【作者】 赵文明；

【导师】 陈国宏；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 本试验对我国大、中、小三个地方鹅种狮头鹅、皖西白鹅和籽鹅的早期生长发育规律进行分析,同时以籽鹅、皖西白鹅、狮头鹅、四季鹅和浙东白鹅为研究对象,对GH基因外显子和内含子、PRL基因编码区全序列以及5’端序列进行PCR-SSCP检测和克隆测序,分别计算5个鹅品种(种群)突变位点的基因型频率和基因频率,比较不同鹅种间的基因型分布规律,并对GH基因和PRL基因多态性与鹅早期增重和屠宰性状进行关联分析。试验结果如下:1.利用Logistic,Gompertz,Bertalanfy和Cubic 4种非线性动物生长模型拟合大、中和小型三个地方鹅种:狮头鹅、皖西白鹅和籽鹅1-11周龄的平均体重,进行早期生长发育规律及遗传参数分析。结果表明:4个方程均能很好地拟合三个鹅种的生长过程,拟合度都在0.99以上,但Gompertz模型更符合实际品种特性,为首选模型,由此模型估计狮头鹅、皖西白鹅和籽鹅生长的拐点时间分别为5.98周龄、5.11周龄和6.16周龄,相应的拐点体重分别为2115.77g、1499.08g和1049.62g。2.对鹅GH基因外显子和内含子进行克隆测序,得到鹅GH基因编码区全序列,长度为651bp。与鸡GH基因cds的同源性为91.4%,与鸭的同源性高达98.5%,演绎成氨基酸后两者同源性分别为97.6%和99.9%;与人、鼠GH基因cds序列同源性分别为63.81%和74.31%,演绎成氨基酸后同源性分别为52.51%和72.81%。并获得了鹅GH基因4个内含子的序列,序列长度分别为1504bp、664bp、362bp和1144bp。3.在鹅GH基因编码区上共检测到4个SNP位点,分别为外显子2的C39T、C74T突变和外显子4的T291C、G297C,仅外显子2的C74T突变导致导致编码的氨基酸由丙氨酸变为缬氨酸,其余位点均为“沉默”突变。外显子2多态性分析结果显示,籽鹅和皖西白鹅具有10种基因型,狮头鹅和四季鹅具有7种基因型,浙东白鹅具有8种基因型,在籽鹅、皖西白鹅和四季鹅中等位基因A为优势等位基因,狮头鹅和浙东白鹅中等位基因D为优势等位基因;外显子4多态性分析结果显示,所有5个鹅品种(种群)均具有3种基因型,除籽鹅外,其它4个品种(种群)中等位基因A均为优势等位基因。外显子多态位点上,所有鹅品种(种群)均处于Hardy– Weinberg平衡状态。4. 4个内含子的7对引物扩增结果具有多态性,测序结果表明,内含子1的P6引物的SNP位点分别为A1066G和C1157T;内含子2的P10引物的SNP位点分别为C548T和T551C,P11引物的SNP位点分别为C548T和T551C;内含子3的P13引物多态性是由该段序列中的5个核苷酸的点突变和缺失造成,分别为A160G和C167G和T264C突变以及176bp处和231bp处分别存在两个碱基的重复和缺失。内含子4的P15的SNP位点为T130C;P18引物的SNP位点分别是G821A和C949T;P19引物的SNP位点为T1137C。在内含子1中只有P6对引物存在多态性,多态性分析结果显示,籽鹅和皖西白鹅具有4种基因型,狮头鹅、浙东白鹅和四季鹅具有3种基因型,在5个鹅品种(种群)中等位基因A均为优势等位基因;内含子2的P10和P11对引物存在多态性且基因型完全对应;所有5个鹅品种(种群)均具有3种基因型,除籽鹅外,等位基因A均为优势等位基因;内含子3的P13对引物存在多态性,狮头鹅、籽鹅和皖西白鹅具有3种基因型,浙东白鹅和四季鹅具有2种基因型,在5个鹅品种(种群)中等位基因B均为优势等位基因;内含子4的P15、P18和P19检测到多态性;引物对P15除四季鹅外,其它4个品种具有3种基因型,在5个鹅品种(种群)中等位基因A均为优势等位基因;内含子4引物对P18皖西白鹅、浙东白鹅和四季鹅具有5种基因型,狮头鹅具有4种基因型,而籽鹅只具有3种基因型,在5个鹅品种(种群)中,狮头鹅等位基因E均为优势等位基因,而籽鹅不具有E等位基因,浙东白鹅E等位基因的频率也相对较高;引物对P19皖西白鹅、浙东白鹅、籽鹅和四季鹅具有3种基因型,而狮头鹅只具有2种基因型,在5个鹅品种(种群)中,籽鹅和四季鹅M为优势等位基因,而皖西白鹅、浙东白鹅和狮头鹅N为优势等位基因。在内含子4引物对P19的多态位点中,只有籽鹅和四季鹅处于Hardy– Weinberg平衡状态(P>0.05);其它内含子多态位点中,所有鹅品种(种群)均处于Hardy– Weinberg平衡状态(P>0.05)。5.对鹅GH基因所有检测到的SNPs的多态性与早期体重和屠宰性状进行关联分析,结果表明鹅GH基因外显子2、内含子3和内含子4的多态位点上对早期增重和屠宰性状存在显著的基因型效应,对于鹅早期增重和屠宰性状,外显子2的多态位点CD和DD基因型是有利基因型,在狮头鹅和浙东白鹅中的优势等位基因D可能是有利基因。内含子3的多态位点上最有利基因型是BB基因型。内含子4上NN基因型对鹅生长具有一定的促进作用,N基因可能是鹅早期增重和屠宰性状的有利基因。6.利用多对引物扩增的方式,本试验克隆测序了鹅PRL基因编码区以及5’端调控区的全序列,cds序列全长为690bp,5’端序列全长为836bp。将所获得序列与鸡和鸭的PRL基因cds序列比较,发现与鸡PRL基因cds序列的同源性为92.8%,与鸭的同源性高达98.7%,演绎成氨基酸后两者同源性分别为93.3%和98.3%。7.在鹅PRL基因的5’端调控区检测到C135G、C201T、C278Abp和T827G突变,在内含子2的32bp和33bp处检测到2个核苷酸GA的缺失/插入,但是在编码区没有发现突变位点。多态性分析结果5′端调控区引物对P3中,籽鹅、皖西白鹅和狮头鹅具有3种基因型,四季鹅和浙东白鹅只具有2种基因型,在5个鹅品种(种群)中等位基因A均为优势等位基因;内含子2多态位点所有5个鹅品种(种群)除浙东白鹅外均具有3种基因型,在5个鹅品种(种群)中等位基因B均为优势等位基因;所有多态位点中,所有鹅品种(种群)均处于Hardy– Weinberg平衡状态(P>0.05).8.对于鹅早期增重和屠宰性状,PRL基因仅仅在5’端调控区的多态位点上存在显著的基因型效应,AA和AB基因型的个体6~10周龄体重显著高于BB基因型个体,AA和AB基因型的个体间6~10周龄体重差异不显著;对于屠宰性状而言,AA和AB基因型的个体活重、屠体重、半净膛重、全净膛重和内脏重也显著高于BB基因型个体,AA和AB基因型的个体间屠宰性状差异不显著。因此,对于鹅早期增重和屠宰性状, AA和AB基因型为有利基因型。更多还原

