CLONING AND SEQUENCING ANALYSIS OF 3 AMYLASE CDNAS IN THE SHRIMP PENAEUS-VANNAMEI (CRUSTACEA-DECAPODA) - EVOLUTIONARY ASPECTS

In Penaeus vannamei, alpha-amylase is the most important glucosidase a nd is present as at least two major isoenzymes which have been purifie d. In order to obtain information on their structure, a hepatopancreas cDNA library constructed in phage lambda-Zap II (Strategene) was scre ened using a synthetic oligonucleotide based on the amino acid sequenc e of a V8 staphylococcal protease peptide of P. vannamei alpha-amylase . Three clones were selected: AMY SK 37 (EMBL sequence accession numbe r: X 77318) is the most complete of the analyzed clones and was comple tely sequenced. It contains the complete cDNA sequence coding for one of the major isoenzymes of shrimp amylase. The deduced amino acid sequ ence shows the existence of a 511-residue-long pre-enzyme containing a highly hydrophobic signal peptide of 16 amino acids. Northern hybridi zation of total RNA with the amylase cDNA con firms the size of the me ssenger at around 1,600 bases. AMY SK 28, which contains the complete mature sequence of amylase, belonged to the same family characterized by a common 3' terminus and presented four amino acid changes. Some ot her variants of this family were also partially sequenced. AMY SK 20 w as found to encode a minor variant of the protein with a different 3' terminus and 57 amino acid changes. Phylogenetic analysis established with the conserved amino acid regions of the (beta/alpha) eight-barrel domain and with the total sequence of P. vannamei showed close evolut ionary relationships with mammals (59-63% identity) and with insect al pha-amylase (52-62% identity). The use of conserved sequences increase d the level of similarity but it did not alter the ordering of the gro upings. Location of the secondary structure elements confirmed the hig h level of sequence similarity of shrimp ol-amylase with pig alpha-amy lase.