猪BMP4基因克

发布时间：2018-07-20 17:24

【摘要】：骨形态发生蛋白4(BMP4)是转化生长因子β(TGF-β)超家族中重要的成员之一,是原始卵泡和卵母细胞生存的必需因子,与哺乳动物的繁殖性能密切相关。目前关于猪BMP4基因的研究较少,其多态性的研究还未见报道。本研究通过PCR扩增和克隆测序技术获得苏淮猪和梅山猪BMP4基因组序列,通过生物信息学方法对其基因组特征和序列特征进行分析:采用池DNA测序技术筛选苏淮猪和梅山猪BMP4基因SNP位点,采用AS-PCR对苏淮猪和梅山猪群体BMP4基因SNP位点多态性;采用RT-PCR技术分析了猪BMP4基因的组织表达特征,并将其克隆入真核表达载体pcDNA3.1中,构建猪BMP4基因过表达载体。研究结果为揭示猪BMP4基因特征和生物学功能提供参考。全文结果如下:1、猪BMP4基因克隆与序列分析通过PCR扩增和克隆测序获得了苏淮猪和梅山猪7147bp的BMP4基因组序列序列,两者核苷酸序列的一致性为98.63%。电子染色体定位发现猪BMP4基因定位在猪1号染色体上,位于203773 kb～203781 kb之间。基因组结构分析猪BMP4基因由4个外显子和3内含子组成。在苏淮猪和梅山猪BMP4基因5'端-1051～-801nt处发现一个潜在的启动子区,含有HEN1、STAT1、AP4等转录因子结合位点,但未发现真核生物启动子典型的TATA框和CAAT框。苏淮猪和梅山猪BMP4基因3'-UTR区序列全长为267bp,同源性为100%,含有AC重复序列、加尾信号、polyA结构和保守的ARE等。苏淮猪和梅山猪BMP4基因编码区仅由2个外显子(外显子3和4)构成,序列全长1230 bp,编码409个氨基酸残基,两者核苷酸和氨基酸序列一致性均为100%,与哺乳动物其它物种的一致性分别在91%和97%以上;与其它哺乳动物一样,苏淮猪和梅山猪BMP4蛋白也含有典型的TGF-β前肽和TGF-β样结构域。结果表明苏淮猪和梅山猪BMP4基因与其它哺乳动物在基因组结构、序列、蛋白结构等具有较高的保守性。2、猪BMP4基因3'UTR多态性分析采用池DNA测序技术在苏淮猪和梅山猪BMP4基因3'UTR 20nt处发现一个SNP,碱基由G突变为C,并将其命名为*20GC。利用建立的AS-PCR方法对苏淮猪和梅山猪群体BMP4基因*20GC位点多态性进行了检测,结果在苏淮猪和梅山猪群体中共检测到GG、GC和CC等3种基因型。在苏淮猪群体中,GG型和G等位基因为优势基因型和优势等位基因,频率分别为为0.815和0.887,而在高繁殖力猪种梅山猪中则相反,CC型和C等位基因为优势基因型和优势等位基因,频率分别为为0.667和0.770。生物信息学方法预测发现猪BMP4基因*20GC突变导致miR-7135结合位点的改变,因此本文构建了含有该多态位点的2种荧光素酶表达载体(pmirGLO-G和pmirGLO-C),与miR-7135mimics共转293细胞,荧光素酶活性分析发现不同荧光素酶表达载体活性无显著差异,说明BMP4基因*20GC突变可能不是通过影响miR-7135与之结合而发挥作用。研究结果表明BMP4基因*20GC位点多态性可能与猪繁殖性能有关。3、苏淮猪BMP4基因组织表达特征与真核表达载体构建利用RT-PCR方法对苏淮猪BMP4基因组织表达特征进行分析,结果显示BMP4基因在苏淮猪心脏、肌肉、卵巢、肝脏、肾脏、脾脏6种组织中均有表达,其中在心脏、肌肉、卵巢和肾脏组织中高表达,而在肝脏和脾脏组织中低表达。将猪BMP4基因编码区克隆入pcDNA3.1载体中,构建BMP4基因真核表达载体,酶切鉴定和质粒测序比对发现连接到载体中的目的片段序列完全正确,说明猪BMP4基因真核表达载体构建成功,并命名为pcDNA3.1-BMP4。将重组质粒pcDNA3.1-BMP4转染体外培养的猪卵泡颗粒细胞,Westerm blot分析发现转染重组质粒pcDNA3.1-BMP4组的颗粒细胞23KD处出现明显BMP4蛋白条带,BMP4蛋白表达水平极显著高于对照组,说明pcDNA3.1-BMP4重组质粒可在猪卵巢颗粒细胞中能够高效表达,为进一步研究其功能奠定了基础。
[Abstract]:Bone morphogenetic protein 4 (BMP4) is one of the important members of the transforming growth factor beta (TGF- beta) superfamily. It is an essential factor for the survival of primitive follicles and oocytes. It is closely related to the reproductive performance of mammals. At present, there are few studies on the BMP4 gene of pigs, and the study of its polymorphism has not been reported. This study was amplified and cloned by PCR. The sequence of BMP4 genome of Suai Huai pig and Meishan pig was obtained by sequencing technology. The genomic characteristics and sequence characteristics were analyzed by bioinformatics methods: using pool DNA sequencing technology to screen the SNP loci of the BMP4 gene of Suai Huai pigs and Meishan pigs, and the SNP polymorphism of BMP4 gene in Suai and Huaihe pigs and Meishan pigs by AS-PCR. The tissue expression characteristics of the porcine BMP4 gene were analyzed and cloned into the eukaryotic expression vector pcDNA3.1 to construct the overexpression vector of porcine BMP4 gene. The results can provide reference for the characterization and biological function of the porcine BMP4 gene. The full text results are as follows: 1, the cloning and sequence analysis of porcine BMP4 gene obtained the Suai Huai pig by PCR amplification and cloning sequencing. And the sequence of BMP4 genome of Meishan pig 7147bp, the consistency of the nucleotide sequence of the two nucleotide sequences is that the porcine BMP4 gene is located on the porcine chromosome 1, located between 203773 KB and 203781 KB. The genome structure analysis of the pig BMP4 gene consists of 4 exons and 3 introns. In the Suai and Huaihe pigs and the Meishan pig BMP4 gene 5' A potential promoter region was found at the end of -1051 to -801nt, containing HEN1, STAT1, AP4 and other transcription factor binding sites, but the typical TATA frame and CAAT frame of the eukaryotic promoter were not found. The 3'-UTR region