一株基因克隆干扰菌的鉴定及其发酵液降解DNA活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.032

生物技术通报 ›› 2016, Vol. 32 ›› Issue (8): 221-225.doi: 10.13560/j.cnki.biotech.bull.1985.2016.08.032

一株基因克隆干扰菌的鉴定及其发酵液降解DNA活性分析

刁文涛^{1, 2}, 王雪妍², 陈晓飞¹, 冯菲¹, 宁萌², 周伏忠^{1, 2}

1. 河南省科学院生物研究所有限责任公司,郑州 450008;
2. 河南省微生物工程重点实验室,郑州 450008

修回日期:2015-12-08 出版日期:2016-08-25 发布日期:2016-08-25
作者简介:刁文涛,男,博士,研究方向：微生物资源和分子遗传;E-mail：diao_wen_tao@163.com
基金资助:
河南省省级重点实验室专项（122300413214）,河南省重点攻关计划项目（142102210087,152102210167）

Isolation and Identification of a Bacterium Disturbing Molecular Cloning,and Analysis of DNA-Degrading Activity of Its Fermentation Broth

DIAO Wen-tao^{1, 2}, WANG Xue-yan², CHEN Xiao-fei¹, FENG Fei¹, NING Meng², ZHOU Fu-zhong^{1, 2}

1. Institute of Biology Co.,Ltd.,Henan Academy of Sciences,Zhengzhou 450008;
2. Key Laboratory of Microbial Engineering of Henan Province,Zhengzhou 450008

Revised:2015-12-08 Published:2016-08-25 Online:2016-08-25

摘要/Abstract

摘要： 旨在寻找导致基因克隆失败的原因,检测整个电泳环境中是否存在对DNA有强降解力的菌株。依据菌体形态,革兰氏反应以及16S rDNA序列对DNA降解菌株进行鉴定,琼脂糖凝胶电泳分析菌株对DNA的降解活性。结果显示,从DNA电泳槽中分离到了一株菌,革兰氏染色鉴定为阴性菌,对该菌进行液体培养,利用质粒pUC19作为底物,检测该菌发酵液对DNA的降解能力,发现该菌发酵液能够迅速并彻底地降解DNA,其最佳降解温度为45℃,将该菌命名为DD（DNA degrading）。对该菌的16S rDNA进行测序比对后,发现其与粘质沙雷氏菌（Serrtia marcescens）的16S rDNA序列同源性高达100% 。结合菌体形态,革兰氏反应以及16S rDNA序列结果,DD菌株为一株粘质沙雷氏菌,DNA降解活性分析显示其具有很强的DNA降解能力。

关键词: DNA降解, pUC19, 粘质沙雷氏菌

Abstract: This work aims to analyze the reason that resulted in the failure of cloning and to check if there are some microorganisms strongly degrading DNA in the environment of electrophoresis chamber. The colony morphology,gram staining and the 16S rDNA sequence were utilized to identify the strain degrading DNA,and agarose gel electrophoresis was for analyzing the DNA-degrading activity. As results,a strain was isolated from DNA electrophoresis chamber,and identified as gram-negative bacterium. Then it was cultured in YPD medium,and the capacity of the fermentation broth degrading DNA was detected using plasmid pUC19 as substrate. The strain quickly and sufficiently degraded DNA at the optimal temperature 45℃,thus this strain was designated DD（DNA degrading）. The sequence alignment of the DD by the 16S rDNA showed that this DD had 100% identity with the that of Serrtia marcescens. Conclusively,the bacterial strain DD isolated from DNA electrophoresis chamber is a Serrtia marcescens strain possessing significant DNA-degrading activity based on the results of colony morphology,gram staining,and 16S rDNA sequence.

Key words: DNA degradation, pUC19, Serratia marcescens

刁文涛, 王雪妍, 陈晓飞, 冯菲, 宁萌, 周伏忠. 一株基因克隆干扰菌的鉴定及其发酵液降解DNA活性分析[J]. 生物技术通报, 2016, 32(8): 221-225.

DIAO Wen-tao, WANG Xue-yan, CHEN Xiao-fei, FENG Fei, NING Meng, ZHOU Fu-zhong. Isolation and Identification of a Bacterium Disturbing Molecular Cloning,and Analysis of DNA-Degrading Activity of Its Fermentation Broth[J]. Biotechnology Bulletin, 2016, 32(8): 221-225.

