多菌灵耐受细菌YF-2~T,YF-3~T的分离鉴定及多菌灵水解酶基因的表达
发布时间：2018-08-24 12:48
【摘要】：多菌灵是一类广谱、内吸性苯并咪唑类杀菌剂,在中国乃至世界范围生产和使用范围极广。多菌灵的化学性质稳定,在环境中半衰期较长,随着多菌灵的使用量逐年增大,在环境中的积累越来越多,其对生态环境造成的不利影响愈加凸显。多菌灵在环境中的降解过程主要通过微生物进行,国内外学者分离得到多株多菌灵降解菌株均为革兰氏阳性细菌。本实验从长期施用多菌灵的土壤中分离纯化得到两株具有多菌灵极强耐受性的革兰氏阴性细菌YF-2~T(1000mg·l-1)、YF-3~T(1200mg·l-1),本文对它们进行了多项分类研究。菌株YF-2~T呈革兰氏阴性,无鞭毛,无运动性,不形成芽孢,细胞呈短杆状(0.4-0.6μm×2.3-2.6μm)。在R2A培养基平板培养8小时后,菌落呈浅黄色,湿润,透明,易于挑取,直径约为1mm,呈饱满的圆形,边缘光滑整齐,具有金属光泽。YF-2~T的生长条件为:25～37℃,pH15.0～8.0,氯化钠浓度0～5%(w/v)。YF-2~T所含脂肪酸的主要成分为 summed feature 3(C16:1ω7c and/or C16:1ω6c),iso-C15:0,iso-C17:03-OH,C16:0 以及anteiso-C15:0;所含醌的主要组成成分为:MK-6;极性脂主要包含磷脂酰乙醇胺(PE),未知的极性脂(L1-L7)以及未知的氨基脂(AL1-AL3)。总DNA G+C mol%含量为35.2mol%。菌株 YF-2~T 与黄杆菌属(Flavobacterium)内模式菌株 Flavobacterium akiainvivensCIP110358T 和 Flavobacterium hauensm KCTC 32147T 的 16S rRNA 基因序列相似性分别为95.99%以及95.92%。综合分析,将YF-2~T鉴定为黄杆菌属(Flavobacterium)的 一个新种,并命名为 Flavobbacterium shanxiense sp.nov.(=CCTCC AB 2014079T= JCM 30153~T)。菌株YF-3~T呈革兰氏阴性,无鞭毛,无运动性,不形成芽孢,细胞呈短杆状(0.5～0.7μm×2.1～2.3μm)。在TSA培养基平板上30℃培养12小时后,菌落呈圆形,橘黄色,透明,中间凸起,边缘光滑整齐,具有金属光泽。YF-3~T的生长条件为:25～37℃,pH 5.0～8.0,氯化钠浓度0～5%(w/v)。YF-3~T所含脂肪酸的主要种类为iso-C15:0,iso-C 17:03-OH,summed feature 9(iso-C17:1 ω9cand/or C16:0 10-methyl)以及 summed feature 3(C16:1aω7c and/or C16:1 ω6c);所含鲲的主要组成成分为:MK-6;极性脂主要包含磷脂酰乙醇胺(PE),未知的极性脂(L1-L5)以及未知的氨基脂(AL1-AL2);所含多胺主要成分为:对称篙精胺以及亚精胺。菌株YF-3~T与参比菌株Chryseobacteriumhispalense AG13~T(16S rRNA基因序列相似性为98.71%)的基因组DNA同源性为31.7±2.1(33.5±3.6),与参比菌株Chryseobacterium t·aiwanense BCRC 17412~T(16S rRNA 基因序列相似性为96.93%)的基因组DNA同源性为28.4±5.4(25.7±4.4),均低于70%。综合分析,将YF-3~T鉴定为金黄色杆菌属(Chryseobacterium)的一个新种,并命名为Chryseobacteriumshandongense sp.nov.(=CCTCC AB 2014060T=JCM 30154T)。多菌灵降解的关键步骤是在多菌灵水解酶基因(mheI)的作用下将多菌灵降解为2-氨基苯并咪唑。以实验室保存的多菌灵降解菌mbc-1的多菌灵水解酶基因(mheI)密码子为基础,通过密码子优化并合成适合在枯草杆菌表达的mheI基因。将优化后mheI克隆到穿梭质粒pP43NMK上,该质粒以强启动子P43和分泌效率较高的nprB基因的信号肽作为表达元件,接着将构建好的重组质粒pP43NMK-mheI转化到枯草杆菌WB800中。成功地实现了mheI 在枯草杆菌中的高效分泌表达,为后期研制酶制剂奠定了基础。
[Abstract]:Carbendazim is a broad-spectrum, endogenous benzimidazole fungicide, which is produced and used widely in China and even in the world. Carbendazim has stable chemical properties and long half-life in the environment. With the increasing use of carbendazim year by year, the accumulation of carbendazim in the environment is increasing, and its adverse effects on the ecological environment are becoming increasingly prominent. The degradation process of carbendazim in the environment is mainly carried out by microorganisms. Many Carbendazim-degrading bacteria were isolated by domestic and foreign scholars. Two carbendazim-resistant gram-negative bacteria, YF-2~T (1000mg l-1), YF-3~T (120) were isolated and purified from the soil with long-term carbendazim application. The strain YF-2~T was gram-negative, flagella-free, motile, spore-free, and short rod-shaped (0.4-0.6 micron *2.3-2.6 micron). After 8 hours of culture on R2A medium, the colony was light yellow, moist, transparent, easy to pick up, about 1 mm in diameter, round and smooth at the edge. The growth conditions of YF-2~T are as follows: 25-37 C, pH 15.0-8.0, NaCl concentration 0-5%(w/v). The main components of fatty acids in YF-2~T are summed feature 3 (C16:17c and/or C16:16c), iso-C15:0, iso-C17:03-OH, C16:0 and ante-C15:0; the main components of quinones are MK-6; the polar lipids mainly contain polar lipids. Phosphatidylethanolamine (PE), unknown polar lipids (L1-L7) and unknown amino lipids (AL1-AL3). The total DNA G+C mol% content was 35.2 mol%. The similarity of 16S rRNA gene sequences between strain YF-2~T and Flavobacterium akiainvivens CIP110358T and Flavobacterium hauensm KCTC 32147T was 95. YF-2~T was identified as a new species of Flavobacterium and named as Flavobacterium shanxiense sp.nov. (= CCTCC AB 2014079T = JCM 30153~T). The strain YF-3~T was Gram-negative, flagella-free, motile, sporeless, and short rod-shaped (0.5-0.7 micron *2.1-2.3 micron) in TSA culture. The colony was round, orange-yellow, transparent, convex in the middle, smooth and neat with metallic luster. The growth conditions of YF-3~T were as follows: 25-37 C, pH 5.0-8.0, concentration of NaCl 0-5% (w/v). The main fatty acids in YF-3~T were iso-C15:0, iso-C 17:03-OH, features 9 (iso-C17:1 cand/or or/or). C16:0 10-methyl and summed feature 3 (C16:1 a_7c and/or C16:1_6c); the main components of the pod are: MK-6; the polar lipids mainly contain phosphatidylethanolamine (PE), the unknown polar lipids (L1-L5) and the unknown amino lipids (AL1-AL2); and the main polyamines are: symmetrical spermine and spermidine; strain YF-3-T and reference strain. The genomic DNA homology of Chryseobacterium hispalense AG13~T (16S rRNA gene sequence similarity was 98.71%) was 31.7 (+2.1) (33.5 (+3.6)) and that of reference strain Chryseobacterium t. aiwanense BCRC 17412~T (16S rRNA gene sequence similarity was 96.93%) was 28.4 (+5.4) (25.7 (+4.4.4%). 3~T was identified as a new species of Chryseobacterium and named Chryseobacterium shandongense sp.nov. (= CCTCC AB 2014060T = JCM 30154T). The key step of carbendazim degradation was to degrade carbendazim to 2-aminobenzimidazole under the action of carbendazim hydrolase gene (mheI). Based on the codon of carbendazim hydrolase gene (mheI) of bacteriolytic mbc-1, the mheI gene suitable for expression in Bacillus subtilis was optimized and synthesized. The optimized mheI was cloned into the shuttle plasmid pP43NMK. The signal peptide of strong promoter P43 and nprB gene with high secretion efficiency was used as the expression element in the plasmid, and then the constructed weight was determined. The recombinant plasmid pP43NMK-mheI was transformed into Bacillus subtilis WB800. The highly secreted expression of mheI in Bacillus subtilis was successfully achieved, which laid a foundation for the later development of enzyme preparation.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X592;X172
本文编号：2200869
[Abstract]:Carbendazim is a broad-spectrum, endogenous benzimidazole fungicide, which is produced and used widely in China and even in the world. Carbendazim has stable chemical properties and long half-life in the environment. With the increasing use of carbendazim year by year, the accumulation of carbendazim in the environment is increasing, and its adverse effects on the ecological environment are becoming increasingly prominent. The degradation process of carbendazim in the environment is mainly carried out by microorganisms. Many Carbendazim-degrading bacteria were isolated by domestic and foreign scholars. Two carbendazim-resistant gram-negative bacteria, YF-2~T (1000mg l-1), YF-3~T (120) were isolated and purified from the soil with long-term carbendazim application. The strain