植物乳杆菌WLPL04的荧光标记及其表征  

Fluorescence labeling and characterization of Lactiplantibacillus plantarum WLPL04

作　　者：钟常城 王祺安 张启蒙 赵香香 王志跃 魏华[1,2,3] 陶雪莹[1,3] ZHONG Changcheng;WANG Qi'an;ZHANG Qimeng;ZHAO Xiangxiang;WANG Zhiyue;WEI Hua;TAO Xueying(State Key Laboratory of Food Science and Resources,Nanchang University,Nanchang 330047,China;Sino German Joint Research Institute,Nanchang University,Nanchang 330047,China;International Institute of Food Innovation,Nanchang University,Nanchang 330299,China)

机构地区：[1]南昌大学食品科学与资源挖掘全国重点实验室,江西南昌330047 [2]南昌大学中德联合研究院,江西南昌330047 [3]南昌大学国际食品创新研究院有限公司,江西南昌330299

出　　处：《南昌大学学报（理科版）》2024年第2期156-165,共10页Journal of Nanchang University(Natural Science)

基　　金：国家自然科学基金资助项目(31800037,32060030)。

摘　　要：植物乳杆菌WLPL04源自健康母乳,能在肠道定植。对植物乳杆菌WLPL04进行荧光标记,为研究其在体内的定植、移位及分布提供重要实验材料。以pMG36e为骨架,含3种不同启动子(P_(X),P_(23)和P_(ldh))的mCherry红色荧光蛋白基因作为标记基因,构建重组质粒pMG36e-P_(X)-mCherry、pMG36e-P_(23)-mCherry和pMG36e-P_(ldh)-mCherry,转化大肠杆菌,通过PCR、质粒DNA酶切、菌落颜色观察和激光共聚焦显微镜观察进行验证。验证构建成功后进一步将3种重组质粒分别导入植物乳杆菌WLPL04。结果显示导入质粒pMG36e-P_(X)-mCherry、pMG36e-P_(23)-mCherry的植物乳杆菌WLPL04,其菌体在激光共聚焦显微镜下均显示明显红色荧光;而导入pMG36e-P_(ldh)-mCherry的菌体其红色荧光较弱。此外,通过生长曲线和扫描电镜观察分析不同重组质粒对菌株生长及形态的影响,结果表明3种质粒对植物乳杆菌WLPL04的生长及菌体形态没有显著影响。以上结果表明植物乳杆菌WLPL04已被红色荧光蛋白mCherry成功标记,可为后续研究其在体内的定植、移位及分布提供重要实验材料。Lactiplantibacillus plantarum WLPL04,originated from healthy breast milk,can colonize the intestine.This article aimed to fluorescently label L.plantarum WLPL04,providing important experimental materials for studying its colonization,translocation,and distribution in vivo.In this study,the mCherry red fluorescent protein gene containing three different promoters(P_(X),P_(23) and P_(ldh))was used as the marker gene,and the recombinant plasmids pMG36e-P_(X)-mCherry,pMG36e-P_(23)-mCherry and pMG36e-P_(ldh)-mCherry were constructed.They were transformed into Escherichia coli and verified by PCR,plasmid DNA digestion,colony color observation and laser confocal microscope observation.After successful validation and construction,the three recombinant plasmids were further introduced into L.plantarum WLPL04.The results showed that L.plantarum WLPL04,which was introduced with plasmids pMG36e-P_(X)-mCherry and pMG36e-P_(23)-mCherry,showed significant red fluorescence under laser confocal microscopy,while those carrying the pMG36e-P_(ldh)-mCherry plasmid displayed weaker red fluorescence.In addition,the effects of different recombinant plasmids on the growth and morphology of L.plantarum WLPL04 were observed and analyzed through growth curves and scanning electron microscopy.The results showed that the three plasmids had no significant effect on the growth and cell morphology of L.plantarum WLPL04.The above results indicated that L.plantarum WLPL04 has been successfully labeled with the red fluorescent protein mCherry,providing important experimental materials for subsequent research on its colonization,translocation,and distribution in vivo.

关 键 词：植物乳杆菌WLPL04 mCherry荧光标记 启动子 表达 

分 类 号：Q786[生物学—分子生物学]