参考文献

[1] 萨姆布鲁克 J, 拉塞尔 DW. 分子克隆实验指南[M]. 第3版. 黄培堂, 译. 北京：科学出版社, 2008.
[2] Eaves GN, Jeffries CD. Isolation and properties of an exocellular nuclease of Serratia Marcescens[J]. J Bacteriol, 1963, 85（2）：273-278.
[3] 黄小龙, 黄东益, 周双清, 等. 粘质沙雷氏菌产灵菌红素培养基的筛选[J]. 生物技术, 2009, 5：65-67.
[4] 韦凤. 产灵菌红素粘质沙雷氏菌发酵条件优化及色素性质的研究[D]. 金华：浙江师范大学, 2012.
[5] 周围. 粘质沙雷氏菌次生代谢产物灵菌红素的生理与生物学活性的研究[D]. 重庆：西南大学, 2014
[6] 张丹峰, 杨培周, 姜绍通. 一株分离自鳜鱼肠道的粘质沙雷氏菌（Serratia marcescens）HFUT 1301的鉴定及灵菌红素的分析[J]. 现代食品科技, 2015, 6：78-83, 204.
[7] Wilfert JN, Barrat FF, Ewing WH, et al. Serratia marcescens：biochemical, serological, and epidemiological characteristics and antibiotic susceptibility of strains isolated at Boston City Hospital[J]. Appl Microbiol, 1970, 19（2）：345-352.
[8] Wilkowske CJ, Washington JA 2nd, Martin WJ, et al. Serratia marcescens. Biochemical characteristics, antibiotic susceptibility patterns, and clinical significance[J]. JAMA, 1970, 214（12）：2157-2162.
[9] Grimont PA, Grimont F. The genus Serratia[J]. Annu Rev Microbiol, 1978, 32：221-248.
[10] Sokalski SJ, Jewell MA, Asmus-Shillington AC, et al. An outbreak of Serratia marcescens in 14 adult cardiac surgical patients associated with 12-lead electrocardiogram bulbs[J]. Arch Intern Med, 1992, 152（4）：841-844.
[11] Suh Y, Alpaugh M, Krause KL, et al. Differential secretion of isoforms of Serratia marcescens extracellular nuclease[J]. Appl Environ Microbiol, 1995, 61（11）：4083-4088.
[12] Matsuo H, Kosaka K, Iwata K, et al. Necrotizing soft tissue infection caused by Serratia marcescens in a patient treated with tocilizumab[J]. Kansenshogaku Zasshi, 2015, 89（1）：53-55.
[13] Bromke BJ, Hammel JM. Regulation of extracellular protease formation by Serratia marcescens[J]. Can J Microbiol, 1979, 25（1）：47-52.
[14] Lyerly DM, Kreger AS. Importance of serratia protease in the pathogenesis of experimental Serratia marcescens pneumonia[J]. Infect Immun, 1983, 40（1）：113-119.
[15] 魏巍, 贺淹才, 方柏山, 等. 粘质沙雷氏菌几丁质酶（ChiC）基因克隆及其生物信息学分析[J]. 江西农业大学学报, 2006, 3：444-448.
[16] 朱绮霞, 陈英, 张搏, 黄日波. 粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究[J]. 生物技术, 2010, 5：12-16.
[17] Filimonova MN, Krause KL, Benedik MJ. Kinetic studies of the Serratia marcescens extracellular nuclease isoforms[J]. Biochem Mol Biol Int, 1994, 33（6）：1229-1236.
[18] 杨海艳. 灵杆菌非特异性核酸酶的原核表达、纯化及活性分析[D]. 杨凌：西北农林科技大学, 2011.
[19] 徐虹, 徐美娟, 杨套伟, 等. 温度对粘质沙雷氏菌合成灵菌红素的影响[J]. 微生物学报, 2014, 5：517-524.
[20] 蔡俊杰. 粘质沙雷氏菌胞外核酸酶重组表达纯化及鉴定[D]. 上海：上海交通大学, 2011.
[21] 邓丹丹. 灵杆菌非特异性核酸酶的固定化及酶学特性研究[D]. 杨凌：西北农林科技大学, 2013.

一株基因克隆干扰菌的鉴定及其发酵液降解DNA活性分析

Isolation and Identification of a Bacterium Disturbing Molecular Cloning,and Analysis of DNA-Degrading Activity of Its Fermentation Broth

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