Construction And Analysis Of CNDA Library From The Sea Cucumber Stichopus Japonicus And Cloning And Characterization Of Cathepsin L Gene

cDNA library was constructed with Gubler-Hoffman according to Takara cDNA LibraryConstruction Kit. The titer of the constructed library is2.0×10~6pfu/ml. The PCR results byrandomly isolated30clones showed that all of them contain recombinant DNA fragments andthe average size of inserts was1.3kb. Clones containing cDNA longer than800bp wereselected for sequencing using T7and T3promoter primers. A lot of expressed sequence tags(ESTs) were obtained by sequencing from the5’ end of the cDNA clones and then the ESTswere compared with sequences in the GenBank and EST databases of NCBI using Blastsearch. Among100ESTs obtained26showed sequence homology to previously identifiedgenes,74matched to uncharacterized genes. The most interesting finds were genes involved inproteolysis and cell apoptosis which may be specific to the autolysis of sea cucumbers,including polyubiquitin and ubiquitin carboxyl-terminal hydrolase, ferritin andmelanotransferrin. Other characteristic features such as genes encoding ribosomal proteinsubunits, collagen, cytochrome c oxidase, NADH dehydrogenase,-amylase and so on werealso observed which are essential to cellular and energy metabolism. The successfulconstruction of cDNA library from Stichopus japonicus and further analysis of ESTs will gaininsight into molecular manifestation of development and metabolism in sea cucumbers andaccelerate functional and regulation gene discovery.Cathepsin L is the most abundant cysteineprotease with only endopeptidase activity, The cathepsin L is responsible for intracellularprotein degradation, and it is also involved in many other important physiological roles suchas antigen presentation, tumor invasion and metastasis, bone resorption and apoptosis.Previous data showed that cathepsin L-like protease is a heterodimeric consisting of acatalytic subunit(CL) and the non-catalytic subunit(CLAP)，cathepsin L-associated protein(CLAP) is a member of the Fas I family of cell adhesion proteins, CLAP confers considerablestability on CL at pH and temperatures normally incompatible with CL activity. Recent datahave demonstrated that CLAP plays an important role in targeting and expression regulationof cathepsin L activity. A cysteine protease gene homologous to the cathepsin L-associatedprotein (CLAP) genes was isolated from cDNA Library(GenBank:EU306878). The full- length cDNA was1335bp，consisting of a5’-terminal untranslated region (UTR) of91bpand489bp3’ untranslated region with an open reading frame of756bp, The ORF encoded251amino acids(aa). The cDNAs encoding cathepsin L in the intestinal and body wall of thesea cucumber Stichopus japonicus have been amplified by PCR using degeneratedoligonucleotide primers and5’-and3’-RACE, cloned and sequenced. The full-length cDNAwas1287bp(GenBank:EU143709), consisting of a5’-terminal untranslated region (UTR) of201bp, a3’-terminal UTR of87bp with a canonical polyadenylation signal sequenceAATAAA and a poly (A) tail, and an open reading frame of999bp. The ORF encoded332amino acids (aa) including a signal peptide of16aa at the N-terminus and a mature peptide of316aa. The putative signal peptide is cleaved at Cys21.