马鹿β-防御素-1cDNA全长克隆、序列信息及表达分析

doi:10.11843/j.issn.0366-6964.2017.02.005

摘要/Abstract

摘要：

为了研究马鹿β-防御素-1（Red deer β-defensin-1，redBD-1）基因的结构与功能，揭示该基因的组织表达规律。本研究利用PCR结合RACE（Rapid-amplification of cDNA ends）技术从马鹿舌黏膜中克隆redBD-1基因的cDNA全长序列并对其进行了生物信息学分析，同时采用Real-time quantitative PCR（RT-qPCR）技术检测该基因在各组织的表达情况。结果表明，redBD-1基因的cDNA全长序列为455 bp，开放阅读框（ORF）为192 bp，编码64个氨基酸。生物信息学分析表明，redBD-1蛋白的理论分子量为6.94 ku，有10个带正电荷的氨基酸残基，无带负电荷的氨基酸残基，理论等电点为10.85。预测redBD-1蛋白有一个分泌信号肽结构，无跨膜区，主要在细胞外发挥生理功能；6 个保守的半胱氨酸残基分别以Cys1-Cys5、Cys2-Cys4和Cys3-Cys6连接形成3个分子内二硫键；成熟蛋白的三级结构是由β-折叠、延伸和无规则卷曲构成。redBD-1基因编码的氨基酸序列同源性最高的是梅花鹿β-防御素（siBD-1）为98.4%，其次是水牛肠β-防御素（BEBD）为92.2%，与人β-防御素-2（HBD-2）同源性最低仅为35.9%。RT-qPCR结果得出，redBD-1在被检器官中均有表达，在消化系统、呼吸系统以及生殖系统的大部分器官表达量较高，肝、肾和脾等实质性器官表达量相对较低。本试验为今后深入研究防御素基因功能以及马鹿黏膜免疫系统提供理论依据。

关键词: 马鹿&beta, -防御素-1, RACE, 基因克隆, 序列分析, 定量表达

Abstract:

This study aimed to study the gene structure and function of red deer β-defensin-1 (redBD-1) in order to better understand the gene's tissue expression pattern. The cDNA full-length sequence of redBD-1 gene was cloned from the red deer's lingual mucous membrane adopting the technology of PCR combining with rapid-amplification of cDNA ends (RACE), the sequence was analysed by bioinformatics, and the expression of the gene in different tissues was determined by Real-time quantitative PCR (RT-qPCR) technology. The results showed that the full-length sequence of redBD-1 gene was 455 bp, and the open reading frame (ORF) was 192 bp encoding 64 amino acids. The bioinformatics analysis indicated that theoretical molecular weight of redBD-1 protein was 6.94 ku containing 10 amino acid residues with positive charges without amino acid residue with negative charges, and its theoretical isoelectric point was 10.85. It could be predicted that redBD-1 protein had a secreting signal peptide structure, had no trans-membrane domain and mainly exereds the ectocellular physiological function; six conserved cysteine residues formed 3 intramolecular disulfide bonds with the connection of Cys1-Cys5, Cys2-Cys4 and Cys3-Cys6; the tertiary structure of maturation protein consisted of β-overlap, extension and random coils. Amino acid sequence of redBD-1 showed highest similarity with that of siBD-1 (98.4%), followed with BEBD (92.2%), HBD-2 (35.9%). RT-qPCR results indicated that redBD-1 was expressed in all detected organs, and the expression level was relatively higher in most organs of digestive system, respiratory system and reproductive system compared with a relatively lower expression level in parenchymatous organs like liver, kidney and spleen. This research could provide a theoretical basis for a better study of defensins gene functions and red deer mucosal immune system.

Key words: red deer β-defensin-1, RACE, gene cloning, sequence analysis, quantitative expression

中图分类号:

S825
S813.1

田巧珍，金鑫，张曼，蔡硕，刘骄，王云鹤，杨银凤. 马鹿β-防御素-1cDNA全长克隆、序列信息及表达分析[J]. 畜牧兽医学报, 2017, 48(2): 225-234.

TIAN Qiao-zhen, JIN Xin, ZHANG Man,CAI Shuo, LIU Jiao, WANG Yun-he, YANG Yin-feng. The Full-length Cloning, Sequence Information and Expression Analysis of Red Deer β-defensin-1 (redBD-1) cDNA[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 225-234.

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