【作者】 王鹏；

【导师】 孙伟； 丁家桐；

【作者基本信息】 扬州大学 ， 特种经济动物饲养， 2010， 硕士

【摘要】 本实验以湖羊为研究对象,以无角陶赛特、萨福克、特克赛尔、小尾寒羊为参照群体,利用候选基因法,采用PCR-RFLP和PCR-SSCP结合DNA测序技术,对GH和MSTN基因部分片段进行了遗传多态性检测,并探讨其与湖羊部分生长性状(初生重、六月龄重、断奶重)间的关系,探索GH和MSTN基因作为影响湖羊肌肉生长发育候选基因的可能性,并对两个基因在湖羊不同生长阶段肌肉组织中表达丰度及其与屠宰性状的相关性进行了研究,旨在揭示湖羊肌肉生长性状的分子遗传学基础,为湖羊肌肉生长性状的选育提供遗传学资料。本研究获得了以下结果:1、GH、MSTN遗传多态及其与湖羊早期肌肉生长性状关联分析(1)GH基因遗传多样性与湖羊早期肌肉生长性状的关系H外显子-1位点发现C→T突变;GH外显子-2位点发现C→G突变;GH外显子-3位点发现A→G突变;GH外显子-4位点发现A→G突变。卡方适合性检验结果表明,研究群体在GH外显子-3位点处于Hardy-Weinberg遗传极度不平衡状态,GH外显子-1位点处于Hardy-Weinberg显著平衡状态,其他位点上均处于Hardy-Weinberg平衡状态。GH外显子-1位点的AA和AB两种基因型在初生重和六月龄重上差异显著;GH外显子-2位点的CC基因型在断奶重上与其他三种基因型间差异显著;GH外显子-3位点的GG和GH基因型以及GH外显子-4位点的MM和LM基因型在三种性状间差异均不显著。GH外显子-3位点的GH基因型和外显子-4位点的LM基因型是生长性状的有利基因型。(2)MSTN基因遗传多样性与湖羊早期肌肉生长性状的关系MSTN启动子位点发现A→G突变;MSTN外显子-2位点发现A→G突变;MSTN 3′端调控区位点发现G→T突变。卡方适合性检验结果表明,研究群体只在MSTN-3`端调控区位点处于Hardy-Weinberg遗传极度不平衡状态,其他位点上均处于Hardy-Weinberg平衡状态。MSTN启动子位点OO、OP和PP三种基因型间对断奶重和六月龄重均差异显著;MSTN外显子-2位点XX和XW两种基因型间对断奶重和六月龄重均差异显著;MSTN 3′端调控区位点YY和YZ两种基因型间对三种性状间差异均不显著。其中,MSTN启动子PP基因型和3′端调控区的YY基因型是生长性状有利基因型。2、GH、MSTN基因表达及其与湖羊屠宰性状关联分析(1)湖羊不同生长阶段GH基因表达分析公羊和母羊GH基因发育变化趋势基本一致。出生后随着日龄的增加,基因相对表达水平在肌肉组织中呈下降趋势。其中在2日龄相对表达水平最高。公羊60日龄和180日龄与其他日龄之间分别存在显著差异,其中60日龄、90日龄和180日龄与其他日龄之间分别存在极显著差异;母羊30与60日龄、120与180日龄与其他日龄之间分别存在显著差异,其中30、60、90、180日龄与其他日龄之间分别存在极显著差异。公母之间在180日龄存在显著差异,在2、30、60、90和120日龄之间存在极显著差异。(2)湖羊不同生长阶段MSTN基因表达分析出生后随着日龄的增加,公羊和母羊MSTN基因相对表达水平在肌肉组织中呈现先上升后下降的趋势。其中在60日龄相对表达水平最高。公羊不同日龄之间分别存在显著差异,其中30与120日龄与其他日龄之间分别存在极显著差异;母羊30与90日龄、60与180日龄与其他日龄之间分别存在显著差异,同时与其他日龄之间分别存在极显著差异。公母之间在60与180日龄之间存在显著差异,在30、90和120日龄之间存在极显著差异。(3) GH、MSTN基因表达丰度与屠宰性状的相关性分析GH mRNA表达水平与MSTN mRNA表达水平存在极显著负相关(r= -0.561)。GH mRNA表达水平与湖羊屠宰前活重、胴体重和净肉重等屠宰性状之间均存在极显著负相关;MSTN mRNA表达水平与宰前活重和胴体重之间不存在相关性,与净肉重间存在显著相关。更多还原

