虎杖PcMYB1基因实时荧光定量PCR检测方法的建立  

Establishment of Real-time Fluorescence Quantitative PCR for Detection of PcMYB1 Gene of Polygonum Cuspidatum

作　　者：林艳丽 李鲁汉 廖辉 覃建兵[1] 伍翔 王岩岩[1] 吴端阳 张鸿杰 柳忠玉[1] Lin Yanli;Li Luhan;Liao Hui;Qin Jianbing;Wu Xiang;Wang Yanyan;Wu Duanyang;Zhang Hongjie;Liu Zhongyu(College of Life Science,Yangtze University,Jingzhou 434025,Hubei)

机构地区：[1]长江大学生命科学学院,湖北荆州434025

出　　处：《长江大学学报（自然科学版）》2020年第3期91-94,I0007,共5页Journal of Yangtze University(Natural Science Edition)

基　　金：国家自然科学基金项目“虎杖中一个新的MYB转录因子在白藜芦醇合成中的功能研究”(81803670);长江大学大学生创新创业训练计划项目“虎杖PcMYB1反义植物表达载体构建及对虎杖毛状根的转化”(2019279)。

摘　　要：为建立实时荧光定量PCR(qRT-PCR)检测虎杖(Polygonum cuspidatum)转录因子PcMYB1基因表达的方法,将PcMYB1基因的ORF片段亚克隆于pUC19载体,得到重组质粒pUC19-PcMYB1,再以pUC19-PcMYB1作为标准样品,建立目的基因的标准曲线,优化qRT-PCR反应体系与反应条件,分析qRT-PCR的灵敏度。结果表明,在qRT-PCR体系中,退火温度为54.1℃时检测结果最好;构建的目的基因标准曲线,其循环阈值与模板拷贝数对数值呈良好的线性关系,扩增效率为92.3%;该方法最低可检测101copies/μL的目的基因。To establish a real-time fluorescent quantitative PCR(qRT-PCR)method for detecting the expression of the transcription factor PcMYB1 of Polygonum cuspidatum,the ORF fragment of PcMYB1 gene was subcloned into pUC19 vector to obtain the recombinant plasmid pUC19-PcMYB1;Then,pUC19-PcMYB1 was taken as the standard sample to establish the standard curve of the target gene,optimize the qRT-PCR reaction system and reaction conditions,and analyze the sensitivity of qRT-PCR.The results show that the best detection results are obtained when the annealing temperature is 54.1℃in qRT-PCR system.There is a good linear relationship between the cyclic threshold and the template copy number logarithm value of the standard curve of constructed target gene,and the amplification efficiency is 92.3%.This method at least can be used to detect 101copies/μL of the target genes.

关 键 词：虎杖(Polygonum cuspidatum) PcMYB1基因 MYB转录因子 实时荧光定量PCR(qRT-PCR) 

分 类 号：Q786[生物学—分子生物学] Q949.744