Cloning and bioinformatics analysis of antifreeze protein from Tenebrio molitor
AIM: To obtain sequence coding gene for the antifreeze proteins (AFP) from local Tenebrio molitor and to elucidate the related bioinformatics data. METHODS: After the total RNA was isolated, from the larva of Tenebrio molitor. cDNA encoding the afpTx was synthesized by RT-PCR, and the PCR products were inserted into the vector pMD19-T simple, which were subcloned into pET-32a( + ) and transformed into E. coli and identified with restriction enzyme analysis. Then the sequencing result was analyzed by MEGA 4. 0 and BioEdit 5. 0. 6 computer program for amino acid sequence homology and evolutionary variance. RESULTS: Sequencing result showed a correctly constructed vector that containing 336 bp antifreeze protein cDNA. Digestion and electrophoresis results confirmed that gene was successfully cloned and subcloned into pET32a ( + ). Sequence similarity analysis indicated that our afpTx sequence showed a sequence homology of 88% to 23 GenBank submitted unique sequences of Tenebrio molitor AFPs, and an average homology of 67% to 11 Dendroides Cana-densis AFPs and 63% to two beetle species. Phylogenetic analysis indicated that two beetle species are homologous and striking feature of this analysis showed that DNA sequence divergence among Dendroides Canndensis were markedly higher that among Tenebrio molitor. CONCLUSION: Successfully cloned local afpTx sequence and proved that this sequence is homologous among two beetle isoforms.
NETL
NSTL
万方数据
维普
