文章摘要
中国明对虾calcineurin B基因的克隆及其在竞争行为中作用的初探
Identification and characterization of the calcineurin B in Fenneropenaeus chinensis: its implications on competitive ability
投稿时间:2019-01-10  修订日期:2019-04-10
DOI:
中文关键词: 中国明对虾  竞争行为  钙调神经磷酸酶  基因克隆  表达分析
英文关键词: Fenneropenaeus chinensis  competitive behavior  calcineurin B  gene cloning  expression analysis
基金项目:国家自然科学基金面上项目 (31572616);国家自然科学基金联合基金项目(U1706203);
作者单位E-mail
孙 犁 上海海洋大学水产与生命学院 1498100076@qq.com 
卢 霞 中国水产科学研究院黄海水产研究所 luxia@ysfri.ac.cn 
孔 杰 中国水产科学研究院黄海水产研究所  
孟宪红 中国水产科学研究院黄海水产研究所  
闫允君 中国水产科学研究院黄海水产研究所  
李波波 上海海洋大学水产与生命学院  
仲伟鹏 中国水产科学研究院黄海水产研究所  
陈宝龙 中国水产科学研究院黄海水产研究所  
曹宝祥 中国水产科学研究院黄海水产研究所  
罗坤 中国水产科学研究院黄海水产研究所  
栾 生 中国水产科学研究院黄海水产研究所 luansheng@ysfri.ac.cn 
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中文摘要:
      中国明对虾(Fenneropenaeus chinensis)在养殖中表现出较强的竞争行为,对其生长性能产生了较大的影响,然而目前为止尚不清楚其发生的分子机制。钙调神经磷酸酶(calcineurin,CN)是高度保守的Ca2+/钙调蛋白(calmodulin, CaM)依赖性丝氨酸/苏氨酸磷酸酶,由催化亚基(CNA)和调节亚基(CNB)组成,是参与许多重要生理过程的多功能蛋白质。CNB在 Ca2+/CaM的介导下主要在动物的中枢神经系统发挥重要的作用。另外,在我们前期通过比较转录组分析筛选出的与中国明对虾竞争行为相关的候选基因中包括提示为CNB的基因。为了进一步明确CNB在中国对虾竞争过程中的作用,本研究通过RACE技术克隆了中国明对虾CN调节亚基B( FcCN-B)的全长cDNA序列,并利用Real-time PCR技术分析了其在高竞争能力组(HCG)和低竞争能力组(LCG)组间不同组织(神经节、心脏、胃、肝胰腺和肠)中的表达情况。结果显示,FcCN-B的全长cDNA序列为2867 bp,包括95 bp 的5' 非编码区(UTR),540 bp的开放阅读框(ORF),和2232 bp 的3' UTR,其中ORF中具有四个保守的EF-hand Ca2+ 结合结构域。蛋白质同源性分析显示,FcCN-B的氨基酸序列与其它物种具有较高的同源性(78.8%-93.8%),其中最高的是中华绒螯蟹(93.8%)和黑腹果蝇(90.5%);系统进化关系分析显示,脊椎动物和无脊椎动物分别独立为一支,而且中国明对虾与中华绒螯蟹单独聚为一支,之后与黑腹果蝇聚类关系最近,提示FcCN-B在对虾中可能具有与其在中华绒螯蟹和果蝇中相类似的功能。Real-time PCR定量结果显示FcCN-B在HCG组的神经节中的表达极显著高于LCG组 (P < 0.01),而其在HCG组的心脏中的表达极显著低于LCG组(P < 0.01)。本研究结果初步证明calcineurin B基因在中国明对虾的竞争行为中可能发挥一定的作用,将为解析中明对虾竞争行为的分子机制奠定重要的基础。
英文摘要:
      Competitive behavior exists widely in animal and plant groups. When groups or individuals have the same needs for the limited resources, competition will occur in intra-species or inter-species. The existence of competitive behavior can significantly affect the growth performance of individuals. There is strong competitive behavior in the breeding of Fenneropenaeus chinensis, and we has proved that the competitive behavior among individuals of F. chinensis has a significant effect on individual growth traits, and this effect can be inherited. However, little information was available for the molecular mechanism of competition behavior in shrimp. Candidate genes involved in competitive behavior of F. chinensis have been identified by comparative transcriptome analysis in our previous study, among which there is a calcineurin (CN) B gene. CN is a highly conserved Ca2+/calmodulin (CaM)-dependent serine/threonine phosphatase, which composes of a catalytic subunit (CNA) and a regulatory subunit (CNB). Previous studies reported that CN-B played an important role in the central nervous system mediated by Ca2+/CaM. In order to further prove the role of CN-B in the competitive behavior, in the present study, the full-length cDNA of CN-B of F. chinensis (FcCN-B) was cloned by RACE technology, and its expression in different tissues (nervous, heart, stomach, hepatopancreas, and intestine) between high competitive group (HCG) and low competitive group (LCG) was analyzed by Real-time PCR. The results show that the full-length cDNA of FcCN-B is 2867 bp, containing 95 bp of 5′ untranslated regions (UTR), 540 bp of open reading frame (ORF), and 2232 bp of 3′ UTR. There are four conserved EF-hand Ca2+ binding domains in the ORF of FcCN-B. Protein homology analysis indicated that the amino acid sequence of FcCN-B has high homology with other species (78.8%-93.8%), among which FcCN-B has the highest homology with Eriocheir sinensis (93.8%) and then with Drosophila. melanogaster (90.5%). The phylogenetic relationship analysis showed that vertebrates and invertebrates were independently clustered into two branches. Furthermore, F. chinensis was clustered with E. sinensis into an independent branches, and then it was clustered to D. melanogaster, which suggesting that FcCN-B might have similar functions as it did in D. melanogaster. The results of Real-time PCR showed that the expression of FcCN-B in the nerve ganglion was significantly higher in HCG than in LCG (P < 0.01), but its expression in heat was significantly lower in HCG than in LCG (P < 0.01). This study preliminarily proved that the calcineurin B may play a certain role in the competition behavior of F. chinensis, and will lay an important foundation for investigating the molecular mechanism of the competition behavior of F. chinensis.
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