MOLECULAR CLONING AND CHARACTERIZATION OF ADENYLOSUCCINATE LYASE cDNA AND GENOMIC DNA IN GRASS CARP

Ctenopharyngodon idellus,which is common known as grass carp,is a kind of large cyprinoid fish in China.It lives in both north and south water of China and is the most important fish in Chinese fresh water aquaculture.Grass carp can efficiently transform vegetable protein into high-quality animal protein.So people call the grass carp "cattle and sheep in water".The intestine of grass carp is the main part for nutrition digestion and the functional genes from the intestinal cell are tightly connected with the efficient transformation of vegetable protein and animal protein.Adenylosuccinate lyase(ADSL) gene encoding enzymes involved in two pathways of purine nucleotide metabolism are of considerable biological and medical importance.It catalyzes two similar but separate reactions in the de novo purine biosynthesis pathway.Now,we are trying to build a connection between grass carp genome project and the industrialization of function feedstuff depended on the information that we have got from this research.In this research,we clone the full length cDNA and genomic DNA of ADSL from grass carp intestine,which is very important in the purine metabolism.The cDNA sequence of Grass Carp ADSL gene contains complete ORF starting at nucleotide 64 with a stop codon at nucleotide 1510—1512.The translated region is of 1446 nucleotides with full open reading frame(ORF) comprising 482 codons preceded by a 5′-untranslated region of 63 nucleotides and followed by 3′-untranslated region of 75 nucleotides.The typical polyadenylation signal AATAAA is found 13bp upstream of the polyA tail.The predicted mass of encoded protein is 54,552 Da with a calculated iso-electric point of 6.72 and-4.11 charges at pH 7.The ADSL amino acid sequence of grass carp has classical "signature" sequence of enzymes that catalyzing β-elimination reactions and two conservative histidine residues as general acid-base catalysis active site.The deduced amino acid sequence shares high homology with other five vertebrates ADSL and has 94.6% similarity with Danio rerio,78.5% with Xenopus laevis,70.8% with Gallus gallus,76.2% with Mus musculus and 76.0% with Homo sapiens.To obtain the grass carp ADSL genomic DNA,we design four pairs primers based on the full length cDNA of ADSL and result 8557bp ADSL genomic DNA of grass carp which encompass 13 exons and 12 introns.The splice sites are well conserved through evolution,and observe the regulation of GT-AG except for the ninth intron whose 5′ site was GC.The alignment of five vertebrates ADSL genomic DNA sequence indicate that they have the same numbers of intron and exon,and the number of base pairs is identical in 2—12 exons.This reveals that the structure of ADSL gene is well conserved through evolution in exon.