Identification of 17β-HSDs gene family and their expression patterns in gonadal development of Sinonovacula constricta
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摘要: 17β-羟类固醇脱氢酶（17β-HSDs）是性类固醇激素合成过程中最后步骤的氧化还原酶，在动物性腺发育、生殖调控中发挥重要作用。本研究基于全基因组数据，利用RACE技术和多种生物信息学软件对缢蛏17β-HSDs基因家族成员进行了鉴定、染色体定位和系统进化分析，并利用qRT-PCR 技术研究了其在不同组织、性腺不同发育时期的表达特征。结果表明，缢蛏17β-HSD6、17β-HSD10、17β-HSD12、17β-HSD14基因分别定位在4条不同染色体上，所编码的蛋白均具有SDR超家族的保守结构域；系统进化结果表明，缢蛏17β-HSDs基因家族成员分化明显，各自聚为独立分支。qRT-PCR分析显示，缢蛏17β-HSDs基因在8个组织中均有表达，且在性腺和肝胰腺中的表达量较高；在缢蛏性腺发育过程中，17β-HSD6基因在精巢和卵巢排放期的表达量较高，17β-HSD10、17β-HSD12、17β-HSD14基因在两性性腺中的表达均呈现先升高再降低的变化趋势，均在成熟期的表达量较高；在成熟期，17β-HSD10基因在精巢的表达量显著高于卵巢（p < 0.05），而17β-HSD12、17β-HSD14基因在卵巢中的表达量显著高于精巢（p < 0.05）。综上结果表明，17β-HSD6基因可能不参与调控缢蛏性腺发育过程，而17β-HSD10、17β-HSD12、17β-HSD14基因的表达与性腺发育过程密切相关，推测其可能通过介导性类固醇激素的合成与代谢调控缢蛏性腺发育过程。研究结果将为深入研究贝类17β-HSDs调控性腺发育、生殖内分泌机制奠定理论基础。
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关键词:
- 缢蛏 /
- 17β-HSDs基因家族 /
- 性腺发育 /
- 表达分析
Abstract: 17β-hydroxysteroid dehydrogenases (17β-HSDs) are the oxidoreductases in the last step of sex steroid hormone synthesis process, and play important roles in animal gonadal development and reproductive regulation. In this study, on the basis of the genome data of the razor clam Sinonovacula constricta, 17β-HSD genes were cloned and identified, and their chromosome distributions, phylogenetic relationships and gene expression patterns in different tissues and under different gonadal developmental stages were analyzed. The results showed that 17β-HSD6, 17β-HSD10, 17β-HSD12 and 17β-HSD14 genes of S. constricta were located on four different chromosomes, and the encoded proteins all contained the conserved SDR superfamily domains. The four 17β-HSD proteins were clearly differentiated and clustered into four independent branches in the phylogenetic trees. The results of qRT-PCR showed that 17β-HSD genes were all expressed in testes tissues, and possessed higher expression levels in gonads and hepatopancreas. During the gonadal development of S. constricta, 17β-HSD6 gene was expressed higher in testis and ovary at the spawning stage. The expression levels of 17β-HSD10, 17β-HSD12 and 17β-HSD14 genes showed a trend of increasing first and then decreasing during the development cycle of testis and ovary, which were all expressed higher at the mature stage. At the mature stage, 17β-HSD10 gene was expressed significantly higher in testis than in ovary (p < 0.05) , while the expression levels of 17β-HSD12 and 17β-HSD14 genes were all significantly higher in ovary than in testis (p < 0.05). Therefore, 17β-HSD6 gene might not be involved in regulating gonadal development process, while the expressions of 17β-HSD10, 17β-HSD12 and 17β-HSD14 genes were closely related to the gonadal development, suggesting that they might affect the gonadal development by regulating the synthesis and metabolism of sex steroid hormones in S. constricta. These findings laid theoretical foundations for further study on mechanism of 17β-HSD genes in regulating gonadal development and reproductive endocrine of mollusks. -
图 1 缢蛏17β-HSDs基因家族成员的氨基酸序列比对
