斑节对虾组织蛋白酶L基因的克隆及其表达分析

Cloning and expression analysis of Cathepsin L cDNA of Penaeus monodon

通过克隆得到斑节对虾(Penaeus monodon)组织蛋白酶L基因(PmCatL)cDNA全长。该基因序列全长1 850bp,包括1 026 bp的开放阅读框,编码341个氨基酸。预测的PmCatL蛋白由信号肽(Met^1-Ala^(18))、前体域(Val^(19)-Gln^(123))和成熟域(Leu^(124)-Val^(341))三部分构成,存在3个半胱氨酸蛋白酶活性位点(Cys^(148)、His^(287)、Asn^(308))和1个潜在的糖基化位点(Asn^(99))。具有组织蛋白酶L的E^(45)X_3RX_3FX_2NX_3IX_3N^(64)和G^(187)CXGG^(192)保守序列。同源性分析显示,PmCatL氨基酸序列与其他物种相似性为36%~75%。进化树显示PmCatL和罗氏沼虾(Macrobrachium rosenbergii)亲缘关系最近,并与其他无脊椎动物的组织蛋白酶L聚成一支。表达结果表明,PmCatL基因在肝胰腺、淋巴、肌肉、性腺等被测组织中均有表达,其中淋巴和肝胰腺的表达量较高;PmCatL基因在斑节对虾仔虾时期表达量较高,可能与斑节对虾在这一时期的生长发育相关;PmCatL在卵巢发育的Ⅳ期表达量最高,可能在斑节对虾的卵巢发育中发挥重要作用。

The cathepsin L c DNA of Penaeus monodon( PmCatL) was cloned by rapid amplification of c DNA ends( RACE). The Pm Cat L c DNA included an open reading frame( ORF) of 1 026 bp encoding a polypeptide of 341 amino acids,which contained a typical signal peptide sequence( Met^1-Ala^(18)),a prodomain( Val^(19)-Gln^(123)) and a mature domain( Leu^(124)-Val^(341)). The preprocathepsin contained a potential N-glycosylation site( Asn^(99)),three catalytic sites( Cys^(148),His^(287) and Asn^(308)) and sequences E^(45)X_3RX_3 F /WX_2NX_3IX_3N^(64) and G^(187)CXGG^(192)were discovered in amino acid sequence. Homology analysis of Pm Cat L reveals that the Pm Cat L shared 36% ~ 75% identity to other known cathepsin L sequences. The phylogenetic tree shows that the Pm Cat L clustered with the invertebrate cathepsin L cysteine proteases and was closely related to Macrobrachium rosenbergii cathepsin L. The expression levels of Pm Cat L in different tissues were analyzed by quantitative real-time PCR. Expression of Pm Cat L was detected in all tested tissues and the highest expression level was in lymph. The expression levels of Pm Cat L in different growth periods show that the expression levels of Pm Cat L with the highest expression level in post-larval,Pm Cat L might be associated with growth and development of P. monodon.Pm Cat L had the highest expression at Stage Ⅳ,which suggests that Pm Cat L might play an important role in ovary development in P. monodon.