1 Provincial Key Laboratory of Wildlife Conservation and Utilization Technology, Zhejiang Normal University, Jinhua 321004, China; 2 College of Life Sciences, Beijing Normal University, Beijing 100875, China; 3 The Management Center of Wuyanling National Natural Reserve in Zhejiang, Wenzhou 325500, China; 4 Xingzhi College, Zhejiang Normal University, Jinhua 321004, China
Abstract：Quasipaa spinosa, a characteristic economic amphibian in China, have both gastronomic and medicinal values. As a sexual dimorphic feature of frogs, the spines of Q. spinosa skin derivatives are of great significance to improve the success rate of holding and also an important classification and identification feature of Tribe Paini. The molecular mechanism of its generation and changes will help to clarify the phylogenetic relationship of the amphibian Tribe Paini. In order to explore the molecular mechanism of the seasonal changes in spines of the Q. spinosa, this study performed transcriptome sequencing on 3 sets of samples from the breast skin of the male Q. spinosa during the breeding period and post-breeding period and the abdomen skin of the male Q. spinosa during the breeding period. A total of 100 312 Unigenes were obtained. After annotation, the result showed that the expression of multiple keratinizing protein genes significantly increased in the breast skin of male Q. spinosa during the breeding period. Genes related to apoptosis, cell proliferation and sex hormone synthesis were found in the differentially expressed genes of chest spiny skin in breeding period and post-breeding period. KEGG analysis showed that multiple differentially expressed genes were found to be enriched in the intercellular lipid metabolism of the skin stratum corneum. It was speculated that the increased keratin gene expression during the breeding period was one of the direct causes of the formation of spines. The reduction of lipid synthesis in the stratum corneum of the skin and the slowdown of cell proliferation might cause the spines to become smaller. In addition, the expression of genes related to sex hormone synthesis decreased during the breedding period to post-breeding period, indicating that the seasonal changes in spines of the Q. spinosa might be regulated by hormone changes. The 15 differentially expressed genes were verified by qPCR, the results showed that the transcriptome results were reliable. This study elucidates the molecular mechanism of spine formation and seasonal changes in Q. spinosa, the findings provide the basis for taxonomic classification of Q. spinosa, and a reference for related candidate genes and metabolic pathways for the seasonal variation and sexual dimorphism of skin derivatives of other vertebrates.
[1] 陈韦君. 2012. 台湾水鹿GH、KRT5、RPS8及COL1A1基因单一核苷酸多态性基因型与产茸量性狀相关性[D]. 博士学位论文, 中兴大学, 导师: 邱奕颖, pp. 56-60. (Chen W J.2012. Correlation between genotypes of single nucleotide polymorphisms of GH, KRT5, RPS8 and COL1A1 genes in Taiwan sambar and velvet yield traits[D]. Thesis for Ph. D., Zhongxing University, Supervisor: Qiu Y Y, pp. 56-60.) [2] 邓泱泱, 荔建琦, 吴松锋, 等. 2006. nr数据库分析及其本地化[J]. 计算机工程, 12(5): 71-73. (Deng Y Y, Li J Q, Wu S F, et al.2006. Analysis of nr database and its localization[J]. Computer Engineering, 12(5): 71-73.) [3] 费梁. 2012. 中国两栖动物及其分布彩色图鉴[M]. 四川科学技术出版社, 成都. pp. 256-257. (Fei L.2012. Chinese Amphibians and Their Distribution Colored Encyclopedia[M]. Sichuan Science and Technology Press, Chengdu. pp. 256-257.) [4] 何飞祥. 2019. 金钱鱼雌雄性腺转录组分析及性类固醇激素水平的研究[D]. 博士学位论文, 广东海洋大学, 导师: 朱春华, pp. 45-50. (He F X.2019. Transcriptome analysis of male and female glands in goldfish and study of sex steroid hormone levels[D]. Thesis for Ph.D., Guangdong Ocean University, Supervisor: Zhu C H, pp. 45-50.) [5] 刘成星, 刘荣臻. 2008. 早期胞质分裂收缩环缢缩机制研究进展[J]. 医用生物力学, 23(06): 480-485. (Liu C X, Liu R Z.2008. Advances in the mechanism of early cytokinesis contraction ring constriction[J]. Medical Biomechanics, 23(06): 480-485.) [6] 梅祎芸. 2019. 基于形态学和分子生物学特征探讨棘胸蛙的分类[D]. 硕士学位论文, 浙江师范大学, 导师: 郑荣泉, pp. 2-7. (Mei Y Y.2019. Exploring the taxonomy of thoracic frogs based on morphological and molecular characteristics[D]. Thesis for M.S., Zhejiang Normal University, Supervisor: Zheng R Q, pp. 2-7.) [7] 闵敏. 2017. 角蛋白24在表皮角质形成细胞和真皮成纤维细胞中的作用研究[D]. 硕士学位论文, 浙江大学, 导师: 郑敏, pp. 45-50. (Min M.2017. The role of keratin 24 in epidermal keratinocytes and dermal fibroblasts[D]. Thesis for M.S., Zhejiang University, Supervisor: Zheng M, pp. 45-50.) [8] 余永胜. 2012. COPS3基因在肝癌细胞增殖中的功能研究 [D]. 博士学位论文, 上海交通大学, 导师: 臧国庆, pp. 55-57. (Yu Y.2012. The role of COPS3 gene in the proliferation of hepatocellular carcinoma[D]. Thesis for Ph. D., Shanghai Jiaotong University, Supervisor: Zang G Q, pp. 55-57.) [9] 张久聪, 聂青和. 2008. 胆汁酸代谢及相关进展[J]. 