摘要
目的:构建菰黑粉菌(Ustilago esculenta P.Henn.)高效的遗传转化体系。方法:基于菰黑粉菌RNA-seq结果,筛选出10个高强度启动子,以pUMa932为基础载体,将异源启动子P_(otef)使用DNA无缝克隆技术替换成所筛选的启动子构建相关载体,以菰黑粉菌UeT55为实验材料,基于PEG介导原生质体转化构建表达菌株。通过qRT-PCR测定不同启动子下eGFP的表达量,并用Image J软件分析测定转化子的荧光强度。结果:启动子P_(mdh1)、P_(mix17)、P_(eno)、P_(qcr8)驱动的eGFP基因表达量和荧光强度与对照P_(otef)无显著差异,启动子P_(mia40)、P_(sti1)、P_(odc2)驱动的eGFP基因表达量和荧光强度均显著低于对照,而启动子P_(hsp)、P_(ef)、P_(cox12)驱动的eGFP基因表达量分别比P_(otef)驱动的高9倍、5倍、2倍以上,荧光强度分别高6倍、4倍、1.5倍以上,均显著高于对照。结论:P_(hsp)启动子表达能力最强,且表达稳定,适宜作为强启动子用于菰黑粉菌遗传转化体系构建。
Aims:This paper aims to construct an efficient genetic transformation system of Ustilago esculenta.Methods:Based on the RNA-seq results of U.esculenta,10 high-strength promoters were screened.Based on pUMa932,the heterologous promoter P_(otef)was replaced by the selected promoter by the DNA seamless cloning technique.The expression strain was constructed based on PEG-mediated protoplast transformation using UeT55 as the experimental material.The expression of eGFP under different promoters was determined by qRT-PCR;and the fluorescence intensity of the transformants was analyzed by Image J.Results:The eGFP gene expression and fluorescence intensity,driven by promoters P_(mdh1),P_(mix17),P_(eno)and P_(qcr8),were not significantly different from the control P_(otef)and were significantly lower than the control driven by promoters P_(mia40),P_(sti1)and P_(odc2);while the expression and fluorescence intensity of eGFP gene,driven by promoter P_(hsp),P_(ef) and P_(cox12),were significantly higher than those in the control group.The expression was 9,5 and 2 times higher than that driven by P_(otef);and the fluorescence intensity was 6,4 and 1.5 times higher than that driven by P_(otef),respectively.Conclusions:P_(hsp)promoter has the strongest expression ability and stable expression.So it is suitable to be used as a strong promoter for the construction of genetic transformation systems of U.esculenta.
作者
卞加慧
胡映莉
汤近天
夏文强
叶子弘
张雅芬
BIAN Jiahui;HU Yingli;TANG Jintian;XIA Wenqiang;YE Zihong;ZHANG Yafen(Zhejiang Provincial Key Laboratory of Biometrology,Inspection and Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,China)
出处
《中国计量大学学报》
2022年第1期106-115,共10页
Journal of China University of Metrology
基金
国家自然科学基金项目(No.U20A2043)。
关键词
菰黑粉菌
启动子
遗传转化
启动子P
荧光强度
Ustilago esculenta
promoter
genetic transformation
Phsp
fluorescence intensity
