斜带石斑鱼IgM、IgZ和IgD重链基因的克隆

Molecular characterization of IgM, IgZ and IgD heavy chain genes in orange-spotted grouper(Epinephelus coioides)

应用RACE方法获得斜带石斑鱼膜结合型免疫球蛋白M（membrane-bound immu-noglobulin M,mIgM）,膜结合型免疫球蛋白D（mIgD）,分泌型免疫球蛋白Z（secretory immu-noglobulin Z,sIgZ）的重链基因。斜带石斑鱼膜结合型IgM重链恒定区包含3个恒定区结构域（μ1,μ2,μ3）以及两个跨膜外显子（TM1,TM2）,TM1外显子与μ3结构域末端相连接。氨基酸序列相似性分析结果显示,斜带石斑鱼mIgM各恒定区与牙鲆mIgM恒定区相似性最高,为53%-78%。mIgD的cDNA全长为3 375 bp,开放阅读框包含3 006 bp,其恒定区由1个μ1外显子,7个δ外显子以及跨膜区组成。斜带石斑鱼IgD恒定区与鳜IgD各恒定区氨基酸序列相似性最高,δ1-δ7的相似性分别为75.5%、75.8%、65.4%、76.6%、88.1%、90.6%、82.8%,TM结构域为82.7%。sIgZ的基因结构与其他硬骨鱼类sIgZ的结构相似,包括4个外显子和3个内含子,内含子长度分别为222、129和458 bp。利用半定量PCR分别检测了这3种基因在斜带石斑鱼各器官/组织中的表达,发现mIgM在头肾、肾脏、脑、脾脏、肠、鳃、心脏和胸腺中均有表达;mIgD的mRNA在头肾、肾脏以及胸腺中有较高的表达,在肠中表达量较低;sIgZ mRNA主要分布于淋巴组织如头肾、肾及脾脏中,而在鳃、心脏和胸腺中的丰度较低。

In the present study, the membrane-bound immunoglobulin M （mlgM）, mIgD and secretory IgZ （slgZ） have been identified by using RACE-PCR from the orange-spotted grouper, Epinephelus coioides. The mlgM and slgZ are comparable with their counterparts in other teleost in terms of their heavy chain eDNA sequences and gene structures. The constant region of the mlgM had three CH domains, i.e., CH1, CH2, CH3, and two transmembrane domains, TM1 and TM2. The TM1 exon is spliced into the regular donor site at the 3＇ terminus of the CH3 domain. When comparing the amino acid sequence of mlgM with those in teleost fish, it is found that the grouper mlgM CH region shares highest identity with that in the oliver flounder （Paralichthys olivaceus）, with CH1, CH2, CH3 having 53% - 78% identity with those in the flounder, respectively. The complete transcript of the grouper mlgD contains 3 375 bp, including a 3 006 bp open reading frame, and the constant region of the mlgD consists serially of a ~tl domain, seven 6 domains （81 to 87）, and a TM region. The degree of amino acid sequence identity, in domain-by-domain comparison with other teleost fishes, reveals an overall higher identity between the IgD in the grouper and that in mandarin fish （Siniperca chuatsi）, with 81 to 57 having 75.5%, 75.8%, 65.4%, 76.6%, 88.1%, 90.6%, 82.8% identity, and the transmembrane domain having 82.7% identity, respectively. The slgZ has a four exon structure in genome, with the introns being 222, 129, and 458 bp in length, respectively. The semi-quantitative RT-PCR was performed to examine the expressions of IgZ, IgM, and IgD in various or- gans / tissues of the grouper. Transcripts encoding the mlgM were detected in all examined samples. IgD was mainly detected in pronephros, kidney, spleen and thymus, with week expression in intestine. IgZ was expressed in lymphoid tissues such as pronephros, kidney and spleen, with relatively low expression detected in gills, thymus and heart.