cDNA cloning, prokaryotic expression and enzymatic characteristics of the glutathione <em>S-</em>transferase GmolGST6 in <em>Grapholita molesta</em> (Lepidoptera: Tortricidae)
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ACTA ENTOMOLOGICA SINICA  2018, Vol. 61 Issue (4): 398-409    DOI: 10.16380/j.kcxb.2018.04.002
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cDNA cloning, prokaryotic expression and enzymatic characteristics of the glutathione S-transferase GmolGST6 in Grapholita molesta (Lepidoptera: Tortricidae)
LI Shuai1,#, SU Li2,#, LI Bo-Liao1, LI Yi-Ping1, LI Guang-Wei3,*, WU Jun-Xiang1,*
 (1. State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China; 2. Agriculture College, Guangxi University, Nanning 530005, China; 3. College of Life Sciences, Yan′an University, Yan′an, Shaanxi 716000, China)
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Abstract 【Aim】This study aims to clone the glutathione S-transferase (GST) gene from the oriental fruit moth, Grapholita molesta, to analyze its structure properties, and to clarify the expression profiles and enzymatic characteristics of protein coded by this gene, so as to provide a theoretical basis for fundamental function research of this gene. 【Methods】 Based on the transcriptome data of female antennae from G. molesta, the complete coding sequence of GST gene was cloned using RT-PCR. The expression levels of this gene in antennae, head (without antennae), thorax, abdomen, leg and wing were measured by real-time fluorescence quantitative PCR (qPCR). The recombinant protein of GST was prokaryotically expressed, and then purified by Ni2+ affinity chromatography. In addition, the stability and kinetic parameters of the recombinant GST were analyzed. 【Results】 The complete coding region sequence of GmolGST6 (GenBank accession no.: MF503496) was obtained, and its ORF is 645 bp in length, encoding 214 amino acids with the predicted molecular mass of 24.21 kD and the theoretical isoelectric point of 5.10. The phylogenetic tree and the three-dimensional structure analysis indicated that GmolGST6 belonges to Delta subfamily of GSTs. qPCR results showed that GmolGST6 was primarily expressed in the antenna of male and female adults, and its expression level in the male antenna was significantly higher than that in the female antenna. The recombinant GmolGST6 showed catalytic activity towards the general substrate CDNB, and had the highest activity at pH 7.5 and 40℃. The Michaelis constant (Km) of the recombinant GmolGST6 was 0.21±0.06 mmol/L, and the maximum reaction rate (Vmax) was 14.02±1.40 μmol/min·mg. 【Conclusion】 Based on the expression profiles of GmolGST6 and the catalytic activities of the recombinant GmolGST6 towards CDNB, we speculate that GmolGST6 may be involved in the catabolism of exogenous substances and protecting the olfactory system from toxification of exogenous substances. 
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LI Shuai
LI Bo-Liao
LI Yi-Ping
LI Guang-Wei
WU Jun-Xiang
Key wordsGrapholita molesta   glutathione S-transferase   olfactory   expression analysis   enzymatic activity     
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LI Shuai,SU Li,LI Bo-Liao et al. cDNA cloning, prokaryotic expression and enzymatic characteristics of the glutathione S-transferase GmolGST6 in Grapholita molesta (Lepidoptera: Tortricidae)[J]. ACTA ENTOMOLOGICA SINICA, 2018, 61(4): 398-409.
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