摘要
为构建灰盖拟鬼伞Coprinopsis cinerea的核定位蛋白重组表达系统,本研究通过蛋白序列比对和信息学分析,预测了灰盖拟鬼伞组蛋白H2B的核定位序列,构建了融合组蛋白H2B核定位序列的绿色荧光蛋白(green fluorescent protein,GFP)重组表达载体,将该载体转入灰盖拟鬼伞AmutBmut菌株,检测组蛋白H2B核定位序列是否可以引导GFP定位在细胞核区域。研究结果显示,在灰盖拟鬼伞β-微管蛋白启动子的驱动下,经过密码子优化的GFP基因可以在鬼伞体内表达、产生绿色荧光。同时来自灰盖拟鬼伞组蛋白H2Bb(CC1G_07639)的核定位序列,可以成功地引导GFP入核。本研究成功地构建了灰盖拟鬼伞核定位蛋白重组表达系统,为后续优化灰盖拟鬼伞的CRISPR/Cas9基因编辑系统、深入研究灰盖拟鬼伞的子实体发育机制奠定了基础。
To construct a recombinant nuclear localization(NL)expression system for Coprinopsis cinerea,the NL sequence(NLS)of histone H2B was determined by amino acid sequence alignment and informatics analysis.The recombinant expression vector of fusion green fluorescent protein(GFP)with the NLS of histone H2B was constructed and transferred into C.cinerea AmutBmut strain to examine whether this NLS can guide GFP expression to the nucleus.The results showed that the codon-optimized GFP gene was expressed to produce green fluorescence under the control of the β-tubulin promoter of C.cinerea.Furthermore,NLS from histone H2Bb(CC1G_07639)can successfully induce GFP expression in the nucleus.In this study,a recombinant NL expression system for C.cinerea with proven efficiency was constructed.Our findings are beneficial to optimizing the CRISPR/Cas9 gene editing system and further exploring the mechanisms of fruiting body formation in C.cinerea.
作者
王宇
赵静
田宣
金盛宇
冉妍妍
黄小娟
袁静
袁生
刘中华
WANG Yu;ZHAO Jing;TIAN Xuan;JIN Shengyu;RAN Yanyan;HUANG Xiaojuan;YUAN Jing;YUAN Sheng;LIU Zhonghua(Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics,Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources,College of Life Sciences,Nanjing Normal University,Nanjing 210023,Jiangsu,China)
出处
《菌物学报》
CAS
CSCD
北大核心
2022年第1期68-77,共10页
Mycosystema
基金
国家自然科学基金(31600053)。
关键词
核定位序列
重组表达
启动子
绿色荧光蛋白
nuclear localization sequence
recombinant expression
promoter
green fluorescent protein
