黄花大苞姜花药发育qRT-PCR内参基因筛选

Selection of reference genes for quantitative real-time PCR during anther development of Caulokaempferia coenobialis (Zingiberaceae)

关键词:  黄花大苞姜, 花药, 内参基因, qRT-PCR, BestKeeper, geNorm, Normfinder

Key words:  Caulokaempferia coenobialis, anther, reference gene, quantitative realtime PCR(qRT-PCR), BestKeeper, geNorm, Normfinder

摘要:

qRT-PCR技术具有定量准确、灵敏度高、重复性好等特点,被广泛用于基因表达分析。内参基因的稳定性对于准确分析实验结果非常重要。该研究以黄花大苞姜(Caulokaempferia coenobialis)花粉母细胞时期(PMC)、四分体时期(TET)、成熟花粉时期(MP)的花药组织为材料,基于3个阶段花药转录组表达谱数据以及常用传统内参基因,筛选出Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)、Malate dehydrogenase (MDH)、α-tubulin3 (TUA3)、β-tubulin7 (TUB7)和Actin6 (ACT6)作为候选内参基因,进行qRT-PCR分析; 并运用BestKeeper、geNorm和Normfinder软件综合分析5个候选内参基因在黄花大苞姜花药发育过程中的表达稳定性。结果表明:MDH和TUB7的表达最稳定,ACT6的稳定性最差; 分别以MDH和TUB7作为内参,分析GBE1在黄花大苞姜花药发育中的表达模式,并与该基因在花药转录组中的表达模式做相关系数分析,3种表达模式结果一致,进一步验证了MDH和TUB7的表达稳定性。这说明MDH和TUB7适合作为qRT-PCR分析黄花大苞姜花药发育过程中相关基因表达模式的内参基因。该研究结果为黄花大苞姜花药发育分子机制相关研究奠定了基础,也为姜科花药发育相关内参基因的选择提供了参考。

Abstract:

Quantitative real-time PCR(qRT-PCR)has been widely used in gene expression analysis with quantitative accuracy, high sensitivity and good repeat ability. The stability of the reference genes is very important to accurate analysis of the target genes expression. Anther tissues from pollen mother cells stage(PMC), tetrad stage(TET)and mature pollen stage(MP)of Caulokaempferia coenobialis (Zingiberaceae)were used to select reference genes. According to the developing anther transcriptome expression profile data and traditional reference genes reported, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Malate dehydrogenase (MDH), alpha tubulin3 (TUA3), beta tubulin7 (TUB7) and Actin6 (ACT6)were selected as candidate reference genes for qRT-PCR analysis. The expression stability of five candidate reference genes during the anther development was comprehensive analysis by BestKeeper, geNorm and Normfinder software. The results showed that the expression of MDH and TUB7 were the most stable, and the ACT6 was the worst. The expression patterns of GBE1 during anther development was obtained based on MDH and TUB7 as reference gene respectively. And the expression stability of MDH and TUB7 was further verified by correlation coefficient analysis(Pearson)with the anther transcriptome expression profile data. The results showed that MDH and TUB7 could serve as qRT-PCR reference gene to analyse the gene expression pattern related to anther developing in C. coenobialis. The study would provide research fundamental data for molecular mechanism research of anther development in C. coenobialis, and reference for the selection of reference genes in other Zingiberaceae species during anther development.