Induction And Gene Cloning Of Glutathione S-transferases In Helicoverpa Armigera (Hübner)

Biochemical properties, induction by allelochemicals and expression differences between tissues or strains, purification and gene cloning of glutathione S-transferases (GSTs) were studied in cotton bollworm Helicoverpa armigera (Htibner).The optical pH of GSTs was 7.5-9 in midguts and 9 in fat bodies; the optical temperature was 50 C in midguts and 45 C in fat bodies; High concentration of relative enzyme (concentration of total protein) had some inhibition on reaction rate of GSTs; and high concentration of substrates (GSH>8 mM, or CDNB>2 mM) showed inhibiting effect on reaction rate of GSTs.In vitro inhibition studies indicated that tannic acid, quercetin and rutin had strong inhibition for the activity of GSTs conjugated with CDNB but with different inhibiting mode among three allelochemicals; the inhibiting effects were dependent on dose. With dose of 0.1 mM, several organophosphate and one carbamate insecticides showed 20-35% inhibition and two pyrethoids decamethrin and alphamethrin had little inhibition on the specific activities of GSTs.The specific activity of GSTs were 554.412.6, 286.6 and 150.5 nmol/mg pro./min in midguts, fat bodies, heads and cuticles respectively. The specific activity of GSTs in midguts of field strain was 66% higher than that of laboratory strain. Since the cotton bollworms of field strain had moderate resistance to several insecticides, the results indicated that there were some relationships between high level of GSTs and insecticide resistance. In all tissues or strains, the affinity of GSTs to both CDNB and GSH had no significant differences.The specific activity of GSTs was increased in midgut and decreased in fat body by different degree after 48 h treatment with 0.01% 2-tridecanone, quercetin and tannic acid. The affinity of GSTs both to CDNB and GSH had no significant changes and the content ofendogenous GSH was not changed either. The results showed that allelochemicals tested induced the expression level of GSTs, but no new isoenzymes induced or there was no variation in isoenzyme composition.Tannic acid with dose of 0.005% had affected the development profile of GSTs activity in cotton bollworms, indicated by its inhibition on GSTs in 5th and 6th instar larvae (59% and 67% of the control respectively). The results of time-effect and dose-effect showed that low dose and/or short time of treatment with tannic acid induced higher level of GSTs specific activity both in midgut and fat body while high dose and/or long time of treatment either depressed GST activity or had no effect on it. When cotton bollworms were continuously fed on artificial diet with tannic acid for 4 generations, their GST specific activity in the 6th instar larvae was all depressed in midgut and either depressed or not different from control in fat body.3 and 5 isoenzymes of GSTs in cotton bollworms were separated by GSH-sepharose 4B and PAGE from midguts and fat bodies respectively. All the isoenzymes were composed of two subunits with the same or similar molecular weight of about 30 kD. There was no change inisoenzyme composition of GSTs in cotton bollwonns treated by tannic acid.Using a pair of primers designed according to the consensus sequences of class I and class III GST genes in Lepidoptera insects, we cloned 219 bp fragment of GST from midguts and fat bodies in cotton bollworms by RT-PCR. Then 3 cDNAs were obtained with 3'-full length by 3'-RACE method. All these sequences are 743 bp and they encoded 198 amino acid residues. There is only 1 amino acid residue difference between two cDNAs from midguts and 3 to 4 amino acid residues difference between two cDNAs from midgut and one from fat body. They are classified as insect class 111 (Epsilon) GST genes based on their high similarity to class III GSTs from Lepidoptera and Diptera insects.