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Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background

文献类型： 外文期刊

作者： Zhou, Bujin 1 ; Chen, Peng 1 ; Khan, Aziz 1 ; Zhao, Yanhong 2 ; Chen, Lihong 1 ; Liu, Dongmei 3 ; Liao, Xiaofang 1 ; Kong 1 ;

作者机构： 1.Guangxi Univ, Coll Agr, Key Lab Plant Genet & Breeding, Nanning, Peoples R China

2.Guangxi Acad Agr Sci, Cash Crop Inst, Nanning, Peoples R China

3.Shangqiu Normal Univ, Coll Biol & Food Sci, Shangqiu, Peoples R China

关键词： cytoplasmic male sterility (CMS);reference genes;RT-qPCR;kenaf;gene expression;pollen development

期刊名称：FRONTIERS IN PLANT SCIENCE （ 影响因子：5.753； 五年影响因子：6.612 ）

ISSN： 1664-462X

年卷期： 2017 年 8 卷

页码：

收录情况： SCI

摘要： Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the production of dysfunctional pollen. Based on reliable reference gene-normalized real-time quantitative PCR (RT-qPCR) data, examining gene expression profile can provide valuable information on the molecular mechanism of kenaf CMS. However, studies have not been conducted regarding selection of reference genes for normalizing RT-qPCR data in the CMS and maintainer lines of kenaf crop. Therefore, we studied 10 candidate reference genes (ACT3, ELF1A, G6PD, PEPKR1, TUB, TUA, CYP, GAPDH, H3, and 18S) to assess their expression stability at three stages of pollen development in CMS line 722A and maintainer line 722B of kenaf. Five computational statistical approaches (GeNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder) were used to evaluate the expression stability levels of these genes. According to RefFinder and GeNorm, the combination of TUB, CYP, and PEPKR1 was identified as an internal control for the accurate normalization across all sample set, which was further confirmed by validating the expression of HcPDIL5-2a. Furthermore, the combination of TUB, CYP, and PEPKR1 was used to differentiate the expression pattern of five mitochondria F1F0-ATPase subunit genes (atp1, atp4, atp6, atp8, and atp9) by RT-qPCR during pollen development in CMS line 722A and maintainer line 722B. We found that atp1, atp6, and atp9 exhibited significantly different expression patterns during pollen development in line 722A compared with line 722B. This is the first systematic study of reference genes selection for CMS and will provide useful information for future research on the gene expressions and molecular mechanisms underlying CMS in kenaf.

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