發(fā)布時(shí)間：2018-07-24 15:09

【摘要】：GDF9和BMP15基因是影響綿羊高繁殖力性狀的主效基因,二者均在卵巢卵母細(xì)胞中特異表達(dá),對(duì)卵泡發(fā)育、顆粒細(xì)胞增殖和排卵均發(fā)揮重要的調(diào)控作用。目前在國外綿羊品種GDF9和BMP15基因編碼區(qū)分別發(fā)現(xiàn)了多個(gè)與高繁殖性狀相關(guān)的SNPs位點(diǎn),但這些SNPs均與我國著名的高繁殖力品種湖羊無顯著關(guān)聯(lián)。為了進(jìn)一步明確GDF9和BMP15基因與湖羊高繁殖力的關(guān)系,本研究以湖羊和巴什拜羊?yàn)檠芯繉?duì)象,分離GDF9基因組序列、BMP15基因編碼區(qū)和5'調(diào)控區(qū)序列,并分析相應(yīng)序列特征;篩選2個(gè)群體中GDF9基因組序列、BMP15基因編碼區(qū)和5'調(diào)控區(qū)序列SNPs位點(diǎn);分析SNPs位點(diǎn)多態(tài)性與湖羊產(chǎn)羔數(shù)的關(guān)系;采用熒光素酶報(bào)告基因系統(tǒng)分析SNPs對(duì)啟動(dòng)子區(qū)的影響和機(jī)制。研究結(jié)果為探索綿羊GDF9和BMP15與綿羊繁殖性能的潛在關(guān)系,篩選影響湖羊高繁殖力性狀的分子標(biāo)記等提供理論依據(jù)。全文主要研究結(jié)果如下:(1)湖羊和巴什拜羊GDF9基因組序列特征通過PCR和測(cè)序獲得了湖羊和巴什拜羊GDF9基因組序列,全長均為5150bp,包括5'調(diào)控區(qū)、2個(gè)外顯子、1個(gè)內(nèi)含子和3'調(diào)控區(qū)。在GDF9基因5'調(diào)控區(qū)預(yù)測(cè)到3個(gè)潛在的啟動(dòng)子區(qū),分別位于-1129nt--1079nt、-633nt--583nt 和-270nt--220nt 處。轉(zhuǎn)錄因子結(jié)合位點(diǎn)預(yù)測(cè)發(fā)現(xiàn)在GDF9基因5'調(diào)控區(qū)中含有多個(gè)轉(zhuǎn)錄因子結(jié)合位點(diǎn),如GATA4、SOX8、YY1、HSF1、AP1、OCT1、SOX1 和 PTF1 等,其中 GATA4、YY1和HSF1等已證實(shí)與卵泡發(fā)育有關(guān)。湖羊和巴什拜羊GDF9基因編碼區(qū)長度均為1362bp,編碼蛋白均含有453個(gè)氨基酸,與其它哺乳動(dòng)物的序列一致性均較高。結(jié)構(gòu)域預(yù)測(cè)發(fā)現(xiàn)GDF9蛋白含有典型的TGF-a結(jié)構(gòu)域。M1-S32位氨基酸為GDF9蛋白可能的信號(hào)肽序列。三級(jí)結(jié)構(gòu)預(yù)測(cè)發(fā)現(xiàn)GDF9蛋白主要由2個(gè)a-螺旋和6個(gè)a-折疊組成。在GDF9基因3'調(diào)控區(qū)預(yù)測(cè)到5處可能的miRNA結(jié)合位點(diǎn),結(jié)合的miRNA包括miR-515-5p、let-7e-3p 和 let-7a-2-3p,這 3 種 miRNA 均與卵泡發(fā)育有關(guān)。(2)湖羊和巴什拜羊GDF9基因SNPs位點(diǎn)多態(tài)性與產(chǎn)羔數(shù)的關(guān)系利用DNA池測(cè)序技術(shù),在湖羊和巴什拜羊GDF9基因組序列中發(fā)現(xiàn)5個(gè)SNPs位點(diǎn),其中編碼區(qū)1個(gè)(1599AG,未引起氨基酸改變)、5'調(diào)控區(qū)3個(gè)(-534AG、-407TG和-332CT,其中-534AG引起轉(zhuǎn)錄因子OCT1結(jié)合位點(diǎn)改變)和3'調(diào)控區(qū)1個(gè)(2547AG,未發(fā)現(xiàn)miRNA結(jié)合位點(diǎn)改變)。在湖羊和巴什拜羊群體中,GDF9基因啟動(dòng)區(qū)3個(gè)SNPs位點(diǎn)完全連鎖,將野生單倍型A-T-C命名為A,突變單倍型G-G-T命名為B。在湖羊中和巴什拜羊群體中均檢測(cè)到3種基因型(AA型、AB型和BB型),其中AA型為優(yōu)勢(shì)基因型(頻率分別為0.6292和0.6420),A為優(yōu)勢(shì)等位基因(頻率分別為0.7921和0.7840)。關(guān)聯(lián)分析發(fā)現(xiàn),AA型和AB型湖羊平均產(chǎn)羔數(shù)均顯著高于BB型。熒光素酶活性型分析發(fā)現(xiàn)A型啟動(dòng)子區(qū)活性與B型無顯著差異,而與轉(zhuǎn)錄因子OCT1表達(dá)載體共轉(zhuǎn)后,B型啟動(dòng)子區(qū)活性比A型啟動(dòng)子區(qū)活性有極顯著降低,說明-534AG突變可能影響轉(zhuǎn)錄因子OCT1與之結(jié)合,進(jìn)而影響啟動(dòng)子區(qū)活性。體外試驗(yàn)發(fā)現(xiàn)過表達(dá)OCT1后卵泡顆粒細(xì)胞凋亡率極顯著升高,說明轉(zhuǎn)錄因子OCT1可促進(jìn)卵泡顆粒細(xì)胞凋亡。(3)湖羊和巴什拜羊BMP15基因組序列特征通過PCR和測(cè)序獲得湖羊和巴什拜羊BMP15基因編碼區(qū)序列,長度均為1182 bp(二者同源性為99.92%),編碼蛋白含393個(gè)氨基酸(二者同源性為100%),與其它哺乳動(dòng)物的序列一致性較高。結(jié)構(gòu)域預(yù)測(cè)發(fā)現(xiàn)BMP15蛋白含有典型的TGF-a結(jié)構(gòu)域。M1-M24位氨基酸殘基為BMP15蛋白可能的信號(hào)肽,三級(jí)結(jié)構(gòu)預(yù)測(cè)發(fā)現(xiàn)BMP15蛋白主要由2個(gè)a-螺旋和6個(gè)a-折疊組成。通過PCR和測(cè)序獲得1806bp湖羊和巴什拜羊BMP15基因5'調(diào)控區(qū)序列,啟動(dòng)子預(yù)測(cè)顯示在BMP15基因-1617 nt--1568 nt處存在可能的啟動(dòng)子區(qū),生物信息學(xué)分析發(fā)現(xiàn)在BMP15基因5'調(diào)控區(qū)中含有多個(gè)轉(zhuǎn)錄因子結(jié)合位點(diǎn),如 HSF、CdxA、OCT-1、SRY、SOX-5、GATA-1、AP-1、NIT-2 和GATA-2等,其中HSF、OCT-1和Sp1等已證實(shí)與卵泡發(fā)育有關(guān)。(4)湖羊和巴什拜羊BMP15基因組c.-1760CA變異與啟動(dòng)子區(qū)活性的關(guān)系通過DNA池測(cè)序方法在湖羊和巴什拜羊BMP15基因編碼區(qū)發(fā)現(xiàn)1個(gè)SNP位點(diǎn)(6355GA,但未引起氨基酸改變),在5'調(diào)控區(qū)發(fā)現(xiàn)5個(gè)突變位點(diǎn)(分別為-1760CA、-1548GA、-1146CT、-1145GA 和-609GA)。BMP15 基因-1760＞A 位點(diǎn)多態(tài)性分析發(fā)現(xiàn)湖羊中均為CC型,巴什拜羊中CC、CA和AA等3種基因型的頻率分別為0.6154、0.3654和0.0192。熒光素酶活性分析發(fā)現(xiàn)野生型(CC)啟動(dòng)子區(qū)活性明顯高于突變型(AA),但未達(dá)到顯著水平(P=0.069),說明-1760CA位點(diǎn)突變對(duì)綿羊BMP15基因的啟動(dòng)子區(qū)活性有一定的影響。
