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

参考文献 38

[1]	CRISPONI L, DEIANA M, LOI A, et al. The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome[J]. Nature Genetics, 2001, 27(2): 159-166.
[2]	COCQUET J, PAILHOUX E, JAUBERT F, et al. Evolution and expression of FOXL2[J]. Journal of Medical Genetics, 2002, 39(12): 916-921.
[3]	WANG D S, KOBAYASHI T, ZHOU L Y, et al. Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1[J]. Molecular Endocrinology, 2007, 21(3): 712-725.
[4]	TUCKER E J. The genetics and biology of FOXL2[J]. Sexual Development, 2022, 16(2/3): 184-193.
[5]	BHAT I A, RATHER M A, DAR J Y, et al. Molecular cloning, computational analysis and expression pattern of forkhead box l2 (Foxl2) gene in catfish[J]. Computational Biology and Chemistry, 2016, 64: 9-18.
[6]	HU Q, GUO W, GAO Y, et al. Molecular cloning and analysis of gonadal expression of Foxl2 in the rice-field eel Monopterus albus[J]. Scientific Reports, 2014, 4: 6884.
[7]	JIANG W B, YANG Y H, ZHAO D M, et al. Effects of sexual steroids on the expression of foxl2 in Gobiocypris rarus[J]. Comparative Biochemistry and Physiology Part B, Biochemistry & Molecular Biology, 2011, 160(4): 187-193.
[8]	ALAM M A, KOBAYASHI Y, HORIGUCHI R, et al. Molecular cloning and quantitative expression of sexually dimorphic markers Dmrt1 and Foxl2 during female-to-male sex change in Epinephelus merra[J]. General and Comparative Endocrinology, 2008, 157(1): 75-85.
[9]	WANG D S, KOBAYASHI T, ZHOU L Y, et al. Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus[J]. Biochemical and Biophysical Research Communications, 2004, 320(1): 83-89.
[10]	周运迪, 吴星星, 赵海萍, 等. 青鳉foxl2基因敲除突变体的构建与表型分析[J]. 广东海洋大学学报, 2019, 39(2): 20-30.
	ZHOU Y D, WU X X, ZHAO H P, et al. Generation and phenotype analysis on foxl2 mutant in medaka, Oryzias latipes[J]. Journal of Guangdong Ocean University, 2019, 39(2): 20-30. (in Chinese with English abstract)
[11]	LI M H, YANG H H, LI M R, et al. Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in Tilapia as demonstrated by TALENs[J]. Endocrinology, 2013, 154(12): 4814-4825.
[12]	吴燕铎, 欧密, 高丹丹, 等. 斑鳢叉头框转录因子基因Foxl2的克隆表达及性类固醇激素刺激下的表达响应[J]. 大连海洋大学学报, 2022, 37(1): 49-60.
	WU Y D, OU M, GAO D D, et al. Molecular cloning, expression and response of Foxl2 gene induced by sex steroid hormones in blotched snakehead Channa maculata[J]. Journal of Dalian Ocean University, 2022, 37(1): 49-60. (in Chinese with English abstract)
[13]	王欣欣. 17α-乙炔基雌二醇对黄颡鱼稚鱼性逆转的诱导作用及关键基因筛选[D]. 扬州: 扬州大学, 2019.
	WANG X X. Induced effects of 17α-ethinylestradiol on sex reversal of yellow catfish larvae (Pelteobagrus fulvidraco) and screening of key genes during sex reversal[D]. Yangzhou: Yangzhou University, 2019. (in Chinese with English abstract)
[14]	胡雪松, 石连玉. 我国三角鲂种质资源的研究进展[J]. 水产学杂志, 2020, 33(3): 84-89.
	HU X S, SHI L Y. A review: research progress on germplasm resource of black bream(Megalobrama terminalis) in China[J]. Chinese Journal of Fisheries, 2020, 33(3): 84-89. (in Chinese with English abstract)
