中国明对虾Dscam基因的克隆及其在免疫致敏(类免疫)诱导反应中的表达分析
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中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所

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中国水产科学研究院黄海水产研究所基本科研业务费专项(20603022017001);国家自然科学基金(31372523);泰山学者种业团队项目;国家现代农业产业技术体系(CARS-48)


Cloning and expression analysis of the Dscam gene during the inducing immune priming (Quasi-immune) response in Fenneropenaeus chinensis
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Yellow Sea Fisheries Research Institute,Yellow Sea Fisheries Research Institute

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    摘要:

    为了研究对虾免疫致敏过程中Dscam基因的表达规律,实验采用同源克隆和RACE技术获得了中国明对虾Dscam基因cDNA全长序列,并对该序列进行生物信息学分析。结果显示,中国明对虾Dscam基因的cDNA全长为6 624 bp,其中包括171 bp的5'端非编码区,459 bp的3'端非编码区,开放阅读框的长度为5 994 bp,编码1 996个氨基酸。推测该基因编码的蛋白含有一个信号肽、10个Ig结构域、6个FNⅢ功能区、1个胞质尾区和1个跨膜结构域。同源性分析及系统进化分析表明,Dscam基因与节肢动物的Dscam基因首先聚为一类,且与凡纳滨对虾的同源性最高,为92.4%。连续投喂6 d热灭活白斑综合征病毒(white spot syndrome virus,WSSV)饵料来诱导免疫致敏反应,在0 、6 和12 d及二次感染后的12 、24、48、72和168 h分别取样,用RT-PCR的方法检测中国明对虾Dscam的相对表达量。结果显示,诱导感染组Dscam基因在第12 天开始上调,且与阴性对照组和未诱导感染组差异显著;二次感染后24 h,Dscam基因的相对表达量达到最大值,与阴性对照组和未诱导感染组差异显著;48 h后基因表达量开始下降,但表达量仍高于阴性对照组和未诱导感染组。实验表明,中国明对虾存在Dscam基因,并在免疫致敏过程中发挥重要作用。

    Abstract:

    To explore the function of Dscam gene of Chinese shrimp Fenneropenaeus chinensis (FcDscam) during the immune priming response, we cloned the cDNA sequence using the rapid amplification of cDNA ends (RACE), and then analyzed the features of this gene based on bioinformatics software. The full length of FcDscam cDNA is 6624 bp, with a 5'-untranslated region (UTR) of 171 bp, a 3'-untranslated region (UTR) of 459 bp, and an opening reading frame (ORF) of 5 994 bp, and it encoded 1 996 amino acids. The protein encoded by FcDscam contains 1 signal peptide, 1 cytoplasmic tail, 1 transmembrane domain, 6 of fibronectin type Ⅲ (FNⅢ) and 10 of immunoglobulin(Ig). As shown by the homology search and neighbor-joining phylogenetic tree, FcDscam gene had the highest homology with that of Litopenaeus vannamei (92.4%). To induce the immune response, test animals were first fed with heat-inactivated WSSV bait for six days and infected again 12 days later. Samples were collected at 0, 6 and 12 d of the first-time infection and 12, 24, 48, 72 and 168 h of the second-time infection, respectively, for analysing the mRNA expression level of FcDscam by real-time PCR. On the 12th day of the first infection, its expression level in induced infection group began to increase, significantly different from those in the negative control group and the non-induced infection group. After 24 h of the second-time infection, its expression level in the infection group reached a maximum, significantly different from those of the negative control group and the non-induced infection group, and the expression level started to decline at the 48 h. Our research demonstrated the existence of the Dscam gene in F. chinensis, and its important role in the process of immune sensitization.

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曹家旺,孟宪红,孔杰,史晓丽,栾生,罗坤,董丽君,陈宝龙.中国明对虾Dscam基因的克隆及其在免疫致敏(类免疫)诱导反应中的表达分析[J].水产学报,2018,42(5):633~645

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  • 收稿日期:2017-05-23
  • 最后修改日期:2017-11-04
  • 录用日期:2017-12-25
  • 在线发布日期: 2018-05-07
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