胃肠病学和肝病学杂志, 25(11): 953-956. (Zhang J C, Nie Q H.2008. Bile acid metabolism and related progress[J]. Journal of Gastroenterology and Hepatology, 25(11).:953-956.) [10] 张武元, 陆宇燕, 史静耸, 等. 2019. 雄性峨眉髭蟾“胡子”的组织结构特征[J]. 四川动物, 38(02): 186-193. (Zhang W Y, Lu Y Y, Shi J S, et al.2019. Transcriptome analysis reveals thegenetic basis underlying the seasonal development of keratinized nuptial spines in Leptobrachium boringii[J]. Sichuan Animals, 38(02): 186-193.) [11] Altschuland S.1997. Gapped BLAST (Basic Local Alignment Search Tool) and PSI-BLAST: A new generation of protein database search programs[J]. Nucleic Acids Research, 25(17): 3389-3402. [12] Amos B, Rolf A.2000. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000[J]. Nucleic Acids Research, 28(1): 325-337. [13] An S, Schorfheide F.2007. Bayesian Analysis of DSGE Models[J]. Econometric Reviews, 26(2-4): 1237-1249. [14] Ashburner M, Ball C A, Blake J A.D.et al.2000. Gene ontology: Tool for the unification of biology[J]. Nature Genetics, 25(1): 254-267. [15] Bert W, Franky B, Sunita J, et al.2013. Frog nuptial pads secrete mating season-specific proteins related to salamander pheromones[J]. Journal of Experimental Biology, 216( 22): 167-176. [16] Bolling M C, Lemmink H H, Jonkman M. F.2011. Mutations in KRT5 and KRT14 cause epidermolysis bullosa simplex in 75% of the patients[J]. The British Journal of Dermatology, 164(3): 468-472. [17] Che J, Hu J D, Zhou W W, et al.2009. Phylogeny of the Asian spiny frog tribe Paini (Family Dicroglossidae) sensu Dubois[J]. Molecular Phylogenetics and Evolution, 50(1): 548-556. [18] Che Jing, Zhou Wei-Wei, Hu Jian-Sheng, et al.2010. Spiny frogs (Paini) illuminate the history of the Himalayan region and Southeast Asia[J]. Proceedings of the National Academy of Sciences of the USA, 107(31): 759-773. [19] Chen F Z, You L J, Yang F, et al.2020. CNGBdb: China National GeneBank DataBase[J]. Hereditas, 42(8): 778-785. [20] Chen Z Z, Lin X L,Ya P, et al.2020. Dysregulated expression of androgen metabolism genes and genetic analysis in hypospadias[J]. Molecular Genetics & Genomic Medicine, 8(8): 577-586. [21] Daniel H, Zhang A, Mohammad A.et al.2018. AR signaling in prostate cancer regulates a feed-forward mechanism of androgen synthesis by way of HSD3B1 upregulation[J]. Endocrinology, 159(8): 658-677. [22] Eddy S R.1998. Profile hidden Markov models (review)[J]. Bioinformatics, 26(7): 693-706. [23] Fadi T, Susan V, Casey A, et al.2010. Detection of gene orthology from gene co-expression and protein interaction networks[J]. BMC Bioinformatics, 11(3): 637-649. [24] Han F F, Ren L L, Ling L, et al.2020. HSD3B1 variant and androgen-deprivation therapy outcome in prostate cancer[J]. Cancer Chemotherapy and Pharmacology, 21(6): 497-517. [25] Hau M.2007. Regulation of male traits by testosterone: Implications for the evolution of vertebrate life histories[J]. BioEssays, 29(2): 364-372. [26] Hayashi Y, Mindy K C, Liu C Y, et al.2010. Monoallelic expression of Krt12 gene during corneal-type epithelium differentiation of limbal stem cells[J]. Investigative Ophthalmology & Visual Science, 51(9): 845-856. [27] Houck L D.2009. Pheromone communication in amphibians and reptiles[J]. Annual Review of Physiology, 71: 161-176. [28] Koonin V E.2004. A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes[J]. Genome Biology, 5(l): 254-264. [29] Manfred G G, Brian J H, Moran Y, et al.2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 29(7): 899-912. [30] Michael J R.1986. Amphibians: Biology of amphibians[J]. Science, 232(4747): 149-169. [31] Minoru K, Susumu G, Shuichi K, et al.2004. The KEGG resource for deciphering the genome[J]. Nucleic Acids Research, 32(4): 278-289. [32] Pascussi J M, Gerbal C S, Drocour L, et al.2003. The expression of CYP2B6, CYP2C9 and CYP3A4 genes: A tangle of networks of nuclear and steroid receptors[J]. BBA-General Subjects, 1619(3): 679-688. [33] Robert D F, Penelope C, Ruth Y, et al.2016. The Pfam protein families database: Towards a more sustainable future[J]. Nucleic Acids Research, 44(1): 523-536. [34] Tanifuji T N, Terai K, Hayashi Y, et al.2006. Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth[J]. Investigative Ophthalmology & Visual Science, 47(2): 755-766. [35] Valuing I.1993. Comparative histochemistry of the sexually dimorphic skin glands of anuran amphibians[J]. Copeia, 1993(1): 133-143. [36] Willaert B, Bossuyt F, Janssenswillen S, et al.2013. Frog nuptial pads secrete mating season-specific proteins related to salamander pheromones[J]. Journal of Experimental Biology, 216(22): 4139-4143. [37] Zhang W, Guo Y, Li J, et al.2016. Transcriptome analysis reveals the genetic basis underlying the seasonal development of keratinized nuptial spines in Leptobrachium boringii[J]. BMC Genomics, 17(1): 249-256.
