【作者】 王存芳；

【导师】 曾勇庆；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2002， 硕士

【摘要】 猪的肉质性状是一个重要的经济性状，与生长性状或胴体性状间常表现为负的遗传相关，产肉力与肉质间存在一定的拮抗关系。如瘦肉率的提高使得猪肉中平均肌内脂肪的含量低于了最佳范围，从而影响了肉的嫩度和风味，肉质变劣，口感变差。而传统的育种手段很难使肉量和肉质均得以提高。因此，育种者们努力寻找影响肉质性状的主效基因及QTL_?候选基因法是研究动物数量性状遗传机制经常采用的方法之一，目前已确定H-FABP和A-FABP基因是影响猪肌内脂肪含量的主要候选基因。本研究选用4个国外猪种杜洛克、汉普夏、长白猪、大约克和1个中国地方品种莱芜猪共207头作为供试群体，利用PCR-RFLP技术和微卫星方法分别对可作为IMF含量候选基因的H-FABP和A-FABP基因进行了研究分析，同时对A-FABP基因的微卫星位点进行克隆测序，并分析了两基因的遗传变异与IMF含量的关系，目的是为确定影响肌内脂肪沉积的主效基因或与其紧密连锁的遗传标记奠定基础。 本课题利用PCR-RFLP技术对五个猪种H-FABP基因的研究发现，H-FABP基因的5’上游区存在HinfⅠ-RFLP，第二内含子区存在HaeⅢ-RFLP和HinfⅠ-RFLP，与文献报道的基本一致，但酶切位点的各等位基因频率明显不同。在莱芜猪、杜洛克、汉普夏、长白猪、大约克中HinfⅠ-RFLP等位基因H的频率分别为：0.6087、0.5227、0.25、0.8511、0.7273；HinfⅠ-RFLP等位基因B的频率分别为0.5937、0.3478、0.6000、0.1154、0.3750；HaeⅢ-RFLP等位基因D的频率分别为0.8158、0.6429、0.9643、0.2778、0.6522。三种酶切位点中，五个猪种之间遗传特性的差异也非常明显，HinfⅠ-RFLP中除长白猪表现为低度多态，PIC＜0.25)外，其余四个猪种均表现为中度多态（0.25＜PIC＜0.5）。在HaeⅢ-RFLP中，汉普夏猪种表现为低度多态（PIC＜0.25），而其余四个猪种均表现为中度多态（0.25＜PIC＜0.5）。杜洛克的HinfⅠ-RFLP表现为明显的高度多态（PIC＞0.5），其余四个猪种均为中度多态（0.25＜PIC＜0.5）。 本研究对A-FABP基因进行了微卫星分析，在五个供试猪种中均检测到了（CA）_?位点，共发现了4个等位基因，10种基因型。经克隆测序发现，4个等位基因微卫星序列的重复数目分别为：A（33）、B（27）、C（23）、D（21）；片段长度依次为A（279bp）、B（265bp）、C（257bp）、D（253bp）。该位点的多态性是由（CA）重复序列的差异引起的，四个等位基因的侧翼序列具有97.58％的同源性，等位基因C与Genebank中注册的野猪A-FABP基因全序列中的微卫星序列（序列号为SSY16039）完全一致。等位基因及基因型在五个猪种中的分布极不均衡，基因C为莱芜猪、长白猪的优势等位基因，基因A在杜洛克、大约克中为优势基因，而汉普夏以基因B为优势基因。莱芜猪、杜洛克在此位点上为明显的高度多态（PIC＞0.5），而其余3个品种均表现为低度多态（PIC＜0.25）。另外，还发现了两个 山乐农业大学顾士学位论文（ZopZ） 碱基的突变：C—T和A一勺。等位基囱B和C中存在这两个碱基突变，而等位基 因D中只有C——T的突变，等位基因A不存在碱基突变。 通过最小二乘分析发现，H－FABP基因的 hh－ddBB单倍体（Hinf．hh，HMlll dd，HinfJ二BB）具有最高的 IMF含量，在各个 PCR－yLP。中，基因型dd、讪和 Bb分别具有最高的最小二乘均值。微卫星（CA）。位点的纯合基因型间，基因型 ＊D的！＊F含量最高（5．肪H％），基因型＊C次之，为4。引08％：基因型＊C的最 小二乘均值最高，其对IMi含量的效应值最大。 综台以上研究结果，在后续工作中，需要更大的群体规模，采用家系设计，联 台更多相关的旭选基因或DNA tie记来进行分析，寻求与IMF沉积紧密连锁的逅传 0记，以便在动物育切实践的标记辅助选择（h4AS ） 中发挥遗传改良作用。更多还原

