三个猪品种SLA-DQA基因多态性与仔猪腹泻的相关性分析
摘要
猪主要组织相容性复合物是已知多态性最丰富的基因簇，在动物的免疫系统中发挥着重要的作用，研究已证明MHC与动物多种疾病的抗性和易感性相关。本研究采用PCR-SSCP及克隆测序的方法对大白、长白和杜洛克三个群体共210头仔猪的SLA-DQA基因4个外显子进行遗传多态性分析，并进行了多态性与仔猪腹泻病的相关性研究,旨在获取相应的分子遗传学信息，明确猪SLA-DQA基因的遗传特性，从遗传本质上来提高猪的抗病力，并分析该基因作为抗病力候选基因的可能性。研究结果如下：
（1）三个猪品种SLA-DQA基因的外显子2、外显子3、外显子4的扩增产物通过PCR-SSCP检测分析，发现均存在多态性，而外显子1未发现多态性。外显子2检测到5个等位基因和8种基因型（AB、BB、BD、BE、BC、CC、CD、DD），CC基因型仅在长白猪中检测到且频率为0.04，CD、DD仅在杜洛克猪中检测到，频率都为0.01，等位基因B为三群体中的优势基因，经测序比对后等位基因C和E各发现一个新的突变位点，分别为（4133bpC→A，3994bpT→A）。
（2）在外显子3中检测到8种等位基因和14种基因型。其中在大白猪中检测到8种基因（A～H），13种基因型，在长白、杜洛克中没有检测到基因D和H，基因型EE、DD、DH在长白中没有被检测到，BC、AB、CC、DD、DH在杜洛克中亦没有检测到，等位基因B为三个猪品种的优势基因。同源性比对后，外显子3共发现14个突变位点，包括6个T→C的转换，1个A→G的颠换，1个A→C和2个C→T的转换，3个G→A的颠换，1个A→G的颠换，其中等位基因B、E、F在4727～4729位发生缺失。核苷酸的突变导致氨基酸发生改变，为错义突变。
（3）外显子4共检测到4种等位基因（A、B、C、D），6种基因型（AA、AB、CC、CD、BB、BC）。在大白猪中只检测到AA、AB、CC3种基因型；在长白猪中未检测到BC基因型，其他5种基因型都检测到，在三个群体中，AA基因型为优势基因型，A等位基因为优势基因。经测序分析，共发现3个核苷酸突变位点，（5126bpA→G，5158bpA→T，5233bpG→A）。
（4）遗传多态性分析结果表明： DQA基因外显子2位点杜洛克和大白猪杂合度分别为0.62和0.79，大于长白猪。三个猪品种的多态信息含量（PIC）介于0.25和0.5之间，属于中度多态；外显子3位点大白猪杂合度为0.54，依次为长白猪0.46，杜洛克0.35，且此位点上三个猪品种的PIC大于0.5，都表现为高度多态；外显子4位点上大白猪、长白猪和杜洛克的杂合度（He）分别是0.37、0.50和0.68。杜洛克多态信息含量（PIC）大于0.5，为高度多态，长白猪、大白猪PIC介于0.25和0.5之间，为中度多态。经卡方检验，三群体在这3个位点上都偏离了Hardy-Weinberg平衡状态(P<0.05)。
（5）最小二乘法分析结果表明，三个位点发现的基因型与仔猪腹泻显著相关(P＜0.05),外显子2上CD基因型个体腹泻评分的最小二乘均值均显著高于AB、BB、BC和BE基因型个体(P<0.05)；外显子3中BB、BC、BE、GF、DD和DH基因型个体腹泻评分的最小二乘均值均显著高于BF、AB、CF、EF和CC基因型个体(P<0.05)；外显子4中CD基因型个体腹泻评分的最小二乘均值显著高于AA、BB、AB、CC。
The swine major histocompatibility complex is the most abundant polymorphicgene cluster, which plays an important role in the animal's immune system, studieshave demonstrated that the MHC is associated with disease resistance andsusceptibility of various animals. Single-strand conformational polymorphism(PCR-SSCP) and sequencing were used in this study to analyse the geneticpolymorphism and its relationship with piglet diarrhea at four exons of the SLA-DQAgenes in three pig breeds in order to gain the molecular genetic informations andexplicit the swine genetic characteristics of the SLA-DQA gene, so as to inprove thedisease resistance of swine from the genetic nature, and analyze the possibility of theDQA gene as candidate gene in disease resistance. The results as follows:
The results showed that exon2, exon3and exon4of SLA-DQA gene from threepig populations were detected polymorphism by PCR-SSCP, but there were nopolymorphism in exon1. Five alleles and eight genotypes (AB, BB, BD, BE, BC, CC,CD, DD) were detected from exon2, genotypes CC was only detected in Landracewith frequency of0.04, CD and DD were only detected in Duroc with frequency of0.01. Allele B is the dominant gene and allele D is the disadvantages gene in the threegroups. By sequencing, the new mutation4133bpC→A and3994bpT→A wasfound in allele C and E, respectively.
