Study On The Glutathione S-transferase Superfamily In The Silkworm, Bombyx Mori

Glutathione S-transferases (GSTs, EC2.5.1.18) are a superfamily of multifunctional enzymes found in almost all living organisms. They were first discovered in animals in 1961 where they were postulated to play a role in the detoxification of drugs. The main function of GSTs is to catalyze nucleophilic attack by reduced glutathione (GSH) on nonpolar compounds that contain an electrophilic carbon, nitrogen, or sulphur atom. Mammalian GSTs are particularly well studied due to their role in cancer epidemiology and treatment. Insect GSTs also are wildly researched, which is focused on insecticide resistance and detoxification of organic hydroperoxide. The previous studies are indicated that insect GSTs played important roles in organophosphate, organochlorine, and pyrethroid insecticides. In insects, there is lack of selenium-dependent glutathione peroxidase (SeGPx). However, GSTs contain the activity of non-selenium-dependent glutathione peroxidase (non-SeGPx), thus, GSTs are very important in the process of antioxidation. Except with the function of catalysis, GSTs can also act as ligandins, etc. The insect GSTs are classified into Delta, Epsilon, Omega, Theta, Sigma, and Zeta classes, as well as unclassified GSTs. The Delta and Epsilon classes are insect specific GSTs, which are mainly related to insecticide resistance.The silkworm is an important economic insect, which is very sensitive to insecticides. Thus, the sericultural production is easily effected by insecticides. In addition, more than 70% agriculture and forestry pests belong to Lepidoptera. Bombyx mori is the model organism of Lepidoptera and the only organism of genome sequencing in Lepidoptera. Thus, study of silkworm GSTs at biochemical and genomic level is not only provided foundation for resistant silkworms, but also established important foundation for biological control of Lepidopteran pests. The main results are followed. 1. Studies on the related physiology of the silkworm GSTsThe physiological characteristic of enzyme is one of the important exhibitions of gene functions. Thus, firstly, we studied on the physiology characteristics of GSTs in various tissues and developmental course in Bombyx mori. We detected GSTs activities toward CDNB in 9 tissues on day three of fifth instar of Dazao strains. The head of the silkworm contained the highest specific activity, and followed by fat body and midgut. The K_m(CDNB) of the GSTs in fat body was higher than that in midgut. It indicated that the GSTs in fat body contained lower affinity for CDNB than those in midgut. While the catalytic efficiency of GSTs in fatbody is higher than that in midgut, due to the fat body GSTs contained higher V₍max(CDNB). But it is interested that the GSTs in head had higher affinity and catalytic efficiency than the GSTs in fat body and midgut. When we used the cumene hydroperoxide (CHP) as substrate to detect the activity of non-selenium-dependent glutathione peroxidases, the GSTs in all the 9 tissues were found the specific activities. It was different from the specific activity toward CDNB that malpighian tubes, ovary, testis, and silkgland had higher specific activities toward CHP. We predicted that the higher specific activities toward CHP in gonad could protect oxidize stress from gonad cell. In development course (egg-larva-pupae-adult), the specific activities toward CDNB presented some certain disciplinary changes. The specific activity of GSTs in eggs was lowest in the development course, newly hatched larva contained a higher activity, and the specific activity reached the highest value on day three of the fifth larva. The activity of GSTs in two-day pupae was 34.505±2.285 nmol/(mg pro·min) and higher than that in newly molted larva of fifth instar, 27.335±2.111 nmol/(mg pro·min). And the activity of one-day adults was similar with the newly molted larva of fifth instar.We found that the LC₅₀ of phoxim treated with the day three of the fifth instar larva could be result in oxidized stress reaction. The content of malondialdehyde (MDA) is often used to evaluate the level of oxidized stress. Thus, we determined the MDA content in fat body and midgut post-treated with phoxim. At 12h post-treatment, MDA content in fat body was reached the highest level, and a multiple of 4.11 relative to control. After 12h, MDA content was decreased, and came back to normal level at 24h. In midgut, MDA content increased at 4h post-treatment, and the ratio between treatment and control reached the highest value (3.156). And then it came back to natural level at 48h. The expression of GSTs was induced by the oxidized stress. The specific activities toward CDNB and CHP in fat body GSTs were remarkably increased at 20h post-treatment, and came back to natural situation at 42h and 48h, respectively. So the increased GST activities may be play important role in oxidized stress.2. The bioinformatics analysis of the silkworm GST genesWe have identified twenty-three putative members of cytosolic GSTs by searching the new assembly of the Bombyx mori genome sequence. The GST gene number in silkworm is similar with Dipteran insects, and larger than that in honey bee (eight members). Delta and Epsilon classes