摘要
【目的】为山麦冬Liriope spicata果实花青素生物合成研究筛选最佳内参基因。【方法】基于山麦冬转录数据,通过变异系数以及变化倍数初步筛选出山麦冬15个候选内参基因。以幼果期和成熟期山麦冬果实为材料,利用实时荧光定量PCR(RT-qPCR)技术测定候选内参基因的表达,采用ΔCt值法、geNorm、NormFinder和BestKeeper等软件包分析候选基因的表达稳定性,最后选取来自6个基因家族(C4H、CHS、MT、UFGT、MYB和bHLH)的10个目的基因(C4H、CHS-1、CHS-2、MT、UFGT-1、UFGT-2、MYB-1、MYB-2、MYB-3、bHLH),对所选内参基因组合进行验证。【结果】4种方法分析得出的候选内参排序存在一定差异,需综合4种方法的几何平均值作为综合排序。根据geNorm软件推荐的最适内参数目为4个,即CNNM、GPR107、EF1-α、G6PD-2最稳定,PDP最不稳定。对10个目的基因表达量标准化验证发现:以CNNM、GPR107为内参组合与选用4个基因作为内参组合无显著差异,相关系数可达0.999 9,且选用双内参组合比4个内参组合的可操作性强,而不适合的内参基因(PDP)会对RT-qPCR结果产生严重偏差。【结论】以CNNM、GPR107作为组合是山麦冬果实花青素生物合成研究的最佳内参基因。
[Objective] The objective is to screen the best reference genes(RGs) for the study of anthocyanin biosynthesis in Liriope spicata fruits. [Method] Based on the transcriptome sequence data(unpublished), 15candidate RGs were selected according to the coefficient of variation and fold change value of gene expression.The expression levels of candidate RGs were determined by RT-qPCR in L. spicata young and mature fruits.The expression stability of these genes was evaluated using software packages and algorithms including ΔCt,geNorm, NormFinder and Bestkeeper. Finally, 10 target genes(C4H, CHS-1, CHS-2, MT, UFGT-1,UFGT-2,MYB-1,MYB-2,MYB-3,bHLH) from 6 gene families(C4H, CHS, MT, UFGT, MYB, and bHLH)were screened to verify the selected reference gene combinations. [Result] There was difference in the ranking of candidate RGs obtained by the 4 methods, so the geometric mean of the 4 methods was used as the comprehensive ranking. Based on geNorm, there were 4 recommended optimal internal parameters, namely CNNM, GPR107, EF1-α and G6PD-2, which were the most stable, while PDP was the most unstable. The standardization verification of the expression levels of 10 target genes showed that there was no significant difference between CNNM and GPR107 as RGs combination and 4 genes as RGs combination, and the correlation coefficient reached 0.999 9. It was more feasible to select the dual RGs combination than 4 RGs combination, while unsuitable RGs(PDP) would cause serious deviation to RT-q PCR results. [Conclusion] The combination of CNNM and GPR107 is the best reference gene for anthocyanin biosynthesis in L. spicata fruit.[Ch, 6 fig. 5 tab. 39 ref.]
作者
干思宸
师悦
梁立军
GAN Sichen;SHI Yue;LIANG Lijun(College of Landscape and Architecture,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China)
出处
《浙江农林大学学报》
CAS
CSCD
北大核心
2022年第2期307-317,共11页
Journal of Zhejiang A&F University
基金
浙江省自然科学基金资助项目(LQ19C160012)。
关键词
山麦冬
实时定量PCR
花青素合成
内参基因
Liriope spicata
quantitative real-time PCR(RT-qPCR)
anthocyanin biosynthesis
reference gene
