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

作　　者：黄孝枭 刘炳正 孙志惠 丛晶晶 苏唯一 魏金亮 HUANG Xiaoxiao;LIU Bingzheng;SUN Zhihui;CONG Jingjing;SU Weiyi;WEI Jinliang(Key Laboratory of Mariculture and Stock Enhancement in North China Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023,China)

机构地区：[1]大连海洋大学农业农村部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《渔业科学进展》2023年第6期203-213,共11页Progress in Fishery Sciences

基　　金：国家重点研发计划“蓝色粮仓科技创新”重点专项(2018YFD0900201)资助。

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c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)获得其全长c DNA序列。通过荧光定量PCR (real-time quantitative PCR,RT-q 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。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 12transcription 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 char

关 键 词：刺参 germ cell-less 生殖细胞 母源因子 

分 类 号：S917[农业科学—水产科学]