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高丽丽, 刁晓明, 李云, 翟旭亮, 周春龙. 中华鳖Foxl2基因克隆及外源性激素对其表达的影响[J]. 水生生物学报, 2019, 43(1): 45-51. DOI: 10.7541/2019.006
引用本文: 高丽丽, 刁晓明, 李云, 翟旭亮, 周春龙. 中华鳖Foxl2基因克隆及外源性激素对其表达的影响[J]. 水生生物学报, 2019, 43(1): 45-51. DOI: 10.7541/2019.006
GAO Li-Li, DIAO Xiao-Ming, LI Yun, ZHAI Xu-Liang, ZHOU Chun-Long. MOLECULAR CLONING AND EXPRESSION OF FOXL2 GENE INDUCED BY EXOGENOUS HORMONE IN THE PELODISCUS SINENSIS[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(1): 45-51. DOI: 10.7541/2019.006
Citation: GAO Li-Li, DIAO Xiao-Ming, LI Yun, ZHAI Xu-Liang, ZHOU Chun-Long. MOLECULAR CLONING AND EXPRESSION OF FOXL2 GENE INDUCED BY EXOGENOUS HORMONE IN THE PELODISCUS SINENSIS[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(1): 45-51. DOI: 10.7541/2019.006

中华鳖Foxl2基因克隆及外源性激素对其表达的影响

MOLECULAR CLONING AND EXPRESSION OF FOXL2 GENE INDUCED BY EXOGENOUS HORMONE IN THE PELODISCUS SINENSIS

  • 摘要: 外源性激素在中华鳖(Pelodiscus sinensis)性别决定有重要作用, 为给中华鳖性别决定机制研究提供生物学信息, 首次克隆和分析了中华鳖Foxl2 cDNA部分序列。为研究其在遗传和生理水平的差异, 以10 mg/kg剂量17α-甲基睾酮(MT)和17β-雌二醇(E2)分别对中华鳖雌雄个体注射, 检测0、6h、12h、24h、48h、7d和14d性腺Foxl2 mRNA表达水平。获得中华鳖Foxl2基因(GenBank登录号: KP734210)部分 cDNA长903 bp, 共编码300个氨基酸, 属于叉头框转录因子家族, 参与卵巢发育和功能维持; 多重序列比对显示, Foxl2具有典型的FH结构域, 与红耳龟的同源性最高, 达到99%; 系统进化树分析显示, 中华鳖Foxl2基因与爬行动物Foxl2基因聚为一个亚支, 且与西部锦龟Foxl2基因距离最近。荧光定量PCR结果显示, 与对照组相比, 注射E2后24h, 卵巢Foxl2 mRNA表达水平被极显著上调(P<0.001), 7d和14d后, 精巢Foxl2 mRNA表达水平极显著上升(P<0.001); 注射MT后24h, 精巢和卵巢Foxl2 mRNA的表达水平均极显著升高(P<0.001)。结果表明, E2和MT促进Foxl2表达, E2促进其表达的性别差异比MT明显。研究可为了解Foxl2的功能及明确外源性激素调控中华鳖Foxl2的分子机制提供基础资料。

     

    Abstract: In order to provide biological information for research on sex-determination mechanism in Pelodiscus sinensis, we here first attempted to clone and analyze the partial-length of Foxl2 cDNA. In addition, to address the differential expression of Foxl2 at genetic and physiological levels, both male and female sexes of Pelodiscus sinensis were treated with 10 mg/kg E2 and 17α-methyltestosterone (MT), respectively; Foxl2 mRNA expression was quantitatively examined in the gonads after injection treatment at 6, 12, 24, and 48h, as well as 7 and 14d, respectively. Foxl2 (GenBank Accession No. KP734210) was achieved, belonging to Forkhead family of transcription factors that is involved in ovarian development and functional maintenance, as well as a 903 bp of open reading frame (ORF) encoding 300 amino acids. Multiple sequence alignment showed that Foxl2 contained typical FH-domain, and the most similar ortholog was Trachemys scripta, reaching up to 99%. Stability analysis of phylogenetic trees showed that Pelodiscus sinensis Foxl2 was clustered with reptile Foxl2 as a sub-branch, and was closely associated with Foxl2 from Chrysemys picta bellii. Results of quantitative reverse transcription PCR (RT-qPCR) indicated that, compared to the control group, E2 significantly up-regulated Foxl2 mRNA repression in ovary after 24h (P<0.001), which in testis was significantly increased after 7 and 14d (P<0.001). MT treatment strongly and equally up-regulated expression levels ofFoxl2 mRNA in ovary and testis at 24h (P<0.001). These results suggested that E2 and MT could up-regulate Foxl2 expression. Moreover, the sex differences in E2 promoting Foxl2 expression is more significant than MT. It can be concluded that the present research contributes to better understanding of the functional role of Foxl2 and provide basic data for further study on how exogenous hormone mediates Foxl2 in Pelodiscus sinensis.

     

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