延黄牛ALDH1A1基因CDS序列克隆及其在各组织中的表达差异研究

doi:10.16431/j.cnki.1671-7236.2019.01.020

摘要/Abstract

摘要：

为探索乙醛脱氢酶1A1（acetaldehyde dehydrogenase 1A1，ALDH1A1）基因功能，本试验以16月龄延黄牛母牛为研究对象，屠宰后采集心脏、肝脏、肺脏、肾脏、胃、十二指肠、皮下脂肪和背最长肌，提取总RNA。根据GenBank上公布的牛ALDH1A1基因mRNA序列（登录号：NM_174239.2），利用Oligo 7.0软件设计引物，应用RT-PCR扩增ALDH1A1基因，将扩增产物连接pMD18-T载体进行克隆测序，获得延黄牛ALDH1A1基因完整CDS序列，应用生物信息学软件分析核苷酸序列及其蛋白结构。以延黄牛不同组织总RNA为模板，通过实时荧光定量PCR技术检测ALDH1A1基因在延黄牛各组织间的表达差异。结果显示，ALDH1A1基因CDS序列全长1 506 bp，编码501个氨基酸；延黄牛ALDH1A1基因序列与野牛、牛的同源性最高（≥99.7%），与猫和豹的同源性分别为89.4%和89.6%，符合物种进化规律。ALDH1A1蛋白分子质量为54.805 ku，理论等电点为7.16，亲水性较强，占86.4%，酸性氨基酸和碱性氨基酸分别占11.4%和12.2%，属于可溶性蛋白，但不是分泌性蛋白，无典型信号肽切割位点；存在31个氨基酸磷酸化位点（分值 > 0.5）。延黄牛ALDH1A1蛋白二级结构含有α-螺旋、延伸链、β-转角和无规则卷曲，分别占42.12%、16.17%、8.18%和33.53%，与该蛋白三级结构预测结果相同。实时荧光定量PCR结果表明，ALDH1A1基因在延黄牛肝脏、胃、皮下脂肪、十二指肠和肾脏组织中极显著表达（P < 0.01）；在背最长肌中显著表达（P < 0.05）。本试验结果为进一步开展延黄牛ALDH1A1基因功能及肉质基因筛选研究提供了参考依据。

关键词: 延黄牛; 乙醛脱氢酶1A1(ALDH1A1)基因; 克隆; 表达差异; 生物信息学

Abstract:

To explore the function of acetaldehyde dehydrogenase 1A1 (ALDH1A1) gene,16-month-old Yanhuang cattle were used as subjects,and total RNA were collected from the heart,liver,lung,kidney,stomach,duodenum,subcutaneous fat and longissimus dorsi after slaughter.According to the ALDH1A1 gene sequence published in GenBank(accession No.:NM_174239.2),primers were designed using Oligo 7.0 software,ALDH1A1 gene was amplified by RT-PCR,and the amplification product was connected into pMD18-T vector for clonging and sequencing.The complete CDS sequence of ALDH1A1 gene in Yanhuang cattle was obtained,the nucleotide sequences and its protein structures were analyzed using bioinformatics softwares.Then using the total RNA from different tissues of Yanhuang cattle as a template,the quantitative expression of ALDH1A1 gene in different tissues of Yanhuang cattle was detected by Real-time quantitative PCR.The results showed that the length of CDS sequence of ALDH1A1 gene was 1 506 bp,which encoded 501 amino acids.The homology of the ALDH1A1 gene sequence with bison and cattle was ≥ 99.7%,and its homology with cats and leopards were 89.4% and 89.6%,respectively,which was in accordance with species evolution rules.The molecular weight of ALDH1A1 was 54.805 ku,the isoelectric point was 7.16;A relatively strong hydrophilicity accounted for 86.4%;acidic and basic amino acids accounted for 11.4% and 12.2%,respectively.ALDH1A1 was soluble proteins,which was not secretory proteins,but there was no typical signal peptide cleavage site.There were 31 amino acid phosphorylation sites (score > 0.5).The secondary structure of ALDH1A1 protein in Yanhuang cattle contained alpha helix,extended chain,beta turn and random coil,which accounted for 42.12%,16.17%,8.18% and 33.53%,respectively,which were identical to the predicted structure of the tertiary structure of ALDH1A1 protein.Real-time quantitative PCR results showed that ALDH1A1 gene was extremely significantly expressed in liver,stomach,subcutaneous fat,duodenum and kidney tissues (P < 0.01),and significantly expressed in longissimus dorsi (P < 0.05) of Yanhuang cattle,respectively.This study results provided a reference for further research on the function of ALDH1A1 gene and the screening of meat gene in Yanhuang cattle.

Key words: Yanhuang cattle; ALDH1A1 gene; clone; expression difference; bioinformatics

中图分类号:

胡忠昌, 曹阳, 吴健, 秦立红, 金海国, 赵玉民. 延黄牛ALDH1A1基因CDS序列克隆及其在各组织中的表达差异研究[J]. 《中国畜牧兽医》, 2019, 46(1): 174-184.

HU Zhongchang, CAO Yang, WU Jian, QIN Lihong, JIN Haiguo, ZHAO Yumin. Cloning of ALDH1A1 Gene CDS Sequence and Its Expression Difference of Different Tissues in Yanhuang Cattle[J]. , 2019, 46(1): 174-184.

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