Cloning and spatio-temporal expression analysis of foxl2 gene and the influence of EE2 on its expression in Megalobrama terminalis

doi:10.3969/j.issn.1004-1524.20230881

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (8): 1789-1799.DOI: 10.3969/j.issn.1004-1524.20230881

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Cloning and spatio-temporal expression analysis of foxl2 gene and the influence of EE2 on its expression in Megalobrama terminalis

TANG Hong¹^,²(), GUAN Wenzhi²^,^*(), XU Xiaojun², NIU Baolong², LOU Bao², SHEN Xiaoming³, GU Zhimin¹^,²^,^*()

1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
2. Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Deqing Haoyuan Aquatic Seed Industry Co., Ltd., Deqing 313200, Zhejiang, China

Received:2023-07-24 Online:2024-08-25 Published:2024-09-06
Contact: GUAN Wenzhi,GU Zhimin

Abstract

Abstract:

To investigate the function of forkhead transcription factor gene 2 (foxl2) during gonadal development of M. terminalis, we cloned the open reading frame (ORF) of M. terminalis foxl2 gene, analyzed the structural characteristics of the encoded protein, and detected its expression at different stages of embryonic development and in different adult tissues in this study. The effect of intraperitoneal injection of sex hormone 17α-ethinylestradiol (EE2) on foxl2 expression was also studied. The results showed that the ORF of foxl2 gene was 921 bp, encoding 306 amino acids, with a conserved forkhead (FH) domain, and had a 96% homology with zebrafish FOXL2. The FOXL2 protein of M. terminalis is closely related to cyprinid fish, but distantly related to humans and mice. The qRT-PCR results showed that foxl2 mRNA was expressed throughout embryonic development, and the expression was higher at 24, 48, 60 h of post-fertilization (P<0.05). The expression level in the ovary was significantly higher than that in the testis and other tissues (P<0.05), and weakly or not expressed in muscle, skin, spleen, kidney and intestine tissues. The expression was tissue-specific. The regulation of foxl2 mRNA expression by EE2 injection showed that foxl2 mRNA in female reached the peak at 24 h after injection and was significantly higher than that in the control and male (P<0.05). In the gonadal tissue, the expression of foxl2 mRNA was highest at 24 h in the ovary and testis after injection, and continued to be high at 36 h in the ovary after injection (P<0.05), suggesting that EE2 could promote the expression of foxl2 mRNA in the brain and ovary of M. terminalis. foxl2 gene is the expression of sexual dimorphism in the gonads of M. terminalis, which may play an important role in the maintenance of ovarian development and function. foxl2 may interact with sex steroid hormones to regulate the gonadal development and function of M. terminalis. This study can provide basic information for the gonadal development and sex regulation mechanism of M. terminalis.

Key words: Megalobrama terminalis, foxl2, gonad development, EE2, mRNA expression analysis

CLC Number:

TANG Hong, GUAN Wenzhi, XU Xiaojun, NIU Baolong, LOU Bao, SHEN Xiaoming, GU Zhimin. Cloning and spatio-temporal expression analysis of foxl2 gene and the influence of EE2 on its expression in Megalobrama terminalis[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1789-1799.

Figures/Tables 8

Table 1 Primers used for gene cloning and qRT-PCR in the experiment

引物 Primer	序列 Sequence(5'-3')	目的 Purpose
foxl2-F	GTTTTGTTTTGGTGTAGGTCTGC	克隆 Cloning
foxl2-R	GTCTTTTAGCGAGTATGTTGGTC	克隆 Cloning
foxl2-QF	AGCATCCGACACAACCTGTC	荧光定量
		PCR Real-time PCR
foxl2-QR	TAGTTGCCCTTTCGCTCACC	荧光定量
		PCR Real-time PCR
18S-F	ACCGCAGCTAGGAATAATGG	内参 Reference
18S-R	GGTCGGAACTACGACGGTAT	内参 Reference

Table 2 Sequence analysis online URL or software

功能 Function	网址或软件 Online URL or software
同源性比对 Alignment of homology	http://www.ncbi.nlm.nih.gov/BLAST/
开放阅读框 Open reading frame	http://www.ncbi.nlm.nih.gov/gorf/gorf.html
信号肽序列 Signal peptide sequence	https://services.healthtech.dtu.dk/services/SingnalP-5.0/
分子量 Molecular mass	http://web.expasy.org/protparam/
等电点 Isoelectric point	http://web.expasy.org/protparam/
蛋白二级结构 Protein secondary structure	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html
蛋白三级结构 Protein tertiary structure	http://www.swissmodel.expasy.org/
蛋白质基序 Protein motif	http://cn.expasy.org/prosite
多序列比对 Multiple sequence alignment	ClustalX; GeneDoc
系统进化树 Phylogenetic tree	MEGA7.0

Fig.1 ORF sequence and predicted amino acid sequence of foxl2 gene in M. terminalis

Fig.2 Predicted secondary structure of FOXL2 protein in M. terminalis (A) and predicted tertiany structure of FOXL2 protein (B) in M. terminalis(left) M. amblycephala(middle) and H. sapiens(right) Blue, α-helix; Red, Extended strand; Green, β-turn; Purple, Random coil.

Fig.3 Comparison of FOXL2 amino acid sequences in M. terminalis with D. rerio and H. sapiens The darker the color, the higher the amino acid sequence similarity. The red box is the Fork head domain.

Fig.4 Phylogenetic tree of FOXL2 amino acid sequences in different species

Fig.5 Diagram of early embryonic development of M. terminalis (A), the relative expression of foxl2 gene in embryonic at different developmental stages (B) and expression of foxl2 gene in tissues of M. terminalis(C) a, 4 h; b,12 h; c, 24 h; d, 36 h; e, 48 h; f, 60 h; g, 72 h. “*” represents significant difference(P<0.05). The same as below.

Fig.6 Effect of EE2 on foxl2 gene expression in the brain (A) and gonads (B) of M. terminalis

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Cloning and spatio-temporal expression analysis of foxl2 gene and the influence of EE2 on its expression in Megalobrama terminalis

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