Cloning and Expression Analysis of the ghr Gene in Channa maculata and Its Response to Sexual Steroid Hormone Induction
WU Yu-Xia1,2, ZHANG Xiao-Tian1,2, ZHANG Yang1,3, ZHAO Jian1,2, LUO Qing1, LIU Hai-Yang1, FEI Shu-Zhan1, CHEN Kun-Ci1,2, OU Mi1,3,*
1 Pearl River Fisheries Research Institute/Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation, Ministry of Agriculture and Rural Affairs, Chinese Academy of Fishery Sciences, Guangzhou 510380, China; 2 College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China; 3 School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, China
Abstract：The growth hormone (GH)-insulin-like growth factor (IGF) axis is the main endocrine axis that regulates fish growth. In order to explore the regulatory mechanism of growth hormone receptor (GHR) in the growth difference between female and male blotched snakehead (Channa maculata), the ghr gene in C. maculata was cloned, gene structure was analyzed, and the expression patterns of ghr in different adult tissues and liver at different developmental stages were clarified, meanwhile, its response to exogenous sex steroid hormones were also analyzed. The results showed that 2 602 bp cDNA sequence of the ghr gene in C. maculata was obtained, including 100 bp 5'-UTR, 1 872 bp ORF, encoding 623 amino acids, and 630 bp 3'-UTR. The genome sequence of ghr gene in C. maculata was 18 118 bp long, including 9 exons and 8 introns. 899 bp 5'-flanking sequence was identified, which contained androgen receptor (AR), bromomain-containing protein 4 (BRD4) and suppressor element-1-silencing transcription factor (REST) and other transcription factor binding sites. ghr was highly expressed in the liver of adult C. maculata, and its expression in males was significantly higher than that in females (P<0.01). The expression analysis of ghr gene at different developmental stages displayed that the highest expression of ghr gene was found in the liver of 365 d male C. maculata, which was significantly higher than that in females at the same period (P<0.01). Hormone induction experiments showed that long-term injection of 17α-ethynylestradiol (EE2) inhibited ghr expression in male and female C. maculata, long-term injection of 17α-methyltestosterone (MT) inhibited ghr expression in female C. maculata, but promoted ghr expression in males within a certain period of time. The results demonstrate that GHR might play a key role in the growth difference between females and males and it is also regulated by sex steroid hormones in C. maculata. This study provides the reference for further exploring the molecular mechanism of growth regulation in blotched snakehead.
吴玉霞, 张啸天, 张扬, 赵建, 罗青, 刘海洋, 费树站, 陈昆慈, 欧密. 斑鳢ghr基因克隆、表达分析及性类固醇激素诱导的响应[J]. 农业生物技术学报, 2024, 32(6): 1371-1384.
WU Yu-Xia, ZHANG Xiao-Tian, ZHANG Yang, ZHAO Jian, LUO Qing, LIU Hai-Yang, FEI Shu-Zhan, CHEN Kun-Ci, OU Mi. Cloning and Expression Analysis of the ghr Gene in Channa maculata and Its Response to Sexual Steroid Hormone Induction. 农业生物技术学报, 2024, 32(6): 1371-1384.
[1] 陈宝定, 贾俊静, 刘丽, 等. 2010. 生长激素受体基因表达的研究进展[J]. 中国畜牧兽医, 37(12): 127-130. (Chen B D, Jia J J, Liu L, et al.2010. Research progress on gene expression of Growth hormone receptor[J]. China Animal Husbandry & Veterinary Medicine, 37(12): 127-130.) [2] 邓利, 林浩然. 2003. 腹腔注射LHRH-A对黑鲷生长激素及其受体的影响[J]. 深圳大学学报, (12): 60-65. (Deng L, Lin H R. 2003. Effect of intraperitoneal injection of LHRH-A on growth hormone and its receptor in Acanthopagrus schlegelii[J]. Journal of Shenzhen University, (12): 60-65.) [3] 高丹丹, 欧密, 吴燕铎, 等. 2021. 斑鳢生长激素基因的克隆及表达分析[J]. 农业生物技术学报, 29(12): 2328-2341. (Gao D D, Ou M, Wu Y D, et al.2021. Cloning and expression analysis of the growth hormone gene in Channa maculata[J]. Journal of Agricultural Biotechnology, 29(12): 2328-2341.) [4] 李雅靖, 王进千, 韦姗姗, 等. 2023. 草鱼GHR2基因生物信息学分析[J]. 现代畜牧兽医, (01): 21-25. (Li Y J, Wang J Q, Wei S S, et al. 2023. Bioinformatics analysis of Ctenopharyngodon idella GHR2 gene[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, (01): 21-25.) [5] 刘少贞, 吕文琪, 吕小也. 2016. 