天祝白牦牛IGF-2基因的全长cDNA克

发布时间：2018-05-06 01:24

本文选题：天祝白牦牛 + IGF-2基因　；参考：《甘肃农业大学》2015年硕士论文

【摘要】：为研究影响白牦牛生长性状的相关基因,本研究选择胰岛素样生长因子2基因(Insμlin-like growth factor-2,IGF-2)为研究对象,采用逆转录多聚链式反应(RT-PCR)技术和cDNA末端快速扩增(RACE)技术克隆获得天祝白牦牛IGF-2基因cDNA全序列;采用生物信息技术对获得的天祝白牦牛IGF-2基因cDNA序列及其编码的氨基酸进行分析,通过同源建模对翻译的蛋白质的3D结构进行预测;根据克隆获得的天祝白牦牛IGF-2基因cDNA全序列,针对其保守区设计特异性引物,以甘油醛-3-磷酸脱氢酶(GAPDH)基因为内参基因,采用SYBR Green 1实时荧光定量PCR方法对IGF-2基因在不同组织的差异表达进行研究;利用DNA混合池、直接测序法和高分辨率溶解曲线(HRM)检测天祝白牦牛IGF-2外显子8和外显子10的多态性,并分析其多态性与天祝白牦牛体重和体尺性状的关联。研究获得如下结果:1.从天祝白牦牛肝脏组织中首次成功得到IGF-2基因的cDNA序列全长1060bp,开放阅读框长540bp,编码179个氨基酸;序列比对和同源性分析显示,在核苷酸水平,该基因与黄牛的同源性最高99%;对比氨基酸序列,该序列与牛、绵羊的同源性高达98%,表明在IGF-2基因在进化过程中的高度保守性。使用PredictProtein在线工具进行二级结构预测发现蛋白有36.31%螺旋,63.69%无规卷曲。运用Swiss model以PBD数据库中人的3D结构为模板预测天祝白牦牛IGF-2蛋白的3D结构,其蛋白结构与人的很相似。2.天祝白牦牛IGF-2基因mRNA在9个组织的表达情况为:在睾丸中表达最高,其次分别为肝脏、肌肉、脾脏、卵巢、大脑、肾脏,而心脏和肺脏未检测到IGF-2 mRNA的表达。3.通过DNA混合池在外显子8和外显子10分别发现两个SNP位点。利用HRM对两个SNP位点进行多态性分析,外显子8检测到TT、CC、TC三种基因型,其中TT为优势基因型;外显子10检测到GG、AA、GA三种基因型,其中GG为优势基因型。通过群体遗传学分析,外显子8的多态位点表现中度多态(PIC=0.25),外显子10的多态位点表现低度多态(PIC=0.192)。χ2检验表明,两个多态位点都处于Hardy-Weinberg平衡状态(P0.05)。多态位点与生长性状相关性分析,外显子8的基因型在体重、胸围、体斜长方面差异显著(P0.05),在管围、体高性状差异不显著,;外显子10的基因型在胸围方面差异显著,其他生长性状差异不显著。
[Abstract]:In order to study the related genes affecting the growth traits of white yaks, insulin like growth factor 2 gene (ins 渭 lin-like growth factor-2) was selected as the research object. The whole cDNA sequence of IGF-2 gene of Tianzhu white yak was cloned by reverse transcription polymerase chain reaction (RT PCR) and rapid amplification of cDNA terminal rase. The cDNA sequence of the IGF-2 gene and the amino acid encoding it were analyzed by bioinformatics, and the 3D structure of the translated protein was predicted by homology modeling. According to the whole cDNA sequence of the IGF-2 gene of Tianzhu white yak, a specific primer was designed for the conserved region of the gene, and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the internal reference gene. The differential expression of IGF-2 gene in different tissues was studied by SYBR Green 1 real-time fluorescence quantitative PCR, and the polymorphism of IGF-2 exon 8 and exon 10 in Tianzhu white yak was detected by DNA mixed cell, direct sequencing and high resolution dissolution curve. The association of polymorphism with body weight and body size of Tianzhu white yak was analyzed. The results of the study are as follows: 1. The cDNA sequence of IGF-2 gene was successfully obtained from liver tissue of Tianzhu white yak for the first time, with an open reading frame of 540 BP, encoding 179 amino acids. Compared with the amino acid sequence, the homology of this gene with cattle and sheep is as high as 98, indicating that the IGF-2 gene is highly conserved in the evolution process. Using PredictProtein on-line tool to predict the secondary structure, 36.31% helical coil was found to be 63.69% random coil. The 3D structure of IGF-2 protein in Tianzhu white yak was predicted by using human 3D structure in PBD database by Swiss model. The protein structure of IGF-2 protein was similar to that of human. The expression of IGF-2 gene mRNA was highest in testis, followed by liver, muscle, spleen, ovary, brain and kidney, but no expression of IGF-2 mRNA was detected in heart and lung. Two SNP loci were found in exon 8 and exon 10 by DNA mixed pool. Using HRM to analyze the polymorphism of two SNP loci, three genotypes of TTG CCTC were detected in exon 8, among which TT was the dominant genotype, and exon 10 was detected as three genotypes of GGG AAGA, among which GG was the dominant genotype. Population genetic analysis showed that the polymorphic loci of exon 8 and exon 10 were moderately polymorphic and low polymorphic, respectively. 蠂 2 test showed that the two polymorphic loci were in the Hardy-Weinberg equilibrium state (P0.05). There were significant differences in body weight, chest circumference and body oblique length between the genotypes of exon 8 and body height, but not in tube circumference, and in chest circumference of exon 10. There was no significant difference in other growth traits.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.85

【共引文献】