Eight alleles and fourteen genotypes were detected in exon3fragment. Eightalleles which formed thirteen genotypes in Large white, alleles D and H were notdetected in Duroc and Landrace. Genotypes EE, DD and DH were not detected inLandrace, and BC, AB, CC, DD, DH were not detected in Duroc, allele B is thedominant gene in three groups. By sequencing and homology analysis that fourteenmutations were found in exon3, included six conversion of T→C, two conversion ofC→T and one conversion of A→C, two transversion of A→G and three transversionof G→A. Alleles B, E and F were deleted in the region of4727～4729bp. Thepolymorphisms would change as amino acid changed, and this mutations was missense mutations.
Gained four alleles and six genotypes（AA, AB, CC, CD, BB, BC）in exon4,which detected in Duroc, but AA, AB, CC genotypes were only detected in Largewhite, BC was not detected in Landrace. AA was the dominant genotype and allele Awas the dominant allele in three groups. By sequencing, there were found three SNPs(5126bpA→G,5158bpA→T,5233bpG→A), respectively, those mutations aresynonymous mutations.
The analysis results of the genetic polymorphisms showed that heterozygosity(He) of Duroc and Large white were0.62and0.79in exon2of DQA gene respectively.Which larger than Landrace, the polymorphism information content of three pigs were0.250.5indicatedHighly polymorphism in three pigs. In exon4locus, heterozygosity (He) of Largewhite, Landrace and Duroc were0.37,0.50and0.68. respectively. The polymorphisminformation content of Duroc was highly polymorphism (PIC>0.5), Large white andLandrace are moderate polymorphism (0.250.05). The piglet diarrhea score of individual genotypes showed that CD wassignificantly higher than that of AB, BB, BC and BE (P <0.05) in exon2,BB, BC, BE,GF, DD and DH was significantly higher than that of BF, AB, CF, EF and CC (P <0.05) in exon3，CD was significantly higher than that of AA, BB, AB and CC (P <0.05) in exon4.
（1）三个猪品种SLA-DQA基因的外显子2、外显子3、外显子4的扩增产物通过PCR-SSCP检测分析，发现均存在多态性，而外显子1未发现多态性。外显子2检测到5个等位基因和8种基因型（AB、BB、BD、BE、BC、CC、CD、DD），CC基因型仅在长白猪中检测到且频率为0.04，CD、DD仅在杜洛克猪中检测到，频率都为0.01，等位基因B为三群体中的优势基因，经测序比对后等位基因C和E各发现一个新的突变位点，分别为（4133bpC→A，3994bpT→A）。
（2）在外显子3中检测到8种等位基因和14种基因型。其中在大白猪中检测到8种基因（A～H），13种基因型，在长白、杜洛克中没有检测到基因D和H，基因型EE、DD、DH在长白中没有被检测到，BC、AB、CC、DD、DH在杜洛克中亦没有检测到，等位基因B为三个猪品种的优势基因。同源性比对后，外显子3共发现14个突变位点，包括6个T→C的转换，1个A→G的颠换，1个A→C和2个C→T的转换，3个G→A的颠换，1个A→G的颠换，其中等位基因B、E、F在4727～4729位发生缺失。核苷酸的突变导致氨基酸发生改变，为错义突变。
（3）外显子4共检测到4种等位基因（A、B、C、D），6种基因型（AA、AB、CC、CD、BB、BC）。在大白猪中只检测到AA、AB、CC3种基因型；在长白猪中未检测到BC基因型，其他5种基因型都检测到，在三个群体中，AA基因型为优势基因型，A等位基因为优势基因。经测序分析，共发现3个核苷酸突变位点，（5126bpA→G，5158bpA→T，5233bpG→A）。
（4）遗传多态性分析结果表明： DQA基因外显子2位点杜洛克和大白猪杂合度分别为0.62和0.79，大于长白猪。三个猪品种的多态信息含量（PIC）介于0.25和0.5之间，属于中度多态；外显子3位点大白猪杂合度为0.54，依次为长白猪0.46，杜洛克0.35，且此位点上三个猪品种的PIC大于0.5，都表现为高度多态；外显子4位点上大白猪、长白猪和杜洛克的杂合度（He）分别是0.37、0.50和0.68。杜洛克多态信息含量（PIC）大于0.5，为高度多态，长白猪、大白猪PIC介于0.25和0.5之间，为中度多态。经卡方检验，三群体在这3个位点上都偏离了Hardy-Weinberg平衡状态(P<0.05)。