17α-甲基睾酮对斑马鱼肝脏vtg基因mRNA表达的影响[J]. 畜牧与饲料科学, 37(5): 9-11. (Liu S Z, Lv W Q, Lv X Y.2016. 17α-methyltestosterone on the expression of vtg gene mRNA in Danio rerio liver[J]. Animal Husbandry and Feed Science, 37(5): 9-11.) [6] 刘士力, 贾永义, 刘加林, 等. 2020. 翘嘴鲌两种生长激素受体基因结构及微卫星多态性与生长性状的相关性[J]. 水产学报, 44(06): 894-906. (Liu S L, Jia Y Y, Liu J L, et al.2020. Correlation between the structure and microsatellite polymorphisms of two growth hormone receptor genes and growth traits in Culter alburnus basilewsky[J]. Journal of Fisheries of China, 44(06): 894-906.) [7] 刘芝亮. 2013. 半滑舌鳎生长轴关键基因的重组表达及对生长与生殖的调控机制研究[D]. 硕士学位论文, 上海海洋大学, 导师: 柳学周, pp. 60-67. (Liu Z L.2013. Recombinant expression of key genes in the growth axis of Cynoglossus semilaevis and its regulation mechanism on growth and reproduction[D]. Thesis for M.S., Shanghai Ocean University, Supervisor: Liu X Z, pp. 60-67.) [8] 马细兰. 2007. 生长激素及其受体调控尼罗罗非鱼生长的分子机理研究[D]. 硕士学位论文, 中山大学, 导师: 林浩然, pp. 46-54. (Ma X L.2007. Molecular mechanism of growth hormone and its receptor regulating growth of Oreochromis niloticus[D]. Thesis for M.S., Sun Yat-sen University, Supervisor: Lin H R, pp. 46-54.) [9] 王海芳. 2014. 鳜生长相关基因的SNPs及其与生长性状的相关性研究[D]. 博士学位论文, 中山大学, 导师: 李桂峰, pp. 13-15. (Wang H F.2014. SNPs identification of growth-related genes and correlation analysis on growth traits in sinipercid species[D]. Thesis for Ph.D., Sun Yat-sen University, Supervisor: Li G F, pp. 13-15.) [10] 王凌宇, 齐飘飘, 陈敏, 等. 2020. 性类固醇激素对黄颡鱼雌雄生长二态性的影响[J]. 水生生物学报, 44(02): 379-388. (Wang L Y, Qi P P, Chen M, et al.2020. Effects of sex steroid hormones on female and male growth dimorphism of Pelteobagrus fulvidraco[J]. Acta Hydrobiologica Sinica, 44(02): 379-388.) [11] 吴雪, 易鸣, 程朝友, 等. 2019. 威宁绵羊GHR基因克隆及组织表达[J]. 基因组学与应用生物学, 38(01): 1-6. (Wu X, Yi M, Cheng C Y, et al.2019. Cloning and tissue expression of Ovis aries GHR gene[J]. Genomics and Applied Biology, 38(01): 1-6.) [12] 熊力, 陈虹, 潘阳阳, 等. 2015. 生长激素受体(GHR)在牦牛不同组织中的表达分析[J]. 农业生物技术学报, 23(09):1234-1239. (Xiong L, Chen H, Pan Y Y, et al.2015. Expression analysis of growth hormone receptor (GHR) in different tissues of Bos grunniens[J]. Journal of Agricultural Biotechnology, 23(09): 1234-1239.) [13] 杨梅. 2020. 昆明裂腹鱼胚胎发育及早期抗氧化能力研究[D]. 硕士学位论文, 贵州大学, 导师: 姚俊杰, pp. 13-15. (Yang M.2020. Study on embryonic development and early antioxidant capacity of Schizothorax grahami[D]. Thesis for M.S., Guizhou University, Supervisor: Yao J J, pp. 13-15.) [14] 张宏叶. 2018. 河川沙塘鳢GH/IGF轴相关基因克隆、表达分析及生长性状相关SNP位点筛查[D]. 硕士学位论文, 南京师范大学, 导师: 尹绍武, 周国勤, pp. 31-32. (Zhang H Y.2018. Cloning, expression analysis, and screening of growth trait related SNP loci of GH/IGF axis related genes in Odontobutis potamophila[D]. Thesis for M.S., Nanjing Normal University, Suppervisor: Yin S W, Zhou G Q, pp. 31-32.) [15] 张雅星, 王滨, 柳学周, 等. 2019. 生长轴对半滑舌鳎早期生长发育的调控作用[J]. 中国水产科学, 26(02): 287-295. (Zhang Y X, Wang B, Liu X Z, et al.2019. Regulation of growth axis on early growth and development of tongue sole Cynoglossus semilaevis[J]. Journal of Fishery Sciences of China, 26(02): 287-295.) [16] 赵建, 陈昆慈, 朱新平, 等. 2019. 用于斑鳢性别鉴定的SNP位点及其检测方法[P]. 中国, CN106119378B. (Zhao J, Chen K C, Zhu X P, et al.2019. SNP loci and their de‐ tection methods for sex identification of Channa maculata[P]. China, CN106119378B.) [17] Alfonso S V, Josep A C G, Jaume P S.2005. Duplication of growth hormone receptor (GHR) in fish genome: Gene organization and transcriptional regulation of GHR type I and I in gilthead sea bream (Sparus aurata)[J]. General and Comparative Endocrinology, 142(1-2): 193-203. [18] Allen C M, Sharman W M, La M C, et al.2002. Attenuation of photodynamically induced apoptosis by an RGD containing peptide[J]. Photochemical & Photobiological Sciences, 1(4): 246-254. [19] Baumann G.2002. Growth hormone binding protein. The soluble growth hormone receptor[J]. Minerva Endocrinology, 27(4): 265-276. [20] Hoegg S, Brinkmann H, Taylor J S, et al.2004. Phylogenetic timing of the fish-specific genome duplication