中国大鲵组织蛋白酶D基因克隆及其表达特征分析

Cloning and its expression characteristics analysis of cathepsin D gene in Chinese giant salamander(Andrias davidianus)

【目的】明确组织蛋白酶D基因(CTSD)在中国大鲵不同组织中的表达模式及其免疫防御功能,为深入探究大鲵抵抗病原入侵的作用机理提供理论依据。【方法】通过RACE克隆中国大鲵CTSD基因cDNA序列,采用ProtParam、SignalP-5.0、TMHMM 2.0、CELLO、NetPhos 3.1及NetNGlyc 1.0等在线软件进行生物信息学分析,并通过实时荧光定量PCR检测健康中国大鲵不同组织及嗜水气单胞菌感染后的CTSD基因表达情况。【结果】中国大鲵CTSD基因cDNA序列全长1992bp,包括1197bp的开放阅读框(ORF)、136bp的5’端非编码区(5’-UTR)和659bp的3’端非编码区(3’-UTR),共编码398个氨基酸残基。中国大鲵CTSD蛋白相对分子量为43 kD,理论等电点(pI)为6.07,属于酸性稳定疏水性蛋白,主要定位于溶酶体,具有跨膜结构和信号肽,包含1个典型的天冬氨酸蛋白酶结构域和2个天冬氨酸蛋白酶活性位点,以及3个N-糖基化位点(131Asn、222Asn和249Asn)和33个潜在的磷酸化位点。中国大鲵CTSD氨基酸序列与东北小鲵CTSD氨基酸序列的相似性最高,达88.68%;基于CTSD氨基酸序列相似性构建的系统发育进化树也显示中国大鲵与东北小鲵的亲缘关系最近。CTSD基因在健康中国大鲵的肺脏、肌肉、皮肤、肾脏、肝脏、脾脏、心脏、胃、脑和肠道等10个组织中均有表达,且以肺脏、肌肉、肠道和脾脏中的相对表达量较高;嗜水气单胞菌感染后72 h,中国大鲵肾脏和肝脏中的CTSD基因相对表达量极显著升高(P<0.01,下同),分别是对照组的13.88和35.67倍;脾脏和皮肤中的CTSD基因相对表达量显著升高(P<0.05),分别是对照组的5.43和1.74倍;肌肉中的CTSD基因相对表达量则在感染后12 h极显著升高至最高值,是对照组的42.96倍。【结论】中国大鲵CTSD氨基酸序列具有高度的保守性,包含1个典型的天冬氨酸蛋白酶结构域和2个天冬氨酸蛋白酶活性位点;CTSD基因呈组成性表达,可能在中国大鲵抗病原菌入侵的免疫反应中发挥作用。

【Objective】To clarify the expression pattern and immune defence function of cathepsin D gene(CTSD)in different tissues of Chinese giant salamander(Andrias davidianus),and to provide a theoretical basis for in-depth investigation of the mechanism of resistance to pathogen invasion in A.davidianus.【Method】The cDNA sequence of CTSD gene of A.davidianus was cloned by RACE,and online softwares such as ProtParam,SignalP-5.0,TMHMM 2.0,CELLO,NetPhos 3.1 and NetNGlyc 1.0 were used for bioinformatics analysis.The expression of CTSD gene in different tissues of healthy A.davidianus and those infected by Aeromonas hydrophila was detected by real-time fluorescence quantitative PCR.【Result】The cDNA sequence of A.davidianus CTSD gene was 1992 bp in length,including an open reading frame(ORF)of 1197 bp,a non-coding region at the 5'-end(5'-UTR)of 136 bp,and a non-coding region at the 3'-end(3'-UTR)of 659 bp,encoding a total of 398 amino acid residues.The A.davidianus CTSD protein had a relative molecular weight of 43 kD and a theoretical isoelectric point(pI)of 6.07.It belonged to the acidic stable hydrophobic proteins,which were mainly located in lysosomes with transmembrane structure and signal peptide,and contained one typical aspartic protease structural domain and two aspartic protease active sites,as well as three N-glycosylation sites(131Asn,222Asn and 249Asn)and 33 potential phosphorylation sites.The amino acid sequence of CTSD of A.davidianus showed the highest similarity with that of the Northeast salamander(Hynobius leechii)at 88.68%,and the phylogenetic tree constructed on the basis of the similarity of the amino acid sequences of CTSD showed that A.davidianus and H.leechii were the closest relatives.CTSD gene was expressed in 10 tissues of healthy A.davidianus,including the lung,muscle,skin,kidney,liver,spleen,heart,stomach,brain,and intestine,and the relative expression was higher in lung,muscle,intestine and spleen.After 72 h of infection with A.hydrophila,the relative expression of the CTSD gene in kidney and liver of A.davidianus was extremely significant(P<0.01,the same below),which were as 13.88 and 35.67 times as that of the control group respectively;and relative expression of spleen and skin of A.davidianus significantly increased(P<0.05),which were as 5.43 and 1.74 times as that of the control group respectively;the relative expression of CTSD gene in muscle increased to the maximum value at 12 h after infection,which was as 42.96 times as that of the control group.【Conclusion】The amino acid sequence of CTSD of A.davidianus is highly conserved,containing one typical aspartic protease structural domain and two aspartic protease active sites;the CTSD gene is constitutively expressed,and it may play a role in the immune response of A.davidianus against pathogenic bacteria invasion.