CRISPR/dCas9在苜蓿中华根瘤菌中的建立与应用  

Establishment and application of CRISPR/dCas9 in Sinorhizobium meliloti 320

作　　者：吴香莹 董会娜[2] 刘振权 张大伟[1,2] WU Xiangying;DONG Huina;LIU Zhenquan;ZHANG Dawei(Dalian Polytechnic University, Dalian 116034, China;Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China)

机构地区：[1]大连工业大学食品学院,辽宁大连116034 [2]中国科学院天津工业生物技术研究所,天津300308

出　　处：《工业微生物》2019年第6期32-38,共7页Industrial Microbiology

基　　金：天津市杰出青年科学基金(17JCJQJC45300);国家自然科学基金(No.31800086)

摘　　要：本文探讨了CRISPR/dCas9系统对苜蓿中华根瘤菌Sinorhizobium meliloti 320内源hemE基因的弱化效果,以提高其维生素B 12的能力。实验首先验证了CRISPR/dCas9系统及CRISPR/dCas9系统不同N20靶向位点在S.meliloti 320中对外源基因gfp的弱化效果。结果显示,当添加诱导剂1%木糖时,单位OD荧光值降低为对照菌株的34.18%,当N20靶位靠近翻译起始位点时,对结构基因弱化效果明显。进一步探究CRISPR/dCas9对S.meliloti 320中内源的弱化效果发现,在加入终浓度为1%木糖诱导后,hemE基因的转录水平与对照相比下降了28.3%。将hemE基因弱化后的工程菌进行摇瓶发酵,其维生素B 12产量较对照株提高了9.86%。研究结果显示,hemE基因的弱化有效促进了代谢流引向维生素B 12合成,CRISPR/dCas9系统对S.meliloti 320内源结构基因具有较好的弱化效果。The weakening effects of the CRISPR/dCas9 system on the endogenous hemE gene of Sinorhizobium meliloti 320 were explored in order to improve its ability to produce vitamin B 12.The experiment first verified the weakening effects of the foreign gene gfp in S.meliloti 320 by different N20 targeting sites in the CRISPR/dCas9 system.The results showed that when the inducer 1% xylose was added,the OD fluorescence value decreased to 34.18% of the control strain,and when the N20 target site was close to the translation initiation site,the structural gene weakening effects was obvious.Further investigation of the weakening effects of CRISPR/dCas9 on the endogenous source of S.meliloti 320 revealed that the transcription level of the hemE gene decreased by 28.3%,when compared with that of the control after the final concentration of 1% xylose was added.The engineered bacteria weakened by the hemE gene were subjected to shake flask fermentation,and the vitamin B 12 yield was increased by 9.86% when compared with that of the control strain.The results showed that the weakening of hemE gene effectively promoted the metabolism of vitamin B 12,and the CRISPR/dCas9 system had good weakening effects on the endogenous structural gene of S.meliloti 320.

关 键 词：维生素B12 SINORHIZOBIUM MELILOTI CRISPR/dCas9 hemE基因 

分 类 号：S54[农业科学—作物学]