sequence of the BMP4 gene of Suai Huai pig and Meishan pig was 267bp and the homology was 100%, containing AC repeat sequence, tail signal, polyA structure and conservatively. E and so on. The BMP4 gene coding region of the Suai and Meishan pigs is composed of 2 exons (exons 3 and 4), with a full length of 1230 BP and 409 amino acid residues. The consistency of both nucleotide and amino acid sequences is 100%, and the consistency between the other species of mammals is 91% and 97%, respectively, and the same as other mammals, Suai Huai pigs and Meishan. The porcine BMP4 protein also contains typical TGF- beta propeptide and TGF- beta like domain. The results show that the BMP4 gene of Suai and Meishan pigs and other mammals have high conserved.2 in genome structure, sequence, protein structure and so on. The 3'UTR polymorphism analysis of pig BMP4 gene uses pool DNA sequencing technology in the BMP4 gene 3'UTR 20nt in Suai and Huaihe pigs and Meishan pigs A SNP was found, the base was mutated from G to C, and it was named *20GC. using the AS-PCR method established by *20GC. to detect the BMP4 gene *20GC polymorphism of the Suai Huai pig and Meishan pig population. The results showed that the 3 genotypes of GG, GC and CC were detected in Suai Huai pig and Meishan pig population. The frequencies of genotypes and dominant alleles were 0.815 and 0.887 respectively, while in the high fecundity porcine Meishan pigs, the CC and C alleles were dominant genotypes and dominant alleles. The frequency of the 0.667 and 0.770. bioinformatics methods predicted the change of miR-7135 binding sites caused by *20GC mutations in porcine BMP4. In this paper, 2 kinds of luciferase expression vectors (pmirGLO-G and pmirGLO-C) containing the polymorphic loci were constructed, and 293 cells were converted to miR-7135mimics. The activity analysis of luciferase showed that there was no significant difference in the activity of different luciferase expression vectors. It was suggested that the *20GC mutation of the BMP4 gene may not play a role by affecting the combination of miR-7135. The results show that the polymorphism of the BMP4 gene *20GC loci may be related to the reproductive performance of the pig.3, the expression characteristics of the BMP4 gene in Suai pig and the eukaryotic expression vector construction and use of RT-PCR method to analyze the expression characteristics of the BMP4 gene in the Suai Huai pig. The results show that the BMP4 gene is in the 6 tissues of the pig heart, muscle, ovary, liver, kidney and spleen of the Suai Huaihe pig. High expression in heart, muscles, ovaries and kidneys, and low expression in the liver and spleen tissues. The porcine BMP4 gene coding region was cloned into the pcDNA3.1 vector to construct the eukaryotic expression vector of the BMP4 gene. The sequence of enzyme digestion and plasmid sequencing was completely correct, and the sequence of the target fragments found in the carrier was completely correct. The eukaryotic expression vector of porcine BMP4 gene was successfully constructed, and the recombinant plasmid pcDNA3.1-BMP4 was named as pcDNA3.1-BMP4. to transfect the cultured porcine follicle granulosa cells in vitro. The Westerm blot analysis found that the 23KD of the recombinant plasmid pcDNA3.1-BMP4 group showed obvious BMP4 protein bands at 23KD, and the expression level of BMP4 protein was significantly higher than that of the control group. It is indicated that pcDNA3.1-BMP4 recombinant plasmid can be highly expressed in porcine ovarian granulosa cells, which lays a foundation for further study of its function.
【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】：S828

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