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牙鲆17β-羟类固醇脱氢酶家族基因特征、表达与功能的初步研究
邹聪聪
学位类型硕士
导师尤锋
2020-05-21
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
关键词牙鲆(paralichthys Olivaceus) Hsd17b 基因特征与表达 性腺发育 性腺分化
摘要

        性类固醇激素在鱼类性腺分化、性腺发育和第二性征形成中起重要作用。性类固醇激素的合成涉及一系列合成酶,如细胞色素P450(cytochrome P450,CYPs)、羟类固醇脱氢酶(hydroxysteroid dehydrogenase,HSDs)等。其中,17β-羟类固醇脱氢酶(17β-hydroxysteroid dehydrogenase,Hsd17b)是关键酶类,主要作用于性激素合成的下游,参与雌酮、雌二醇和雄烯二酮、睾酮的相互转化。本研究以具有重要经济价值的海水养殖鱼类牙鲆(Paralichthys olivaceus)为研究对象,克隆获得了10个Hsd17b家族基因。联合本实验室前期发表的Hsd17b1基因,对这些基因的结构进行了分析,预测了这些基因所编码蛋白的生化特性,研究了各基因在雌雄成体12种组织和雌雄性腺I-V期的表达谱。并以Hsd17b1-3为例,研究了这两个基因在性类固醇激素合成途径中的作用,以期对性类固醇激素的合成和调控机制有更深入地认识,为牙鲆等鱼类单性养殖提供参考。

        1、从牙鲆基因组和转录组数据中筛选到Hsd17b3、-4、-7、-8、-9、-10、-12a、-12b、-14和-15共10个基因的序列,通过克隆与测序进行验证,经序列比对获得ORF区。其中,Hsd17b9-15在鱼类中未见报道。结构域和基序分析结果表明,这10个基因都属于短链脱氢酶/还原酶(short-chain dehydrogenase/reductase,SDR)超家族,均具有保守的基序,如辅因子结合位点基序TGxxxGxG、反应决定方向基序PGxxxT、结构稳定基序NNAG和活性中心基序YxxxK。氨基酸序列分析预测Hsd17b1、-4、-7、-12a和-14是亲水蛋白,其余为疏水蛋白;Hsd17b1、-3和-12b蛋白的稳定性较低。染色体定位分析结果显示,Hsd17b基因分布在8条染色体上(chr 1、7、10、17、19、22、23和24),其中Hsd17b12a-12b分别位于19号和7号染色体上,表明其是两个不同基因,仅序列高度相似,而不是源自同一基因的不同转录本。

        2、利用实时定量PCR(real-time quantitative PCR,qPCR)技术检测了Hsd17b家族基因在牙鲆雌雄成体不同组织及雌雄性腺I-V期的表达模式。结果表明,该家族基因大多数为广谱表达,Hsd17b9-10-12a-12b在牙鲆雌雄性腺中有差异表达,其中Hsd17b10-12a-12b在卵巢中高表达,Hsd17b9在精巢中的表达高于卵巢。在整个性腺发育过程中,Hsd17b3-9在精巢中高表达,Hsd17b1-4-8-10-12a-12b在卵巢中高表达,推测它们在牙鲆性腺发育雌雄激素合成中发挥重要作用。

        3、筛选Hsd17b1-3进行了初步功能研究。首先对Hsd17b3在牙鲆性腺分化过程的表达进行了qPCR分析。结果显示,在对照组、高温组(28 °C ± 0.3 °C,HT)和雌激素受体抑制剂(他莫西芬,100 ppm,TM)组中,全长2 cm(Total length,TL)时,Hsd17b3的表达水平最高,与对照组相比,HT组和TM组的Hsd17b3表达较低。HEK 293T(human embryonic kidney 293T)细胞转染分析表明,Hsd17b1和-3蛋白分布在细胞质和细胞核中。然后,酶比活力测定结果显示,Hsd17b1在精巢I-V期中的比活力高于同期的卵巢,并且除IV期外,其差异均为极显著(P < 0.01);与基因表达模式类似,在I-V期,Hsd17b3在精巢中的比活力显著高于卵巢(P < 0.05,P < 0.01)。同时,进一步分别对成体牙鲆进行了腹腔注射他莫昔芬(10 mg/mL)和雄激素受体抑制剂(氟他胺,50 mg/mL)。实验结果显示,他莫昔芬腹腔注射后,卵巢和精巢中Hsd17b1的表达无明显变化;精巢中Hsd17b3的表达水平显著降低(P < 0.01),而卵巢中未观察到明显变化。氟他胺注射后,Hsd17b1在精巢和卵巢中表达上调,并且在卵巢中上调显著(P < 0.05);精巢和卵巢中Hsd17b3的表达下调,但与对照组相比无显著差异。