（5）最小二乘法分析结果表明，三个位点发现的基因型与仔猪腹泻显著相关(P＜0.05),外显子2上CD基因型个体腹泻评分的最小二乘均值均显著高于AB、BB、BC和BE基因型个体(P<0.05)；外显子3中BB、BC、BE、GF、DD和DH基因型个体腹泻评分的最小二乘均值均显著高于BF、AB、CF、EF和CC基因型个体(P<0.05)；外显子4中CD基因型个体腹泻评分的最小二乘均值显著高于AA、BB、AB、CC。
The swine major histocompatibility complex is the most abundant polymorphicgene cluster, which plays an important role in the animal's immune system, studieshave demonstrated that the MHC is associated with disease resistance andsusceptibility of various animals. Single-strand conformational polymorphism(PCR-SSCP) and sequencing were used in this study to analyse the geneticpolymorphism and its relationship with piglet diarrhea at four exons of the SLA-DQAgenes in three pig breeds in order to gain the molecular genetic informations andexplicit the swine genetic characteristics of the SLA-DQA gene, so as to inprove thedisease resistance of swine from the genetic nature, and analyze the possibility of theDQA gene as candidate gene in disease resistance. The results as follows:
The results showed that exon2, exon3and exon4of SLA-DQA gene from threepig populations were detected polymorphism by PCR-SSCP, but there were nopolymorphism in exon1. Five alleles and eight genotypes (AB, BB, BD, BE, BC, CC,CD, DD) were detected from exon2, genotypes CC was only detected in Landracewith frequency of0.04, CD and DD were only detected in Duroc with frequency of0.01. Allele B is the dominant gene and allele D is the disadvantages gene in the threegroups. By sequencing, the new mutation4133bpC→A and3994bpT→A wasfound in allele C and E, respectively.
Eight alleles and fourteen genotypes were detected in exon3fragment. Eightalleles which formed thirteen genotypes in Large white, alleles D and H were notdetected in Duroc and Landrace. Genotypes EE, DD and DH were not detected inLandrace, and BC, AB, CC, DD, DH were not detected in Duroc, allele B is thedominant gene in three groups. By sequencing and homology analysis that fourteenmutations were found in exon3, included six conversion of T→C, two conversion ofC→T and one conversion of A→C, two transversion of A→G and three transversionof G→A. Alleles B, E and F were deleted in the region of4727～4729bp. Thepolymorphisms would change as amino acid changed, and this mutations was missense mutations.
Gained four alleles and six genotypes（AA, AB, CC, CD, BB, BC）in exon4,which detected in Duroc, but AA, AB, CC genotypes were only detected in Largewhite, BC was not detected in Landrace. AA was the dominant genotype and allele Awas the dominant allele in three groups. By sequencing, there were found three SNPs(5126bpA→G,5158bpA→T,5233bpG→A), respectively, those mutations aresynonymous mutations.
The analysis results of the genetic polymorphisms showed that heterozygosity(He) of Duroc and Large white were0.62and0.79in exon2of DQA gene respectively.Which larger than Landrace, the polymorphism information content of three pigs were0.25
引文
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