中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (6): 2196-2206.doi: 10.16431/j.cnki.1671-7236.2023.06.003

• 生物技术 • 上一篇    下一篇

藏猪CDKN1B基因克隆、生物信息学及表达分析

韦明邦1, 边巴琼达2, 肖青青1, 段梦琪1, 强巴央宗1, 商鹏1   

  1. 1. 西藏农牧学院动物科学学院, 林芝 860006;
    2. 萨迦县扯休乡农牧综合服务中心, 日喀则 857000
  • 收稿日期:2022-10-12 出版日期:2023-06-05 发布日期:2023-05-30
  • 通讯作者: 商鹏 E-mail:nemoshpmh@126.com
  • 作者简介:韦明邦,E-mail:Bangbangbang962@163.com。
  • 基金资助:
    西藏自治区重大科技专项项目(XZ202101ZD0005N);中国农业大学基本科研业务费专项资金项目(2022TC125);林芝市巴宜区重点研发计划(2021-GX-SY-01)

Cloning,Bioinformatics and Expression Analysis of CDKN1B Gene in Tibetan Pigs

WEI Mingbang1, BIANBA Qiongda2, XIAO Qingqing1, DUAN Mengqi1, CHAMBA Yangzom1, SHANG Peng1   

  1. 1. College of Animal Science, Tibet Agricultural and Animal Husbandry University, Linzhi 860006, China;
    2. Agriculture and Animal Husbandry Comprehensive Service Center in Laxiu Village, Sa'gya County, Rekaze 857000, China
  • Received:2022-10-12 Online:2023-06-05 Published:2023-05-30

摘要: 【目的】探究细胞周期蛋白依赖性激酶抑制剂1B (CDKN1B)基因在低氧适应中发挥的作用。【方法】对藏猪CDKN1B基因CDS区进行克隆并测序,并对其序列进行相似性比对及系统进化树构建。采用在线软件对CDKN1B蛋白进行生物信息学分析,利用实时荧光定量PCR和Western blotting方法分别检测藏猪和大约克猪心脏、肾脏及肺脏组织中CDKN1B基因的mRNA和蛋白相对表达量。【结果】藏猪CDKN1B基因CDS区序列长597 bp,与野猪的相似性高达100%,且与野猪的亲缘性最近,与斑马鱼的亲缘性最远。CDKN1B蛋白由198个氨基酸组成,共有30个磷酸化位点:17个丝氨酸位点、9个苏氨酸位点、4个酪氨酸位点;氨基酸不稳定指数(Ⅱ)为66.53;该蛋白为亲水性蛋白,疏水性最大值为1.089,最小值为—2.867。藏猪CDKN1B蛋白二级结构由无规则卷曲(73.23%)、α-螺旋(15.66%)、延伸链(10.10%)及β-转角(1.01%)组成,三级结构预测结果与二级结构一致。该蛋白为非跨膜蛋白和非分泌蛋白,主要定位于细胞核中,占比约为82.6%,在细胞质中约占8.7%,在线粒体和细胞骨架中均占4.3%。CDKN1B蛋白与CDK2、CCND1和CDK4相关性高达99.9%。藏猪和大约克猪CDKN1B mRNA表达量与蛋白表达量一致,均为在心脏中表达量最高,其次是肾脏和肺脏;藏猪心脏、肾脏和肺脏组织中CDKN1B基因mRNA表达量极显著或显著高于大约克猪(P<0.01;P<0.05),蛋白表达量只在肾脏组织中显著高于大约克猪(P<0.05)。【结论】本试验成功克隆出藏猪CDKN1B基因CDS区序列,CDKN1B基因在心脏、肾脏及肺脏组织中均具有较高的表达量,且在藏猪心脏、肾脏及肺脏组织中表达量高于大约克猪。该结果为进一步研究藏猪CDKN1B基因及其编码蛋白在低氧适应性中的作用奠定基础。

关键词: 藏猪; CDKN1B基因; 生物信息学; 表达

Abstract: 【Objective】 The purpose of this study was to explore the role of cyclin-dependent kinase inhibitor 1B (CDKN1B) gene in hypoxia adaptation.【Method】 The CDS region of CDKN1B gene of Tibetan pigs was cloned and sequenced, and its sequence was compared and phylogenetic tree was constructed.The bioinformatics analysis of CDKN1B protein was carried out using online software.The relative expression of CDKN1B gene mRNA and protein in heart, kidney and lung tissues of Tibetan pigs and Yorkshire pigs were detected by Real-time quantitative PCR and Western blotting.【Result】 The CDS region of CDKN1B gene in Tibetan pigs was 597 bp, and was 100% similar to Sus scrofa.It was the closest to Sus scrofa and the farthest to Barchydanioreriovar.CDKN1B protein was composed of 198 amino acids and had 30 phosphorylation sites, including 17 serine sites, 9 threonine sites and 4 tyrosine sites, and the amino acid instability index (Ⅱ) was 66.53.The protein was hydrophilic with a maximum hydrophobicity of 1.089 and a minimum of -2.867. The secondary structure of CDKN1B protein in Tibetan pigs was composed of random coil (73.23%), alpha helix (15.66%), extended chain (10.10%) and beta turn (1.01%).The prediction of the tertiary structure was consistent with that of the secondary structure.The protein was a non-transmembrane protein and non-secretory protein, which was mainly located in the nucleus, accounting for about 82.6%, 8.7% in the cytoplasm, and 4.3% in the mitochondria and cytoskeleton, respectively.The correlation between CDKN1B protein and CDK2, CCND1 and CDK4 was as high as 99.9%.The expression of CDKN1B mRNA in Tibetan pigs and Yorkshire pigs was the same as that of protein, which was the highest in heart, followed by kidney and lung.The mRNA expression of CDKN1B gene in heart, kidney and lung of Tibetan pigs were extremely significantly or significantly higher than that of Yorkshire pigs (P<0.01 or P<0.05), and the expression of protein only in kidney tissue was significantly higher than that in Yorkshire pigs (P<0.05).【Conclusion】 The CDS sequence of CDKN1B gene in Tibetan pigs was successfully cloned.The expression of CDKN1B gene was high in heart, kidney and lung, and the expression in Tibetan pigs were higher than that in Yorkshire pigs.These results laid a foundation for further study on the role of CDKN1B gene and its encoded protein in hypoxia adaptation in Tibetan pigs.

Key words: Tibetan pigs; CDKN1B gene; bioinformatics; expression

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