed.LQY-18T cells are Gram-negative, non-motile, non-spore-forming, non-flagellated, strictly aerobic rods, approximately 0.6-1.4μm in length and 0.3-0.6μm in diameter. After 3 days of incubation on TSA, colonies are 1.0-2.0 mm in diameter, yellowish, convex, circular and smooth with entire margins. The almost complete 16S rRNA gene sequence of strain LQY-18T indicated that strain LQY-18T belongs to the genus Sphingobacterium of the phylum Bacteroidetes and showed sequence similarities of 89.1-92.9% with recognized species of the genus Sphingobacterium. Phylogenetic analysis demonstrates strain LQY-18T belongs to the genus Sphingobacterium and forms a subclade with Sphingobacterium shayense CCTCC AB 209006T. The major respiratory quinone is MK-7. The major polar lipid is phosphatidylethanolamine and an unknown aminolipid and several unknown polar lipids are also detected. Sphingolipid is present. The major cellular fatty acids are summed feature 3 (C16:1ω6c and/or C16:1ω7c), iso-C15:0 and iso-C17:0 3-OH. The DNA G+C content of the type strain is 40.3 mol%. On the basis of phenotypic, genotypic and phylogenetic properties. Strain LQY-18T should be classified as a novel species of the genus of Sphingobacterium, for which the name Sphingobacterium wenxiniae sp. nov. is proposed.BA-3T cells are aerobic, Gram-negative, catalase-and oxidase-positive, non-motile, non-sporulating rods with rounded ends, approximately 0.6-0.8μm wide and 1.0-1.2μm long. Colonies on 10-fold-diluted LB agar are circular (0.2-0.4 cm in diameter), convex and cream-white. The major quinone is ubiquinone Q-10. The Major fatty acids(>5%) are summed feature 8 (C18:1ω6c and/or C18：1ω7c), C16:0, summed feature 3 (C16:1ω6c and/or C16:1ω7c) and C14:0 2-OH. The polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), sphingoglycolipids (SGL1-SGL2), glycolipid (GL) and phosphatidylcholine (PC). The major cellular polyamine is spermidine. DNA G+C content is 63.8 mol%. Strain BA-3T should be classified as a novel species of the genus of Sphingobium, for which the name Sphingobium jiangsuense sp. nov.BA-3Tis proposed.The primers were constructed based on the published sequence of the pyrethroid-hydrolying gene. The pyrethroid-hydrolying gene(pytH) was cloned by PCR from JZ-2. The analysis and alignment of the pyrethroid-hydrolying gene revealed that this gene has 840 bases, which coding for the pyrethroid hydrolase contained 280 amino acids. The pytH gene from the strain JZ-2 was linked to the expression plasmid vector pET29a, the expression strain of the gene was gained by transfer of the expression plasmid into strain BL21 (DE3).Error Prone PCR was used to generate PytH variants improvement in hydrolysis of substrates and a mutation was obtained. The variant, LB37 which had two site mutations (Y19F, H127R), exhibited increase energy for bifenthrin hydrolysis.According to the results we obtained two site-mutation variants by site-directed mutagenesis. Study of their kinetic parameters for hydrolysis of bifenthrin was performed. Results showed that M19 exhibited a 5.5-fold increase in kcat/Km values and H127R exhibited a 14-fold increase in kcat/Km values. The best variant Mn7 displayed a 6-fold increase in kcat and 70-fold increase in kcat/Km values, this is synthetical effect of two mutants.The proposal metabolic pathway of 3-Phenoxybenzoic acid was deduced as follows: strain JZ-2 metabolized 3-Phenoxybenzoic acid to m-hydroxybenzoic acid and catechol; catechol then metabolized by ortho cleavage pathway, m-hydroxybenzoic acid could not be degraded.The reported catechol 1,2-dioxygenasegenes were aligned using Clustalx software and degenerated primers were designed according to the conserved sequence. A 400bp length segment was amplified from JZ-2 genomic DNA, and sequence analysis showed a significant level of homology (98%) to catechol 1,2-dioxygenase of Sphingomonas sp. KA1 plasmid pCAR3 (GeneBank Aeeession No.AB270530). Gene catA was cloned into the suicide vector PJQ200SK for knocking out of the catA in JZ-2 and the resultant strain JZ-2-dcatA lost the ability to degrade catechol and the feedback control of hydroquinone bring on the consequence that JZ-2-AcatA can just degrade 3-PBA slightly, about 30%in 24 hours. When used strain JZ-2 to degrade the SPs residue in Tea in Lishui, Zhejiang Province, we found that the degradation rate reached 55-75%, showed that strain JZ-2 has a strong prospect.A genetically engineered microorganism(GEM) designated as JZ-2-mbhDHIM, capable of mineraling 3-PBA was successfully constructed by homologous recombination. The 3-HBA-degrading genes mbhDHIM was inserted into the chromosome of JZ-2 by using a homologous recombination vector with sacB gene as counter selectable marker. The GEM was relatively stable and without bringing any antibiotic marker. The homologous recombination events were confirmed by PCR detection.更多还原