【Abstract】 The growth and development parameters about early bodyweight of three local goose breeds of large, medium and small type including Shitou goose, Wanxi White goose and Zi goose were analyzed, and the exons and introns of growth hormone(GH) gene and the 5’-regulatory region and exons of prolactin (PRL) gene were cloned and sequenced. Based on the sequence obtained, the single nucleotide polymorphism (SNP) of all these sequence was investigated in Zi goose, Wanxi White goose, Shitou goose, Siji goose and Zhedong White goose with PCR-SSCP method. After Calculated the frequency of genotypes and genes and compared the distribution of genotype frequencies among different goose breeds, the polymorphism of GH and PRL Gene and its relationship with body weight and carcass traits were analyzed. The results were as followed.1. Growth and development regularity and genetic parameters of average week bodyweight of local goose breeds of large, medium and small type from 1 week-old to 11 week-old were fitted with four non-lineared growth models, namely, Logistic model, Gompertz model, Bertalanfy model and Cubics model. The results showed that growth process of three geese could be demonstrated very well by four models, and all indexes of fitness were very high (more than 0.99). But Gompertz model was the best model because of its less bias of feed practice. The inflection age of growth were 5.98 weeks, 5.11 weeks and 6.16 weeks in Shitou goose, Wanxi White goose and Zi goose, respectively, and bodyweight at inflection age were 2115.77g, 1499.08g and 1409.62g, respectively.2. The exons and introns of GH gene were cloned and sequenced, and obtained the coding sequence(cds) of goose, whose length was 651bp. Homologous comparison showed that the identity of cds of GH gene was 91.4% between goose and chicken, the identity of cds of GH gene was 98.5% between goose and duck, and that was 97.6% and 99.9% in amino acid sequences, respectively. Further analysis showed that the identities of cds of goose GH gene with human and mouse were 63.81% and 74.31% and that was 52.51% and 72.81% in amino acid level, respectively. Meanwile, the sequence of four introns were obtained and their length were 1504bp, 664 bp, 362bp and 1144bp, respectively.3. Four SNPs were found in the coding region of GH gene, which are C39T and C74T mutation whith exon 2, and T291C and G297C mutation within exon 4. However, except from the C74T mutation in exon 2, where coding amino acid was changed from alanine to valine, other mutations were slient mutation. The result of polymorphism analysis on exon 2 showed that all kinds of ten genotypes appeared in the populations of Zi goose and Wanxi White goose, seven kinds of genotypes appeared in the populations of Shitou goose and siji goose and eight kinds of genotypes appeared in the population of Zhedong White goose. The allele A was dominant allele in the populations of Zi goose, Siji goose and Wanxi White goose, while allele D was dominant allele in the populations of Shitou goose and Zhedong White goose. The result of polymorphism analysis on exon4 showed that all five populations appeared three kinds of genotypes. The allele A was dominant allele in all populations except for Zi goose. Within all populations the frequencies of alleles in exons fited with Hardy-Weinbery equilibrium (P>0.05). 