生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (4): 49-.doi: 10. 3969 / j. issn. 2095-1736. 2021. 04. 049

• 研究报告 • 上一篇    下一篇

42Sp50/eEF1a在正常和gsdf缺失型青鳉性腺中的表达差异

  

  1. 1.上海海洋大学农业部淡水水产种质资源重点实验室,上海201306; 2.上海海洋大学水产种质资源发掘与利用教育部重点实验室,上海201306; 3.上海海洋大学水产科学国家实验教学示范中心,上海201306
  • 出版日期:2021-08-18 发布日期:2021-08-18
  • 通讯作者: 关桂君,教授,博士生导师,研究方向为鱼类性别分化调控机制,E-mail:gjguan@shou.edu.cn
  • 作者简介:武小雯,硕士研究生,研究方向为鱼类性别分化调控机制,E-mail:1165033441@qq.com
  • 基金资助:
    国家重点研发计划项目(2018YFD0900601)

Identification of 42Sp50 and eEF1a expression in normal and gsdf-deficientovaryinmedaka(Oryziaslatipes)

  1. 1. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University,Shanghai 201306, China; 2. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education, Shanghai Ocean University, Shanghai 201306, China; 3. National DemonstrationCenter for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
  • Online:2021-08-18 Published:2021-08-18

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摘要： 青鳉(Oryziaslatipes)性腺体细胞衍生因子gsdf敲除可导致XY雌性化,但Gsdf的生物学作用和靶基因仍不摘清楚要。用Label Free蛋白组学结合MS质谱分析,定量比对正常的XX卵巢、XY精巢及gsdf缺失XY卵巢中的蛋白质表达谱,发现真核翻译延伸因子1(eEF1)的卵巢异构型42Sp50的表达量,在gsdf缺失卵巢中显著升高。通过实时定量PCR扩增,定量分析了42Sp50在正常XXgsdf++卵巢、XX和XY型gsdf--卵巢中的表达。用抗人EEF1A抗体的免疫荧光技术,鉴定了青鳉eEF1a和/或42Sp50在生/殖细胞性分化及性成熟期/,gsdf缺失卵巢同正常卵巢对照组的细胞内表达变化,验证了蛋白组学发现的在gsdf-/ -卵巢中有大量卵细胞高表达42Sp50。青鳉eEF1a和42Sp50氨基酸序列高度同源,但在氨基酰基结合囊和核糖体结合部位有显著差异,为此推测Gsdf信号可能直接或间接地影响XY生殖细胞表达42Sp50和雌性分化。同时建立了双色细胞荧光免疫技术用于鉴别青鳉XX型和gsdf缺失的XY型卵细胞,为研究脊椎动物配子发生的分子机制提供了新线索。

关键词: 性腺体细胞衍生因子, 真核翻译延伸因子1, 42Sp50基因, 卵巢发育, 青鳉

Abstract: XY female can be created by genetic manipulation of the gonadal soma derived factor (gsdf), through transgenic over-ex-pression or targeted disruption ofgsdfin medaka (Oryziaslatipes), however, the bio-function and target genes ofgsdfare still unclear. We used Label Free proteomics combined with Mass Spectrometry (MS) to quantitatively compare the protein expression profiles of nor-mal XX ovaries, XY testis, andgsdf-deficient XY ovaries. Result showed that the protein level of ovarian isoform (42Sp50) of eukary-otic translation elongation factor 1a (eEF1a) was significantly enhanced ingsdfdepletion ovaries, in contrast to the normal male or fe-male. The transcripts of42Sp50were also significantly enriched in XYgsdfdeficiency ovaries by real-time PCR quantitative analysis, in contrast to less expression in normal XX ovaries or rare in XY testis. The amino acid sequence of medaka eEF1a was highly homolo-gous to human EEF1A, but significantly different from medaka42sp50in the region of amino-acyl binding pocket. The expression ofeEF1aincluding42Sp50was examined in gonads during sexual differentiation, and mature gonads of normal female, male andgsdfde-pletion ovary by immunofluorescence using anti-human EEF1A antibody. Evidence of normal XX distinct fromgsdf-deficient XY oo-cytes by dual-color immunofluorescent analysis provides a new clue for the mechanism of vertebrate gametogenesis.

Key words: gsdf;eEF1a;42Sp50, oogenesis, medaka

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