【摘要】：GPR54基因是牛性成熟狀的重要候選基因之一,本文選擇42頭安徽本地黃牛、44頭西門塔爾牛及其雜交育種群116頭作為試驗(yàn)群體,分析牛GPR54基因5’調(diào)控區(qū)的SNPs以及不同基因型啟動子的啟動效率和牛性成熟狀之間的關(guān)聯(lián)性。試驗(yàn)提取牛血液基因組后,通過測序鑒別SNP位點(diǎn),雙熒光素酶報(bào)告基因驗(yàn)證其SNP位點(diǎn)對啟動子效率的影響。結(jié)果表明:1.GPR54基因的啟動子區(qū)GPR973序列存在兩個(gè)SNPs位點(diǎn),分別是距離CDS起點(diǎn)上游第816位和754位:C-816T和T-754C,西門塔爾牛和安徽地方黃牛分別只有1種基因型,西門塔爾牛的基因型全部為CCTT,安徽地方黃牛的基因型全部為TTCC,分析其單倍型結(jié)果顯示連鎖不平衡。2.C-816T突變位點(diǎn)附近存在TCF-1和AP-3(2)轉(zhuǎn)錄因子結(jié)合位點(diǎn),T-754C附近及其本身存在TFIID、NF-1、CAC-binding_pro、TCF-1轉(zhuǎn)錄因子以及F位點(diǎn)等。從C-816T到T-754C形成一個(gè)以TCF-1開頭結(jié)尾的復(fù)合轉(zhuǎn)錄因子結(jié)合區(qū)域,其中還包含一個(gè)TFIID結(jié)合的G/TATAAA盒。經(jīng)過預(yù)測,本目的片段含有一個(gè)從-878bp至-638bp的啟動子區(qū)域。3.通過雙熒光素酶報(bào)告基因驗(yàn)證了西門塔爾牛和安徽地方黃牛不同GPR973基因型的啟動子效率,報(bào)告基因驗(yàn)證兩種單倍型的啟動子存在明顯差異,其中-816CT-754啟動子的效率要比-816TC-754提高34.31%(p0.01)。4.通過對不同品種牛GPR54進(jìn)行實(shí)時(shí)熒光定量PCR反應(yīng),結(jié)果顯示與雙報(bào)告基因檢測結(jié)果一致。C-816CT-754T牛和T-816TC-754C牛的表達(dá)差異明顯(p0.05),其中C-816CT-754T牛是T-816TC-754C牛的2.6倍。5.通過對不同品種牛進(jìn)行關(guān)聯(lián)分析,基因型為816CC754TT牛的初情期與基因型為816TT754CC牛的初情期相比提前了1.28月,且差異極顯著(p0.01)。
[Abstract]:GPR54 gene is one of the important candidate genes for sexual maturation of cattle. 42 native Anhui cattle, 44 Simmental cattle and their cross breeding population were selected as experimental populations. The relationship between the SNPs of the 5 'regulatory region of bovine GPR54 gene, the promoter efficiency of different genotypes and the sexual maturity of cattle was analyzed. After the bovine blood genome was extracted, the SNP sites were identified by sequencing, and the effect of the SNP locus on the promoter efficiency was verified by double luciferase reporter gene. The results showed that there were two SNPs loci in the promoter region of 1.GPR54 gene, 816 and 754 from the upstream of CDS: C-816T and T-754C. Simmental and Anhui yellow cattle had only one genotype, respectively. The genotypes of Simmental cattle were all CCTT,. All the genotypes of Anhui native cattle were TTCC,. The results of linkage disequilibrium showed that there were TCF-1 and AP-3 (2) transcription factor binding sites near the 2.C-816T mutation site, and T-754C region and its own existence. In TFIID,NF-1,CAC-binding_pro,TCF-1 transcription factors and F loci, and so on. From C-816T to T-754C, a complex transcription factor binding region ending with TCF-1 is formed, which also contains a TFIID binding G/TATAAA box. It is predicted that this fragment contains a promoter region from-878bp to-638bp. The promoter efficiency of different GPR973 genotypes in Simmental cattle and Anhui native cattle was verified by double luciferase reporter gene. The promoter efficiency of 816CT-754 promoter was increased by 34.31% (p0.01) compared with that of 816TC-754. The results of real-time fluorescence quantitative PCR reaction on GPR54 of different breeds of cattle showed that the results were consistent with the results of double reporter gene detection. The difference between C-816CT-754T cattle and T-816TC-754C cattle was significant (p0.05), and C-816CT-754T cattle were 2.6 times as much as T-816TC-754C cattle. According to the correlation analysis of different breeds of cattle, the initial oestrous period of 816CC754TT cattle was 1.28 months earlier than that of 816TT754CC cattle, and the difference was very significant (p0.01).
【學(xué)位授予單位】：安徽農(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

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