【作者】 刘宇；

【导师】 刘小林；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2012， 硕士

【摘要】 胰岛素诱导诱导基因（Insulin induced gene INSIG）包括INSIG1基因和INSIG2基因两个成员，参与机体脂质代谢，抑制胆固醇合成，同时也参与能量平衡和体重的调节。本试验以INSIG1基因和INSIG2基因为候选基因，以秦川牛为研究对象，采用RT-PCR法成功克隆了秦川牛生长和胴体性状候选基因INSIG2，并对其进行了生物信息学分析；采用实时荧光定量PCR技术研究了秦川牛INSIG1基因和INSIG2基因在不同组织中的表达规律；采用PCR-RFLP、PCR-SSCP技术和DNA测序技术相结合，研究了秦川牛INSIG1基因和INSIG2基因在秦川牛群体中的遗传多样性及其与生长和胴体性状的相关性，主要结果如下：1.秦川牛INSIG2基因的克隆及生物信息学分析分别以三头20±2月龄的秦川牛肝脏组织为材料，成功克隆得到秦川牛INSIG2基因完整编码区序列，大小为362bp，提交GenBank获得登录号JQ951942，该片段开放阅读框包括351个核苷酸，共编码116个氨基酸，对INSIG2基因编码蛋白质的生物信息学分析发现该蛋白相对分子质量12.8kD，等电点为7.73；INSIG2蛋白蛋白质疏水性预测结果显示疏水值最大为2.444，位于第54和第55个氨基酸处，疏水值最小为-1.533，位于第40个氨基酸处；跨膜结构预测显示该蛋白可能有三个由内向外的跨膜螺旋，分别位于第22~39aa处、45~65aa处和76~97aa处；在该蛋白第16、44和第78个氨基酸处，可能有三个蛋白质磷酸化位点；信号肽区域预测发现该蛋白没有信号肽锚定位点；2.秦川牛INSIG1、INSIG2基因组织表达规律采用用实时荧光定量技术研究了3头20±2月龄秦川牛INSIG1基因和INSIG2基因在肝脏、肺脏、脾脏、瘤胃、心脏、腹脂、肌肉以及小肠8个组织中的相对表达量。结果发现：秦川牛INSIG1基因在肝脏中高度表达，在其他7个组织中表达量很低；秦川牛INSIG2基因在8个组织中均有表达，在肝脏中表达量最高，其次是在肺脏中，在其他6个组织中表达量较少。3.秦川牛INSIG1基因多态性及其与生长和胴体性状的关联分析利用DNA测序技术和PCR-RFLP技术研究了215头20±2月龄秦川牛INSIG1基因多态性及其与生长和胴体性状的关联分析，发现了INSIG1基因A4366G、T4534C、T5001C和A5235G四个突变位点，遗传多样性分析结果表明四个位点均为中度多态位点；分别研究了各位点与秦川牛生长和胴体性状的相关性，从研究结果可以看出，部分基因型个体在生产性能方面要优于其他基因型个体，在A4366G位点，GG型个体腰角宽和胴体重显著高于AA型个体（P<0.05），而GG型个体宰前活重极显著高于AA型个体（P<0.01），不同基因型之间体高和体长间差异不显著；在T4534C位点，CC型个体体长和腰角宽显著高于TT型个体（P<0.05），而CC型个体和TC型个体在体高方面极显著高于TT型个体（P<0.01），；在T5001C位点，CC型个体体长显著高于TT型个体（P<0.05），在体高、宰前活重、胴体重和腰角宽方面，各基因型之间差异不显著。在A5235G位点，各基因型之间生长和胴体性状无显著差异，总体上来说，AA型个体在各个性状上要优于AG型个体和GG型个体（P>0.05）。4.秦川牛INSIG2基因多态性及其与生长和胴体性状的关联分析利用DNA测序技术和PCR-SSCP技术研究了215头20±2月龄秦川牛INSIG2基因多态性及其与秦川牛肉质和胴体性状的关联分析，发现了秦川牛INSIG2基因T15570C突变位点，遗传多样性分析结果表明该位点为中度多态位点；关联分析结果显示TT型个体体高显著高于TC型个体（P<0.05），TT型为优势基因型个体，在其他性状上不同基因型与生产性状之间无显著相关（P>0.05）。本研究成功克隆获得秦川牛INSIG2基因完整编码区序列并对其进行了生物信息学分析，为进一步研究INSIG2基因在秦川牛生长发育过程中的作用奠定了理论基础。并对INSIG1基因和INSIG2基因在秦川牛不同组织中的表达规律进行了研究，为进一步研究相关性状的表达提供了科学依据。对秦川牛INSIG1基因和INSIG2基因SNP位点进行检测，并对其与生长和胴体性状的相关性进行了研究，部分位点发现了显著相关，为进一步开展秦川牛本品种选育工作提供了理论依据。更多还原

