中山大学学报自然科学版 ›› 2018, Vol. 57 ›› Issue (1): 7-20.

• 论文 • 上一篇    下一篇

尼罗罗非鱼主要组织相容性复合体Ⅱ β(MHC Ⅱ β)基因的克隆、表达和多态性

何佳男,罗文娜,刘晓春,林浩然   

  1. 有害生物控制与资源利用国家重点实验室∥中山大学生命科学学院,广东 广州 510275
  • 收稿日期:2017-05-31 出版日期:2018-01-25 发布日期:2018-01-25
  • 通讯作者: 刘晓春(1968年生),男;研究方向:水生生物学;E-mail:lsslxc@mail.sysu.edu.cn

Molecular cloning, expression and polymorphism analysis of major histocompatibility complex class Ⅱβ genes in Nile tilapia(Oreochromis niloticus)

HE Jianan, LUO Wenna, LIU Xiaochun, LIN Haoran   

  1. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
  • Received:2017-05-31 Online:2018-01-25 Published:2018-01-25

摘要:

MHCⅡβ 基因在鱼类免疫反应中起重要作用。采用同源克隆和末端快速扩增(RACE)技术,克隆了尼罗罗非鱼的MHCⅡβ基因。MHCⅡβ 基因含有5个外显子和4个内含子,其cDNA全长为1 142 bp,开放读码框为750 bp,编码249个氨基酸。通过同源性分析和进化树分析发现,与其他硬骨鱼类和哺乳类的相似性在30.6%~78.6%,和鲈形目的鱼类同源性最高。从25尾罗非鱼的50个开放读码框的阳性克隆中共获得10条不同的核苷酸序列,分别编码不同的氨基酸。氨基酸序列比对分析发现,多态性位点主要集中在 MHCⅡβ 基因的第2外显子上。通过对海豚链球菌感染后易感个体和抗病个体的PCR-SSCP电泳分析,发现电泳条带出现2~12条不同的单带,测序结果表明个体存在1~6个等位基因,暗示至少存在3个 MHCⅡβ 基因座。qPCR检测表明,MHCⅡβ 基因在鳃、心脏、脾脏等组织高度表达,在肾脏、肌肉、脑等组织中中度表达,而在皮肤、肠、肝脏等组织中表达最弱。在攻毒后的头肾组织中,MHCⅡβ 基因的表达量呈现下降-上升-下降的变化趋势,表明 MHCⅡβ 参与了罗非鱼的免疫反应。

关键词: 尼罗罗非鱼;MHCⅡ&beta, ;组织分布;多态性

Abstract:

The major histocompatibility complex (MHC) class Ⅱβplay an important role in the immune response of teleost. In this study,full-length MHC Ⅱβ cDNA and genomic sequences were obtained from Nile tilapia (Oreochromis niloticus) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR).The full length of cDNA was 1 142 bp and the open reading frame (ORF) was 750 bp encoding 249 amino acid. The genomic structure of MHC Ⅱβ genes was similar with other teleost species including 5 exons and 4 introns. By homology comparison and phylogenetic analysis, MHCⅡβ genes of Nile tilapia had a higher identity and a closer evolutionary relationship with perciform fishes than other fishes. The putative protein had 30.6%~78.6% identity with those of other teleosts and mammals. Ten MHC Ⅱβ alleles were obtained from the fifty positive clones of ORF from 25 individuals. The amino acid sequence analysis showed that polymorphism was mostly focused on exon 2.PCR-SSCP and sequencing results revealed one to six different alleles for two to twelve bands in susceptibile or resistant fishes that after Streptococcus iniae challenge,which suggested there were at least 3 loci.qPCR indicated that high expression of MHC Ⅱβ was detected in gill, heart, spleen, and lower expression in kidney, muscle, brain and the lowest expression in skin, intestine and liver. After Streptococcus iniae challenge, the expression presented the tendency of the down-up-down in head and kidney, which demonstrated MHC Ⅱβ involved in tilapia immune response.

Key words: Nile tilapia, MHC Ⅱ&beta, tissue expression, polymorphism

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