其他摘要

        Sex steroid hormones play an important role in fish sex differentiation, gonadal development and secondary sexual characteristics. Their synthesis involves a series of catalyzing enzymes, such as cytochrome P450 (CYPs), hydroxysteroid dehydrogenase (HSDs) and so on. Among them, 17β-hydroxysteroid dehydrogenase (Hsd17b) is a key class of enzyme that acts in downstream part of the steroidogenesis pathway and possesses the capacity for oxidation and reduction between estrone and estradiol and between androstenedione and testosterone. The olive flounder Paralichthys olivaceus, a commercially valuable marine aquaculture fish, was studied in this study. A total of 10 flounder Hsd17b family genes were obtained. Combined with the Hsd17b1, one gene published previously in our laboratory, we analyzed their genetic structure and the protein biochemical characteristics. The expression profiles of Hsd17b family genes were detected in 12 different tissues of adult female and male flounders and in gonads at I-V gonadal development stages. Hsd17b1 and -3 were used as examples to further study their function in the flounder steroidogenesis pathway. These results could contribute to our understanding of the regulation of sex steroid hormone synthesis in fish gonadal development and provide a reference for the monoculture of Paralichthys olivaceus and other fishes.

        1. Hsd17b3, -4, -7, -8, -9, -10, -12a, -12b, -14 and -15 gene sequences were screened within the flounder genome and transcriptome data. After cloning and sequencing, ORF regions were obtained. Hsd17b9 and -15 have never been reported in a fish. According to the domain and motif analyses, they all belong to the short-chain dehydrogenase/reductase (SDR) superfamily and contain conserved motifs, such as the cofactor binding site TGxxxGxG, reaction direction determining motif PGxxxT, structural stable motif NNAG and active center motif YxxxK. Analysis of amino acid sequences predicted that Hsd17b1, -4, -7, -12a and -14 were hydrophilic proteins, and the remaining proteins were hydrophobic. The stability of Hsd17b1, -3 and -12b proteins was predicted to be low. Chromosome distribution results showed that the flounder Hsd17b genes were distributed on eight chromosomes (chr 1, 7, 10, 17, 19, 22, 23 and 24), of which Hsd17b12a and -12b were located on chromosomes 19 and 7, respectively. It was demonstrated that they were two genes and had just highly similarity rather than different transcripts derived from the same gene.

        2. Real-time quantitative PCR (qPCR) was used to detect the expression patterns of the various Hsd17b family genes in different tissues of adult flounder and in gonads at I-V gonadal developmental stages. The results showed that expression of most genes in tissues was widely distributed. Hsd17b9, -10, -12a and -12b were differently expressed in the testis and ovary, and among them Hsd17b10, -12a and -12b were highly expressed in the ovary, and Hsd17b9 was highly expressed in the testis than that in the ovary. Moreover, throughout gonadal development, the expression of Hsd17b3 and -9 was higher in the testis than that of the ovary, and Hsd17b1, -4, -8, -10, -12a and -12b expression was higher in the ovary than that of the testis, suggesting that they might play an important role in androgen and estrogen synthesis in the flounder gonadal development.

        3. Functional studies on Hsd17b1 and -3 were carried out. Firstly, the expression levels of Hsd17b3 in the gonadal differentiation were detected by qPCR. The results showed that in the control, high temperature (28 °C ± 0.3 °C, HT) and estrogen receptor inhibitors (tamoxifen, 100 ppm, TM) groups, the expression level of Hsd17b3 was highest when the flounder was at 2 cm total length (TL), and compared with the control group, the expression of Hsd17b3 was decreased in the HT and TM groups. Transfection analysis in the HEK 293T (human embryonic kidney 293T) cells showed that Hsd17b1 and -3 were located in the cytoplasm and nucleus. Then, the results of enzyme specific activity determination showed that the activities of Hsd17b1 at stages I-V were higher in the testis than those in the ovary, and except for the stage IV, the difference between testis and ovary was extremely significant (P < 0.01). Similar to the gene expression pattern, the specific activities of Hsd17b3 at stages I-V was significantly higher in the testis than that of the ovary (P < 0.05, P < 0.01). At the same time, the in vitro intraperitoneal injection on the flounder was performed with estrogen tamoxifen (10 mg/mL) and androgen receptor inhibitor (flutamide, 50 mg/mL). The results showed that after challenge with tamoxifen, the expression levels of Hsd17b1 in the testis and the ovary were no significantly difference. The Hsd17b3 expression level in the testis decreased significantly (P < 0.01), and in the ovary no significant expression change was observed. Moreover, after challenge with flutamide, the expression levels of Hsd17b1 in the ovary and testis were up-regulated, and in the ovary, the increase was distinct (P < 0.05). Hsd17b3 expression was down-regulated in the testis and ovary without significant difference.

学科领域生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/164632
专题实验海洋生物学重点实验室
推荐引用方式
GB/T 7714
邹聪聪. 牙鲆17β-羟类固醇脱氢酶家族基因特征、表达与功能的初步研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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