【作者】 杨帆；

【导师】 洪青；

【作者基本信息】 南京农业大学 ， 微生物学， 2015， 硕士

【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。更多还原

【Abstract】 Carbendazim is a broad spectrum,systemic benzimidazole fungicide,which has been widely used in China and abroad.Chemical character of carbendazim is stable and its half-life period is long.As the usage amount of carbendazim has increased year by year,itsnegative influence on environment becomes more obvious and serious.Carbendazim is degradaed mainly by microorganisms in the environment;so far strainscapable of degrading carbendazimwere Gram-staining positive.Two carbendazim tolerant strains YF-2~T(1000mg·L-1)andYF-3~T(1200mg·L-1)were isolated from the soilsubjected to the long-term application of carbendazim.Polyphasic classification was carried out on these two strains.Cells of YF-2~T are Gram-staining negative,non-motile,non-spore-forming,rod-shaped,approximately 0.4-0.6 μm width,2.3-2.6 μm in length.Colonies on R2A agar inoculated 8h are circular with a shiny surface and entire edges,light-yellow-pigmented(flexirubin-type),translucent and mucoid.Growth occurs at between 25-37℃,at pH 5.0-8.0,and with 0-5%(w/v)NaCl.The strain contains menaquinone-6 as the major respiratory quinone.Polar lipids consist of phosphatidylethanolamine,seven unidentified lipids and three unidentified aminolipids.The predominant fatty acids(≥5%)are summed feature 3(comprising C16:1 ω7c and/or C16:1 ω6c),iso-C15:0,iso-C17:0 3-OH,C16:0 and anteiso-C15:0.The G+C contents of strain YF-2~T is 35.2%.Analysis based on sequence of the 16S rRNA gene showed that strain YF-2~T was closely related to strains Flavobacterium akiainvivens IK-1T and Flavobacterium hauense BX12~T with 95.99%and 95.92%sequence similarity,respectively.In conclusion,strain YF-2~T shouldrepresent a novel species of the genus Flavobacterium,for which the name Flavobacterium shanxiense sp.nov.is proposed.Cells of YF-3~T are Gram-stainingnegative,non-motile,non-spore-forming,rod-shaped,approximately 0.5-0.7 μm in width,2.1-2.3 μm in length.Colonies grown 12h on TSA are circular with a shiny surface and entire edges,yellow-orange-pigmented(flexirubin-type,non-diffusible),translucent and mucoid.Growth conditions are 25-37℃,at pH 5.0-8.0,with 0-5%(w/v)NaCl.Menaquinone-6 is the main respiratory quinone.The predominant fatty acids(≥50%)are iso-C15:0,iso-C17:0 3-OH,summed feature 9(comprising iso-C17:1ω9cand/or C16:0 10-methyl)and summed feature 3(comprising C16:1ω7c and/or C16:1 ω6c).Polar lipids consist of phosphatidylethanolamine,five unidentified lipids and two unidentified aminolipids.Sym-homospermidine is the predominant polyamine but minor amounts of spermidine arealso present.The DNA G+C content of strain YF-3~T is 37.0mol%.The 16S rRNA gene sequence of strain YF-3~T showed similarity withChryseobacterium hispalense AG13~T and Chryseobacterium taiwanense BCRC 17412~T are 98.71%and 96.93%respectively.DNA-DNA relatedness to reference strains Chryseobacterium hispalense AG 13~T and Chryseobacterium taiwanense BCRC 17412~T are 31.7±2.1(33.5±3.6)and 28.4±5.4(25.7±4.4)respectively,which are well below the threshold of 70%recommended for the delineation of bacterial species.In conclusion,strain YF-3~T shouldrepresent a novel species of the genus Chryseobacterium,for which the name Chryseobacterium shandongense sp.nov.is proposed.The key step of carbendazim degradation is the hydrolysis of carbendazimto2-Aminobenzimidazole,which is catalyzed by MheI.The codons of the mhel gene from the carbendazim-degrading strain mbc-1 was optimized and synthesized in order to be suitable for the its expression in Bacillus subtilis WB800,and then the optimized gene(mhel)was cloned intoshuttle expression vector pP43NMK,which has strong promoter P43 and nprB signal peptide.Then the constructed plasmidpP43NMK-mheIis transformedinto Bacillus subtilis WB800.Gene(mhel)is expressed successfully and efficiently,which provides the foundationfor the production of enzymeMheI.更多还原