平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立

doi:10.3864/j.issn.0578-1752.2017.12.020

Abstract

Abstract: 【Objective】 The objective of this article is to construct a reference gene screening system of real-time qPCR in Ping’ou hybrid hazelnut (C. heterophylla Fisch × 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 RefFinder 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 Ch18S 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 Ch18S 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 Ch18S rRNA were ranked as the most stable reference genes in 8 samples.

Key words: Ping’ou hybrid hazelnut(C. heterophylla Fisch. ×, C. avellana L.), quantitative real-time PCR, reference gene, stability, system establishment

YANG Dan, LI Qing, WANG GuiXi, MA QingHua, ZHU LiQuan. Reference Genes Selection and System Establishment for Real-time qPCR Analysis in Ping’ou Hybrid Hazelnut (C. heterophylla Fisch. × C. avellana L.)[J].Scientia Agricultura Sinica, 2017, 50(12): 2399-2410.

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Reference Genes Selection and System Establishment for Real-time qPCR Analysis in Ping’ou Hybrid Hazelnut (C. heterophylla Fisch. × C. avellana L.)

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