【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胞中特異表達,對卵泡發(fā)育、顆粒細胞增殖和排卵均發(fā)揮重要的調控作用。目前在國外綿羊品種GDF9和BMP15基因編碼區(qū)分別發(fā)現(xiàn)了多個與高繁殖性狀相關的SNPs位點,但這些SNPs均與我國著名的高繁殖力品種湖羊無顯著關聯(lián)。為了進一步明確GDF9和BMP15基因與湖羊高繁殖力的關系,本研究以湖羊和巴什拜羊為研究對象,分離GDF9基因組序列、BMP15基因編碼區(qū)和5'調控區(qū)序列,并分析相應序列特征;篩選2個群體中GDF9基因組序列、BMP15基因編碼區(qū)和5'調控區(qū)序列SNPs位點;分析SNPs位點多態(tài)性與湖羊產(chǎn)羔數(shù)的關系;采用熒光素酶報告基因系統(tǒng)分析SNPs對啟動子區(qū)的影響和機制。研究結果為探索綿羊GDF9和BMP15與綿羊繁殖性能的潛在關系,篩選影響湖羊高繁殖力性狀的分子標記等提供理論依據(jù)。全文主要研究結果如下:(1)湖羊和巴什拜羊GDF9基因組序列特征通過PCR和測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'調控區(qū)、2個外顯子、1個內含子和3'調控區(qū)。在GDF9基因5'調控區(qū)預測到3個潛在的啟動子區(qū),分別位于-1129nt--1079nt、-633nt--583nt 和-270nt--220nt 處。轉錄因子結合位點預測發(fā)現(xiàn)在GDF9基因5'調控區(qū)中含有多個轉錄因子結合位點,如GATA4、SOX8、YY1、HSF1、AP1、OCT1、SOX1 和 PTF1 等,其中 GATA4、YY1和HSF1等已證實與卵泡發(fā)育有關。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個氨基酸,與其它哺乳動物的序列一致性均較高。結構域預測發(fā)現(xiàn)GDF9蛋白含有典型的TGF-a結構域。M1-S32位氨基酸為GDF9蛋白可能的信號肽序列。三級結構預測發(fā)現(xiàn)GDF9蛋白主要由2個a-螺旋和6個a-折疊組成。在GDF9基因3'調控區(qū)預測到5處可能的miRNA結合位點,結合的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。(2)湖羊和巴什拜羊GDF9基因SNPs位點多態(tài)性與產(chǎn)羔數(shù)的關系利用DNA池測序技術,在湖羊和巴什拜羊GDF9基因組序列中發(fā)現(xiàn)5個SNPs位點,其中編碼區(qū)1個(1599AG,未引起氨基酸改變)、5'調控區(qū)3個(-534AG、-407TG和-332CT,其中-534AG引起轉錄因子OCT1結合位點改變)和3'調控區(qū)1個(2547AG,未發(fā)現(xiàn)miRNA結合位點改變)。在湖羊和巴什拜羊群體中,GDF9基因啟動區(qū)3個SNPs位點完全連鎖,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到3種基因型(AA型、AB型和BB型),其中AA型為優(yōu)勢基因型(頻率分別為0.6292和0.6420),A為優(yōu)勢等位基因(頻率分別為0.7921和0.7840)。關聯(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型啟動子區(qū)活性與B型無顯著差異,而與轉錄因子OCT1表達載體共轉后,B型啟動子區(qū)活性比A型啟動子區(qū)活性有極顯著降低,說明-534AG突變可能影響轉錄因子OCT1與之結合,進而影響啟動子區(qū)活性。體外試驗發(fā)現(xiàn)過表達OCT1后卵泡顆粒細胞凋亡率極顯著升高,說明轉錄因子OCT1可促進卵泡顆粒細胞凋亡。(3)湖羊和巴什拜羊BMP15基因組序列特征通過PCR和測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個氨基酸(二者同源性為100%),與其它哺乳動物的序列一致性較高。結構域預測發(fā)現(xiàn)BMP15蛋白含有典型的TGF-a結構域。M1-M24位氨基酸殘基為BMP15蛋白可能的信號肽,三級結構預測發(fā)現(xiàn)BMP15蛋白主要由2個a-螺旋和6個a-折疊組成。通過PCR和測序獲得1806bp湖羊和巴什拜羊BMP15基因5'調控區(qū)序列,啟動子預測顯示在BMP15基因-1617 nt--1568 nt處存在可能的啟動子區(qū),生物信息學分析發(fā)現(xiàn)在BMP15基因5'調控區(qū)中含有多個轉錄因子結合位點,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等已證實與卵泡發(fā)育有關。(4)湖羊和巴什拜羊BMP15基因組c.-1760CA變異與啟動子區(qū)活性的關系通過DNA池測序方法在湖羊和巴什拜羊BMP15基因編碼區(qū)發(fā)現(xiàn)1個SNP位點(6355GA,但未引起氨基酸改變),在5'調控區(qū)發(fā)現(xiàn)5個突變位點(分別為-1760CA、-1548GA、-1146CT、-1145GA 和-609GA)。BMP15 基因-1760＞A 位點多態(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)啟動子區(qū)活性明顯高于突變型(AA),但未達到顯著水平(P=0.069),說明-1760CA位點突變對綿羊BMP15基因的啟動子區(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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S826

