野生抗病牙鲆MHC II B内含子1和外显子2序列多态性

Polymorphism of MHC II B intron 1 and exon 2 in wild disease-resistance Japanese flounder (Paralichthys olivaceus)

通过对牙鲆(Paralichthys olivaceus)MHC II B基因内含子1和外显子2直接测序的方法,从83个抗病牙鲆个体中共得到了85个等位基因,其中76个为新发现的等位基因。在扩增得到的263bp的外显子2中,共发现了60个多态性位点,其中33个是新发现的。不同的等位基因均以较低的频率出现,基因频率范围从0.006到0.069,基因型频率范围从0.012到0.169。通过序列分析,共发现了6种内含子1,其中4种是新发现的。内含子1中均包含了一个12bp的重复单元,其3'端含有一个富含CT/GT的区域。牙鲆MHC II B基因的高多态型和低频率揭示了牙鲆群体MHC基因资源的丰富性,为选择育种提供了可能。

MHC II B genes play a crucial role in immune responses and self-tolerance. MHC and antigen form peptide： MHC （pMHC） complex which could be bound by T-cell receptor （TCR） and initiates immune responses. MHC genes are characterized by their high level of polymorphism. The polymorphism occurs not only in coding region but also in promoter region. There are a lot of researches about the association between different MHC IIB alleles and resistance or susceptibility to pathogens. As frequency of MHC IIB alleles in Japanese flounder stock which we studied was low, more individuals were analyzed to find more alleles. Resistant stock was obtained by natural selection and pathogen injection. Fin was cut and stored in ethanol. DNA was extracted by salt-out method. PCR was performed with gene specific primer. The PCR fragment was purified, cloned and sequenced. The sequences were analyzed by DNAMAN and some of them were eliminated according to SNP definition. Eighty five alleles were obtained from 83 resistant individuals based on sequence of MHC IIB intron 1 and exon 2 in Japanese flounder, among which there were 76 new alleles. Sixty polymorphic sites were discovered from 263 bp exon 2, of which 33 were first discovered. All alleles appeared as low frequency, with gene frequency ranging from 0.006 to 0.069 and genotype frequency ranging from 0.012 to 0.169. Six types of intron 1 were discovered with a 12 bp repeat element while four of them were first discovered. There was a CT/GT rich region at 3＇ end of intron 1. It is proved that MHC IIB genes of Japanese flounder were high polymorphic and appeared as low frequency. So the flounders we studied have no close relationship with each other, and are suitable for family selection which could be resistant to pathogen. Micro-recombination exists in MHC IIB genes of Japanese flounder which could produce polymorphism.