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Exploring reliable reference genes for gene expression normalization in Karenia mikimotoi using real-time PCR

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Abstract

The dinoflagellate Karenia mikimotoi is one of the most noxious species that can form harmful algal blooms in coastal areas worldwide, causing serious damage to aquaculture and fisheries. In the present study, twelve commonly reported candidate genes (cob, cyc, eif4e, ef2, mdh, sam, α-tub, β-tub, actin, cal, 18s, and gapdh) were selected to evaluate their expression stability under different temperatures, salinities, phosphorus and nitrogen limitation, and different time points over a diel cycle in K. mikimotoi. In addition, three algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate and select the most suitable reference genes. It was observed that α-tub and eif4e were the most suitable reference genes for all samples. In contrast, ef2 and 18s genes showed least stability. Further, a list of stable genes, including α-tub, eif4e, gapdh, and mdh was generated that can be used as reference genes in specific environmental condition in K. mikimotoi. The gene expression profile of KFcp gene was evaluated over a diel cycle using the most stable reference genes. Results revealed that the expression of KFcp with transcripts abundances was higher in light period and lower in dark period, indicating similar gene expression profile of this gene in typically reported algae. This study provides suitable reference genes for further research on the expression and regulation of genes related to important biochemical processes in K. mikimotoi, improving our understanding of the molecular mechanism of its behind toxin secretion and bloom formation

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Funding

This work was funded by the National Natural Science Foundation of China (grant nos. 41976130 and 41606121), the Demonstration Project for Innovative Development of Fuzhou’s Marine Economy during the 13th Five-Year Plan, China (nos. FZHJ15 and FZHJ04), Fuzhou Administration of Science and Technology, China (no. 2018-G-47), and the Special Foundation for Yong Scientists of Fuzhou University, China (no. XRC-18008).

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Authors and Affiliations

  1. Fujian Engineering Research Center for Comprehensive Utilization of Marine Products Waste, Fuzhou University, Fujian, 350116, China

    Xinguo Shi, Lemian Liu, Youping Xie, Ruijuan Ma & Jianfeng Chen

  2. Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fujian, 350116, China

    Xinguo Shi, Yuchun Xiao, Lemian Liu, Youping Xie, Ruijuan Ma & Jianfeng Chen

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  1. Xinguo Shi
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  2. Yuchun Xiao
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  3. Lemian Liu
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  4. Youping Xie
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  5. Ruijuan Ma
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  6. Jianfeng Chen
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Correspondence to Xinguo Shi or Jianfeng Chen.

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Fig. S1

Diel pattern of KFcp expression normalized against two most stable genes (α-tub and gapdh). Black bar denoted dark period and other time points are in light period. Mean ± standard deviation (PDF 29 kb)

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Shi, X., Xiao, Y., Liu, L. et al. Exploring reliable reference genes for gene expression normalization in Karenia mikimotoi using real-time PCR. J Appl Phycol 32, 431–440 (2020). https://doi.org/10.1007/s10811-019-01961-z

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  • DOI: https://doi.org/10.1007/s10811-019-01961-z

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