中国大鲵组织蛋白酶D基因克隆及其表达特征分析OA北大核心CSTPCD

[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 investi-gation 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 molecu-lar 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 as-partic 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 con-structed 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,in-testine 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 pro-tease 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.