Molecular Cloning And Sequence Analysis Of CDNAs Encoding Partial Putative Molt-inhibiting Hormone From Four Kind Of Crustaceans

Total RNAs were extracted from eyestalks of the shrimps (including Penaeus chinenszs, Trachypenaeus Curvirostris, and Penaeus slylirostris) and the crab Eriocheir sinensis. Eyestalk complementary DNAs were gotten from these DNA-free total RNAs by reverse transcription. Based on the amino acid sequence of the molt- inhibiting hormone from Penaeus japonicus, two primers P1 and P2 were designed. Polymerase Chain Reaction (PCR) was performed using P1 and P2 as primers and these eyestalk cDNAs as templates. Four specific cDNAs were cloned under proper reaction conditions. At the same time in a control of PCR whose template is genomic DNA of P stylirostris, a specific DNA fragment of the same size as the specific eDNA from eyestalk of P stylirostris was cloned. These specific cDNAs and DNA were subcloned into vector for sequencing. The sequencing results demonstrated that the specific cDNA from P chinensis eyestalk consists of 203 base pairs, and all the specific cDNAs from the shrimps T curvirostris, P sfylirostris and the crab E. sinensis consist of 215 base pairs. The specific DNA from genomic DNA of the shrimp P stylirostris is almost as same as the specific eDNA from P stylirostris eyestalk. Using the on-line sequence similarity searching tool Fasta3 on internet web, many neuropeptides from crustacean CHH family of high similarity with these deduced amino acid sequences from eDNA of eyestalks from these four kinds animals were found. The aligmnents of the deduced amino acid sequences from eyestalk cDNAs of the II / cDNA J~Ij~Ji shrimps P chinensis, T curvirosfris, and P stylirostris and the crab E. sinensis with neuropeptides of CHH family from various crustaceans demonstrate that they have the highest identity and homology with most MIHs from crustaceans. It suggests that the deduced amino acid sequences from eyestalk eDNA of the shrimps P chinensis, T curvirostris, and P stylirostris and the crab E. sinensis are the part of MIH neuropeptides of corresponding animals. By using nucleotide acid sequences and amino acid sequences data, The relationship among MIHs from P chinensis, E. sinensis, T curvirosris, and P styliroswis and six published MIHs from various crustacean was analyzed. The gene tree of MIH disagrees with phylogenetic tree of species. The relationship among E. sinensis, T curvirostris, and P stylirosU-is is very near.