黄喉拟水龟Cathelicidin基因克隆及表达分析

Cloning and expression analysis of Cathelicidin gene in Mauremys mutica

  • 摘要: 【目的】克隆黄喉拟水龟(Mauremys muticaCathelicidin基因(MmCath)并分析其组织表达特征,为深入研究MmCath基因在黄喉拟水龟先天免疫中的潜在作用提供理论依据。【方法】运用RACE克隆黄喉拟水龟MmCath基因cDNA序列,利用生物信息学软件进行序列特征分析,并通过实时荧光定量PCR检测MmCath基因在黄喉拟水龟不同组织中的表达特征及细菌攻毒后的表达情况。【结果】 MmCath基因cDNA序列全长777 bp,包括63 bp的5'非编码区、483 bp的开放阅读框(ORF)和231 bp的3'非编码区。MmCath基因编码160个氨基酸残基,包括N端的信号肽区,含有4个保守半胱氨酸(Cys)残基的Cathelin肽区和C端的成熟肽区,符合Cathelicidins蛋白家族典型特征。MmCath前体蛋白相对分子质量为17.74 kD,理论等电点(pI)为5.27,其二级结构中α-螺旋占31.25%,无规则卷曲占20.63%,β-折叠占48.12%。黄喉拟水龟MmCath前体蛋白氨基酸序列与西部锦龟(Chrysemys picta bellii)Cathelicidin氨基酸序列的相似性最高,达80.63%,亲缘关系最近。MmCath基因在黄喉拟水龟的表达具有组织差异性,以脾脏和肝脏中的相对表达量较高,在表皮、心脏、肾脏、肺脏、脑、肠道和肌肉等组织中的相对表达量较低;嗜水气单胞菌(Aeromonas hydrophila)攻毒后MmCath基因表达上调,在攻毒后第3和第6 h其相对表达量极显著高于攻毒前(0 h)的相对表达量(P<0.01,下同),之后有所下降,在攻毒后第36 h再极显著上升,随后下降。【结论】从黄喉拟水龟中克隆的MmCath基因属于Cathelicidins基因家族,参与了黄喉拟水龟抗病原感染过程,可为有效控制黄喉拟水龟细菌性疾病提供新思路。

     

    Abstract: 【Objective】 The Cathelicidin gene of Mauremys mutica (MmCath) was cloned and its tissue expression pattern was analyzed, so as to provide theoretical foundation for further studying the potential function of MmCath gene in the innate immunity of M. mutica.【Method】 The cDNA sequence of the MmCath gene of M. mutica was cloned by RACE. Sequence analysis was conducted by bioinformatics software. The expression patternof MmCath gene in different tissues of M. mutica and its expression change after bacterial challenge were identified by real-time fluorescence quantitative PCR. 【Result】 The full length of the cDNA sequence of MmCath gene was 777 bp, including a 5' noncoding region of 63 bp, an open reading frame(ORF) of 483 bp, and a 3' noncoding region of 231 bp. The MmCath gene encoded 160 amino acid residues, which contained the signaling peptide region at the N terminus, the Cathelin peptide region with 4 conserved cysteine (Cys) residues, and the mature peptide region at C terminus, in accordance with the typical family characteristics of Cathelicidins protein family. MmCath precursor protein had a relative molecular weight of 17.74 kDa and theoretical isoelectric point(pI) of 5.27. In its secondary structure, α-helix accounted for 31.25%, random coil 20.63% and β-turn 48.12%. The amino acid sequence of MmCath precursor protein shared a highest sequence similarity(80.63%) and closest genetic relationship with the amino acid sequence of Cathelicidin in Chrysemys picta bellii. Expressions of MmCath gene were different in various tissues in M. mutica. Its higher relative expressions were shown in spleen and liver, while the relative expressions were lower in the tissues like skin, heart, kidney, lung, brain, intestine, and muscle. Up regulation of expression was shown in MmCath gene after the challenge of Aeromonas hydrophila. The relative expression levels at 3 and 6 h were extremely significantly higher than that at before challenge(0 h) (P<0.01, the same below), and then decreased. It markedly increased again at 36 h post challenge and decreased afterwards.【Conclusion】 The MmCath gene cloned from M. mutica belongs to the Cathelicidins gene family. It involves in the process of resisting attack of invading pathogen of M. mutica and can provide new ideas for effective control of bacterial diseases of M. mutica.

     

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