Fig. 1 Multiple alignment of the amino acid sequences encoded by 17β-HSDs gene family in Sinonovacula constricta
图 2 缢蛏17β-HSDs基因家族蛋白的motif分析
Fig. 2 Motif analysis of the 17β-HSDs gene family protein in Sinonovacula constricta
图 3 缢蛏17β-HSDs基因家族的染色体定位
Fig. 3 Chromosomal localization of the 17β-HSDs gene family in Sinonovacula constricta
图 5 缢蛏17β-HSDs基因在不同组织中的表达特征（n = 4，不同小写字母代表差异显著p < 0.05）
Fig. 5 Relative expression of 17β-HSDs gene family in different adult tissues of S. constricta (n = 4, superscript letters represent statistically significant differences at p < 0.05)
图 6 缢蛏性腺不同发育时期生殖细胞形态学观察
M：精巢；M1：增殖期；M2：生长期；M3：成熟期；M4：排放期；F：卵巢；F1：增殖期；F2：生长期；F3：成熟期；F4：排放期；Sg：精原细胞；Sc：精母细胞；St：精细胞；Sz：精子；Og：卵原细胞；Oc：卵母细胞；Mo成熟卵细胞；标尺：50 μm
Fig. 6 Observation of various gonadal development stages from S. constricta
M: testis; M1: proliferative stage; M2: growing stage; M3: mature stage; M4: spawning stage; F: ovary; F1: proliferative stage; F2: growing stage; F3: mature stage; F4: spawning stage; Sg: spermatogonium; Sc: spermatocyte; St: spermatid; Sz: spermatozoon; Og: oogonium; Oc: oocyte; Mo: mature oocyte; scale bar: 50 μm
图 7 缢蛏17β-HSDs基因家族在性腺不同发育时期的表达差异分析（n = 6，不同小写字母代表差异显著p < 0.05）
Fig. 7 Relative expression of 17β-HSDs gene family in gonads of Sinonovacula constricta at different gonadal developmental stages (n = 6, superscript letters represent statistically significant differences at p < 0.05)
表 1 实验所用引物序列信息
Tab. 1 Information of primers used in experiments
引物名称 引物序列（5′-3′） 用途 RACE-17β-HSD6-5 CCATAGACCTTTGTCGGCGAGTTGCTGT 基因克隆 RACE- 17β-HSD6-3 TGGCGACACCTACTTCAGGGCTTTTATT RACE-17β-HSD10-5 CTGCCACGCTGGAGGTGTTGATGATGAC RACE- 17β-HSD10-3 CACGCCCCTGATGGAATCTTTACCTGAG RACE-17β-HSD12-5 CCACACCTTTCCGTCGTTCAGCCAT RACE- 17β-HSD12-3 CCAGGGATGGCTGAACGACGGAAAGGTG RACE-17β-HSD14-5 CAGACACGCCTTTCCACATTCCTCCGAT RACE- 17β-HSD14-3 TGGACCCCTATGTGGGACAGTCTGGCAA 17β-HSD6-F ATCTGGGTTGTGAATAGACTGCT 序列验证 17β-HSD6-R CTGAGAACCTGGATTTTAGCGT 17β-HSD10-F TAACAGGAGGAGCGTCAGGA 17β-HSD10-R CCCATTGATGAAAGGATTCTCTAT 17β-HSD12-F GGAGGCTGTATTGGGAAGATTT 17β-HSD12-R CTCCATCTTTTTCTTAGCCCTTT 17β-HSD14-F TGGAGGGAGTAAGGGTATCGG 17β-HSD14-R CTGCTGCTAAAAACAGACACGC Real-17β-HSD6-F CGCAAGAATCCTCCATAACAACT 荧光定量 Real-17β-HSD6-R GCGAACA CAGCCACTCCG Real-17β-HSD10-F CTCCCCACATCCAAAGGC Real-17β-HSD10-R CAATGGCAATACCAGCACAA Real-17β-HSD12-F TGTTCAGTGTGTGAGCCCATAC Real-17β-HSD12-R TCCATCTTTTTCTTAGCCCTTT Real-17β-HSD14-F GGAAGACAAAGGGCAACATAAT Real-17β-HSD14-R TCCCACATAGGGGTCCAAAT 18s rRNA-F TCGGTTCTATTGCGTTGGTTTT 18s rRNA-R CAGTTGGCATCGTTTATGGTCA 
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表 2 缢蛏17β-HSDs基因家族的理化性质分析
Tab. 2 The physicochemical properties of 17β-HSDs gene family in Sinonovacula. constricta
登录号ID 基因名 染色体 CDS/bp 氨基酸数 分子量/kDa 等电点 亲水性 不稳定指数 亚细胞定位 OP903386/ ctg553.20 17β-HSD6 Chromosome16 988 329 36.84 9.59 0.043 36.71 膜结合线粒体 OP903387/ ctg304.93 17β-HSD10 Chromosome10 720 239 25.53 8.19 0.028 20.90 膜结合线粒体 OP903388/ ctg492.3.1 17β-HSD12 Chromosome3 963 320 34.86 9.42 0.184 39.98 膜结合内质网 OP903389/ ctg220.10 17β-HSD14 Chromosome14 810 269 28.99 5.28 –0.227 40.38 细胞质
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