【Abstract】 Meat quality trait is an important economic character. A negative genetic correlation is found between meat quality and growth traits or carcass traits. There is antagonism between the meat production and meat quality. The improvement of lean meat percentage lows the IMF content of pork out of the best range, and the tender and flavo.’ is also decreased. Thus meat is of poor quality. But it is difficult to improve both the quantity and quality of meat by the traditional breeding methods. Therefore, the major genes and QTL, affecting meat quality are studied. Candidate gene method is one of the approaches which are often used to study quantity trait in animals. At present, H-FABP and A-FABP genes are certain to be the major candidate genes affecting the IMF content in swine. In this tad>. DNA samples from 207 pigs including four imported breeds: Duroc. Hampshire, Landrace and Yorkshire and one native breed of China: Laiwu, were analyzed. H-FABP and A-FABP genes were studied by PCR-RFLP, and microsatellite, respectively. Meanwhile the microsatellite loci of A-FABP gene was cloned and sequenced. The effect of genetic variation of the two genes on IMF content was analyzed. The aim of this study was to determine the major genes affecting IMF deposition or the genetic markers linked with them tightly.The study of H-FABP gene in the five breeds by PCR-RFLPS showed one Hint" I -RFLP in 5’-upstream and one Haelll-RfLP and one Hinf I -RFLP in the second intron. The result was accorded with the previous reports. But the allele frequency of every locus was different significantly. The frequencies of H allele for Hinf I -RFLP in Laiwu, Duroc, Hamipire, Landrace, and Yorkshire, were 0.6087, 0.5227, 0.25, 0.8511 and 0.7273, respectively; the frequencies of B allele for Hinf I ’-RFLP were 0.5937, 0.3478, 0.6000, 0.1153 and 0.3750, respectively; the frequencies of D for Haelll-RFLP were 0.8158, 0.6429. 0.9643, 0.2778 and 0.6522, respectively. The difference of genetic properties among the five breeds was also significant. In Hinf-RFLP. except Landrace that had lowpolymorphism(PIC<0.25), the others had intermediate polymorphism(0.25<PICO.5).ln Haelll-RFLP. Hampire had low polymorphism(PIC<0.25). but the others had intermediate polymorphism (0.25<PIC<0.5). In Hinf I ’-RFLP, Duroc had high polymorphism(PIO0.5) obviously, while the others had intermediate polymorphism(0.25<PIC<0.5).The amplified fragment length polymorphism of A-FABP gene in five breeds was investigated. There were four alleles and ten genotypes. Cloning and sequencing showed that the number of repeated sequences for the four alleles was A (33), B (27), C (23) and D (21.): the lengthes of fragment were A (279bp), B (265bp), C (257bp) and D (253bp). respectively. The polymorphism of (CA)r was caused by the difference in the number of repeated sequence. Identity for the flanking sequences of four alleles was 97.58%. Allele C was accorded with microsatellite sequence of A-FABP Genome sequences in Sus Scrofa inscripited in Gene Bank. The distribution of alleles and genotypes was of disequilibration. Alleie C was the superiority gene for Laiwu and Landrace. allele A for Duroc and Yorkshire, but allele B for Hampshire. The result showed that Laiwu and Duroc had high polymorphism(PIC>0.5). but the other three breeds were of low polymorphism(PIC<0.25). In addition, the mutations of two bases was discovered. One was C ?T. the other was A-G. Allele B and C had these two mutations, nevertheless, allele D had C-*T only, allele A had no mutation.The effect of RFLPS for H-FABP gene on IMF content was analyzed by Least Square .Means. IMF content of the monoploid genotypes dd-hh-BB was the highest. For the homozygous genotypes. IMF content of genotype DD was the highest (5.0622%) and thai of genotype CC was next to DD; the LSM value of genotype CC was the highest and i; had the topmost effect.In summary, it is necessary to further explore genetic markers linked with major gene affecting IMF deposition by using more animals, applying family design, and combinin更多还原