Cloning and expression of cathepsin L in Cherax quadricarinatus and its regulation by dietary vitamin C

Australian redclaw crayfish,Cherax quadricarinatus,belongs to Decapoda order,and Parastacidae family.Because it grows rapidly and can reach commercial size in a few months at optimal conditions,it has been one of the most important economy shrimps in China.However,the physiological mechanisms of redclaw crayfish have not been well studied,especially the basic information of key nutrients and the related enzymes or proteins are missing.Cathepsin L(CatL) is an important enzyme related to digesting process in crustaceans,however,its molecular structure and correlation with dietary nutrients have not been investigated.In the present study,in order to obtain insights into the regulatory effects of key dietary nutrients on expression of CatL gene,the CatL gene of C.quadricarinatus was cloned first using RACE-PCR methods.The tissue-specific CatL gene expression was also performed,and the C.quadricarinatus were fed with artificial diets containing different levels of vitamin C(0,100,200,400,800,1 600,3 200 mg /kg) for 8weeks,and the expression of CqCatL mRNA in different tissues and different groups were examined.The CatL cDNA was isolated from the hepatopancreas of C.quadricarinatus using RACE-PCR methods,which was designated CqCatL(Genbank accession number:KJ913663).The full-length cDNA of CqCatL obtained from the present study was 1 810 bp in length with an open reading frame of 1 026 bp,encoding a putative peptide of 341 amino acids,and the predicted molecular mass and isoelectric point were 37.63 ku and 5.17.By alignment,the amino acid sequence of CqCatL of C.quadricarinatus showed high homology with those of other animals.For example,the deduced amino acids sequence of C.quadricarinatus CqCatL shared 79%homology with Penaeus monodon,and 74%,59%,59%,58% and 58% with Macrobrachium nipponense,Eriocheir sinensis,Metapenaeus ensis,Metapenaeus ensis and Litopenaeus vannamei,respectively.This suggested that CqCatL was highly conservative.The tissue-specific expression pattern study indicated that CqCatL mRNA was widely expressed in hemocytes,gill,hepatopancreas,muscle,ovary,intestine,stomach,heart,brain and antennal gland,indicating CqCatL may play important roles in many tissues of the C.quadricarinatus.The highest expression was observed in hepatopancreas,and the second was seen in hemocytes,while only very low expressions of CqCatL mRNA were found in intestine and antennal gland.After feeding with seven levels of vitamin C,the mRNA expression of CqCatL was the highest in the 400 mg /kg dietary vitamin C group and significantly higher than the other groups.The results showed that CqCatL might play an important role in the physiological process of C.quadricarinatus,and its gene expression and functions were likely regulated by nutrients,such as vitamin C.