多菌靈耐受細(xì)菌YF-2~T,YF-3~T的分離鑒定及多菌靈水解酶基因的表達(dá)
發(fā)布時間：2018-08-24 12:48
【摘要】：多菌靈是一類廣譜、內(nèi)吸性苯并咪唑類殺菌劑,在中國乃至世界范圍生產(chǎn)和使用范圍極廣。多菌靈的化學(xué)性質(zhì)穩(wěn)定,在環(huán)境中半衰期較長,隨著多菌靈的使用量逐年增大,在環(huán)境中的積累越來越多,其對生態(tài)環(huán)境造成的不利影響愈加凸顯。多菌靈在環(huán)境中的降解過程主要通過微生物進(jìn)行,國內(nèi)外學(xué)者分離得到多株多菌靈降解菌株均為革蘭氏陽性細(xì)菌。本實驗從長期施用多菌靈的土壤中分離純化得到兩株具有多菌靈極強(qiáng)耐受性的革蘭氏陰性細(xì)菌YF-2~T(1000mg·l-1)、YF-3~T(1200mg·l-1),本文對它們進(jìn)行了多項分類研究。菌株YF-2~T呈革蘭氏陰性,無鞭毛,無運動性,不形成芽孢,細(xì)胞呈短桿狀(0.4-0.6μm×2.3-2.6μm)。在R2A培養(yǎng)基平板培養(yǎng)8小時后,菌落呈淺黃色,濕潤,透明,易于挑取,直徑約為1mm,呈飽滿的圓形,邊緣光滑整齊,具有金屬光澤。YF-2~T的生長條件為:25～37℃,pH15.0～8.0,氯化鈉濃度0～5%(w/v)。YF-2~T所含脂肪酸的主要成分為 summed feature 3(C16:1ω7c and/or C16:1ω6c),iso-C15:0,iso-C17:03-OH,C16:0 以及anteiso-C15:0;所含醌的主要組成成分為:MK-6;極性脂主要包含磷脂酰乙醇胺(PE),未知的極性脂(L1-L7)以及未知的氨基脂(AL1-AL3)。總DNA G+C mol%含量為35.2mol%。菌株 YF-2~T 與黃桿菌屬(Flavobacterium)內(nèi)模式菌株 Flavobacterium akiainvivensCIP110358T 和 Flavobacterium hauensm KCTC 32147T 的 16S rRNA 基因序列相似性分別為95.99%以及95.92%。綜合分析,將YF-2~T鑒定為黃桿菌屬(Flavobacterium)的 一個新種,并命名為 Flavobbacterium shanxiense sp.nov.(=CCTCC AB 2014079T= JCM 30153~T)。菌株YF-3~T呈革蘭氏陰性,無鞭毛,無運動性,不形成芽孢,細(xì)胞呈短桿狀(0.5～0.7μm×2.1～2.3μm)。在TSA培養(yǎng)基平板上30℃培養(yǎng)12小時后,菌落呈圓形,橘黃色,透明,中間凸起,邊緣光滑整齊,具有金屬光澤。YF-3~T的生長條件為:25～37℃,pH 5.0～8.0,氯化鈉濃度0～5%(w/v)。YF-3~T所含脂肪酸的主要種類為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);所含鯤的主要組成成分為:MK-6;極性脂主要包含磷脂酰乙醇胺(PE),未知的極性脂(L1-L5)以及未知的氨基脂(AL1-AL2);所含多胺主要成分為:對稱篙精胺以及亞精胺。菌株YF-3~T與參比菌株Chryseobacteriumhispalense AG13~T(16S rRNA基因序列相似性為98.71%)的基因組DNA同源性為31.7±2.1(33.5±3.6),與參比菌株Chryseobacterium t·aiwanense BCRC 17412~T(16S rRNA 基因序列相似性為96.93%)的基因組DNA同源性為28.4±5.4(25.7±4.4),均低于70%。綜合分析,將YF-3~T鑒定為金黃色桿菌屬(Chryseobacterium)的一個新種,并命名為Chryseobacteriumshandongense sp.nov.(=CCTCC AB 2014060T=JCM 30154T)。多菌靈降解的關(guān)鍵步驟是在多菌靈水解酶基因(mheI)的作用下將多菌靈降解為2-氨基苯并咪唑。以實驗室保存的多菌靈降解菌mbc-1的多菌靈水解酶基因(mheI)密碼子為基礎(chǔ),通過密碼子優(yōu)化并合成適合在枯草桿菌表達(dá)的mheI基因。將優(yōu)化后mheI克隆到穿梭質(zhì)粒pP43NMK上,該質(zhì)粒以強(qiáng)啟動子P43和分泌效率較高的nprB基因的信號肽作為表達(dá)元件,接著將構(gòu)建好的重組質(zhì)粒pP43NMK-mheI轉(zhuǎn)化到枯草桿菌WB800中。成功地實現(xiàn)了mheI 在枯草桿菌中的高效分泌表達(dá),為后期研制酶制劑奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X592;X172
本文編號：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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X592;X172
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 楊帆;多菌靈耐受細(xì)菌YF-2~T,YF-3~T的分離鑒定及多菌靈水解酶基因的表達(dá)[D];南京農(nóng)業(yè)大學(xué);2015年
，本文編號：2200869
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