南京农业大学学报2024，Vol.47Issue(2)：205-212,8.DOI:10.7685/jnau.202301001
甜瓜属异源多倍体不同倍性材料内参基因的筛选及评估
Reference gene selection and evaluation for Cucumis allopolyploids with different ploid levels
摘要
Abstract
[Objectives]Polyploidy is widespread in plants kingdom.However,the lack of reference genes has severely limited the research progress of polyploidy-related gene expression.The present study aimed to improve the accuracy and reproducibility of gene expression analyses in polyploids by establishing a set of stable reference genes.[Methods]The abundance of 10 candidate reference genes(UBI-ep,ACT,ACT3,TUA,EF-1α,CACS,TIP41,F-box,CYP and UBQ)were compared by real-time fluorescence quantitative analysis(qPCR)at different ploidy levels of Cucumis including artificial allotetraploid,their diploid parents and triploid offsprings.The stability of these candidate reference genes expression was analyzed by geNorm and NormFinder software.Furthermore,the correlation of relative transcript abundance estimated by qPCR analysis and transcriptome sequencing(RNA-seq)data was calculated to evaluate the stability of candidate reference genes.[Results]The qPCR results showed that the reference gene CYP in cucumber had the highest expression with a CT value of 15.8,and the reference gene F-box in tetraploid material had the lowest expression with a CT value of 30.5.Four stable reference genes were selected by combining the results of geNorm and NormFinder,which were F-box,TIP41,ACT3 and ACT.Among them,F-box and TIP41 had a lower stability value M than ACT3 and ACT genes at the polyploid level,but their expression abundance were low.When ACT3 was used as the internal reference,the relative transcript abundance correlation coefficient between qPCR and RNA-seq was extremely significant(R2=0.844,P＜0.01).But the correlation was minimum when F-box was an internal parameter.[Conclusions]Attention should be paid to the selection of reference genes when comparing transcript abundance across different ploidy levels.TIP41 could be selected as the internal reference gene for the low abundance expression genes of multiple ploidy materials in melon.For highly expressed genes,ACT3 could be used as an internal reference gene.关键词
甜瓜属/多倍体/内参基因/实时荧光定量PCR/稳定性Key words
Cucumis/polyploidy/reference gene/quantitative real-time PCR/stability分类
农业科技引用本文复制引用
王盼乔,虞夏清,翟于菲,赵勤政,孟雅,朱早兵,李季,陈劲枫..甜瓜属异源多倍体不同倍性材料内参基因的筛选及评估[J].南京农业大学学报,2024,47(2):205-212,8.基金项目
国家自然科学基金项目(32102388,31430075) （32102388,31430075）
江苏省自然科学基金项目(BK2022148) （BK2022148）
河南农业大学博士启动基金(30501121) （30501121）
