The Polymorphism Study By PCR-RFLP And Sequence Analysis Of SLA-DQA,DQB Gene In Laboratory Tibet Mini-pigs

Major histocompatibility complex(MHC),a genic system located in chromosome which made of a group of close linkage and highly polymorphism gene locus.The MHC made an important contribution in immune response of vertebrate,it not only encode major histocompatibility antigen and regulate graft rejective reaction, but also make an effect in regulation of immune response and immune discrimination, the most important one is antigen presentation.The gene of MHC have affluent polymorphism,and have intimate correlation with affectability of disease and production trait of animals,it was usually used as marker gene of origin of species, evolution and anti-disease and breeding in animals.The MHC of swine also named leucocyte antigen(SLA),which mainly classified asâ… ,â…¡andâ…¢category.SLAâ…¡mainly takes immunal function on exogenous antigen,and have compact correlation with the ability of anti-disease in pigs.At present,there were many genes in SLAâ…¡be physical located,such as:DRA,DRB, DQA and DQB,and so on.Human major compatibility antigen was also named as HLA,while mice's were named as H-2.For the past few years,many foreign scholars have studied the SLA of laboratory pig,HLA of human and H-2 of mice, and found there were some similarities among them.The similary study in our country started relatively late.Presently,there were few reports on the structure of SLA fits for heterogenic transplantation of Chinese pig breeds and the role of which in pig-human heterogenic transplantation.60 ones of laboratory Tibet mini-pigs blood samples which provided by Laboratory Animal Centre of Southern Medical University were used as study subject.PCR-RFLP and comparative genome method were used to analyze the SLA-DQA and DQB gene which located in the seventh chromosome of swine,we got the PCR sequence fragment which include partial sequences of intron 1 and intron 2 and the complete sequence of extron 2 of DQA and DQB.PCR products were digested by restriction enzyme,and then use electrophoresis to analyze the RFLP patterns of the two genes and distinguish the frequences of different genotypes and alleles.Chi square test were applied to detect whether the genotype frequences of different RFLP sites were consistent with Hardy-Weiberg equilibrium. In order to detect whether Tibet mini-pigs have mutation in these sites or distinct genetic diversity,we compared our result with other breeds of reported mini-pigs. After purification,cloning and direct sequencing(DS) of SLA-DQA,SLA-DQB gene extron 2,we compared the sequence of these two genes with those of other breeds of pigs,human,mice and Canines',so as to investigate their homology as well as the origin and heredity evolution mechanism of Tibet mini-pigs.In this study,we got three types of genotype and alleles respectively of SLA-DQA after digested by EcoRâ… and Aluâ… .The restriction enzyme map of EcoRâ… -RFLP site showed that the genotype frequency of BB(45.000%) was higher than that of AB(31.667%) and AA(23.333%),B was the predominant allele. As for the Aluâ… site,the genotype frequency of MN(50.000%) was higher than MM(30.000%) and NN(20.000%),M was the predominant allele.Combined with double enzyme digestion results,there were 7 PCR-RFLP patterns of SLA-DQA gene in Tibet mini-pigs as follows:AAMM,AANN,ABMM,ABMN,ABNN,BBMM and BBMN.The genotype frequency of BBMM(30.000%) was higher than other ones, while the genotype AANN or BBNN was not found in our study.Four different types of alleles(A,B,C,D) and five different types of genotypes (AA,BB,BC,DD,AD) were obtained from SLA-DQB after digested by restriction enzyme Haeâ…¢,and the result showed that the genotype frequency of BC(33.333%) was more than others at Haeâ…¢-RFLP site,B was the predominant allele(33.333%); Mean while,three different types of alleles(E,F,G) and five different types