were also obvious duplicated in silkworm genome, contained 4 and 8 members, respectively. In addition, Omega class was also duplicated, which 4 members were identified. Silkworm GST genes were similar with other insect that GSTs genes present the characteristic of cluster distribution. However, with the exception of e1, e4 and e5, the other Epsilon GSTs were dispersed in genome. It may be indicated that the molecular mechanism of duplication for Epsilon GSTs was different from the local duplication. Relative to Dipteran GSTs, silkworm GSTs contained more and larger introns. We analyzed the long introns of silkworm GSTs and found many transposons located in it. Thus, we speculated than the multiple of transposons or its remains located in introns of silkworm GST genes could result in more and larger introns.3. Expression pattern and cloning of the silkworm GSTsWith the exception of BmGSTe2, BmGSTe6, BmGSTe8, and BmGSTo4, all the silkworm GSTs contained ESTs evidences. And in the developmental course (egg-larva-pupae-adult), there had expression of the silkworm GSTs. The ESTs number and tissue distribution of different classes had obvious differences, which may be reflected their distinct function during the development of the silkworm. Based on the gene microarray data during the course of mature silkworm to adult, the GST gene cluster was obviously distinguished two groups, which one contained high expression and another was low expression or no expression. Four Delta and Epsilon GSTs showed high expression, meanwhile, BmGSTd3 and BmGSTe5 presented differences in development course and sex. The other high-expression genes were non-specific class GSTs, which showed persistent expression in development course and similar expression in female and male. The low or no expressions of GSTs were belonged to insect specific classes. Thus, the expression of insect specific GSTs showed differences in development course and sex may be related to its important function.RT-PCR experiments detected the expression signals of all the GSTs in multiple tissues on day three of the fifth instar larvae. The GSTs of non-specific classes expressed in almost all the tissues, and its expression level was very high. While, insect specific GSTs showed specific expression in tissues, especially, BmGSTe2 and BmGSTe6 were only detected in midgut. In addition, we found no expression evidence or low expression levels of most Delta and Epsilon GSTs in the fat body which was thought to be the main detoxification organ. This may explain the sensitivity of the silkworm to certain insecticides. After the LC₅₀ of phoxim was used to treat with the day three of fifth instar larva, BmGSTe8 was detected up-regulated expression in fat body by RT-PCR and real time quantitative PCR. It indicated that BmGSTe8 could be induced in the course of oxidized stress and may play important role in the stress reaction. In addition, we cloned 12 silkworm GST genes by RT-PCR and deposited to GenBank.4. GSTs expression in the silkworm selected with insecticides The silkworms were selected with phoxim and fenpropathrin, respectively. After selection for five and four generations, respectively, the 50 % lethal dosages (LD₅₀) of the silkworm selected with phoxim and fenpropathrin were a multiple of 1.78 and 4.18 relative to control (F₀). The resistance of the silkworm selected with fenpropathrin was rapidly developed. In the F₅ (the fifth generation selected with phoxim) silkworms, the specific activities toward CDNB and CHP of fat body GSTs were higher than control (F₀), and in midgut, the GST activity toward CHP was also increased. In the F₄ (the fourth generation selected with fenpropathrin) silkworms, the fat body and midgut GST activities toward CDNB and CHP were also increased, respectively. The K₍m (CDNB) of GSTs in insecticides selected silkworms was similar with control, but V₍max(CDNB) was increased similar with GST activities. Thus, the increased activities of GSTs might be regulated by the transcription of GST genes. And we found two up-regulated GST genes (BmGSTe3and BmGSTe2) in the fat body and midgut through RT-PCR and real time quantitative PCR. So the up-regulated expression of BmGSTe3and BmGSTe2 might be resulted in the increase of GST activities, and it might be one of the important mechanisms for the increase of insecticide resistance in the silkworm selected with phoxim and fenpropathrin.5. Expression of BmGSTe3 in Bac-to-Bac baculovirus expression systemBmGSTe3 might be an important insecticide resistant gene in the silkworm. We cloned the cDNA of BmGSTe3 and obtained the recombinant of Bacmid-BmGSTe3 to use to express the recombinant protein of BmGSTe3. The recombinant baculovirus was transfected into sf-9 cells by Cellfectin reagent. At 72h post-transfection, sf9 culture medium and cell pellet were used for SDS-PAGE, and objective bands were obtained in the two samples. It indicated that BmGSTe3 expressed in the sf-9 cells. And then, we used P4 generation of recombinant baculovirus to transfect into sf-9 cells. We detected the GST activities toward CDNB in culture medium and cell lysate, and both were higher than those transfected with wild Bacmid. Thus, the activities of GSTs were further validated that the BmGSTe3 expressed correctly in sf-9 cells.