Study On The Crustacean Trypsin Gene

In this paper, five crustaceas are utilized as reseach objects, including Chinese Prawn(Penaews chinensis), Japanese Prawn(Penaeus japonicus), Red Swamp Crayfish(Procambarus claikii), Japanese Mantis Shrimp(0ratosquilla oratorio) and grass shrimp(Neocaridina denticulate sinensis). Their partial genomic trypsin gene are cloned and their sequence are analyzed. Their genomic DNA are extracted firstly by a improved method of genomic DNA extraction. The purity and quality of DNA obtained by this method is satisfying. The ratio of OD260 to OD280 is over 1.7. These genomic DNA are then amplified by PCR with two oligoneclotide primers synthesised to obtain partial nucleotide sequences of trypsin gene. 5 trypsin gene clones were obtained after a series of manipulation: purification of PCR production, ligation and transformation of recombinant plasmids, blue-white selection and so on. DNA sequence analysis indicates that these 5 partial genomic gene sequence are highly identical to the trypsin gene sequence of Pacific White Shrimp(Litopenaeus vannamei) and that they all consist of one intron and two incomplete extrons. The analysis of deduced amino acid sequences based on the trypsin gene fragments shows that these 5 deduced protein sequences are over 56% identical to the sequence of Pacific White Shrimp. They are also relatively highly identical(30%-50%) to the sequences of verteibrates such as bony fish, chicken, africa clawed frog and mammals. All the deduced protein sequences possesses all the key amino acids characteristic of the serine protease family. The catalytic active site is composed of the canonical triad of His, Asp and Ser and a specificity pocket occupied by Asp and the residues Gly which define the trypsin specificity pocket. There are other conservative sites such as 8 Cys residues comprising 3 disulphide bridges at least. Therefore, these 5 genomic DNA fragments can prove to be the incomplete trypsin gene sequences of these 5 crustaceas. Phylogenetic analysis is processed with these 5 crustaceas' and other 11species' trypsin gene sequences and deduced protein sequences. Polygenetic analysis trees are obtained. The result of phylogenetic analysis is the same as the present taxonomy according as the characteristic surface configuration. This certify that the method holding phylogenetic analysis with conservative trypsin gene sequences and deduced protein sequences is heplful to test present taxonomy and provides an accurate melocular marker to tell the taxology station and relationship of different species.