[Abstract]:GDF9 and BMP15 genes are the main genes affecting the Prolificacy of sheep. All of the two are specifically expressed in ovarian oocytes. They play an important role in follicle development, granulosa cell proliferation and ovulation. At present, many SNPs loci related to high reproductive traits are found in the foreign sheep breeds, GDF9 and BMP15 gene coding regions. In order to further clarify the relationship between GDF9 and BMP15 gene and the high fecundity of Hu sheep, this study took Hu sheep and bahshibai sheep as the research object to separate the sequence of GDF9 genome, BMP15 gene coding region and 5'regulation region, and analyze the sequence characteristics of the BMP15 gene and the corresponding sequence of the SNPs, and screened 2. The GDF9 genome sequence, the BMP15 gene coding region and the SNPs locus in the 5'regulatory region, the relationship between the polymorphism of the SNPs locus and the lambing of the lake sheep, and the effect and mechanism of SNPs on the promoter region by the luciferase reporter gene system. The result is to explore the potential relationship between the reproductive performance of sheep GDF9 and BMP15 and the sheep and screen the shadow. The main research results are as follows: (1) the genomic sequence characteristics of the GDF9 genome of Hu sheep and bashbai sheep were obtained by PCR and sequencing, and the genome sequence of Hu sheep and bashbai sheep GDF9 was obtained. The whole length of the genome sequence was 5150bp, including the 5'regulatory region, 2 exons, 1 introns and 3' regulation areas. In GDF9 The gene 5'regulatory region was predicted to 3 potential promoter regions, located at -1129nt--1079nt, -633nt--583nt and -270nt--220nt, and the transcription factor binding sites were predicted to contain multiple transcription factor binding sites in the GDF9 gene 5' regulatory region, such as GATA4, SOX8, YY1, HSF1, AP1, OCT1, etc. The length of the GDF9 gene coding region of Hu sheep and bashbai sheep is 1362bp, the encoding protein contains 453 amino acids, and the conformance of all the other mammals is high. The domain prediction found that GDF9 protein contains the typical TGF-a domain.M1-S32 bit amino acid as the possible signal peptide sequence of GDF9 protein. It was predicted that the GDF9 protein was mainly composed of 2 a- helices and 6 a- folds. The possible miRNA binding sites were predicted in the 3'regulatory region of the GDF9 gene, and the miRNA included miR-515-5p, let-7e-3p and let-7a-2-3p, and the 3 miRNA were related to the follicle development. (2) the polymorphism of the GDF9 gene locus of the Hu and bahbai sheep and the number of lambs were related. Using DNA pool sequencing technology, 5 SNPs loci were found in the GDF9 genome sequence of Hu sheep and bashbai sheep, of which 1 (1599AG, no amino acid change) in the coding region, 3 (-534AG, -407TG and -332CT, -534AG caused by OCT1 binding site