Molecular Cloning Of Triosephosphate Isomerase Gene From Monochamus Alternatus And Its Expression Analysis By Real-time PCR

Monochamus alternatus Hope(Coleoptera: Cerambycidae), an important forest pest in China, is a woodboring insect pest of Pinus massoniana and other species of the genus Pinus. Monochamus alternatus is an efficient vector of pine wood nematode,which causes pine wilt disease.The homodimeric enzyme triosephosphate isomerase, a kind of glycolytic enzyme, exists widely in many organisms. Triosephosphate isomerase(TPI) plays an important role in glycolysis, gluconeogenesis, fatty acid synthesis and the pentose shunt, and converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate. In this study, we screened TPI gene sequence of 5' end, from M. alternatus c DNA library. Then, we amplified 3'-terminal sequence of TPI gene by rapid amplification of c DNA ends(RACE) technology.Bioinformatics analysis showed that the full-length c DNA of M.alternatus triosephosphate isomerase gene was 1450 bp in length, which contained a 744 bp open reading frame(ORF) encoding 247 amino acids with a calculated molecular mass of 26.71 k Da and an isoelectric point of 9.26. We designated it as Ma TPI(Gen Bank: KX024698). The molecular formula of deduced amino acid from Ma TPI is C1192H1892N320O363S6,which is a hydrophil protein. There was no transmembrane region and signal peptide cleavage site in the protein. There were 7 serine phosphorylation sites and 4 threonine phosphorylation sites. Prediction of secondary structure and tertiary structure of Ma TPI protein found that the protein was mainly composed of alpha helix and beta folding structure. We also found Ma TPI contained a triosephosphate isomerase domain integrity, which suggests that Ma TPI belongs to the TPI family. Ma TPI contains TPI active functional sequence(AYEPVWAIGTG), and shows the highest homology with Tribolium castaneum and Tenebrio molitor(80%), and has amino acid sequence homology with the homodimeric enzyme triosephosphate isomerase from 12 species of insects such as Helicoverpa armigera(70-73%). Phylogenetic analysis shows it is in the same branch as M. alternatus, T. castaneum and T. molitor. The genetic distance between M. alternatus and Wasmannia auropunctata is far.The relative expression levels of Ma TPI detected by RT-qPCR indicated that the expression levels of Ma TPI in adults was higher than that in pupae and larvae, and there was a significant difference in expression level among different developmental stages(P < 0.05). The relative expression of Ma TPI in the adult abdomen was significantly higher than that in the head, thorax, legs, antennae and wings(P < 0.05). The expression of Ma TPI in different tissues of larvae was: fat body > body wall > hemolymph> hemolymph midgut > Malpighian tube(P < 0.05). Our results also showed that the expression of Ma TPI was upregulated after treated by three pesticides(Beauveria bassiana, Acetamiprid, Malathion), and downregulated after treated by eight pesticides(Bacillus thuringiensis, Metarrhizium anisopliae, Nimbin, Thiacloprid, Methomyl, Chlorpyrifos, Dimehypo, Decamethrin).