缢蛏<i>17β-HSDs</i>基因家族鉴定及其在性腺发育过程中的表达分析

李琳; 董迎辉; 付璐璐; 孟钇萍; 林志华; 姚韩韩

doi:10.12284/hyxb2023137

缢蛏17β-HSDs基因家族鉴定及其在性腺发育过程中的表达分析

doi: 10.12284/hyxb2023137

1.
浙江万里学院生物与环境学院浙江省水产种质资源高效利用技术研究重点实验室，浙江宁波 315100
2.
宁海海洋生物种业研究院，浙江宁海 315604

基金项目: 国家自然科学青年基金项目（31902393）；国家现代贝类产业技术体系项目（CARS-49）；宁波市重大科技攻关暨“揭榜挂帅”项目（2021Z114）；国家海洋水产种质资源库项目（2023）；国家级大学生创新创业训练计划项目（202310876019）。

详细信息

作者简介:
李琳（2001—），女，浙江省宁波市人，主要从事贝类分子遗传学研究。E-mail：2492947101@qq.com

通讯作者:
姚韩韩（1984—），女，高级实验师，主要从事贝类分子遗传学研究。E-mail：yaohanhan1020@126.com

中图分类号: [P735]
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出版历程
- 收稿日期: 2023-02-13
- 修回日期: 2023-04-26
- 网络出版日期: 2023-11-02
- 刊出日期: 2023-10-30

Identification of 17β-HSDs gene family and their expression patterns in gonadal development of Sinonovacula constricta

1.
Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China
2.
Ninghai Marine Biological Seed Industry Research Institute, Ninghai 315604, China

摘要

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。
- 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.
- Sinonovacula constricta /
- 17β-HSDs gene family /
- gonadal development /
- expression analysis

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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

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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

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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

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图 4 17β-HSDs基因家族的系统进化树

Fig. 4 Phylogenetic tree of the 17β-HSDs gene family

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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)

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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

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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)

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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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