平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立

Reference Genes Selection and System Establishment for Real-Time q PCR Analysis in Ping'ou Hybrid Hazelnut(C. heterophylla Fisch. × C. avellana L.)

【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。

[ Objective] The objective of this article is to construct a reference gene screening system of real-time qPCR inPing＇ou hybrid hazelnut （C. heterophylla Fisch x C. avellana L, main cultivars of Corylus in China） for gene expression analysis, and provide a theoretical basis for the study of plant resource utilization and innovative breeding. [Method] Eight candidate genes were selected from the transcriptome sequencing data of the non-pollination, compatible pollination and incompatible pollination stigma of Ping＇ou hybrid hazelnut in authors＇ previous study. Four candidate genes were selected from related articles. Eight different tissues or organs, such as the blooming styles, the catkins before elongation, the young leaves, the pollen, the cambium of annual branch, the green stem, the root tip and the sucker of the main cultivar of Ping＇ou hybrid hazelnut ＇Dawei＇ were used as the samples in reverse transcriptional PCR and real-time qPCR experiments. The expression stability of twelve candidate reference genes was analyzed by geNorm, NormFinder, BestKeeper, Delta Ct and RetFinder programs. [Result] Reverse transcriptional PCR showed that the amplification of twelve primers was specific, there were significant differences in the expression of ChaSTP5 and ChaTF in different materials, and the remaining candidate reference genes were expressed in eight tissues. Real-time qPCR showed that the expression of ChlSS rRNA was at the highest level and ChaSTP5 was at the lowest level, and the remaining ten candidate reference genes belong to moderate expression. As for the stability of the candidate genes, ChaSTP5 and ChaTF were the least stable, and the stability of the remaining ten candidate genes was at a moderate level. The results of geNorm, NormFinder, BestKeeper and Delta Ct showed that ChaActin was the most stable reference gene and Ch18S rRNA ranked in the top five, while the ranking of other candidate reference genes was different. The stability analysis indicated that ChaActin and Chl8S rRNA are suitable as reference genes. The pairwise variation （V） calculated by geNorm showed that six reference genes could accurately normalize the date of real-time qPCR. There was a significant correlation between the four programs at 0.01 level, and the correlation between NormFinder and Delta Ct was the highest, Delta Ct and BestKeeper was the lowest. [Conclusion] The reference gene screening system for real-time qPCR in Ping＇ou hybrid hazelnut was set up including four main steps： primers were screened first by reverse transcriptional PCR, real-time qPCR analysis was based on primer properties and gene expression, primer stability was evaluated using four programs （geNorm, NormFinder, BestKeeper, Delta Ct） and the optimal stable reference gene was selected by the comprehensive analysis of RefFinder. As for the reference genes selection, ChaActin and ChlSS rRNA were ranked as the most stable reference genes in 8 samples.