菰黑粉菌细胞核和线粒体的荧光标记菌株构建
Construction of Fluorescently Labeled Strains of Nucleus and Mitochondria for Ustilago esculenta
汤近天 1杨芙容 1章蕾蕾 1李钰康 1张雅芬 1傅慧兰 1夏文强 1崔海峰 1叶子弘1
作者信息
- 1. 中国计量大学生命科学学院/浙江省生物计量与检验检疫技术重点实验室,杭州 310018
- 折叠
摘要
菰(Zizania latifolia)的茎部膨大形成可食用的茭白,与菰黑粉菌(Ustilago esculenta)的侵染有关,而侵染与否取决于菰黑粉菌的二型态转换.在二型态转换过程中,细胞核与线粒体的变化动态仍然未知.本研究将定位于细胞核的组蛋白H1编码基因和线粒体定位信号(mitochondrial targeting signa,MTS)编码序列,分别连接GFP和mCherry荧光蛋白基因后,构建细胞核和线粒体荧光标记载体.采用根癌农杆菌介导遗传转化法(Agrobacterium tumefaciens-mediated transformation,ATMT),以菰黑粉菌野生型菌株UeT14和UeT55为出发菌株来进行遗传转化,得到细胞核或线粒体中稳定表达红、绿色荧光蛋白的重组菌株:UeT55-H1-GFP、UeT14-H1-mCherry、UeT55-MTS-GFP和UeT14-MTS-mCherry.在UeT55-H1-GFP和UeT14-H1-mCherry菌株中,孢子中央有明亮的荧光点,与DAPI染色所形成的蓝色荧光共定位.在UeT55-MTS-GFP、UeT14-MTS-mCherry菌株中,孢子内可见小点状荧光,且绿色荧光与MitoTracker形成的红色荧光共定位.本研究获得了细胞核及线粒体能稳定表达荧光蛋白标记的菰黑粉菌,为研究菰黑粉菌生长发育、融合和侵染的分子机制提供了重要的参考和材料.
Abstract
Stem swollen of Zizania latifolia was associated with the successful infection of Ustilago esculenta,which depended on the dimorphic transition of U.esculenta.The dynamic changes of the nucleus and mitochondria during the process of dimorphic transition remain unknown.This study will focus on the coding gene for histone H1 and the coding sequence for the mitochondrial targeting signal(MTS)in the nucleus,respectively.After connecting them with the GFP and mCherry fluorescent protein genes,respectively,fluorescent labeling vectors for the nucleus and mitochondria was constructed.Wild-type strains UeT14 and UeT55 of U.esculenta as experimental materials to genetic transformation by Agrobacterium tumefaciens-mediated transformation(ATMT),and the recombinant strains that stably expressed red and green fluorescent proteins in nucleus or mitochondria were obtained:UeT55-H1-GFP,UeT14-H1-mCherry,UeT55-MTS-GFP and UeT14-MTS-mCherry.In UeT55-H1-GFP and UeT14-H1-mCherry strains,bright and round fluorescent spots could be seen in the center of the spores,which were co-located with the blue fluorescence formed by DAPI.In UeT55-MTS-GFP and UeT14-MTS-mCherry strains,small dot green or red fluorescence could be seen in the spores,and the green fluorescence was co-located with the red fluorescence formed by MitoTracker.In this study,the stable expression of fluorescent protein in nucleus or mitochondria was obtained,which provides an important reference and material for studying the molecular mechanism of growth,development,fusion and infection of U.esculenta.
关键词
菰黑粉菌/菰/细胞核/线粒体/荧光蛋白标记Key words
Ustilago esculenta/Zizania latifolia/Nucleus/Mitochondria/Fluorescent protein labeling引用本文复制引用
基金项目
国家自然科学基金(32100154)
国家自然科学基金联合基金(U20A2043)
中国计量大学基本科研业务费项目(220016)
出版年
2024
国家科技期刊平台
NSTL
万方数据