【作者】 汪代华；

【导师】 吴登俊； 徐刚毅；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 天府肉羊是在成都麻羊与萨能、努比亚、吐根堡、波尔山羊复杂杂交基础上.按品系培育要求,采用群体继代选育法进行同质选配和横交固定,辅助分子生物技术育成的专门化肉用山羊品系。本试验对该品系的种质特性进行了系统研究,并以CAST、POUIFI、MyoG、MSTN、PRLR、PRL作为其生产性的候选功能基因,筛选出了与该品系的肌肉品质、生长发育、繁殖与泌乳性状显著相关的遗传效应位点和分子标记,结果表明：天府肉羊生产性能优良,肉用特征明显,胸部宽深,肋骨开张良好,背腰平直,臀、股部肌肉丰满,四肢粗壮,鼻梁稍隆起,耳大下垂,公、母羊均有角,头部为黄色或黑色或白色,体躯被毛白色。公羊的初生、6月龄和成年体重分别为3.70kg、29.36kg、77.38kg,母羊分别为3.32kg、26.41kg、54.15kg,公羊的生长拐点时间和休重分别为4.7月龄、24.39kg,母羊分别为4.3月龄、19.36kg。经产母羊哺乳期(60d)产奶量83.49kg,产羔率197.5%。6月龄和周岁阉羊的胴体重分别为13.90kg、22.46kg,屠宰率分别为51.25%、52.64%,肌纤维直径分别为28.14μm、31.44μm,嫩度剪切力分别为2.79kg、3.98kg。肌肉必需氨基酸含量9.90%,不饱和脂肪酸含量51.94%,C6、C8、C10等低级挥发性脂肪酸含量低,肉质好,肉用性能突出,具有生产肥羔优势。首次成功克隆出天府肉羊钙蛋白酶抑制蛋白基因(CAST),该基因(Genbank:GU944861) cDNA全长2435bp,完整开放阅读框为31-2217bp,编码728个氨基酸,与绵羊、牛、猪的CAST基因同源性分别为88.8%-95.6%、83.8%-92.2%、73.3%-81.8%。经荧光定量检测,该基因在多个组织中均有表达,肌肉组织的表达量显著高于内脏组织,在背最长肌中的表达量最高。生物信息学分析发现,该蛋白氨基酸序列中存在4个保守结构域,二级结构以无规卷曲为主,富含亲水区域,存在多个磷酸化位点。天府肉羊CAST基因外显子6具有丰富的多态性,存在AA、BB、CC、FF4种纯合丛因型和AB、AC、BD、EF4种杂合基因型。AA型为72位TG缺失,BB型为59位G→C突变、65位G→T突变,CC型为153位A→C突变,FF型为52位C→G突变、66位G→T突变、68位GTG插入。同时,该基因内含子6第943位T碱基缺失,造成Xmn I酶切位点改变,形成MM、MN、NN3种基因型。FF基因型个体的嫩度剪切力和肌纤维直径比CC型分别高1.35kg和8.05μm, NN型比MM型分别高1.06kg和6.96μm,均达到差异显著水平(P<0.05)。试验提示,CAST基因突变对天府肉羊肌肉品质有显著影响,其中CC和MM基因型个体的肌肉品质更好。POUIFI丛因内含子4存在2个变异位点,分别为528位G→A突变和708位A缺失,形成GG、GK、KK3种基因型。GG基因型个体的2月龄胸围显著大于GK型,GK型个体的2月龄体高显著大于KK型,GG型个体的初生重和2月龄体重、体长、体高、胸围比KK型分别高0.61kg、2.83kg、4.01cm、4.75cm、5.41cm,均达到差异显著水平(P<0.05)。试验提示,该基因突变对天府肉羊早期生长发育有显著影响,其中GG基因型个体的生长性能更突出。MyoG基因内含子第422位C→T突变,形成PP、PQ、QQ3种基因型,PP基因型个体的2月龄体重和成年体重显著高于PQ、QQ型，PP、PQ型个体的6月龄体重显著高于QQ型。PP基因型个休的2月龄、6月龄和成年体重比QQ型分别高2.37kg、4.05kg、9.76kg,均达到差异显著水平(P<0.05)。试验提示,该基因位点突变对天府肉羊生长发育有显著影响,PP基因型个体的生长性能更突出。MSTN基因内含子2的第3783位T→C颠换突变,导致EcoRV酶位点缺失,形成WW、WX、XX3种基因型。WW、WX基因型个体的2月龄胸围显著高于XX型,WW型个体的成年体重显著高于WX、XX型,WW型个体的成年胸围显著高于XX型。WW丛因型个体的2月龄、6月龄和成年体重分别比XX型个体高3.17kg、4.91kg、10.71kg,均达到差异显著水平(P<0.05)。试验提示,该基因位点突变对天府肉羊生长发育有显著影响,WW丛因型个体的生长性能更突出。PRLR丛因外显子10发现2个突变位点,52bp处G→A突变形成HH、HL、LL型3种基因型,220bp处T→C突变形成了SS、ST、TT另3种基因型。HH丛因型经产母羊的产羔数比LL型高0.39只(P<0.05),产奶量比LL型高11.53kg(P<0.05)；SS丛因型经产母羊的产羔数比ST型高0.68只(P<0.05),产奶量比TT型高13.35kg(P<0.05)。试验提示,该基因突变对天府肉羊繁殖和泌乳性能有显著影响,HH和SS基因型母羊的繁殖和泌乳性能更突出。PRL基因外显子5的576bp发生C→A突变,检测到RR、RZ、ZZ3种些因型。RR基因型经产母羊的产羔数和产奶量显著高于RZ、ZZ型,RR丛因型个体的产羔数比ZZ型高0.60只(P<0.05),RR基因型个休的产奶量比RZ型高7.88kg(P<0.05)。试验提示,该基因位点对繁殖和泌乳性能有显著影响,RR丛因型母羊的繁殖和泌乳性能更突出。试验表明：天府肉羊CAST、POU1F1、MyoG、MSTN、PRLR、PRL基因中分别存在与其生产性状显著相关的遗传效应位点和分子标记,可以作为候选基因用于该品系生产性能的进一步选育提高。同时,研究结果为天府肉羊新品种培育的标记辅助选择提供了重要试验依据。更多还原

【Abstract】 A specialized goat strain, named Tianfu, was formed by closed herd breeding on the basis of hybrids resulting from Chengdu Ma goat crossbred with Saanen, Nubian, Toggenburg and Boer goat. Characteristics, production performance, meat quality traits and the related candidate genes, including CAST, POU1F1, MyoG. MSTN. PRLR and PRL of the goat were revealed in the study. Results were shown as follows.This new population has a good physique for mutton production. Body weight (BW) of the goat at birth,6 months, adult are 3.70kg,29.36kg,77.38kg for male, and 3.32kg,26.41kg,54.15kg respectively for female. The growth inflection point of the goat is 4.3-4.7 months, the milk yield of lactation (60d) and litter size of ewe is 83.49kg and 197.5% respectively. The dressed weight, dressing percentage, muscle fiber diameter, tenderness shear force of castrated male slaughtered at 6 months and 12 months are 13.90kg,51.25%,28.14μm,2.79kg and 22.46kg,52.64%,31.44μm,3.98kg respectively. The essential amino acids and unsaturated fatty acid (UFA) content of muscle is 9.90% and 51.94% respectively.Calpastatin (CAST) is an important gene for meat quality traits in livestock and poultry. Caprine CAST gene was amplified for the first time using RACE-PCR. The full-length cDNA of the gene (Accession No.GU944861) was 2435 base pair (bp) and contained a 2187bp open reading frame (ORF) encoding a protein with 728 amino acids residues. Bioinformatic analysis indicated that the gene was 89.8%-95.4%,83.5%-92.2% and 72.8%-81.8% identical to that of sheep, cattle, pig. It was predicted that caprine CAST contained four conserved domains with 42 serine phosphorylation loci,18 threonine phosphorylation loci,1 tyrosine phosphorylation locus and 5 specific PKC phosphorylation loci. Quantitative analysis showed that the gene was expressed in various tissues with the highest expression in longissimusdorsi.Three genotypes MM, MN, NN, which resulting from T nucleotide base deletion in 943th locus, were found in intron 6 of CAST gene in Tianfu goat. Four pure genotypes AA, BB, CC and FF were found in exon 6 of the gene too. AA type resulted from TG deletion in 72th locus, BB type from G→C mutation in 59th locus and G→T mutation in 65th locus, CC type from A→C mutation in 153th locus, and FF type from C→G mutation in 52th locus, G→T mutation 66th locus and GTG insertion in 68th locus. The tenderness and muscle fiber of goat with CC genotype was 1.35kg and 8.05μm significantly less than that of FF genotype, MM genotype was 1.06kg and 6.96μm significantly less than NN genotypes.Two mutations,IVS4+528A>G and IVS4+708delA, which produced three types GG, GK and KK, were found in intron 4 of POU1F1 gene in Tianfu goat. The birth weight and body weight, body length, body height, chest girth of 2 months of goat with GG genotype were 0.61kg,2.83kg,4.01cm,4.75cm, 5.41cm significantly higher than that of KK genotype respectively. It suggested the polymorphism can be as the candidacy molecular marker for early growth of Tianfu goats.Three genotypes of PP, PQ and QQ of MyoG gene in Tianfu goats were detected. The polymorphism was caused by C→T transversion at 422th locus. The body weight of 2,6 months and adult of goat with PP genotype were 2.37kg,4.05kg and 9.76kg significantly higher than that of QQ genotype. Similarly, EcoRV polymorphisms, which resulting to three genotypes WW, WX and XX, of MSTN gene were detected in Tianfu goats. Body weight of 2,6 months and adult of goat with WW genotype were 3.17kg, 4.91kg and 10.71kg significantly higher than that of XX genotype. It suggested the polymorphism of MyoG gene and MSTN gene can be as the candidacy molecular marker for growth of Tianfu goats.PRLR and PRL gene were studied as candidate genes for the prolificacy and milk yield of Tianfu ewes. Results indicated that there were two SNP sites in exon 10 of PRLR gene, the G→A mutation in 52bp locus brought about three genotypes HH, HL, LL, and T→C mutation in 220bp locus brought about other three genotypes SS, ST, TT. The litter size and milk yield of Tianfu goats with HH genotype were 0.39 and 11.53kg significantly more than that of goat with LL genotype, and SS genotype were 0.68 and 13.35kg significantly more than those with ST genotype. Similarly, the C→A mutation in 576 locus in exon 5 of PRL gene formed three genotypes RR、RZ、ZZ. The litter size of RR genotype goat was 0.60 significantly more than that of ZZ genotypes, and the milk yield of RR genotype goat was 7.88kg significantly more than those with RZ genotype. These results preliminarily showed that PRLR and PRL gene are correlated with litter size and milk yied in Tianfu goats, and they can be as candidate genes to promote prolificacy and milk yield of the breed.In conclusion, some mutations of the related candidate genes, incluing CAST, POU1F1、MyoG、MSTN、PRLR、PRL which controled production performances and meat quality traits were found in Tianfu goat, and it will play a key role in Marker-Assisted Selection (MAS) for Tianfu goat breeding.更多还原