correlates with the diversification of teleost fish[J]. Journal of Molecular Evolution, 59(2): 190-203. [21] Jia Y D, Jing Q Q, Gao Y H, et al.2019. Involvement and expression of growth hormone/insulin-like growth factor member mRNAs in the ovarian development of turbot (Scophthalmus maximus)[J]. Fish Physiology and Biochemistry, 45(3): 955-964. [22] Jiao B W, Huang X G, Chan C B, et al.2006. The co-existence of two growth hormone receptors in teleost fish and their differential signal transduction tissue distribution and hormonal regulation of expression in sea bream[J]. Journal of Molecular Evolution, 36(1): 23-40. [23] John A H B, Cox R M.2007. Development of sexual size dimorphism in lizards: Testosterone as a bipotential growth regulator[J]. Sex Size & Gender Roles, (01): 195-205. [24] Lee L T O, Nong G, Chan Y H, et al.2001. Molecular cloning of a teleost growth hormone receptor and its functional interaction with human growth hormone[J]. Gene, 270(1-2): 121-129. [25] Li Y, Zhu Q F, Huang Y, et al.2022. Cloning and characterization of two types of growth hormone receptors in tomato clownfish (Amphiprion frenatus), and their expression under different light spectra and photoperiods[J]. Aquaculture International, 30: 483-500. [26] Lori K D, Andrew L P, Naoshi H, et al.2008. Gender-specific expression of multiple estrogen receptors, growth hormone receptors insulinlike growth factors and vitellogenins, and effects of 17bete-estradiol in the male tilapia (Oreochromis mossambicus)[J]. General and Comparative Endocrinology, 156(3): 544-551. [27] Mao H H, Chen K C, Zhu X, et al.2017. Identification of suitable reference genes for quantitative real-time PCR normalization in blotched snakehead Channa maculata[J]. Journal of Fish Biology, 90: 2312-2322. [28] Nguyen N H.2016. Genetic improvement for important farmed aquaculture species with a reference to carp, tilapia and prawns in Asia: Achievements, lessons and challenges[J]. Fish and Fisheries, 17(02): 483-506. [29] Norbeck L A, Sheridan M A.2011. An in vitro model for evaluat ing peripheral regulation of growth in fish: Effects of 17β-estradiol and testosterone on the expression of growth hormone receptors, insulin-like growth factors, and insulinlike growth factor type 1 receptors in rainbow trout (Oncorhynchus mykiss)[J]. General and Comparative Endocrinology, 173(2): 270-280. [30] Ou M, Huang, R, Yang C, et al.2021. Chromosome-level genome assemblies of Channa argus and Channa maculata and comparative analysis of their temperature adaptability[J]. Gigascience, 10(10): giab070. [31] Ou M, Chen K, Gao D, et al.2022. Characterization, expression and CpG methylation analysis of Dmrt1 and its response to steroid hormone in blotched snakehead (Channa maculata)[J]. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 257: 110672. [32] Ozaki Y, Fukada H, Kazet o Y, et al.2006. Molecular cloning and characterization of growth hormone receptor and its homologue in the Japanese eel Anguilla japonica[J]. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 143(4): 422-431. [33] Ross R J M.1999. The GH receptor and GH insensitivity[J]. Growth Hormone & IGF Research,(Suppl B)2: 42-46. [34] Tanaka M, Hayashida Y, Wakita M, et al.1995. Expression of aberrantly spliced grow th hormone receptor mRNA in the sex-linked dwarf chicken, Gifu 20[J]. Growth Regulation, 5(4): 218-223. [35] Venter J C, Adams M D, Myers E W, et al.2001. The sequence of the human genome[J]. Science, 291(5507): 1304-1351. [36] Wang J A, Li D F, Zhao X, et al.2020. GH and GHR gene cloning, expression and their associations with growth-related traits of the barbel chub (Squaliobarbus curriculus)[J]. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 243-244: 110429. [37] Yuan X, Lin Y X, Qin J K, et al.2020. Molecular identification, tissue distribution and in vitro functional analysis of growth hormone and its receptors in red-spotted grouper (Epinephelus akaara)[J]. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 250: 110488.
