猪CBR1基因不同拷贝形式克隆及其多态性分析

doi:10.3864/j.issn.0578-1752.2018.08.018

摘要/Abstract

摘要： 【目的】获得猪CBR1基因不同拷贝形式编码区序列，了解其序列特征，分析猪CBR1基因不同拷贝形式的多态性及其与繁殖性能间的相关性。【方法】以五指山猪睾丸组织的cDNA为模板，采用克隆测序技术获得猪CBR1基因不同拷贝形式编码区序列。利用DNASTAR对CBR1不同拷贝形式编码的氨基酸序列进行分析。利用DNAMAN软件分析猪CBR1基因不同拷贝形式间的同源性，及其与多个哺乳动物间的同源性。利用ExPaSy提供的Protparam 在线软件分析猪CBR1基因不同拷贝的蛋白质理化特性。采集136头大白和47头长白繁殖母猪耳组织样，提取基因组DNA，采用PCR-测序检测猪CBR1基因不同拷贝形式的多态位点，分析其与繁殖性能的相关性。【结果】猪CBR1基因的3个不同拷贝形式均编码3个外显子，其编码区序列全长分别为870，870和846bp，其12—18位氨基酸残基均为保守的GlyXXXGlyXGly序列，符合CBRs家族的结构特性。同源性比对结果显示，CBR1基因不同拷贝形式的CDS序列同源性达87%以上，不同哺乳动物间的CBR1基因的CDS序列同源性达82%以上，可见哺乳动物CBR1基因在进化过程中比较保守。CBR1的3种不同拷贝形式编码蛋白质的理化特性较为相近，均不稳定，且为亲水性蛋白，说明其3个不同拷贝形式可能在体内发挥着类似的功能。多态性分析结果显示，拷贝V1中发现5个SNP位点，拷贝V2中发现4个SNP位点，拷贝V3中发现2个SNP位点。大白猪中，拷贝V1的启动子上游153 bp处的A/T突变和外显子3的379 bp处的C/T突变，拷贝V2上游10 bp处的AC插入突变、外显子1—63 bp处C/T突变和内含子1—76 bp处G/T突变与产活仔数显著相关。长白猪中的SNP位点则与繁殖性能不相关。【结论】获得了猪CBR1基因不同拷贝形式的编码区序列，发现了5个与大白猪经产仔数显著相关的SNP位点，CBR1基因可能作为有价值的候选基因应用于大白猪的繁殖性能选育。

关键词: 猪, 羰基还原酶1基因, 重测序, de-novo组装

Abstract: 【Objective】 In order to obtain and analysis the different copy forms of CBR1 gene coding region, and analysis the correlation between polymorphism and reproductive performance of different copy forms of CBR1 gene in pigs.【Method】 Based on the cDNA of the Wuzhishan pig’s testis , the different copy sequences of CBR1 gene were obtained by cloning sequencing technology. The amino acid sequences encoded by different copy forms of CBR1 gene were analyzed by DNASTAR software. Homology between different copy forms of porcine CBR1 gene, and that compared with other mammals were analyzed using DNAMAN software. The physical and chemical properties of different proteins of porcine CBR1 gene were analyzed by Protparam online software on ExPaSy. 136 Yorkshire and 47 Landrace pig ears samples were selected for DNA extraction and PCR-sequencing to explore the relationship between polymorphism of CBR1 gene and porcine reproductive performance. 【Result】 All the three different copies of CBR1 gene encoded 3 exons, and the sequence length of the coding region were 870bp, 870bp and 846bp respectively. Their 12-18th amino acid residues were GlyXXXGlyXGly, which were in accordance with the CBRs family structural characteristics. The results of homology comparison showed that the CDS sequence homology among different copies of CBR1 gene was more than 87%, and more than 82% among different mammals, indicated that the CBR1 gene of mammal was conserved in evolution process. The physical and chemical properties of 3 different copies of CBR1 were similar, all of them were unstable and belong to hydrophilic proteins, which indicated that they may play a similar role in vivo. Polymorphism analysis showed that 5 SNPs were found in copy V1, 4 SNPs were found in copy V2, and 2 SNPs were found in copy V3. In the Yorkshire pigs, the A/T mutation at the promoter upstream region 153 bp and the C/T mutation at exon3 379 bp in V1; AC insertion at the promoter upstream region 10 bp, C/T mutation at exon 1 63 bp and G/T mutation at intron1 76 bp in V2, were significantly correlated with the multiparous alive litter size, while the SNPs in Landrace pigs were not associated with reproductive performance. 【Conclusion】 Coding region sequence of 3 different copies of CBR1 gene were obtained; 5 SNPs were found significantly associated with multiparous alive litter size , suggested that CBR1 gene may be used as a valuable candidate gene for improving breeding performance of Yorkshire pigs.

Key words: swine, CBR1 gene, re-sequencing, de-novo assembly

齐传翔，徐奎，牟玉莲，杨述林，李奎，吴添文. 猪CBR1基因不同拷贝形式克隆及其多态性分析[J]. 中国农业科学, 2018, 51(8): 1607-1616.

QI ChuanXiang, XU KUI, MU YuLian, YANG ShuLin, LI Kui, WU TianWen. The Cloning of Porcine CBR1 Gene’s Different Copy Forms and Analysis of Its Polymorphism[J]. Scientia Agricultura Sinica, 2018, 51(8): 1607-1616.

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