[15]	江仁党, 肖剑平, 黄毅春, 等. 三角鲂规模人工繁殖和苗种培育技术要点[J]. 动物科学与动物医学, 2002, 19(5): 59-60.
	JIANG R D, XIAO J P, HUANG Y C, et al. Key points of large-scale artificial propagation and fry cultivation of Siniperca chuatsi[J]. Animal Science and Veterinary Medicine, 2002, 19(5): 59-60. (in Chinese)
[16]	任席林, 梁帅淇, 钟艳芳, 等. 泰国斗鱼foxl2基因的分子克隆及组织表达模式[J]. 基因组学与应用生物学, 2021, 40(1): 109-116.
	REN X L, LIANG S Q, ZHONG Y F, et al. Molecular cloning and tissue expression patterns of foxl2 gene in Bettas plendens[J]. Genomics and Applied Biology, 2021, 40(1): 109-116. (in Chinese with English abstract)
[17]	HE Y F, WANG X G, WU X B, et al. Expression profiles of amh and foxl2 in Schizothorax kozlovi, and their response to temperature during the early developmental stage[J]. Journal of Genetics, 2018, 97(1): 127-136.
[18]	WANG D D, ZHANG G R, WEI K J, et al. Molecular identification and expression of the Foxl2 gene during gonadal sex differentiation in northern snakehead Channa argus[J]. Fish Physiology and Biochemistry, 2015, 41(6): 1419-1433.
[19]	唐露, 胡琼, 赵瑞阳, 等. 白斑狗鱼FOXL2基因克隆及其表达[J]. 贵州农业科学, 2020, 48(5): 97-101.
	TANG L, HU Q, ZHAO R Y, et al. Cloning and expression of FOXL2 gene in Esox lucius[J]. Guizhou Agricultural Sciences, 2020, 48(5): 97-101. (in Chinese with English abstract)
[20]	ZHOU H Q, ZHONG D M, ZHANG M Q, et al. Molecular characterization and expression profiling of foxl2 in zig-zag eel (Mastacembelus armatus)[J]. Gene Reports, 2023, 33.
[21]	叶容晖, 任海波. 三角帆蚌(Hyriopsis cumingii)叉头蛋白2(foxl2)基因和性别决定框14(sox14)基因克隆与表达分析[J]. 海洋与湖沼, 2018, 49(1): 160-167.
	YE R H, REN H B. Molecular cloning and gene expression of foxl2 and sox14 gene from Hyriopsis cumingii[J]. Oceanologia et Limnologia Sinica, 2018, 49(1): 160-167. (in Chinese with English abstract)
[22]	ASHIDA H, UEYAMA N, KINOSHITA M, et al. Molecular identification and expression of FOXL2 and DMRT1 genes from willow minnow Gnathopogon caerulescens[J]. Reproductive Biology, 2013, 13(4): 317-324. [PubMed]
[23]	ZHOU Y Y, LI Y Q, LIAO Q L, et al. Examination of the role of the forkhead box L2 (Foxl2) in gonadal and embryonic development in the boring giant clam Tridacna crocea[J]. Aquaculture, 2022, 560.
[24]	NAKAMOTO M, MATSUDA M, WANG D S, et al. Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes[J]. Biochemical and Biophysical Research Communications, 2006, 344(1): 353-361.
[25]	YARMOHAMMADI M, POURKAZEMI M, KAZEMI R. Differential expression of foxl2 and cyp19a1a mRNA during gonad developmental stages in great sturgeon Huso huso[J]. Journal of Fish Biology, 2017, 90(3): 1104-1111.
[26]	MU W J, WEN H S, LI J F, et al. Cloning and expression analysis of Foxl2 during the reproductive cycle in Korean rockfish, Sebastes schlegeli[J]. Fish Physiology and Biochemistry, 2013, 39(6): 1419-1430.
[27]	SHU C, WANG L J, ZOU C C, et al. Function of Foxl2 and Dmrt1 proteins during gonadal differentiation in the olive flounder Paralichthys olivaceus[J]. International Journal of Biological Macromolecules, 2022, 215: 141-154.