4. Seven pairs of primers of introns existed polymorphisms. Sequencing results revealed 13 SNPs, which were as follows: A1066G and C1157T amplied by P6 primers, and C548T and T551C amplied by P10 primers from intron 1; five point mutations and deletions caused polymorphism in intron 3 amplied by P13 primers, they were A160G, C167G, T264C and two bases insertion/deletion mutations after position 176 and 231 bp; T130C amplied by P15 primers, G821A and C949T amplied by P18 primers, and T1137C amplied by P15 primers from intron 4.Only fragment amplied by P6 primers of intron 1 existed polymorphism, and the result of polymorphism analysis showed that all kinds of four genotypes appeared in the populations of Zi goose and Wanxi White goose, while three kinds of genotypes appeared in the populations of Shitou goose, siji goose and Zhedong White goose, and the allele A was the dominant allele in all the goose populations. P10 and P11 primers of intron 2 existed polymorphisms and had complete corresponding genotypes, and all the goose populations had all the three genotypes, in which the allele A was the dominant allele except in Zi goose. P13 primers of intron 3 also existed polymorphism, all the three genotypes appeared in the populations of Shitou goose, Zi goose and Wanxi White goose, while two kinds of genotypes appeared in the populations of Siji goose and Zhedong White goose, and the allele B was the dominant allele in all the goose populations. Polymorphisms were detected from fragments amplied by P15, P18 and P19 primers of intron 4. Except Siji goose, the other four populations had all the three genotypes, and the allele A was the dominant allele in all populations; The result of polymorphism analysis on P18 showed that all kinds of five genotypes appeared in the populations of Wanxi White goose, Zhedong White goose and siji goose, four kinds of genotypes appeared in the populations of Shitou goose, and three kinds of genotypes appeared in the population of Zi goose. In the all five populations, the allele E was the dominant allele in Shitou goose, the frequency of E allele in Zhedong White goose also showed relative high, while there was no E allele existing in Zi goose. Wanxi White goose, Zhedong White goose, Zi goose and siji goose had all the four genotypes and Shitou goose only had two genotypes in P19, M was the dominant allele in Zi goose and siji goose, while N was the dominant allele in Wanxi White goose, Zhedong White goose and Shitou goose. In P19 polymorphic loci, only Zi goose and siji goose were in Hardy-Weinberg equilibrium (P>0.05); But in the other polymorphic loci in introns, all the goose populations were in Hardy-Weinberg equilibrium (P>0.05).5. Association analysis on polymorphism of GH gene and