【作者】 罗婷颖；

【导师】 肖天放；

【作者基本信息】 福建农林大学 ， 动物遗传育种与繁殖学， 2010， 硕士

【摘要】 本研究以莆田黑猪和国寿黑猪作为试验材料,设计了15对引物,通过PCR-SSCP技术在试验群体中对猪H-FABP、A-FABP和AMPD1基因的部分外显子中进行了多态性筛选。对SSCP分析结果中的每种纯合子基因型进行测序,从而进一步确定各多态位点存在及变异情况,结果表明:(1)经过SSCP检测,结果只在H-FABP基因的三个位点找到多态性,其余的位点均未找到多态性。莆田黑猪在H-FABP基因的5′-调控区检测出AA、BB和AB三种基因型,而国寿黑猪只有AA和AB两种。扩增的外显子2中国寿黑猪有CC、DD和CD三种基因型,莆田黑猪有DD和CD两种。外显子4处莆田黑猪有EE、FF和EF三种基因型,而在国寿黑猪中检测到EE、EF、EG及FG四种基因型,两个猪种的差异较大。(2)莆田黑猪在5′-调控区、外显子2处属于中度多态(0.25<P<0.5),外显子4为低度多态(P<0.25)。国寿黑猪在5′-调控区、外显子4处属于中度多态,外显子2为低度多态。Hardy-Weinberg平衡适合性检验结果表明:两个猪种在5′-调控区、外显子2中均处于非平衡状态(P<0.01)。在外显子4处莆田黑猪处于Hardy-Weinberg平衡状态(P>0.05),而国寿黑猪处于非平衡状态(P<0.01)。(3)测序结果表明,在5′-调控区AA、BB型的第6bp和第15bp都检测到碱基A和T的缺失,在第103bp都发生C→T的突变。AA型中第7bp和11bp分别发生G→T和C→A的突变。外显子2的CC型第2bp处出现A→C的突变,第62bp、65bp都是T→C的突变,推导氨基酸序列发现第一个氨基酸是由组氨酸(His)转变成脯氨酸(Pro),第21和22个都是异亮氨酸(Ile)转变成苏氨酸(Thr)。外显子4中每一个基因型变异碱基数都达到80个以上,其中FG型在内含子3第28bp出现了一段“AGCTTTTTTT”10bp的插入。(4)将莆田黑猪和国寿黑猪H-FABP基因外显子2的核苷酸序列与GenBank中野猪、牦牛、驴、人、大鼠、鸡和斑马鱼等7个物种的核苷酸序列进行同源比对,分别存在0、3、8、10、16、23、29、43个碱基差异,其同源性介于100%～67.2%之间;推导不同物种的氨基酸序列,分别发生0、4、4、4、9、8、13、11个氨基酸的改变,同源性介于100%～69.7%之间。更多还原

【Abstract】 In this paper, DNA sample was extracted from 70 pigs including Putian black pig and Guoshou black pig. Several exons of H-FABP, A-FABP and AMPD1 gene were detected by PCR-SSCP with 15 pairs primers, in order to find some new polymorphism loci. Each homozygote found in SSCP of gene was sequenced. The main results were as follows:(1) The results of SSCP showed that polymorphism were only found in three loci of H-FABP gene. In 5′regulatory region there were three genotypes ( AA, BB and AB ) in Putian black pig, but two genotypes ( AA and AB ) in Guoshou black pig. Three ( CC, DD and CD ) and two ( DD and CD ) genotypes were respectively detected in two breeds in exon2. In exon4, there were three genotypes ( EE, FF and EF ) in Putian black pig, however, four genotypes ( EE, EF, EG and FG ) in Guoshou black pig. Every loci had two or three alleles.(2) Putian black pig had intermediate polymorphism ( 0.25<P<0.5 ) in 5′regulatory region and exon2 but low polymorphism ( P<0.25 ) in exon4. Guoshou black pig had intermediate polymorphism in 5′regulatory region and exon4 but low polymorphism in exon4. A chi-square analysis suggested that 5′regulatory region and exon2 of two breeds were not in Hardy-Weinberg Equilibrium ( P<0.01 ). Exon4 of Putian black pig was in Hardy-Weinberg Equilibrium ( P>0.05 ), Guoshou black pig was not ( P<0.01 ).(3) In 5′regulatory region, two nucleotides A and T deleted at the site of 6th bp and 15th bp, a nucleotide mutation C→T was found at 103bp both of AA and BB genotypes. Besides, AA genetype existed two mutations of G→T at 7 th bp and C→A at 11th bp. In exon2, nucleotide mutation A→C was tested at 2nd bp, T→C was detected at 62 nd bp and 65 th bp. The mutation sites made the change of His→Pro at the 1st amino acid; Ile→Thr at the 21st and the 22nd amino acids. In exon4, there were five genotypes in all and inserted a 10bp sequence“AGCTTTTTTT”at the site of 28th bp of FG genetype.(4) The consequence of nucleotide sequence alignments in H-FABP exon2 indicated that there were 0, 3, 8, 10, 16, 23, 29, 43 base mutations between Putian black pig and eight species ( Guoshou black pig, Sus, Bos, Equus, Homo, Mus, Gallus and Danio ) , homology ranging from 100% to 67.2%; amino acid alterations at 0, 4, 4, 4, 9, 8, 13, 11 positions, homology ranging from 100% to 69.7%.更多还原