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
河南省科学院生物研究所有限责任公司,郑州 450008; 河南省微生物工程重点实验室,郑州 450008 河南省微生物工程重点实验室,郑州,450008 河南省科学院生物研究所有限责任公司,郑州,450008
2016-08-25
DNA 降解 pUC19 粘质沙雷氏菌
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。%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.
