| 黄孝枭,刘炳正,孙志惠,丛晶晶,苏唯一,魏金亮.刺参germ cell-less基因表达分析及转录因子预测.渔业科学进展,2023,44(6):203-213 |
| 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：10.19663/j.issn2095-9869.20220511002 |
| 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 Germ cell Maternal factor |
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| 中文摘要: |
| 不同性别的刺参(Apostichopus japonicus)在生长速度、免疫能力等方面具有显著差异，解析其性别决定和性别分化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中起重要作用，但无脊椎动物中关于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 (real-time quantitative PCR, RT-qPCR)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的表达量呈现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。在整个胚胎发育过程中均能检测到Ajgcl转录本，意味着Ajgcl作为母源因子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 structural domain plays an important role in the occurrence of mammalian gonads as it specifically combines with other DNA proteins. In this study, a unigene annotated as germ cell-less (Ajgcl) was screened from the gonadal genome of Apostichopus japonicus (sea cucumber) through homology. The full-length Ajgcl cDNA sequence was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). Subsequently, we analyzed the expression characteristic of germ cell-less in A. japonicus using several methods, including multiple sequence alignment, phylogenetic analysis, and real-time quantitative PCR (RT-qPCR). The results showed that germ cell-less attaches to the BTB/POZ superfamily. The full-length cDNA of Ajgcl was 2 316 bp, which contained a 725 bp 3′UTR, a 139 bp 5′UTR, and a 1 455 bp open reading frame (ORF), encoding a protein of 484 amino acids including a conservative BTB/POZ domain. RT-qPCR analysis revealed that germ cell-less was expressed in the ovary, testis, intestines, stomach, longitudinal muscle, respiratory tree, tube feet, and coelomocyte. Remarkably, the expression level in the ovary was the highest and 2.25 times that in the testis. Moreover, as a maternal factor, germ cell-less was expressed throughout embryonic development. During ovarian development, the expression of germ cell-less in growing stage 2 was slightly elevated, but when the ovum developed into mature 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 did not change significantly throughout the developmental stages of the testis. In addition, the online tool Match was used to predict the transcription factor binding sites in the germ cell-less promoter of A. japonicus. Subsequently, a total of 12 transcription factor binding sites were projected, namely Oct-1, FOXD3, PAX-6, CRP, c-Myb and NF-1. Notably, there were four Oct-1 binding sites between -1890 and -608. The characteristics of germ cell-less gene promoter were obtained, and germ cell-less was shown to be a germ cell marker gene for A. japonicus. This study provides data for future studies related to germ cell development in A. japonicus. |
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