| Enolase(2-phospho-D-glycerate hydrolyase)is a metalloenzyme that catalyzes the dehydration of 2-phospho-D-glycerate(PGA)to phosphoenolpyruvate(PEP)in the last steps of the catabolic glycolytic pathway,which plays a vital role in the progress of cellular energy metabolism.Enolase belonging to a new class of protein,called moonlighting protein,has being extensively demonstrated.Upon stimulatory signals,externalization of enolase contributes to different pathologies including cell apoptosis,injury,autoimmunity,infection,inflammation.Helicoverpa armigera,(Hubner)is one of the most important worldwide agricultural insect pests in cotton.Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis(Bt)has been deployed for combating cotton bollworm.However,with the widespread popularization of Bt cotton,the resistance to Bt toxin will influence on its long-term and effective application,so it is vital to find out resistance mechanism of Bt.In this study,Helicoverpa armigera was chosen as material.Analysis of the transcriptome of H.armigera was carried out,with the use of RT-PCR technology,we for the first time characterized the complete ORF of enolase gene from H.armigera.The ORF was 1302 bp,which encoded 432 amino acids residues.Sequences analysis indicated that H.armigera enolase protein was a conserved-protein in many species,and showed more than 97%amino acid sequnence identity to the enolases of Spodoptera litura and Spodoptera frugiperda.Bioinformatics analyses reavealed the predicted molecular weight and theoretical pI were 151.69 kDa and 8.24,respectively.H.armigera enolase lacks N-terminal signal peptide and membrane anchor signal sequence.qRT-PCR analysis indicated that enolase was diffentially expressed in various tissues of the 5th-instar larvae.A high expression level was detected in the head,but trace mRNA levels was detected in fregut,midgut,hindgut and malpighian tubule.Meanwhile,enolase was transcribed in all the life stages of H.armigera with the lowest expression level during the egg and pupal stage,and reached the maxium at 4th-instar larvae.We investigated the subccllular localization of enolase proteins by expressing eGFP-fused protein in BTI-Tn-5B1-4(Hi5).GFP-Enolase fusion expression analysis showed that enolase was distributed in cytoplasm and nucleus of Hi5 cells,and riched mainly in the nucleus.The protein in the cytoplasm is a punctual distribution.Enolase protein was expressed by prokaryotic expression system and the protein was successfully purified.Then western blot and Mass spectrometry analysis confirmed the expressed enolase protein(50kDa).When the nucleotide sequnence and amino acids residues sequence were compared between the sensitive strains and resistant strains,there were no amino acids residues change among the two sensitive strains and five resistant strains,but LF5.0 and LF240.0 has 1 and 6 different amino acid residues,respectively.The result of qRT-PCR showed that the expression of H.armigera enolase gene from all of the resistant strains was much higher than the sensitive strains,and the highest expression occurred in the LF20.0 and LF30.0.And also in the midgut,the expression of the gene in resistant strains(BtR)was higher than that in sensitive strains.Therefore,the expression of enolase varies according to the metabolic,development or pathophysiological conditions of cells,and may influence growth,development,and metabolism in H.armigera.Either the higher level of enolase in Bt resistant strains may devote energy to adapt to the various environment.The definite mechanism needs further study. |