modification) and 1 in 3'regulation region (2547AG, no change of binding site found) in the 5' regulatory region. In the group of Hu sheep and bashbai sheep, 3 SNPs loci in the GDF9 gene promoter region are completely linked, and the wild haplotype A-T-C is named A. The mutation haplotype G-G-T is named as B. in the lake sheep and the basbai sheep population, and 3 genotypes (AA, AB and BB) are detected, and the AA type is the dominant genotype (frequency 0.6292 and 0.6420 respectively) and A is superior. The potential alleles (frequencies were 0.7921 and 0.7840 respectively). Correlation analysis showed that the average number of lambs in AA and AB sheep was significantly higher than that of BB. The activity of luciferase found no significant difference between A promoter activity and B type, but the activity of B type promoter region was more active than that of A type promoter after the transcription factor OCT1 expression vector was converted. The results showed that the -534AG mutation could affect the binding of the transcription factor OCT1 and the activation of the promoter region. In vitro, the apoptosis rate of follicle granulosa cells was significantly increased after the overexpression of OCT1, indicating that the transcription factor OCT1 could promote the apoptosis of follicle granulosa cells. (3) the sequence characteristics of BMP15 genome of Hu and bahash sheep through PCR and The sequence of the BMP15 gene coding region of Hu sheep and bashbai sheep was sequenced, the length of which was 1182 BP (two homology was 99.92%), the encoded protein contained 393 amino acids (two homology 100%), which was higher in consistency with the sequence of other mammals. The domain prediction found that BMP15 protein contained a typical TGF-a domain.M1-M24 bit amino acid residue of BMP The possible signal peptide of 15 protein and three stage structure prediction found that BMP15 protein was mainly composed of 2 a- helices and 6 a- folds. Through PCR and sequencing, the 5'regulation region sequence of 1806bp sheep and bashbai sheep BMP15 gene was obtained. The promoter prediction showed that there was a viable promoter region in BMP15 gene -1617 nt--1568 NT. Bioinformatics analysis found that the B was in B. There are multiple transcription factor binding sites in the MP15 gene 5'regulatory region, such as HSF, CdxA, OCT-1, SRY, SOX-5, GATA-1, AP-1, NIT-2 and GATA-2. 1 SNP loci (6355GA, but no amino acid change) were found in the BMP15 gene coding region of Shibai sheep, and 5 mutation sites (-1760CA, -1548GA, -1146CT, -1145GA and -609GA).BMP15 gene -1760 > A loci polymorphism found in the 5'regulatory region were found in the frequency of 3 genotypes in lake sheep. The activity of 0.6154,0.3654 and 0.0192. luciferase showed that the activity of the wild type (CC) promoter region was significantly higher than that of the mutant type (AA), but it did not reach a significant level (P=0.069), indicating that the mutation of the -1760CA site had a certain effect on the promoter activity of the sheep BMP15 gene.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S826

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