Dataset Information

Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments.

ABSTRACT: Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC8224294 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Selection and Verification of Appropriate Reference Genes for Expression Normalization in <i>Cryptomeria fortunei</i> under Abiotic Stress and Hormone Treatments.

Zhang Yingting Y Zhu Lijuan L Xue Jinyu J Yang Junjie J Hu Hailiang H Cui Jiebing J Xu Jin J

Genes 20210521 6

<i>Cryptomeria fortunei</i> has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization i ...[more]

PMID: 34064253

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Project description:Salsola ferganica is a natural desert herbaceous plant in the arid area of western and northwestern China. Because of its salt tolerance and drought resistance, it is of great significance in desert afforestation and sand-fixing capacity. There has been much research on the genes involved in plants under desert stresses in recent years. The application of the best internal reference genes for standardization was a critical procedure in analyzing the gene expression under different types. Even so, the reference gene has not been reported in the application of gene expression normalization of S. ferganica. In this study, nine reference genes (TUA-1726, TUA-1760, TUB, GAPDH, ACT, 50S, HSC70, APT, and U-box) in S. ferganica were adopted and analyzed under six different treatments (ABA, heat, cold, NaCl, methyl viologen (MV), and PEG). The applicability of candidate genes was evaluated by statistical software, including geNorm, NormFinder, BestKeeper, and RefFinder, based on their stability values in all the treatments. These results indicated that the simultaneous selection of two stable reference genes would fully standardize the optimization of the normalization research. To verify the feasibility of the above internal reference genes, the CT values of AP2/ERF transcription factor family genes were standardized using the most (ACT) and least (GAPDH) stable reference genes in S. ferganica seedlings under six abiotic stresses. The research showed that HSC70 and U-box were the most appropriate reference genes in ABA stressed samples, and ACT and U-box genes were the optimal references for heat-stressed samples. TUA-1726 and U-box showed the smallest value in gene expression levels of cold treatment. The internal reference groups of the best applicability for the other samples were U-box and ACT under NaCl treatment, ACT and TUA-1726 under MV stress, HSC70 and TUB under PEG treatment, and ACT in all samples. ACT and U-box showed higher stability than the other genes based on the comprehensive stability ranking of RefFinder, as determined by the geometric mean in this study. These results will contribute to later gene expression studies in other closely related species and provide an important foundation for gene expression analysis in S. ferganica.

Dataset Information

Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments.

Publications

Selection and Verification of Appropriate Reference Genes for Expression Normalization in <i>Cryptomeria fortunei</i> under Abiotic Stress and Hormone Treatments.

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