of genotypes(EE,EF,EG,FF,GG) were obtained from SLA-DQB after digested by restriction enzyme Rsaâ… ,the result showed that the genotype frequency of EF (33.333%) was more than others at Rsaâ… -RFLP site,E was the predominant allele (48.333%).Combined with double enzyme(Haeâ…¢and Rsaâ… ) digestion results, there were 7 PCR-RFLP patterns of SLA-DQB gene in Tibet mini-pigs as follows: AAEE,AAFF ADEG,ADGG,BBEE,BCEF and DDGG.Among then,the BC genotype at Haeâ…¢-RFLP site was corresponded with the EF genotyope at Rsaâ… -RFLP site,and the composite genotype(BCEF) of them was the highest one (33.333%).Ploymorphism information content(PIC) results showed that the SLA-DQA and SLA-DQB gene of Tibet mini-pigs presented moderate polymorphic(0.25＜PIC_DQA＜0.5) and high polymorphic PIC_DQB＞0.5) respectively.According to the values of x² test(chi-square test),the genotypes at EcoRâ… site didn't reach the significance level(x²=4.300,P＞0.05),while the Aluâ… site has significant difference (x²=8.400,P＜0.05);The x² value of gene frequencies of SLA-DQB digested by Haeâ…¢and Rsaâ… are 7.200 and 2.933 respectively,show no significance atα=0.05 level,which mean the two RFLP sites are consistent with Hardy-Weiberg Law.Sequencing the target gene product of second extron in SLA-DQA and DQB,we found restriction enzyme sites of EcoRâ… and Aluâ… site respectively (5'...G↓AATTC...3' and 5'...AG↓CT...3'),as well as restriction enzyme sites of Haeâ…¢and Rsaâ… (5'...GG↓CC...3' and 5'...GT↓AC...3'),which indicated that the PCR products were the target gene fragment.Compared the sequence results with the whole SLA-DQA and DQB sequence and haman HLA-DQA,DQB genee in Genbank by BLAST,the sequence homology of Tibet mini-pigs' SLA-DQA and the pig with an accession number of BX088590 in GenBank reach up to 99%,which only has 2 different bases.Compared the DQA sequence with those of Taihu pig (AY303988),Wuzhishan pig(AY243104),human(XM₀01129369),dog (NM₀01011726.1)and mouse(NM₀10378.2),the homology are 97%,91%,83 %,84%,71%respectively.The result is little higher than Wuzhishan Mini-pig Inbred and Xishuangbanna Miniature Pig Inbred Line compared with corresponding HLA high-frequency allele(HLA-DQA^*03011) reported by Wuqun(2004)(80.87% and 81.96%respectively).The sequence homology of Tibet mini-pigs' SLA-DQB and the Zang pig and Jinhua pig with an accession number of AY769655 and AY769646 respectively in GenBank reaches up to 98.2%.The sequence comparing results of HLA-DQB1^*0305GR and HLA-DQB1^*03011 alleles were 82.4%and 81.7%.The former result was higher than Wuzhishan mini-pig compared with corresponding HLA high-frequency allele(HLA-DQB^*03032) reported by Wuqun (79.31%).Conclusions are obtained from this research as follows:The polymorphism of SLA-DQB exon 2 in laboratory Tibet mini-pigs is more abundant than DQA.Homozygote RFLP patterns are predominant patterns in the two genes,which illustrate the group of Tibet mini-pigs have a high homozygosity. It is essential to do system breed conservation work and scientific selection of this group of pigs.According to the chi-square test,only Aluâ… RFLP site doesn't consistent with Hardy-Weinberg equilibrium.More research are suggested to carry out in order to make sure whether this result has direct correlation with the high stress resistance of Tibet mini-pigs.Nucleotide sequences of SLA-DQA,DQB exon 2 were obtained by PCR product direct sequencing.By comparison with other pig breeds we found some mutation of bases,also there sequence homology with huam HLA are higher than Wuzhishan mini-pigs and Xishuangbanna Miniature Pig,as well as H-2.Thus,we conclude that laboratory Tibet mini-pigs are more suitable for heterotransplant research.Based on the cluster analysis of cladgram,it's known that Tibit mini-pigs have a closer genetic relationship with Taihu pig breed and Yunnan wiled pigs.This is very important for the evolution mechanism research of Tibet mini-pigs.