三角鲂foxl2基因克隆和时空表达特征及EE2对其表达的影响

doi:10.3969/j.issn.1004-1524.20230881

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (8): 1789-1799.DOI: 10.3969/j.issn.1004-1524.20230881

三角鲂foxl2基因克隆和时空表达特征及EE2对其表达的影响

唐红¹^,²(), 关文志²^,^*(), 许晓军², 牛宝龙², 楼宝², 沈小明³, 顾志敏¹^,²^,^*()

1.上海海洋大学水产与生命学院,上海 201306
2.浙江省农业科学院水生生物研究所,浙江杭州 310021
3.德清昊源水产种业有限公司,浙江德清 313200

收稿日期:2023-07-24 出版日期:2024-08-25 发布日期:2024-09-06
作者简介:顾志敏,E-mail: guzhimin2006@163.com
*关文志,E-mail: gwzguan@163.com;
唐红(1997—),女,重庆人,硕士研究生,研究方向为鱼类遗传育种。E-mail: 2247643273@qq.com
通讯作者: 关文志,顾志敏
基金资助:
国家重点研发计划(2022YFD2400605)

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

摘要/Abstract

摘要：

为探究foxl2基因(forkhead transcription factor gene 2)在三角鲂(Megalobrama terminalis,M. terminalis)性腺发育过程中的功能,该研究克隆了三角鲂foxl2基因的开放阅读框(open reading frame,ORF),分析了其编码蛋白的结构特征,检测了其在胚胎发育不同时期和成体不同组织中的表达情况,并研究了腹腔注射性激素17α-乙炔基雌二醇(17α-ethinylestradiol,EE2)对foxl2表达的影响。结果表明:三角鲂foxl2基因ORF为921 bp,共编码306个氨基酸,具有保守的叉头(forkhead,FH)结构域,与斑马鱼(Danio rerio)FOXL2的同源性较高达到96%。三角鲂FOXL2蛋白与鲤科鱼类亲缘关系较近,而与人类和小鼠相距较远。qRT-PCR结果显示,foxl2 mRNA在三角鲂整个胚胎发育过程中均表达,在受精后24、48、60 h表达量较高(P<0.05);在卵巢中的表达量显著高于精巢和其他组织(P<0.05),在肌肉、皮肤、脾、肾和肠组织中弱表达或不表达,表达具有组织特异性。腹腔注射EE2对三角鲂foxl2 mRNA表达调控结果显示,在脑组织中,雌鱼foxl2 mRNA在注射24 h达到峰值且显著高于对照组和雄鱼(P<0.05);在性腺组织中,foxl2 mRNA在注射24 h后的卵巢和精巢中表达量最高,且在36 h的卵巢中持续高表达(P<0.05),表明EE2能够明显促进三角鲂脑和卵巢foxl2 mRNA的表达。研究表明,foxl2基因在三角鲂性腺中呈性别二态性表达,推测其在卵巢发育和功能维持方面起重要作用,foxl2可能是与性类固醇激素相互作用来调节三角鲂性腺发育和功能维持。该研究可为三角鲂性腺发育及性别调控机制提供基础资料。

关键词: 三角鲂, foxl2, 性腺发育, EE2, mRNA表达分析

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

中图分类号:

唐红, 关文志, 许晓军, 牛宝龙, 楼宝, 沈小明, 顾志敏. 三角鲂foxl2基因克隆和时空表达特征及EE2对其表达的影响[J]. 浙江农业学报, 2024, 36(8): 1789-1799.

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.

图/表 8

表1 本实验基因克隆和qRT-PCR所用引物

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

表2 序列分析在线网址或软件

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

图1 三角鲂foxl2基因ORF序列及其预测的氨基酸序列

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

图2 三角鲂FOXL2蛋白二级结构预测(A)和三角鲂(左)、团头鲂(中)和人类(右)FOXL2蛋白三级结构预测(B) 蓝色,α螺旋;红色,延伸链;绿色,β转角;紫色,无规卷曲。

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.

图3 三角鲂FOXL2与斑马鱼FOXL2、人类FOXL2氨基酸序列比对颜色越深表示氨基酸序列相似性越高,红色方框为叉头框区域。

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.

图4 不同物种FOXL2氨基酸序列构建的系统进化树

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

图5 三角鲂早期胚胎发育图(A),foxl2基因在三角鲂不同胚胎发育时期中的相对表达(B)及foxl2基因在三角鲂各组织中的表达(C) a, 4 h; b,12 h; c, 24 h; d, 36 h; e, 48 h; f, 60 h; g, 72 h。“*”代表显著差异(P<0.05),下同。

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.

图6 EE2对三角鲂脑(A)和性腺(B)中foxl2基因表达的影响

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

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三角鲂foxl2基因克隆和时空表达特征及EE2对其表达的影响

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

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