【相似文獻】

相關期刊論文 前10條

1 決肯·阿尼瓦什,哈米提·哈凱莫夫,買買提明·巴拉提;巴什拜羊質量性狀遺傳的初步研究[J];草食家畜;1998年02期

2 劉武軍;余雄;李建華;王旭光;趙福賓;馬杰;趙澤;;巴什拜羊春季牧食行為研究[J];新疆農(nóng)業(yè)科學;2008年01期

3 依明·蘇來曼;;新疆巴什拜羊經(jīng)濟效益分析[J];現(xiàn)代畜牧獸醫(yī);2008年06期

4 ;巴什拜羊的由來[J];新疆畜牧業(yè);2010年03期

5 海拉提·庫爾曼;提力克;;巴什拜羊幾種表型性狀及其利用[J];草食家畜;2011年02期

6 決肯·阿尼瓦什;克木尼斯汗;海拉提;季文憑;杜曼;;巴什拜羊瘦肉型新品系的培育及其生產(chǎn)性能分析[J];新疆農(nóng)業(yè)科學;2010年02期

7 ;巴什拜羊標準[J];新疆畜牧業(yè);1990年01期

8 才蔡;;裕民縣巴什拜羊走進內蒙古大草源[J];山西農(nóng)經(jīng);2008年02期

9 克木尼斯?jié)h·加漢;決肯·阿尼瓦什;;巴什拜羊羔羊體重增長規(guī)律的研究[J];草食家畜;2010年03期

10 克木尼斯?jié)h·加漢;決肯·阿尼瓦什;;引入1/4野盤羊血的瘦肉型巴什拜羊羔羊產(chǎn)瘦肉性能的初步研究[J];黑龍江畜牧獸醫(yī);2010年17期

相關會議論文 前4條

1 韓業(yè)東;決肯·阿尼瓦什;帕娜爾;李齊發(fā);謝莊;;巴什拜羊群體遺傳多樣性與遺傳分化的研究[A];中國動物遺傳育種研究進展——第十五次全國動物遺傳育種學術討論會論文集[C];2009年

2 孟慶龍;李曰俊;敬兵;師駿;馮曉軍;;塔里木盆地西部巴什托普斷裂的主要特征及其油氣勘探意義[A];中國科學院地質與地球物理研究所2008學術論文匯編[C];2009年

3 吳勇聰;應詩家;閆益波;王子玉;張艷麗;徐曉亮;王鋒;;綿羊BMP15基因的多態(tài)性及其與湖羊產(chǎn)羔數(shù)的相關性[A];中國畜牧獸醫(yī)學會動物繁殖學分會第十五屆學術研討會論文集（上冊）[C];2010年

4 肖敏;習欠云;施振旦;楊林;蔡偉光;江青艷;張永亮;;慢病毒介導的水牛BMP15及GDF9干涉質粒的篩選[A];全國動物生理生化第十一次學術交流會論文摘要匯編[C];2010年

相關重要報紙文章 前10條

1 本報記者　王慧敏;巴什拜羊回來了[N];人民日報;2005年

2 本報記者 龐旭民 整理;裕民之子——巴什拜[N];塔城報;2008年

3 本報記者 李明勝;愛國民主典范巴什拜[N];塔城日報(漢);2011年

4 記者 李明勝;科技引領巴什拜羊產(chǎn)業(yè)蓬勃發(fā)展[N];塔城日報(漢);2012年

5 記者 李建華;我區(qū)巴什拜羊示范區(qū)項目建設初見成效[N];塔城報;2007年

6 記者 范春海;巴什拜羊成裕民名牌[N];塔城報;2007年

7 通訊員 李明勝;裕民巴什拜羊大出風頭[N];伊犁日報(漢);2008年

8 記者紀洲;裕民縣做大做強巴什拜羊產(chǎn)業(yè)[N];新疆日報(漢);2009年

9 本報記者 紀洲;走出新疆的巴什拜羊[N];新疆日報(漢);2009年

10 記者 龐旭民 李明勝;裕民巴什拜羊尾巴變小了[N];塔城報;2009年

相關博士學位論文 前3條

1 冷青文;盤羊和巴什拜羊MHC-DRB1、DRB3基因多態(tài)性與綿羊肺炎支原體感染相關性研究[D];石河子大學;2015年

2 決肯·阿尼瓦什;巴什拜羊生物學特性及其遺傳多樣性研究[D];南京農(nóng)業(yè)大學;2010年

3 巴吐爾·阿不力克木;不同貯藏條件下新疆巴什拜羊肉品質變化機制的研究[D];南京農(nóng)業(yè)大學;2016年

相關碩士學位論文 前9條

1 金雯雯;湖羊和巴什拜羊GDF9、BMP15基因克隆與多態(tài)性分析[D];南京農(nóng)業(yè)大學;2016年

2 張曉麗;新疆巴什拜羊重組SPLUNC1蛋白的體外生物學功能研究[D];石河子大學;2016年

3 林東旭;玉龍縣納西族丁巴什羅舞變遷研究[D];云南藝術學院;2017年

4 邵勇鋼;新疆巴什拜羊血液生理生化指標與體重相關性的研究[D];新疆農(nóng)業(yè)大學;2001年

5 韓業(yè)東;巴什拜羊微衛(wèi)星位點的遺傳多樣性及其與體重體尺的相關性研究[D];南京農(nóng)業(yè)大學;2009年

6 李俊;RARG及BMP15基因與從江香豬繁殖性能關聯(lián)分析及表達規(guī)律研究[D];貴州大學;2016年

7 金恒;寧都黃雞BMP15、INHA基因多態(tài)性與生長、繁殖性狀的關聯(lián)分析[D];江西農(nóng)業(yè)大學;2016年

8 柳春云;塔里木盆地巴什托油氣田石炭系儲層預測[D];西安石油大學;2011年

9 李迪;塔里木盆地巴什托普油田晚喜馬拉雅期油氣藏動態(tài)調整機制研究[D];貴州大學;2017年

，

本文編號：2141771