文章摘要
刺参germ cell-less基因表达分析及转录因子预测
Expression of Germ cell-less and Its Putative Regulatory Region in the Sea Cucumber, Apostichopus japonicus
投稿时间:2022-05-11  修订日期:2022-07-11
DOI:
中文关键词: 刺参  germ cell-less  生殖细胞  母源因子  
英文关键词: Apostichopus japonicus  Germ cell-less gene  Generative cell  Maternal factor
基金项目:国家重点研发计划“蓝色粮仓科技创新”重点专项(2018YFD0900201)水产养殖生物性别和发育的分子基础与调控机制
作者单位邮编
黄孝枭 大连海洋大学 116023
刘炳正 大连海洋大学 
丛晶晶 大连海洋大学 
苏唯一 大连海洋大学 
魏金亮 大连海洋大学 
孙志惠 大连海洋大学 116023
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中文摘要:
      刺参(Apostichopus japonicus)是我国重要的海水养殖品种,具有极高的经济价值。不同性别的刺参在生长速度、免疫能力等方面具备显著差异,解析其性别决定和性别分化的机制具有重要的理论和经济价值。目前刺参的性腺发育机制尚不清晰,挖掘性腺发育相关基因是解析其发育机制的重要途径。研究表明, Germ cell-less基因在哺乳动物性腺发生起重要作用,但是无脊椎动物中关于Germ cell-less基因的研究较少。本研究从刺参基因组中鉴定了germ cell-less (Ajgcl)基因片段,随后通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE)获得其全长cDNA序列。通过荧光定量PCR揭示了Ajgcl在成体组织中呈现泛表达状态,性腺中表达量最高,且雌性性腺中的表达量是雄性性腺中表达量的2.25倍。随着卵子的发生, Ajgcl的表达发生先上升后下降的动态表达变化,而在精子发生的过程中其表达量变化不大,意味着其可能在卵子发生过程中发挥重要作用。Ajgcl是母源因子,在整个胚胎发育过程中均能检测到其转录本,意味着Ajgcl可能与原始生殖细胞的形成有关。此外,Ajgcl基因启动子中具有Oct-1、FOXD3、PAX-6、CRP、c-Myb和NF-1等转录因子的结合位点。本研究为深入解析germ cell-less基因在刺参等无脊椎动物性腺发育中发挥的功能奠定了基础。
英文摘要:
      The germ cell-less gene belongs to the BTB/POZ protein-family, whose structure domain plays an important role in the occurrence of mammalian gonad as it is combined with other DNA protein specifically. In this study, A unigene annotated as germ cell-less(Ajgcl) was screened from the gonadal genome of Apostichopus japonicus through homology, the full-length Ajgcl cDNA sequence was obtained by 5′ and 3′RACE (rapid amplification of cDNA ends). Subsequently, we analyzed the expression characteristic of germ cell-less gene in Apostichopus japonicus by multiple sequence alignment, phylogenetic analysis, real-time quantitative PCR (RT-qPCR) and etc. The results proves: germ cell-less gene attaches to the BTB/POZ superfamily. The full-length cDNA of Ajgcl was 2316 bp, which contains a 725 bp 3’UTR, a 139 bp 5’UTR and a 1455 bp opening reading frame(ORF), encoding a protein of 484 amino acids including a conservative BTB/POZ domain. Real-time quantitative PCR(RT-PCR) analysis revealed that germ cell-less gene was expressed in the ovary, testis, intestines, stomach, longitudinal muscle, respiratory tree, tube feet, and coelomocyte. Remarkably, the expression level in ovary was the highest and was 2.25 times that in testis. Moreover, as a maternal factor, germ cell-less gene was expressed in the whole embryonic development. During the development of ovary, the expression of germ cell-less in growing stage(stage 2) was slightly elevated, but when ovum developed to mature stage(stage 3), the expression level was significantly down-regulated. It was not until the end of gametogenesis that its expression level returned to its original level; the expression of germ cell-less gene didn’t change significantly throughout the developmental stages of the testis. In addition, Online tool Match was used to predict the transcription factor binding sites in germ cell-less gene promoter of Apostichopus japonicus. Then, a total of 12 transcription factor binding sites were projected, namely Oct-1, FOXD3, PAX-6, CRP, c-Myb and NF-1. Notably, there are four Oct-1 binding sites between -1890 and -608. Thus obtaining the characteristics of germ cell-less gene promoter. The germ cell-less gene has been shown to be a germ cell marker gene for Apostichopus japonicus. Study provides data for studies related to germ cell development in Apostichopus japonicus.
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