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Validation of reference genes for accurate normalization of gene expression with quantitative real-time PCR in Haloxylon ammodendron under different abiotic stresses

Abstract

Haloxylon ammodendron plays an important role in maintaining the structure and function of the entire ecosystem where it grows. No suitable reference genes have been reported in H. ammodendron plants to date. In this study, a total of 8 reference genes (18S, ACT1, ACT7, UBC18, TUA5, GAPDH, EF- and UBQ10) were selected from the available trancriptome database, and the expression stability of these 8 candidate genes was validated under different abiotic stress with three different statistical algorithms (geNorm, NormFinder and BestKeeper). The results produced from different models were in agreement with each other essentially: 18S and TUA5 were the most stable genes under drought stress, 18S, the most stable gene under heat stress and mechanical damage, ACT7 and UBC18, stable under salt stress while TUA5 and GAPDH expressed constantly under mechanical damage, and ACT1 expressed steadily under cold conditions. Expression profiles of several stress response genes, including FT-5, FT-9, DREB2A and DREB2C, were further confirmed with various candidate reference genes. None of the candidate genes showed a constant expression among all tested samples. Hence, it’s essential to use more than one reference gene in order to guarantee the accuracy of quantitative real-time PCR. The results of this study will contribute to the accuracy and reliability in transcripts quantification, which is of significance to transcription-based studies and applications in this important shrub H. ammodendron.

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Funding

This work was funded by the National Natural Science Foundation of China (No. 31260181).

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Correspondence to Hua Zhang or Hao Ma.

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Supplementary Fig. 1

Phenotypic characteristics of one-year old seedlings of H. ammodendron. (a) General view of an untreated one-year old seedling growing in a tube filled with sand. (b) Assimilation branches separated from an untreated one-year old seedlings (Bar = 2 cm) (JPEG 218 kb)

Supplementary Fig. 2

Amplification of full length coding domains of ACT1, 18S, TUA5, ACT7, EF-, GAPDH, UBC18 and UBQ10. M:DL 2000 DNA marker. Unit: bp (JPEG 722 kb)

Supplementary Fig. 3

Specificity of qRT-PCR amplicons. (a) 1.8% agarose gel electrophoresis showing amplification of a single product at the expected size for each reference gene. M represents 100 bp and 250 bp DNA Ladder. Lane 1-8 indicate ACT1, 18S, TUA5, ACT7, EF-, GAPDH, UBC18 and UBQ10, respectively. (b) Dissociation curves with single peak were generated from all amplicons (JPEG 1060 kb)

Supplementary Table 1

Primers for cloning full length coding domains (ACT1, 18S, TUA5, ACT7, EF-, GAPDH, UBC18 and UBQ10) and qRT-PCR (FT-5, FT-9, DREB2A and DREB2C) (DOCX 39 kb)

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Wang, B., Du, H., Yao, Z. et al. Validation of reference genes for accurate normalization of gene expression with quantitative real-time PCR in Haloxylon ammodendron under different abiotic stresses. Physiol Mol Biol Plants 24, 455–463 (2018). https://doi.org/10.1007/s12298-018-0520-9

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Keywords

  • Haloxylon ammodendron
  • Reference genes
  • Abiotic stress
  • Expression stability