【Abstract】 There are two members in insulin induced gene family: INSIG1and INSIG2, both ofthem involved in lipogenesis, cholesterol metabolism and the regulation of energy and bodyweight. In this experiment, we take both INSIG1and INSIG2as candidate gene to study thefunction of them in Qinchuan beef cattle. In this study, we collected the liver samples ofQinchuan cattle to clone the INSIG2gene, using bioinformatics methods to predict thestructure and function of this gene. Analyzed the expression pattern in different tissues ofQinchuan cattle by real-time fluorescent quantitative PCR. Used PCR-RFLP, PCR-SSCP andDNA sequencing to detect the SNP of the two genes in Qinchuan cattle and their associationwith the growth and carcass traits. The main results were showed as following:1. Cloning and bioinformatics analysis of the INSIG2gene in Qinchuan cattleThe362bp of INSIG2gene was cloned by RT-PCR, including completely CDS region.We submitted it to GenBank and obtained the accession number of the sequence(JQ951942).The ORF of INSIG2gene including351bp nucleotides, encoding116amino acids. The relativemolecular mass and PI of INSIG2protein are12.8kD and7.73, respectively. The maximum hydrophobicityvalue is2.444and minimum value is-1.533, located at54th,55th and40th amino acids. Three strongtransmembrane helices located at22-39aa,45-65aa and76-97aa. Three phosphorylation sites are located at16th,44th and78th amino acids. No signal peptide domain was found in INSIG2protein.2. Expression pattern of the INSIG1and INSIG2genes in Qinchuan cattleExpression pattern of INSIG1and INSIG2genes in liver, luang, spleen, stomach, heart,abdominalfat, muscle and intestine of Qinchuan cattle by real-time fluorescent quantitativePCR. The results showed that the expression of INSIG1mRNA was highest in liver and alllower levels in other seven tissues. For INSIG2gene, the expression of liver was the highestand then lungs, the expression of the other6tissues were all lower than the two tissues.3. Effects of polymorphisms of INSIG1gene on growth and carcass traits of bovineWe used the PCR-RFLP and DNA sequencing to detect the SNPs in INSIG1gene of21520±2month age Qinchuan cattle, and the association between the polymorphisms and growthand carcass traits were analyzed. A total of4SNPs were found, they were A4366G, T4534C,T5001C and A5235G, respectively. Genetic diversity results showed all of them were mediumpolymorphism. The associations between the mutation at locus A4366G with the growth andcarcass traits showed that genotype had a significant effect on hip width, slaughter weight and carcass weight(P<0.05). The genotypes at locus T4534C had a significant effect on bodylength, wither height and hip width(P<0.05).. The genotypes at locus T5001C had asignificant effect on hip width(P<0.05).. In addition, there were no significant differencesbetween genotypes at locus A5235G(P>0.05).4. Effects of polymorphisms of INSIG2gene on growth and carcass traits of bovinePCR-SSCP and DNA sequencing were used to detect the SNPs in INSIG12gene of21520±2month age Qinchuan cattle. A SNP of T15570C was found. Genetic diversity resultsshowed it was medium polymorphism. The association study showed that TT genotype hadsignificant taller body height than TC genotype(P<0.05), there are no significant differencesin other traits(P>0.05).In conclusion, we successfully cloned the INSIG2gene of Qinchuan cattle, it is usefulfor the further research on the function of this gene in the growth process of Qinchuan cattle.The expression patterns of INSIG1and INSIG2genes analysis provide a scientific theory onthe expression of relative traits. The purpose of this study is to settle the theory foundationson the native breeding work of Qinchuan beef cattle.更多还原