新西兰白兔CTSB、CTSS基因克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2023.09.006

摘要/Abstract

摘要： 【目的】克隆新西兰白兔组织蛋白酶B(cathepsin B,CTSB)与CTSS基因CDS区序列并进行生物信息学分析,探究其在新西兰白兔不同组织中的表达规律。【方法】采用PCR法扩增并克隆CTSB、CTSS基因CDS区序列,使用Mega 7.0软件构建系统进化树并分析氨基酸序列相似性;利用生物信息学软件分析CTSB、CTSS蛋白理化性质、跨膜结构、信号肽位点及亚细胞定位情况;用实时荧光定量PCR检测CTSB、CTSS基因在新西兰白兔不同组织中的表达量。【结果】新西兰白兔CTSB、CTSS基因CDS区序列长分别为1 020、1 023 bp,共编码339、340个氨基酸,蛋白分子质量分别为37.627、38.253 ku,理论等电点为5.53、8.40,不稳定系数为30.76、32.38,分别为酸性及碱性蛋白。新西兰白兔CTSB、CTSS基因氨基酸序列分别与猪、马的亲缘关系最近,与鸡亲缘关系较远。CTSB、CTSS蛋白二级结构均主要由α-螺旋和无规则卷曲构成,二者三级结构预测结果与二级结构一致,均为亲水性蛋白;CTSB蛋白信号肽剪切位点位于第17—18位氨基酸处,含有1个N-端Propeptide-C1结构域与1个Peptidase-C1A-C结构域;CTSS蛋白信号肽剪切位点位于第25—26位氨基酸处,含有1个Inhibitor-129结构域与1个Peptidase-C1结构域。亚细胞定位结果表明,CTSB、CTSS蛋白均主要分布于细胞质(包括细胞壁)内。实时荧光定量PCR结果显示,CTSB基因在腹脂和肝脏组织中表达量较高,CTSS基因在脾脏组织中表达量最高,均显著高于其他组织(P＜0.05)。【结论】成功克隆新西兰白兔CTSB、CTSS基因CDS全长序列,揭示了CTSB、CTSS基因在新西兰白兔不同组织中的表达水平并初步分析了其生物学功能,为进一步研究家兔CTSB、CTSS基因功能及调控机制提供了基础和参考。

关键词: 新西兰白兔; CTSB基因; CTSS基因; 克隆; 生物信息学分析; 组织表达

Abstract: 【Objective】 The CDS regions of cathepsin B (CTSB) and CTSS genes in New Zealand White rabbits were cloned and bioinformatics analysis was performed,and explore the expression regularity in different tissues of New Zealand White rabbits.【Method】 PCR was used to amplify and clone the CDS region of CTSB and CTSS genes,and Mega 7.0 software was used to construct the phylogenetic tree and analyze the amino acid similarity.Bioinformatics software was used to analyze the physical and chemical properties,cross-membrane structures,signal peptides and sub-cellular locations of CTSB and CTSS proteins.The expressions of CTSB and CTSS genes in different tissues of New Zealand White rabbits were detected by Real-time quantitative PCR.【Result】 The sequence length of CDS region of CTSB and CTSS genes in New Zealand White rabbits was 1 020 and 1 023 bp,encoding 339 and 340 amino acids,the molecular weight was 37.627 and 38.253 ku,the theoretical isoelectric point was 5.53 and 8.40,and the instability coefficient was 30.76 and 32.38,which were acidic and basic proteins respectively.The amino acid sequences of CTSB and CTSS genes of New Zealand White rabbits were closely related to pigs and horses,respectively,but were far related to chickens.The secondary structure of CTSB and CTSS proteins was mainly composed of alpha helix and random coil.The predicted tertiary structure of both proteins was consistent with the secondary structure,and both were hydrophilic proteins.CTSB signal peptide splicing sites was located at 17-18 amino acids,containing 1 N-end Propeptide-C1 domain and 1 Peptidase-C1A-C domain,while CTSS signal peptide splicing sites was located at 25―26 amino acids,which contained 1 Inhibitor-129 domain and 1 Peptidase-C1 domain.The results of subcellular localization showed that CTSB and CTSS proteins were mainly distributed in the cytoplasm (including the cell wall).Real-time quantitative PCR results showed that the expression of CTSB gene was higher in abdominal fat and liver,and the expression of CTSS gene was the highest in spleen,both of which were significantly higher than other tissues (P<0.05).【Conclusion】 The full-length CDS sequences of CTSB and CTSS genes in New Zealand White rabbits were successfully cloned,and the expression in different tissues of New Zealand White rabbits were revealed and the biological functions were initially analyzed,which providing a basis and reference for further research on the functions and regulatory mechanisms of CTSB and CTSS genes in rabbits.

Key words: New Zealand White rabbits; CTSB gene; CTSS gene; cloning; bioinformatics analysis; tissues expression

中图分类号:

S829.1

袁冬冬, 宋国华, 陈孟娟, 王智通, 柏中峰, 蔡含芳, 李明, 许会芬. 新西兰白兔CTSB、CTSS基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50(9): 3499-3509.

YUAN Dongdong, SONG Guohua, CHEN Mengjuan, WANG Zhitong, BAI Zhongfeng, CAI Hanfang, LI Ming, XU Huifen. Cloning,Bioinformatics Analysis and Tissues Expression of CTSB and CTSS Genes in New Zealand White Rabbits[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3499-3509.

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