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Your Position :Home->Past Journals Catalog->2016年53 No.4

cDNA cloning, expression and characterization ofglucose oxidase from Heliothis viriplaca
Author of the article:LEI Lei** ZHAO Kui-Jun GAO Yan-Ling LI Li-Li FAN Dong***
Author's Workplace:College of Agronomy, Northeast Agricultural University, Harbin 150030, China
Key Words: Heliothis viriplaca, glucose oxidase, cloning, expression, enzyme activity
Abstract:

[Objectives]  To obtain the full-length cDNA sequence of glucose oxidase (GOX) from Heliothis viriplacaand determine the GOX activity in the prokaryotic expression system, mRNA expression levels in different tissues and the characterization of glucose induction. [Methods]  Total RNA was isolated from the larva of H. viriplaca. The RT-PCR and rapid amplification of cDNA ends techniques were used to clone the full-length GOX cDNA sequence. The E. coli prokaryotic expression system was used to express the cDNA sequence. The recombinant protein, with a His-tag, was purified by Ni-NAT affinity chromatography. After purification, the recombinant protein was renatured using the gradient dialysis technique. The activity of GOX protein was determined with glucose as the substrate. Real-time PCR was used to analyze mRNA expression levels in different tissues and the reaction of glucose induction. [Results]  The cDNA sequence of GOX was 2 154 bp, including an open reading frame of 1 824 bp encoding a polypeptide of 607 amino acids with an estimated molecular mass of 67.04 ku and an estimated isoelectric point of 5.13. The cDNA sequence has been deposited in GeneBank (accession No. KT907054) and designated as HvGOX. SDS-PAGE revealed that HvGOX was successfully expressed in E. coli. The expressed protein was active after denaturation, purification and renaturation. Sequence analysis indicates that HvGOX shares extensive similarities with that of other insects. Real-time PCR results showed that the mRNA transcripts were expressed in different tissues, with the highest expression in the salivary gland. GOX expression in larvae was induced by feeding them soybean leaves soaked in 0.01%, 0.1%, 1% and 10% glucose solution; expression was highest when larvae were fed leaves soaked in 10% glucose solution. [Conclusion]  A novel GOX cDNA sequence was obtained from H. viriplaca. The results provide a foundation for further research on the biological function of GOX in insects.

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