Cloning And Expression Analysis Of Elastase CDNA From Grass Carp (Ctenopharyngodon Idella)

Grass Carp(Ctenopharyngodon idellus) is one of the special freshwater fishes in China with important economic value.Most studies of grass carp focus on culturing, breeding,pathogenic biology,disease prevention and treatment in present,other than on basic research.Even basic research focus mainly on traditional biological fields such as taxonomy,histology,and ecology,fewer reports involved in modern molecular biological approaches.In this context,elastase,which was related to digestion and immunity,was used as an object to study the roles in digestive and immune using molecular biological techniques,bioinformatics to clone the elastase gene cDNAs and sequence analysis,and phylogeny of the pancreatic elastase of teleost so as to provide theoretical basis for digestive and immune mechanism research.1 The pancreatic elastase-1,2,3 precursor cDNAs were cloned from grass carpTotal RNA was isolated from grass carp hepatopancreas.Three pairs of gene specific primers were designed based on the ESTs(GenBank AC No:CK233019; CF887854;CF887855) to clone the full-length cDNAs of three pancreatic elastase isoforms by rapid amplification cDNA end.The result showed:①The full-length of Ela-1(GenBank AC No:EU622520) was 938 bp,which contained a 798 bp of open reading frame(ORF) encoding a putative pancreatic elastase precursor of 266 amino acids,flanked by a 19 bp of 5' un-translated region(UTR) and a 118 bp of 3'-UTR.②The full-length of Ela-2(GenBank AC No:EU622522 ) was 926 bp,which composed of a 798 bp of ORF encoding 266 amino acids,sided by a 24 bp of 5'-UTR and a 101 bp of 3'-UTR.③The full-length of Ela-3(GenBank AC No:EU622521 ) was 964 bp, including a 807bp of ORF,encoding 269 amino acids,a 31 bp of 5'-UTR and a 123 bp of 3'-UTR.④Deduced amino acid sequences of Ela-1,2 and 3 had theoretical isoelectric point of 7.02,6.97 and 6.14,respectively,and had calculated molecular mass of 28875.49 Da,28512.34 Da and 28784.68 Da,respectively. 2 Deduced amino acid sequence homology analysis and phylogenetic analysis of grass carp ElastaseTo reveal the phylogenic relationship of grass carp elastase with those of other mammals and of teleosts,we performed amino acid sequence homology alignment using the Clustal W computer program and constructed unrooted phylogenic tree of Ela-1,2 and 3 by the neighbor-joining method,basing on the paired alignment of all amino acid sequences from different species,From the tree,we realized:①The group of Ela-1 contained two clusters:the one was composed of mammals,birds and amphibians,and the other one was composed of fishes.This indicated that the divergence between fishes and other species began at an early period.Ela-2 and Ela-3 of different species clustered together in a big group,then,separated from each other at the first divergence point.②In the tree of Ela-1,the grass carp Ela-1 was clustered with that of gilthead seabream,bastard halibut and Atlantic salmon.The cluster composed of fishes Ela-1 was separated from that of mammals such as human,house mouse,Norway rat,cattle and pig.③In the tree of Ela-2,the grass carp Ela-2 was clustered with that of bastard halibut,gilthead seabream,zebrafish and Atlantic cod. The cluster composed of fish Ela-2 was separated from that of mammals such as human,house mouse,Norway rat,cattle and pig.As we seen from the tree,house mouse and Norway rat,cattle and pig,human and rhesus monkey,African clawed frog and silurana tropicalis were clustered together,respectively.④In the tree of Ela-3,the Grass Carp Ela-3 was clustered with that of zebrafish,yellowfin seabream, glithead seabream and Atlantic cod.Of note,Ela-3 of fishes was separated from those of other species.As a matter of fact,mammals such as human,pig,cattle and amphibians clustered together independently.These evidences showed that the pancreatic elastase was conserved,but the evolutionary divergence between different species exist indeed,and began at an early evolutionary period.⑤As we can see from the tree,Ela-2 and 3 had close genetic distance.It was presumed that:three isoforms derived from the common ancestral gene and they were members of one gene family. The difference in construction,function and evolution were probably due to factors such as gene repetition and mutation.3 The expression of elastase-1,2 and 3 in grass carp were analyzed by RT-PCRThe results of RT-PCR revealed:①Grass carp Ela-1,2 and 3 expressed at a high level in hepatopancrea,foreintestine and spleen and at a low level in kidney.The result indicated:elastase was secreted mainly by pancreatic cells,and foreintestine was responsible for most proteins hydrolysis.What is more,high level expression in spleen and low expression in kidney revealed that elastase played an important role in immunity.②In brain,only Ela-1 expressed weakly.②Ela-1,2 and 3 all had no positive belt in heart,leukocyte and muscle.Of note,no or low mRNA expression in leukocyte was due to mature neutrophil elastase which had no ability to synthesize elastase.④As we can see from the expression results,Ela-2 and 3 were similar in expression pattern,but they were different from Ela-1 in expression pattern just as they were in evolution analysis.So,we obtained results as follows:the genetic distance between Ela-2 and 3 was close and we could presume that the function of Ela-1 was different from the other two isoforms.