【作者】 董斌科；

【导师】 蒋思文；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 妊娠期是猪重要的繁殖性状之一。在生产过程中缩短母猪的妊娠期可以缩短其繁殖周期,提高母猪的繁殖效率,降低生产成本。妊娠期是数量性状,由多基因的控制的,且单个基因的效应值较小,易受环境因素的影响,用常规的育种方法对其进行遗传改良进展缓慢。有研究报道影响猪妊娠期的QTL位于猪的第1、9和15号染色体上,根据猪-人基因组比较图谱和基因的生理生化功能选取了两个候选基因:肾素（Renin）和白介素-10（IL-10）。应用生物信息学和比较基因组的方法对这两个基因进行了克隆,经不同品种间序列的比对发现这两个基因的突变位点,并选择三个突变位点与妊娠期和产仔数进行相关性分析:1.根据与人的Renin基因有较高同源性的猪EST以及人和小鼠等物种该基因cDNA相似性较高的区域设计了5对引物,以梅山猪cDNA作为模板扩增、克隆并测序,拼接后获得1333 bp的整合cDNA序列,包含全部CDS和部分5’和3’非翻译区。利用获得的cDNA序列设计18对引物对其DNA序列进行扩增,克隆、测序和拼接后得到了13332bp的DNA序列。2.利用Clustal X、ORF Finder、Spidey、MEGA4.0、PSORTⅡ等软件对猪Renin基因结构、编码的蛋白质分子量、等电点、系统进化树等特征进行了预测和分析。我们发现该基因共有9个外显子和8个内含子且内含子的剪切方式符合“GT-AG”法则,进化树分析得出该基因与反刍动物的相似性最高,与啮齿类的次之,与灵长类的最低。3.根据猪IL-10基因的cDNA序列设计7对引物,克隆得到了该基因4062 bp的DNA序列,经过对该基因的结构进行分析,该基因包含有4个内含子,内含子的剪切方式符合“GT-AG”法则。4.根据已获得的Renin基因的cDNA序列设计引物,用RT-PCR的方法检测其在不同组织中的表达情况,发现在肾、卵巢和输卵管中有表达,在肾中的表达最高。5.根据获得的这两个基因的序列,清平猪、大白猪、长白猪和杜洛克猪的DNA作为模板进行扩增、克隆并测序。经序列比对,发现了在Renin的第6内含子228bp处发现一个C/T转换,第7内含子的114bp处发现一个A/G转换和IL-10基因第3内含子的835bp处发现一个A/G转换,据此建立适宜的PCR-RFLP SNP分型方法。6.Renin的第6内含子内的突变可以被HhaⅠ酶切,建立该基因的PCR-HhaⅠ-RFLP检测方法,对该位点在清平猪、新清平猪、长白猪、大白猪,杜洛克猪和中国瘦肉猪新品系DⅣ₁系、DⅣ₂系7个中外猪群体中进行基因型分型,分析了它们的基因型频率和基因频率,并与妊娠期和产仔数进行相关性分析,结果表明该位点TT基因型在国外猪种中占有优势,地方品种和培育品种与外种猪的基因型频率之间均存在显著地差异,且都达到了极显著的水平。该位点在新清平猪中对于妊娠期的显性效应达到了显著地水平。在DⅣ₁系中CC基因型的妊娠期要显著高于TT和CT基因型的妊娠期。该位点在不同的群体中对于产仔数的加性效应和显性效应均不显著。7.Renin基因第7内含子内的突变可以被bstxⅠ酶切,建立该基因的PCR-bstxⅠ-RFLP检测方法,在清平猪、新清平猪、长白猪、大白猪杜洛克猪和中国瘦肉猪新品系DⅣ₁系、DⅣ₂系7个中外猪群体中进行基因型分型,分析了它们的基因型频率和基因频率,并与妊娠期和产仔数进行相关性分析,结果表明该位点A等位基因在各个猪群中占有优势,在杜洛克猪中除外。大部分猪群之间基因型频率差异显著。在新清平猪中该位点对于妊娠期的显性效应差异显著,为0.93d。该位点在杜洛克猪中对于产仔数的加性效应达到极显著地水平,GG基因型的产仔数要显著地高于AG和AA基因型的产仔数。8.IL-10的第3内含子内的突变可以被Eco81Ⅰ酶切,建立该基因的PCR-Eco81Ⅰ-RFLP检测方法,在清平猪、新清平猪、长白猪、大白猪杜洛克猪和中国瘦肉猪新品系DⅣ₁系、DⅣ₂系7个中外猪群体中进行基因型分型,分析了它们的基因型频率和基因频率,并与妊娠期进行相关性分析,结果表明该位点GG基因型在清平猪、新清平猪和杜洛克猪中占有优势,而在长白猪、大白猪、DⅣ₁系和DⅣ₂系中AA基因型占有优势。大部分猪群之间基因型频率差异显著。该位点在杜洛克猪中对于妊娠期的显性效应显著,致使AG基因型的妊娠期要显著长于AA基因型的妊娠期。该位点在大白猪中对于产仔数的加性效应显著,GG基因型的产仔数要显著少于AG和AA基因型的产仔数。更多还原

【Abstract】 The gestation length serves as one of the most importent reproductive traits. Reducing the gestation length,taking swine breeding into account,directly results in a shorter reproductive cycle;correspondingly reduce the production cost by increasing the reproductive efficiency of sows.Gestation length is a quantitative trait which is affected by several genes’ cooperation.Due to the amount of genes and every single effect it provides,as well as environmental factors,regular breeding methods have scarcely any apparent genetic improvement on this character.The QTL related with gestation length is located at the 1,9,15 chromosome in sus scrofa.According to swine- human genome comparison and both physiological and biochemical function,two candidate genes renin and interleukin-10 have been selected in this experiment.In this experiment several methods like bioinformatics and conservation of linkage with swine and man are being applied to clone these genes and polymorphic sites have been made in order to analyze the relativity with the gestation length and and total number of piglet born.1.Based on swine’s EST which has a high homology with human renin gene and the cDNA of other species like mice,five primers have been designed on the sequences with great similarity within several animals.Meishan’s cDNA has been chosen as the template which is sequenced and cloned.After splicing and integrating,the cDNA, 1333 base pair,has been acquired including the whole CDS and parts of 5’ and 3’ untranslationed region.Later,the DNA sequence has amplified,cloned and sequenced which was examined as 13332 base pairs.2.With the help of software Clustal X、ORF Finder、Spidey、MEGA4.0、PSORTⅡ, renin’s gene structure,the structure of protein,property,the phylogenetics