湖羊和巴什拜羊GDF9、BMP15基因克隆与多态性分析

发布时间：2018-07-24 15:09

【摘要】：GDF9和BMP15基因是影响绵羊高繁殖力性状的主效基因,二者均在卵巢卵母细胞中特异表达,对卵泡发育、颗粒细胞增殖和排卵均发挥重要的调控作用。目前在国外绵羊品种GDF9和BMP15基因编码区分别发现了多个与高繁殖性状相关的SNPs位点,但这些SNPs均与我国著名的高繁殖力品种湖羊无显著关联。为了进一步明确GDF9和BMP15基因与湖羊高繁殖力的关系,本研究以湖羊和巴什拜羊为研究对象,分离GDF9基因组序列、BMP15基因编码区和5'调控区序列,并分析相应序列特征;筛选2个群体中GDF9基因组序列、BMP15基因编码区和5'调控区序列SNPs位点;分析SNPs位点多态性与湖羊产羔数的关系;采用荧光素酶报告基因系统分析SNPs对启动子区的影响和机制。研究结果为探索绵羊GDF9和BMP15与绵羊繁殖性能的潜在关系,筛选影响湖羊高繁殖力性状的分子标记等提供理论依据。全文主要研究结果如下:(1)湖羊和巴什拜羊GDF9基因组序列特征通过PCR和测序获得了湖羊和巴什拜羊GDF9基因组序列,全长均为5150bp,包括5'调控区、2个外显子、1个内含子和3'调控区。在GDF9基因5'调控区预测到3个潜在的启动子区,分别位于-1129nt--1079nt、-633nt--583nt 和-270nt--220nt 处。转录因子结合位点预测发现在GDF9基因5'调控区中含有多个转录因子结合位点,如GATA4、SOX8、YY1、HSF1、AP1、OCT1、SOX1 和 PTF1 等,其中 GATA4、YY1和HSF1等已证实与卵泡发育有关。湖羊和巴什拜羊GDF9基因编码区长度均为1362bp,编码蛋白均含有453个氨基酸,与其它哺乳动物的序列一致性均较高。结构域预测发现GDF9蛋白含有典型的TGF-a结构域。M1-S32位氨基酸为GDF9蛋白可能的信号肽序列。三级结构预测发现GDF9蛋白主要由2个a-螺旋和6个a-折叠组成。在GDF9基因3'调控区预测到5处可能的miRNA结合位点,结合的miRNA包括miR-515-5p、let-7e-3p 和 let-7a-2-3p,这 3 种 miRNA 均与卵泡发育有关。(2)湖羊和巴什拜羊GDF9基因SNPs位点多态性与产羔数的关系利用DNA池测序技术,在湖羊和巴什拜羊GDF9基因组序列中发现5个SNPs位点,其中编码区1个(1599AG,未引起氨基酸改变)、5'调控区3个(-534AG、-407TG和-332CT,其中-534AG引起转录因子OCT1结合位点改变)和3'调控区1个(2547AG,未发现miRNA结合位点改变)。在湖羊和巴什拜羊群体中,GDF9基因启动区3个SNPs位点完全连锁,将野生单倍型A-T-C命名为A,突变单倍型G-G-T命名为B。在湖羊中和巴什拜羊群体中均检测到3种基因型(AA型、AB型和BB型),其中AA型为优势基因型(频率分别为0.6292和0.6420),A为优势等位基因(频率分别为0.7921和0.7840)。关联分析发现,AA型和AB型湖羊平均产羔数均显著高于BB型。荧光素酶活性型分析发现A型启动子区活性与B型无显著差异,而与转录因子OCT1表达载体共转后,B型启动子区活性比A型启动子区活性有极显著降低,说明-534AG突变可能影响转录因子OCT1与之结合,进而影响启动子区活性。体外试验发现过表达OCT1后卵泡颗粒细胞凋亡率极显著升高,说明转录因子OCT1可促进卵泡颗粒细胞凋亡。(3)湖羊和巴什拜羊BMP15基因组序列特征通过PCR和测序获得湖羊和巴什拜羊BMP15基因编码区序列,长度均为1182 bp(二者同源性为99.92%),编码蛋白含393个氨基酸(二者同源性为100%),与其它哺乳动物的序列一致性较高。结构域预测发现BMP15蛋白含有典型的TGF-a结构域。M1-M24位氨基酸残基为BMP15蛋白可能的信号肽,三级结构预测发现BMP15蛋白主要由2个a-螺旋和6个a-折叠组成。通过PCR和测序获得1806bp湖羊和巴什拜羊BMP15基因5'调控区序列,启动子预测显示在BMP15基因-1617 nt--1568 nt处存在可能的启动子区,生物信息学分析发现在BMP15基因5'调控区中含有多个转录因子结合位点,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等已证实与卵泡发育有关。(4)湖羊和巴什拜羊BMP15基因组c.-1760CA变异与启动子区活性的关系通过DNA池测序方法在湖羊和巴什拜羊BMP15基因编码区发现1个SNP位点(6355GA,但未引起氨基酸改变),在5'调控区发现5个突变位点(分别为-1760CA、-1548GA、-1146CT、-1145GA 和-609GA)。BMP15 基因-1760＞A 位点多态性分析发现湖羊中均为CC型,巴什拜羊中CC、CA和AA等3种基因型的频率分别为0.6154、0.3654和0.0192。荧光素酶活性分析发现野生型(CC)启动子区活性明显高于突变型(AA),但未达到显著水平(P=0.069),说明-1760CA位点突变对绵羊BMP15基因的启动子区活性有一定的影响。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S826

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