RNA-seq Based Discovery of New Reference Genes and Primers in Brassica napus under Aluminum Stress

doi:10.7668/hbnxb.20191565

Abstract

Abstract: In order to study the stability of the endogenous reference genes in Brassica napus under aluminum stress, and discover new reference genes in the root tissues that were mainly poisoned by aluminum, the root tissues of aluminum-tolerant variety Ganyouza 7 and aluminum-sensitive variety Rongyou 18 in Brassica napus at seedling stage were used as the research materials, and the control groups(0 h) and 2 time gradients(3, 24 h) of aluminum stress(100 μmol/L AlCl₃, pH 4.5) treatment groups were set respectively in nutrient solution culture. Firstly, five candidate endogenous reference genes(Actin7, ARFA1E, SAP5, UXS3, UBC9) which met the characteristics of housekeeping genes were selected by transcriptome sequencing with reference genome(RNA-seq) and differential gene expression analysis(FPKM). Subsequently, real-time fluorescence relative quantitative PCR analysis, amplification plot and melt curve analysis, amplification efficiency analysis(LinRegPCR), gene expression stability analysis(geNorm, NormFinder, BestKeeper) and relative expression level analysis(REST 2009) were used successively. Results indicated that new primers of candidate endogenous reference genes developed in this study had high specificity. The expression stability of the four candidate endogenous reference genes(Actin7, ARFA1E, SAP5, UXS3) selected from the transcriptome sequencing data had been verified. According to the comprehensive ranking of expression stability from high to low, the order was UXS3, SAP5, ARFA1E, Actin7, and the optimal combination was SAP5 and UXS3. In summary, the new primers of candidate endogenous reference genes developed had high specificity, and could improve the accuracy of real-time fluorescence relative quantitative PCR experiments in aluminum stress experimental system, and newly discovered endogenous reference genes(Actin7, ARFA1E, SAP5, UXS3) could be used in real-time fluorescence relative quantitative PCR experiments of aluminum stress experimental system as new endogenous reference genes.

Key words: Brassica napus, Endogenous reference gene, RNA-seq, Real-time fluorescence relative quantitative PCR, Expression stability analysis, Aluminum stress

CLC Number:

Q786

DING Ge, HUANG Yang, CHEN Lunlin, LI Shuyu, SONG Laiqiang, XIONG Jie. RNA-seq Based Discovery of New Reference Genes and Primers in Brassica napus under Aluminum Stress[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(1): 1-9. doi: 10.7668/hbnxb.20191565.

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