生物技术通报 ›› 2016, Vol. 32 ›› Issue (4): 94-101.doi: 10.13560/j.cnki.biotech.bull.1985.2016.04.012

• 研究报告 • 上一篇    下一篇

三疣梭子蟹nm23基因克隆及在卵巢发育中的表达分析

徐晴,朱冬发,谢熙,邱锡尔,沈锡权   

  1. 宁波大学海洋学院,宁波 315211
  • 收稿日期:2015-07-10 出版日期:2016-04-25 发布日期:2016-04-26
  • 作者简介:徐晴,女,研究方向:甲壳动物内分泌学;E-mail:634605585@qq.com
  • 基金资助:
    国家自然科学基金项目(41376152,40976098),浙江省自然科学基金项目(LY13C190006)

Cloning and Expression Analysis of Gene nm23 during Ovarian Development of Portunus trituberculatus

XU Qing ZHU ,Dong-fa, XIE Xi ,QIU Xi-er, SHEN Xi-quan   

  1. The School of Marine Science,Ningbo University,Ningbo 315211
  • Received:2015-07-10 Published:2016-04-25 Online:2016-04-26

摘要: Nm23基因编码的核苷二磷酸激酶(NDPK)参与调控生物体的多项生理功能,包括生长、发育、分化和肿瘤转移等。根据三疣梭子蟹(Portunus trituberculatus)转录组数据库中的nm23同源序列,利用cDNA末端快速扩增技术(RACE)首次克隆获得了三疣梭子蟹nm23基因的全长cDNA序列(GenBank登录号:KP027331)。该序列全长777 bp,编码一个151个氨基酸的蛋白,该蛋白包含一个典型的核苷二磷酸激酶(NDPK)区域,具有NDPK活性位点和多肽结合位点。推导的氨基酸序列与已知甲壳动物的nm23一致性达到90%以上,与脊椎动物第一类nm23的一致性也达到70%以上。系统进化树分析发现第一类nm23与第二类nm23分别聚为一支,甲壳动物nm23属于第一类,且与nm23-1和nm23-2亲缘关系较近。采用实时荧光定量PCR(qPCR)技术分析了三疣梭子蟹nm23的组织差异表达及其在卵巢发育过程中的表达水平变化,结果表明nm23在三疣梭子蟹各组织内均有表达,其表达水平在眼柄、肌肉和Y器中最高,卵巢和肝胰腺次之。在三疣梭子蟹卵巢发育过程中,卵巢中nm23的表达水平在Ⅰ期最高,之后随着卵巢发育的进行,其表达水平逐渐下降,并在成熟期降至最低,这表明nm23可能参与调控三疣梭子蟹的卵巢发育。

关键词: 三疣梭子蟹, nm23, 基因克隆, 卵巢发育, 基因表达

Abstract: Nm23 is a family of genes encoding the nucleoside diphosphate(NDP)kinase,which functions in a wide variety of biological processes,including growth,development,differentiation,and tumor metastasis. In this study,based on the nm23 homologous sequence in the transcriptome dataset of Portunus trituberculatus,the full-length cDNA of P. trituberculatus nm23 was obtained using the rapid amplification of cDNA ends(RACE). The full-length cDNA(GenBank accession number KP027331)was 777 bp,encoding for a protein of 151 amino acids with one typical NDP kinase domain that harbored all the crucial residues for nucleotide binding and enzymatic activity. The deduced amino acid sequence of P. trituberculatus nm23 exhibited more than 90% identities with that of other crustaceans,and more than 70% identities with the nm23 of group I of vertebrates. The phylogenic tree constructed based on nm23’s amino acid sequences indicated that the crustacean nm23 belonged to the group I(nm23-1),which was separated from the nm23 of group II(nm23-2),moreover,the nm23-1 and nm23-2 were close. The expression levels of P. trituberculatus nm23 in different tissues and during the ovarian development were detected by quantitative real-time PCR(qPCR). The results showed that nm23 was ubiquitously expressed in all tissues examined,its mRNA level was the most abundant in eyestalk,muscle and Y-organs,and second in ovary and hepatopancreas. During the ovarian development,the nm23 was mostly expressed at stage I,then declined along with the development of ovaries,and to the minimum at stage V,suggesting that the nm23 had the potential role in the ovarian development of P. trituberculatus.

Key words: Portunus trituberculatus, nm23, gene cloning, ovarian development, gene expression