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Molecules. 2018 Sep 12;23(9). pii: E2331. doi: 10.3390/molecules23092331.

Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella.

Zhang Q1,2, Liu W3,4, Cai Y5,6, Lan AF7, Bian Y8,9.

Author information

1
Institute of Applied Mycology, Plant Science and Technology College, Huazhong Agricultural University, Wuhan 430070, Hubei, China. qzhang196@163.com.
2
Key Laboratory of Agro-Microbial Resource Comprehensive Utilization, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China. qzhang196@163.com.
3
Institute of Applied Mycology, Plant Science and Technology College, Huazhong Agricultural University, Wuhan 430070, Hubei, China. zhenpingliuwei@163.com.
4
Key Laboratory of Agro-Microbial Resource Comprehensive Utilization, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China. zhenpingliuwei@163.com.
5
Institute of Applied Mycology, Plant Science and Technology College, Huazhong Agricultural University, Wuhan 430070, Hubei, China. loveylcai@163.com.
6
Key Laboratory of Agro-Microbial Resource Comprehensive Utilization, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China. loveylcai@163.com.
7
School of Biological Science and Engineering, Shanxi University of Technology, Hanzhong 723001, Shaanxi, China. lanafeng2004@126.com.
8
Institute of Applied Mycology, Plant Science and Technology College, Huazhong Agricultural University, Wuhan 430070, Hubei, China. bianyinbing@mail.hzau.edu.cn.
9
Key Laboratory of Agro-Microbial Resource Comprehensive Utilization, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China. bianyinbing@mail.hzau.edu.cn.

Abstract

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

KEYWORDS:

gene expression analysis; housekeeping gene; morel; normalizer genes; qRT-PCR

PMID:
30213125
PMCID:
PMC6225436
DOI:
10.3390/molecules23092331
[Indexed for MEDLINE]
Free PMC Article

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