葡萄实时定量PCR中稳定内参基因的筛选  

Identification of the appropriate reference gene through using a realtime quantitative PCR in grapes

作　　者：查倩[1,2] 奚晓军[1] 蒋爱丽[1] 田益华[1] 王世平[2] 

机构地区：[1]上海农业科学院林木果树研究所,上海201403 [2]上海交通大学农学与生物技术学院,上海200240

出　　处：《果树学报》2016年第3期268-274,共7页Journal of Fruit Science

基　　金：国家葡萄产业技术体系项目(CARS-30-9);上海市农委科技兴农推广项目[沪农科推字(2012)第1-2号]

摘　　要：【目的】通过ge Norm软件筛选实时荧光定量PCR稳定内参基因,基于多内参基因评价体系进行目的基因相对表达量的计算。【方法】获得不同葡萄品种叶片和果皮中6个候选内参基因的循环阈值(Ct),利用ge Norm软件计算内参基因平均表达稳定性数值M和基因配对差异值V,从而判断内参基因最适组合。【结果】发现候选内参基因表达稳定性由高到低的排列顺序为Vv EF1r=Vv EF1-α>Vv GAPDH>Vv ACTIN>Vv ACT1>Vv UBQ,不同组织分别分析均发现Vv EF1-α和Vv EF1r的稳定性最高,且基因配对差异值V2/3为0.104,所以内参基因的最适组合为Vv EF1-α和Vv EF1r。利用ge Norm软件计算得到的多内参基因评价体系的标准化因子可应用于目的基因的相对表达量分析。【结论】ge Norm软件筛选实时荧光定量PCR稳定内参基因的方法可以应用到多条件和多组织的不同植物样本中,并用于目的基因表达谱的研究,具有重要的广泛应用价值。【Objective】A real-time quantitative PCR（q RT-PCR） is an efficient and reliable molecular technique to determine changes in a gene＇s m RNA expression. Compared with earlier methods, q RTPCR has the advantages of high speed, high sensitivity, high degrees of automation and reproducibility.The most common procedure used for q RT-PCR is the relative measurement of gene expression of interest after normalization using endogenous reference genes. The selection of suitable reference genes for q RTPCR is important for accurate normalization of the target gene expression found in specific experimental materials or conditions. Although the reference gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene. Beginning in 2002, ge Norm has been developed to select stably expressed reference genes, and to use multiple reference genes for normalization in q RT-PCR. ge Norm determines the expression stability of candidate reference genes by gene stability measure value（M）. ge Norm determines the number of candidate reference genes through using a pairwise variation V value. Reference genes are selected and analyzed by using ge Norm to calculate the target gene expression in various tissues of different grape cultivars.【Methods】Experiment Material consisted of eight different grape cultivars including：‘Jufeng＇‘Jumeigui＇‘Zuijinxiang＇‘Shenfeng＇‘Shenyu＇‘Shenhua＇‘Xiahei＇and‘Hupei 1#＇. Total RNA was extracted using a RNA extraction kit（Omega） according to the manufacturer＇s instructions, and the DNA was digested at 37 ℃ for 30 min using DNase I（Takara） according to the manufacturer＇s instructions. The concentration of each RNA sample was determined using a spectrophotometer. Only samples with an absorbance ratio at 260 and 280 nm（A260/A280） ≥ 1.9 were used. To obtain the first-strand cDNA, 1 μg of each DNA-free RNA sample was used for reverse trans

关 键 词：葡萄 ge NORM 内参基因 标准化因子 实时荧光定量PCR 

分 类 号：S663.1[农业科学—果树学]