YF-2~T was gram-negative, flagella-free, motile, spore-free, and short rod-shaped (0.4-0.6 micron *2.3-2.6 micron). After 8 hours of culture on R2A medium, the colony was light yellow, moist, transparent, easy to pick up, about 1 mm in diameter, round and smooth at the edge. The growth conditions of YF-2~T are as follows: 25-37 C, pH 15.0-8.0, NaCl concentration 0-5%(w/v). The main components of fatty acids in YF-2~T are summed feature 3 (C16:17c and/or C16:16c), iso-C15:0, iso-C17:03-OH, C16:0 and ante-C15:0; the main components of quinones are MK-6; the polar lipids mainly contain polar lipids. Phosphatidylethanolamine (PE), unknown polar lipids (L1-L7) and unknown amino lipids (AL1-AL3). The total DNA G+C mol% content was 35.2 mol%. The similarity of 16S rRNA gene sequences between strain YF-2~T and Flavobacterium akiainvivens CIP110358T and Flavobacterium hauensm KCTC 32147T was 95. YF-2~T was identified as a new species of Flavobacterium and named as Flavobacterium shanxiense sp.nov. (= CCTCC AB 2014079T = JCM 30153~T). The strain YF-3~T was Gram-negative, flagella-free, motile, sporeless, and short rod-shaped (0.5-0.7 micron *2.1-2.3 micron) in TSA culture. The colony was round, orange-yellow, transparent, convex in the middle, smooth and neat with metallic luster. The growth conditions of YF-3~T were as follows: 25-37 C, pH 5.0-8.0, concentration of NaCl 0-5% (w/v). The main fatty acids in YF-3~T were iso-C15:0, iso-C 17:03-OH, features 9 (iso-C17:1 cand/or or/or). C16:0 10-methyl and summed feature 3 (C16:1 a_7c and/or C16:1_6c); the main components of the pod are: MK-6; the polar lipids mainly contain phosphatidylethanolamine (PE), the unknown polar lipids (L1-L5) and the unknown amino lipids (AL1-AL2); and the main polyamines are: symmetrical spermine and spermidine; strain YF-3-T and reference strain. The genomic DNA homology of Chryseobacterium hispalense AG13~T (16S rRNA gene sequence similarity was 98.71%) was 31.7 (+2.1) (33.5 (+3.6)) and that of reference strain Chryseobacterium t. aiwanense BCRC 17412~T (16S rRNA gene sequence similarity was 96.93%) was 28.4 (+5.4) (25.7 (+4.4.4%). 3~T was identified as a new species of Chryseobacterium and named Chryseobacterium shandongense sp.nov. (= CCTCC AB 2014060T = JCM 30154T). The key step of carbendazim degradation was to degrade carbendazim to 2-aminobenzimidazole under the action of carbendazim hydrolase gene (mheI). Based on the codon of carbendazim hydrolase gene (mheI) of bacteriolytic mbc-1, the mheI gene suitable for expression in Bacillus subtilis was optimized and synthesized. The optimized mheI was cloned into the shuttle plasmid pP43NMK. The signal peptide of strong promoter P43 and nprB gene with high secretion efficiency was used as the expression element in the plasmid, and then the constructed weight was determined. The recombinant plasmid pP43NMK-mheI was transformed into Bacillus subtilis WB800. The highly secreted expression of mheI in Bacillus subtilis was successfully achieved, which laid a foundation for the later development of enzyme preparation.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X592;X172
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1 杨帆;多菌灵耐受细菌YF-2~T,YF-3~T的分离鉴定及多菌灵水解酶基因的表达[D];南京农业大学;2015年
，本文编号：2200869
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