【作者】 胡冬利；

【导师】 谢庄；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 家畜的繁殖性状是重要的经济性状之一。由于绵羊产羔数性状遗传力低只有0.1左右,通过常规选择方法难以提高,因此寻找控制排卵数乃至产羔数的主效基因研究,已成为各国绵羊育种学家关注的焦点。湖羊作为我国著名的多胎性地方绵羊品种,具有四季发情,每胎的产羔率高(平均为230%)等特点。因此本研究以湖羊为主要研究对象,以GDF9、INHBB和Smad2基因作为湖羊高繁殖力性状的候选基因进行分析,并从分子水平探索湖羊多胎性状的分子遗传机理,以充分利用湖羊的多胎性能,为绵羊繁殖力的标记辅助选择和育种提供理论依据。本文主要采用RT-PCR和实时荧光定量PCR对湖羊GDF9、INHBB、Smad2的组织表达、发情期单羔和多羔湖羊卵巢组织mRNA表达水平进行了研究；用PCR-RFLP法对湖羊GDF9和INHBB的多态性进行了分析,结果如下：1.组织表达结果显示：GDF9、INHBB和Smad2基因在湖羊下丘脑、垂体、卵巢、子宫、输卵管、心、肝、脾、肺、肾组织中均有表达,说明GDF9、INHBB和Smad2基因不仅在湖羊的生殖器官中起着重要作用,可能对其他器官更具有普遍意义的作用。2.定量研究结果表明GDF9mRNA和INHBBmRNA在发情期多羔湖羊卵巢中的表达水平显著高于单羔湖羊(P<0.05),表明GDF9mRNA和INHBBmRNA表达水平与湖羊的繁殖性状有一定的关系,GDF9mRNA和INHBBmRNA的高表达量可能是引起多羔湖羊较高的产羔数的原因之一。3.定量研究结果表明Smad2mRNA在发情期单羔和多羔湖羊卵巢组织中表达差异不显著(P>0.05),说明Smad2mRNA表达水平与湖羊的繁殖性状没有直接关系。4.本研究设计了9对引物扩增了GDF9基因的几乎全长,克隆测序没有发现突变位点。李碧侠等(2003)采用PCR-SSCP方法发现在小尾寒羊、湖羊、多赛特羊和萨福克羊的GDF9cDNA的第152bp处发生单碱基改变(A→G),并导致了氨基酸的改变(天冬酰胺→天冬氨酸),本研究用PCR-RFLP方法对GDF9基因此处的突变进行了检测,在所研究的47只湖羊群体中并无发现此突变,另外测序结果中也未发现该突变。5.克隆测序发现INHBB基因外显子2第391bp处有1个A→C的单碱基突变,该突变导致第131位氨基酸由丝氨酸改变为精氨酸；INHBB基因外显子2第766bp处和第767bp处分别发生了C→G突变和G→C突变,此两处突变导致第256位氨基酸由精氨酸改变为丙氨酸。本研究用PCR-RFLP方法对INHBB基因外显子2第391bp处的突变进行了单核苷酸多态性分析,在所研究的47只湖羊群体中均发现了此突变,排除了INHBB基因外显子2第391bp位点突变影响湖羊产羔数的可能性。更多还原

【Abstract】 Livestock breeding trait is one of important economic traits. It’s difficult to improve the properties according traditional methods because the number of sheep lambing is low genetic traits only about 0.1.Finding control mechanism for ovulation rate and litter size of the major genes (or mutation) aroused widespread concern of breeding sheep jurists throughout the world.As the famous local multi-fetus sheep,Hu sheep are non-seasonal estrus and have a high rate of multi-fetus (average of 230%).So we choose Hu sheep as the main analyse animal, GDF9、INHBB and Smad2 were studied as candidate genes on the high productivity of Hu sheep,and research the molecule heredity mechanism of multi-fetus trait for Hu sheep from molecule level,in order to make the best of the Hu sheep multi-fetus,to provide theory evidence for MAS and breeding of reproduction power in Hu sheep.The tissue expression pattern and the mRNA expression levels of GDF9,INHBB and Smad2 genes in the ovary between single fetuses and multiplets Hu sheep during oestrum were analysed using RT-PCR and real time PCR,The polymorphism of GDF9 and INHBB genes were identified using PCR-RFLP. The results are as follows:1. The tissue expression results indicated that GDF9,INHBB and Smad2 genes expresse in hypothalamus,pituitary,ovary,uterus,fallopian tube,heart, liver, spleen,lung and kidney of Hu sheep,showed that GDF9,INHBB and Smad2 genes not only play important role in reproductive organs,and may have some universal significance in other organs.2. Real time PCR results indicated that GDF9mRNA and INHBBmRNA expression levels in the ovary of multiplets Hu sheep during oestrum is prominent higher than single fetuses Hu sheep(P<0.05), showed that GDF9mRNA and INHBBmRNA expression levels have some relation with the reproductive traits in Hu sheep,the high expression levels of GDF9mRNA and INHBBmRNA may the one causes of high rate of multi-fetus for multiplets Hu sheep.3. Real time PCR results indicated that there’s no obvious difference of Smad2mRNA expression levels in the ovary of single fetuses Hu sheep and multiplets Hu sheep during oestrum(P>0.05), showed that Smad2mRNA expression levels have no direct relation with the reproductive traits in Hu sheep.4. In this study,we designed 9 pairs of primers amplified almost the whole length of GDF9 gene, clone Sequencing results showed that there were no mutation sites founded,Li Bi Xia(et al.,2003) found one single nucleotide mutation:A→G at cDNA 152 of GDF9 gene in Small Tail Han sheep,Hu sheep,Dorset sheep and Suffolk sheep analyzed by PCR-SSCP,and this mutation resulted in an amino acid change:asparagine→aspartic acid,the single nucleotide polymorphism at this site of GDF9 gene was identified using PCR-RFLP,but didn’t found this mutation in 47 Hu sheep, Moreover,the sequencing results also didn’t found this mutation.5. Clone Sequencing results showed that there was one single nucleotide mutation:A→C at exon2 391bp of INHBB gene, this mutation resulted in an amino acid change:serine→arginine acid at 131;there were two nucleotide mutations:C—G and G→C at exon2 766bp and 767bp of INHBB gene,the two mutations resulted in an amino acid change:arginine→alanine acid at 256.The single nucleotide polymorphism of mutation:A→C at exon2 391bp of INHBB gene was identified using PCR-RFLP, and found this mutation in all 47 Hu sheep, so eliminate the possibility of mutation:A→C at exon2 391bp of INHBB gene effects the litter size in Hu sheep.更多还原