body weight and carcass traits showed that the polymorphic loci of exon 2,intron 3 and intron 4 of GH gene had significant genotype effect on body weight (the age of 1-10 weeks) and carcass traits. For body weight and carcass traits of goose, the CD and DD genotypes in exon 2 were favorable genotypes, while the allele A which is dominant allele in populations of Shitou goose and Zhedong White goose maybe was the favorable gene.BB genotype was the most favorable genotype for body weight and carcass traits of goose among all genotypes of intron 3, NN genotype was also favorable genotype for growth of goose and allele N maybe was the favorable gene for body weight and carcass traits of goose.6. The the 5’-regulatory region and exons of PRL gene were amplified by some premiers, cloned and sequenced, and then obtained the coding sequence of goose were obtained, the length of which was 690bp. Homologous comparision show that the identity of cds of PRL gene was 92.8% between goose and chicken, the identity of cds of PRL gene was 98.7% between goose and duck, and that was 93.3% and 98.3% in amino acid sequences, respectively. Meanwile, the sequence of 5’-regulatory region was obtained and its length was 836bp.7. Six SNPs were found in the PRL gene, which are C135G, C201T, C278Abp and T827G mutation whith the 5’-regulatory region, and GA InDel in the site of 32bp and 33bp within intron 2, while no polymorphism was detected in the exons of PRL. The result of polymorphism analysis on 5’-regulatory region showed that all kinds of three genotypes appeared in the populations of Zi goose and Wanxi White goose and Shitou goose, two kinds of genotypes appeared in the populations of siji goose and Zhedong White goose, allele A was dominant allele in all five populations. The result of polymorphism analysis on intron 2 showed that all kinds of three genotypes appeared in all populations except for Zhedong White goose and allele B was dominant allele in all five populations. Within all populations the frequencies of 5’-regulatory region and intron 2 fited with Hardy-Weinbery equilibrium (P>0.05).8. Association analysis on polymorphism of PRL gene and body weight and carcass traits showed that the only polymorphic loci of 5’-regulatory region of PRL gene had significant genotype effect on body weight and carcass traits. 6-10 week bodyweight of individuals with AA and AB genotype were significant higher(P<0.05) than the of individuals with BB genotype, but were insignificant difference(P>0.05) in 6-10 week bodyweight between geese with AA and BB genotype. The carcass traits such as live weight, eviscerated weight and semi-eviscerated weight of individuals with AA and AB genotype were also significant higher (P<0.05) than the of individuals with BB genotype, and insignificant difference (P>0.05)in carcass traits between geese with AA and AB genotype. The results indicated that AA and AB was favorable genotype for body weight and carcass traits of goose.更多还原