[28]	KOBAYASHI Y, HORIGUCHI R, NOZU R, et al. Expression and localization of forkhead transcriptional factor 2 (Foxl2) in the gonads of protogynous wrasse, Halichoeres trimaculatus[J]. Biology of Sex Differences, 2010, 1(1): 3.
[29]	李法君, 付春鹏, 罗永巨. Dmrt基因在水生生物中的研究进展[J]. 水生生物学报, 2016, 40(5): 1068-1077.
	LI F J, FU C P, LUO Y J. Research progress of Dmrt genes in hydrobiontes[J]. Acta Hydrobiologica Sinica, 2016, 40(5): 1068-1077. (in Chinese with English abstract)
[30]	张红燕, 朱文旭, 赵诚, 等. 河川沙塘鳢DMRT1基因的克隆及其表达分析[J]. 海洋渔业, 2018, 40(5): 569-580.
	ZHANG H Y, ZHU W X, ZHAO C, et al. Cloning and expression analysis of DMRT1 gene in Odontobutis potamophila[J]. Marine Fisheries, 2018, 40(5): 569-580. (in Chinese with English abstract)
[31]	李林, 梁宏伟, 李忠, 等. 黄颡鱼DMRT1基因cDNA全长克隆及其表达分析[J]. 华中农业大学学报, 2012, 31(2): 220-226.
	LI L, LIANG H W, LI Z, et al. Cloning and expression analysis of DMRT1 gene in Pelteobagrus fulvidraco[J]. Journal of Huazhong Agricultural University, 2012, 31(2): 220-226. (in Chinese with English abstract)
[32]	黄洋, 黎金海, 王文基, 等. 泰国斗鱼Dmrt1基因的部分cDNA克隆及表达分析[J]. 水产科学, 2021, 40(4): 618-623.
	HUANG Y, LI J H, WANG W J, et al. Molecular cloning, partial sequence and tissue expression of Dmrt1 gene in Siamese fighting fish Betta splendens[J]. Fisheries Science, 2021, 40(4): 618-623. (in Chinese with English abstract)
[33]	PARK M, SHIN E, WON M, et al. FOXL2 interacts with steroidogenic factor-1 (SF-1) and represses SF-1-induced CYP17 transcription in granulosa cells[J]. Molecular Endocrinology, 2010, 24(5): 1024-1036.
[34]	GARDNER L, ANDERSON T, PLACE A R, et al. Sex change strategy and the aromatase genes[J]. Fish Physiology and Biochemistry, 2003, 28(1): 147-148.
[35]	PARK M, SUH D S, LEE K, et al. Positive cross talk between FOXL2 and antimüllerian hormone regulates ovarian reserve[J]. Fertility and Sterility, 2014, 102(3): 847-855.e1.
[36]	ARIS A Z, SHAMSUDDIN A S, PRAVEENA S M. Occurrence of 17α-ethynylestradiol (EE2) in the environment and effect on exposed biota: a review[J]. Environment International, 2014, 69: 104-119.
[37]	田华, 汝少国, 邴欣. 环境激素对鱼类内分泌轴线的干扰作用及其机制研究进展[J]. 安全与环境学报, 2009, 9(2): 10-14.
	TIAN H, RU S G, BING X. A review on the advances in the research of dis-ruption effects and mechanisms of environmen-tal hormone on endocrine axes in fish[J]. Journal of Safety and Environment, 2009, 9(2): 10-14. (in Chinese with English abstract)
[38]	吴风瑞. 南方鲇Foxl2基因克隆、表达及其在性腺分化中作用的初步研究[D]. 重庆: 西南大学, 2007.
	WU F R. Molecular cloning, gene expression of Foxl2 and its possible roles in gonadal sex differentiation in southern catfish, Silurus meridionnalis Chen[D]. Chongqing: Southwest University, 2007. (in Chinese with English abstract)

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 38

相关文章 3

编辑推荐

Metrics

本文评价

[1]	刘凯, 谢楠, 郭炜, 马恒甲. 三角鲂MHCⅠα基因全长cDNA克隆与生物信息学分析[J]. 浙江农业学报, 2022, 34(6): 1162-1174.
[2]	刘凯, 冯晓宇, 马恒甲, 谢楠. 钱塘江三角鲂线粒体基因组测序及其结构特征分析[J]. 浙江农业学报, 2020, 32(9): 1591-1608.
[3]	徐增洪;赵朝阳;周鑫;*. 克氏原螯虾的体色变化与其生长发育的关系[J]. , 2010, 22(6): 839-842.