本文選題：天祝白牦牛 + IGF-2基因��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：為研究影響白牦牛生長性狀的相關(guān)基因,本研究選擇胰島素樣生長因子2基因(Insμlin-like growth factor-2,IGF-2)為研究對象,采用逆轉(zhuǎn)錄多聚鏈?zhǔn)椒磻?yīng)(RT-PCR)技術(shù)和cDNA末端快速擴(kuò)增(RACE)技術(shù)克隆獲得天祝白牦牛IGF-2基因cDNA全序列;采用生物信息技術(shù)對獲得的天祝白牦牛IGF-2基因cDNA序列及其編碼的氨基酸進(jìn)行分析,通過同源建模對翻譯的蛋白質(zhì)的3D結(jié)構(gòu)進(jìn)行預(yù)測;根據(jù)克隆獲得的天祝白牦牛IGF-2基因cDNA全序列,針對其保守區(qū)設(shè)計(jì)特異性引物,以甘油醛-3-磷酸脫氫酶(GAPDH)基因?yàn)閮?nèi)參基因,采用SYBR Green 1實(shí)時(shí)熒光定量PCR方法對IGF-2基因在不同組織的差異表達(dá)進(jìn)行研究;利用DNA混合池、直接測序法和高分辨率溶解曲線(HRM)檢測天祝白牦牛IGF-2外顯子8和外顯子10的多態(tài)性,并分析其多態(tài)性與天祝白牦牛體重和體尺性狀的關(guān)聯(lián)。研究獲得如下結(jié)果:1.從天祝白牦牛肝臟組織中首次成功得到IGF-2基因的cDNA序列全長1060bp,開放閱讀框長540bp,編碼179個(gè)氨基酸;序列比對和同源性分析顯示,在核苷酸水平,該基因與黃牛的同源性最高99%;對比氨基酸序列,該序列與牛、綿羊的同源性高達(dá)98%,表明在IGF-2基因在進(jìn)化過程中的高度保守性。使用PredictProtein在線工具進(jìn)行二級結(jié)構(gòu)預(yù)測發(fā)現(xiàn)蛋白有36.31%螺旋,63.69%無規(guī)卷曲。運(yùn)用Swiss model以PBD數(shù)據(jù)庫中人的3D結(jié)構(gòu)為模板預(yù)測天祝白牦牛IGF-2蛋白的3D結(jié)構(gòu),其蛋白結(jié)構(gòu)與人的很相似。2.天祝白牦牛IGF-2基因mRNA在9個(gè)組織的表達(dá)情況為:在睪丸中表達(dá)最高,其次分別為肝臟、肌肉、脾臟、卵巢、大腦、腎臟,而心臟和肺臟未檢測到IGF-2 mRNA的表達(dá)。3.通過DNA混合池在外顯子8和外顯子10分別發(fā)現(xiàn)兩個(gè)SNP位點(diǎn)。利用HRM對兩個(gè)SNP位點(diǎn)進(jìn)行多態(tài)性分析,外顯子8檢測到TT、CC、TC三種基因型,其中TT為優(yōu)勢基因型;外顯子10檢測到GG、AA、GA三種基因型,其中GG為優(yōu)勢基因型。通過群體遺傳學(xué)分析,外顯子8的多態(tài)位點(diǎn)表現(xiàn)中度多態(tài)(PIC=0.25),外顯子10的多態(tài)位點(diǎn)表現(xiàn)低度多態(tài)(PIC=0.192)。χ2檢驗(yàn)表明,兩個(gè)多態(tài)位點(diǎn)都處于Hardy-Weinberg平衡狀態(tài)(P0.05)。多態(tài)位點(diǎn)與生長性狀相關(guān)性分析,外顯子8的基因型在體重、胸圍、體斜長方面差異顯著(P0.05),在管圍、體高性狀差異不顯著,;外顯子10的基因型在胸圍方面差異顯著,其他生長性狀差異不顯著。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823.85

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