tree,has been predicted and analyzed.We find that this gene with 9 extrons and 8 introns matching the GT-AG rule,is extremely resemble with rumen animals,less similar with rodents,and least conformity with primate.3.Relied on cDNA of IL-10 gene,seven primers have designed and the whole gene has been cloned with 4062bp.After analyzing the structure of the gene,4 introns have been included and complied with the GT-AG rule.4.RT-PCR has been used to exam renin expression in different tissues,the results demonstrate that kidney,ovaries,oviduct have expressed this gene with the highest one founded in kidney. 5.Different DNA of Qingping,Large white,Landrace and Duroc have been amplified, cloned and sequenced.Transvertion of C/T at the 6 intron,288bp,and A/G at the 7 intron 114bp in renin as well as the transvertion A/G in the third intron of IL-10 at 835bp have been found based on sequences comparison.Appropriate PCR-RFLP SNP typing methods have been established.6.The mutation in the sixth intron of renin can be cut by enzyme HhaⅠ.Qingping,New Qingping,large white,landerace,Duroc and Chinese lean meat pig strain DⅣ₁、DⅣ₂,altogether seven kinds of swine have been typed.The genetype frequency and gene frequency were analyzed in order to dig out the relativity with gestation length and and total number of piglet born.The result shows that the specific site with genetype TT plays more prominent effect in foreign pigs,whereas local breed and cultivated strain has significant variation from foreign swine even achieved the extremely significant variation.The dominant effect on gestation length in this site in Qingping has reached significant level as well.In the DⅣ₁ strain,hogs’ gestation length with gene type CC were significantly longer than that of TT and CT gene type. The dominant effect and the additive effect on total number of piglet born in this site in all the groups are not significant.7.The mutation in seventh intron of renin can be cut by enzyme bstxⅠ.Qingping,New Qingping,large white,landerace,Duroc and Chinese lean meat pig strain DⅣ₁、DⅣ₂,altogether seven kinds of swine have been typed.The genetype frequency and gene frequency were analyzed in order to dig out the relativity with gestation length and total number of piglet born.The result shows that allele gene A has a more conspicuous advantage in any swinery,besides duroc.The gene frequency has significant variation and the dominant effect on gestation length in Qingping was significant,0.93day.The additive effect on total number of piglet born in this site in Duroc has reached significant level,hogs’ total number of piglet born with gene type GG were significantly more than that of AG and AA gene type.8.The mutation in third intron of IL-10 can be cut by enzyme Eco81Ⅰ.Qingping,New Qingping,large white,landerace,Duroc and Chinese lean meat pig strain DⅣ₁、DⅣ₂,altogether seven kinds of swine have been typed.The genetype frequency and gene frequency were analyzed in order to dig out the relativity with gestation length and total number of piglet born.The result shows that the specific site with gene type GG plays more prominent effect in Qingping,New Qingping and duroc,whereas within large white,landerace,DⅣ₁,and DⅣ₂ AA acted a more important role.Most swinery has significant variation from each other.The dominant effect on gestation length in this site in duroc has reached significant level,consequently pigs with gene type AG have a longer gestation length than those of AA gene type.The additive effect on total number of piglet born in this site in large white has reached significant level,hogs’ total number of piglet bom with gene type GG were significantly fewer than that of AG and AA gene type.更多还原