Construction Of Spodoptera Exigua (H�'bner) CDNA Library And Molecular Characterization Of Glutathione S-transferase

Spodoptera exigua (Hiibner) Lepidoptera, Noctuidae, an agricultural pest. It is also one kind of the pests which has become seriously resistant to insecticides in these years. In this study, the S. exigua cDNA library was constructed, and SeGSTe and gene SeGSTo were cloned from the library and their molecular characteristics were studied.1、The construction of the S. exigua cDNA libraryThe construction of the cDNA library database was based on the larvae and adults of different development stages of S. exigua in order to clone the resistance-related genes of S. exigua, and the analysis of genes on the molecular level encoding related proteins and enzymes of S. exigua were conducted by bioinformatics and EST techniques. Then monoclonal was randomly picked and sequenced, and EST of 782 independent genes were obtained by blast on NCBI.The statistical results showed:of all 782 independent ESTs,157 (20.1%) were genes coding unknown proteins,279 (35.7%)were highly homologous with genes coding known proteins and the rest were genes coding proteins which have been functionally annotated, mainly including the catalytic enzymes, proteins of metabolism, cell transport, energy cycle, transcription, translation and signal transduction.2、Study on the molecular characteristion of SeGSTe gene and SeGSTo geneTwo glutathione S-transferase genes were screened from the cDNA library of S. exigua and cloned, named as GSTe and GSTo respectively. The cDNA full-length of GSTe is 672 bp,5 ’UTR 23 bp,3’UTR 51 bp and encodes 224 amino acids. The cDNA full-length of GSTo is 747 bp,5’UTR 80 bp,3’UTR 184 bp and encodes 249 amino acids. The results of homology analysis of amino acid sequence showed:S. exigua GSTe is highly homologous with known GSTe and the homology with Spodoptera litura is the highest at 98%. SeGSTo is also highly homologous with known GSTo and the homology with Bombyx mori is the highest at 79%. Phylogenetic analysis revealed that SeGSTe belongs to the epsilon gene family of glutathione S-transferase and SeGSTo belongs to the omega gene family of glutathione S-transferase.The mRNA expression levels of two genes in different tissues and different stages were analyzed with RT-PCR and qRT-PCR, the results showed:the expression level of SeGSTe gene and SeGSTo gene was high at its 4th and 5th instar larvae in the midgut of S. exigua. Contrarily, the expression levels were relatively low during the pre-pupal stage. The expression level of SeGSTe gene and SeGSTo gene in fat body were lower than that in midgut. The expression level was highest on the first day, then gradually decreased on the second and third day. Similarly, the fifth instar larvae had a high expression level on the first and second day of and reduced from the third day. However, the expression level was higher during the pre-pupal stage. The pupal period witnessed a gradual increase in the expression level from the first day to the sixth day and peaked at the sixth day, followed by a decrease in the expression level in the adults stage. The soluble recombinant proteins SeGSTe and SeGSTo were obtained by applying the baculovirus expression system. The SDS-PAGE analysis showed that the molecular weight of SeGSTe was approximately 25 ku and that of SeGSTo was approximately 29 ku. Recombinant SeGSTe and SeGSTo both have DNA protective activity. Meanwhile, Km and Vmax values of recombinant SeGSTe and SeGSTo were measured with the substrate of CDNB, showing that Km and Vmax values of SeGSTe was 1.07 mM and 106.4 mmol/mg pro./min, respectively, and Km and Vmax values of SeGSTo was 0.85 mM and 769.2 nmol/mg pro./min, respectively.The result showed that the recombinant protein SeGSTo substrate affinity is better than that of recombinant protein SeGSTe.