平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立

发布时间：2018-12-16 09:06

【摘要】：【目的】构建中国榛属植物主要栽培种平欧杂种榛实时荧光定量PCR内参基因的筛选体系,并筛选稳定的内参基因,为榛属植物的基因表达分析提供内参基因,进而为其植物资源利用和创新育种研究提供理论基础。【方法】利用课题组前期对平欧杂种榛不同亲和性授粉、授粉后不同时间的雌蕊转录组测序数据,结合相关文献搜索,共选取12个候选内参基因;以平欧杂种榛主栽品种‘达维’的盛花期雌蕊、未伸长期雄花序、幼嫩叶片、花粉、一年生枝形成层、嫩茎、根尖、根蘖等8个不同组织器官为研究材料;通过反转录PCR初筛,实时荧光定量PCR检测表达量,并利用ge Norm、Norm Finder、Best Keeper、Delta Ct程序和Ref Finder在线网站评价内参基因的稳定性。【结果】反转录PCR初筛表明,12个候选内参基因引物的特异性良好,Cha STP5和Cha TF在不同组织器官材料中表达存在明显差异,其余候选内参基因在8个组织中均有表达。实时荧光定量PCR的表达谱分析表明,Ch18S r RNA的表达量最高,Cha STP5表达量最低,其余10个候选内参基因均为中等表达量;Cha STP5和Cha TF的稳定性最差,其余10个候选内参基因的稳定性处于中等水平。ge Norm、Norm Finder、Best Keeper和Delta Ct的结果表明,Cha Actin均排名第一,为最稳定的内参基因,Ch18S r RNA的排名均在前五,而其他候选内参基因在不同程序分析结果中的排名存在差异。稳定性综合分析表明,Cha Actin和Ch18S r RNA的稳定性良好,即在8个不同组织器官中表达量均一且在4个稳定性分析程序中均排名靠前,适合作为内参基因;ge Norm程序的变异系数分析则表明,选取6个内参基因便可对RT-q PCR的数据进行精准的标准化处理;相关性分析表明,4个程序均在0.01水平上呈现显著的相关性,Norm Finder和Delta Ct程序的相关性最高,Delta Ct与Best Keeper的相关性最低。【结论】建立了平欧杂种榛实时荧光定量PCR内参基因的筛选体系:以反转录PCR初筛引物,荧光定量PCR分析引物特性及基因表达,4个程序单独评价引物稳定性,Ref Finder综合分析选出最适稳定内参基因,并选出榛属植物8个不同组织器官中最为稳定的2个内参基因Cha Actin和Ch18S r RNA。
[Abstract]:[objective] to construct the screening system of real-time fluorescence quantitative PCR for the internal reference gene of the main cultivated species of Corylus in China, and to screen the stable internal reference gene, and to provide the internal reference gene for the gene expression analysis of the genus Corylus. Then it provides a theoretical basis for the utilization of plant resources and the study of innovative breeding. [methods] using the data of different compatibility pollination and different time after pollination of Fructus heterophylla, combining with the relevant literature search, A total of 12 candidate internal reference genes were selected. Eight different tissues and organs, such as pistil, immature male inflorescence, young leaves, pollen, annual shoot cambium, tender stem, root tip, root tiller, etc. By reverse transcription PCR screening, real-time fluorescence quantitative PCR was used to detect the expression, and ge Norm,Norm Finder,Best Keeper,Delta Ct program and Ref Finder online website were used to evaluate the stability of internal reference gene. [results] reverse transcription PCR screening showed that, The specificity of 12 candidate endogenous reference gene primers was good, the expression of Cha STP5 and Cha TF in different tissues and organs was different, and the other candidate endogenous reference genes were expressed in 8 tissues. The expression profile of real-time quantitative PCR showed that the expression of Ch18S r RNA was the highest, and that of Cha STP5 was the lowest, and the other 10 candidate internal reference genes were all moderately expressed. The stability of Cha STP5 and Cha TF was the worst, and the stability of the other 10 candidate genes was at the medium level. The results of ge Norm,Norm Finder,Best Keeper and Delta Ct showed that, Cha Actin was the most stable gene. Ch18S r RNA ranked in the top five, while other candidate internal reference genes were ranked differently in different program analysis results. The stability analysis showed that the stability of, Cha Actin and Ch18S r RNA was good, that is, the expression levels of, Cha Actin and Ch18S r RNA were uniform in 8 different tissues and organs, and ranked first among the four stability analysis programs, so they were suitable as internal reference genes. The coefficient of variation analysis of ge Norm program showed that six internal reference genes could be used to standardize the data of RT-q PCR. The correlation analysis showed that the correlation between, Norm Finder and Delta Ct was the highest at 0. 01 level. The correlation between Delta Ct and Best Keeper was the lowest. [conclusion] A screening system for real-time fluorescent quantitative PCR gene of Corylus heterophylla was established: reverse transcription PCR primer, fluorescent quantitative PCR analysis primer characteristics and gene expression were established. Four programs were used to evaluate the stability of primer by comprehensive, Ref Finder analysis. The most stable internal reference genes Cha Actin and Ch18S r RNA. were selected from 8 different tissues and organs of Hazel.
【作者单位】：西南大学农学与生物科技学院;中国林业科学研究院林业研究所/国家林业局林木培育实验室/林木遗传育种国家重点实验室;
【基金】：国家自然科学基金(31500560) 中国林科院基本科研业务费专项经费(CAFYBB2016QB002)
【分类号】：S664.4

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