红花檵木实时荧光定量PCR分析内参基因的选择  

Reference Genes Selection for Real-time Fluorescence Quantitative PCR in Lorpetalum chinense var.rubrum

作　　者：张力[1] 于晓英[1] 符红艳[1] 陈己任[1] 李达[1] 陈海霞[1] 

机构地区：[1]湖南农业大学园艺园林学院,长沙410128

出　　处：《中国农学通报》2013年第10期11-17,共7页Chinese Agricultural Science Bulletin

基　　金：国家自然基金面上项目"DREB和XET基因聚合表达的分子基础"(31071826);湖南省高等学校科学研究项目重点项目"红花檵木叶色高温返青的分子机理研究"(10A057)

摘　　要：实时荧光定量PCR(qRT-PCR)是目前基因定量表达分析的重要方法,而适宜内参基因的选择对目的基因表达特征的准确分析至关重要,研究目的是筛选适宜于红花檵木目的基因qRT-PCR分析的校正内参基因。以红花檵木‘大叶红’(Lorpetalum chinense var.rubrum‘Dayehong’)的芽、嫩茎、叶、花瓣、种子和愈伤组织为材料,采用qRT-PCR技术比较了4个常用看家基因微管蛋白基因(α-tubulin)、肌动蛋白基因(β-actin)、18S核糖体RNA(18SrRNA)与甘油醛-3-磷酸-脱氢酶基因(GAPDH)的表达情况。4个看家基因均能特异扩增并显示较高的扩增效率;经geNorm和NormFinder程序综合分析,当采用qRT-PCR分析红花檵木不同器官和组织中的基因表达差异时,以GAPDH表达最为稳定;在比较不同发育阶段的叶片及明暗处理的愈伤组织中的基因表达差异时,β-actin表达最为稳定。当采用qRT-PCR分析红花檵木不同器官和组织及不同发育阶段的叶片与明暗处理的愈伤组织基因表达差异时,可以分别选择GAPDH和β-actin作为校正内参基因。Real-time fluorescence quantitative PCR（qRT-PCR） has been widely used in gene expression analysis to date, and selection of suitable reference genes for qRT-PCR according to specific experimental materials or conditions is very important for accurate normalization of target gene expression. The goal of this study was to selection the suitable reference genes for qRT-PCR in L. chinense var. rubrum. In this study, expression of four commonly used housekeeping genes such as α-tubulin, β-actin, 18S ribsomal RNA （18S rRNA） and glyceraldehyde-3-phosphate dehydrogenase （GAPDI-1） were assessed by qRT-PCR in various tissues （buds, young stems, leaves, petals, seeds and callus）. All the candidate reference genes could amplify specifically with high efficiency in qRT-PCR reaction. To determine the expression stability of these genes, the data were determined by two commonly used applets （geNorm and NormFinder）, which produced highly comparable results depending on the experimental parameters. GeNorm and NormFinder algorithms revealed that β-actin and GAPDH were both in the highest stability. GAPDH could be used as a reference gene for qRT-PCR in various organs and tissues of L. chinense var. rubrum, and β-actin could be used as a reference gene for qRT-PCR in leaves at different stages and callus among diverse treatments of L. chinense var. rubrum.

关 键 词：红花檵木 实时荧光定量PCR 内参基因 选择 

分 类 号：S687.2[农业科学—观赏园艺]