【Abstract】 In this experiment, Hu sheep was the researched object and Poll Dorset,Texel,Suffolk and Xiao Wei Han sheep were the referenced groups.Methods of candidate gene,PCR-RFLP, PCR-SSCP and DNA sequencing techniques were applied to analyze the genetic variation of partly GH and MSTN gene fragments and approach the relation between genetic variation and partly growth traits and analyse the possibiliti of GH and MSTN gene to be the candidate genes that effecting the growth and development of muscle in Hu sheep.At the same time, investigate the allocation and express abundance of the two genes at different stages of days in muscle in Hu sheep.The purpose is to reveal the genetic technology of growth in muscle in Hu sheep by using candidate gene method and provide genetic information for the selection of muscle growth in Hu sheep. The results were as follows:1 Association analysis of genetic polymorphism and early muscle growth traits of GH and MSTN gene in Hu sheep(1) Association analysis of genetic polymorphism and early muscle growth traits of GH gene in Hu sheepA single nucleotide substitution of C to T was found on the GH Extron-1 site. There was a single nucleotide substitution of C to G was found on the GH Extron-2 site and a A to G single nucleotide substitution was found on the GH Extron-3 site. There was also a single nucleotide substitution of A to G was found on the GH Extron -4 site. The result of chisquare-suitability test showed:site of GH Extron-3 was in extreme imbalance, site of GH Extron-1 was significant balanced condition and other sites were balanced condition.There were significant differences on Birth weight and 6-month-age weight between the two genotypes Aa and AB of GH Extron-1. There was significant difference on Post weaning weight for CC genotype of GH Extron-2 than other three grnotypes. There was no significant difference on the three growth traits for GH Extron-3 between GG and GH genetypes and GH Extron-4 between MM and LM genetypes.GH genetype in Extron-3 and LM genetype in Extron-4 were advantageous genetype for growth traits.(2) Association analysis of genetic polymorphism and early muscle growth traits of MSTN gene in Hu sheepA single nucleotide substitution of A to G was found in MSTN promoter site,a A to G single nucleotide substitution was found in Extron-2 site,a single nucleotide substitution of G to T was found in 3′control region of MSTN site. The result of chisquare-suitability test showed: 3′control region site was in extreme imbalance and other sites were in balanced condition. There were significant differences on Post weaning weight and 6-month-age weight between the three genotypes OO,OP and PP of MSTN promoter.There were significant differences on Post weaning weight and 6-month-age weight between the two genotypes XX and XW site of MSTN Extron-2.There was no significant difference on the three growth traits between the two genotypes YY and YZ of 3′control region site of MSTN. YY genetype in 3′control region and PP genetype in promoter were advantageous genetype for growth traits.2 Association analysis between GH,MSTN gene expression and butcher traits in Hu sheep (1)Expression of GH gene at different early days in Hu sheepThere was fundamental same tendency of developed changes of GH gene between rams and ewes.Followed by the increase of days after birth,the relative expression levels took on descend tendency in muscular tissue.On the 2 days,there was the highest expression level.For rams:there were significant difference between 60,180 days and other days.Among that,there were extreme significant difference between 60,90,180 days and other days.For ewes:there were significant difference between 30,60days and 120,180 days and other days.Among that, there were extreme significant difference between 30,60,90,180 days and other days. Between rams and ewes,there was significant difference on 180 days and extreme significant difference between 2,30,60,90 and 120 days.(2)Expression of MSTN gene at different early days in Hu sheepFollowed by the increase of days after birth,the relative expression levels of MSTN gene in muscular tissue took on the tendency of first increased and then descended. There was the highest expression level on the 60 days. For rams: there were significant difference between defferent days.Among that, there were extreme significant difference between 30,120 days and other days.For ewes:there were both significant and extreme significant difference between 30,90 days and 60,180 days and other days. Between rams and ewes,there was significant difference between 60 and 180 days and extreme significant difference between 30,90 and 120 days.(3) Correlation analysis of expression abundance of GH and MSTN gene and butcher traitThe results of correlation analysis showed that: there were extreme significant negative correlation between GH gene expression and MSTN gene expression.There were extreme significant negative correlation between GH gene expression and Live weight,Carcass weight and Meat percentage.At the same time, there was significant correlation between MSTN gene expression and Meat percentage.There was no significant correlation between MSTN gene expression and Live weight and Carcass weight.更多还原