Characterization Of Glutathione S-Transferase Genes From The Aquatic Midege Chironomus Tentans And Application Of Insecticidal Activity Of The Total Alkaloid From Tripterygium Wilfordii Hook

Heavy uses of chemical fertilizers and synthetic chemical pesticides have led to significant water eutrophication and pesticide residue contaminations, which have not only affected non-target organisms in the ecosystem, but also harmed to the residents' health through water and food, and become a growing global concern. Environmental pesticides can affect non-target organisms harmfully through modifying many metabolic pathways involving many enzymes and proteins. As an ecologically important bioindicator species due to the significant role in food webs and occurrence in various aquatic habitats worldwide, the aquatic midge (Chironomus tentans) is a well recognized and widely used insect species for studying the impact of environmental pollutants in aquatic systems in the past decades. Although the application of the aquatic midge as bioindicator species has been explored by domestic and international scientists, glutathione S-transferases as an important phase II detoxification enzyme family have not been characterized in detail. To comprehensively characterize midge's toxicological and molecular responses to environmental pesticides and other toxic stressors, we have developed an expressed sequence tag (EST) database containing over 10,000 sequences from a C. tentans cDNA library. From the EST database, we have identified a great deal of important detoxification enzymes and physiologically important heamglobin. Specifically, a total of 11 cDNAs encoding full-length GSTs were obtained and a relatively detailed analysis of these genes was carried out. We explored molecular characteristics of the 11 GST genes, their developmental stage- and tissue-specific expression patterns, and the effect of alachlor on total GSTs activities and gene expressions. Our study provides the first insight into molecular characteristics of GSTs and their transcriptional response to alachlor exposures in C. tentans. Furthermore, to encounter public health nuisance caused by the outbreak of non-biting mosquito, we studied larvicidal ctivity of alkaloids from Tripterygium. wilfordii against aquatic midges,optimized extracting technology and micro-emulsifier(ME)formulation processing. The main results and conclusions were outlined as follows:1. EST from the aquatic midge was analyzed by using Blast2go, NCBI blastx and other online programs. A number of important ESTs putatively encoding detoxification enzymes and physiologically important heamglobin, including 29 cytochrome P450, 11 GSTs, 14 esterases, 7 alcohol dehydrogenases, 3 metallothionein and 89 hemoglobin were identified and analyzed. Those findings provided a basis for further investigation on genes of aquatic midge.2. 11 full-length GSTs cDNA sequences were obtained and phylogenetically analyzed. Phylogenetic analysis of these 11 GSTs deduced from their cDNAs revealed 7 GSTs that belong to three different cytosolic classes, including 2 in delta, 4 in sigma and 1 in omega, based on their sequence similarities to other insect GSTs, particularly those from Anopheles gambiae and Drosophila melanogaster. The remaining four GSTs were unclassified due to the lack of homologies to the currently known class. The nomenclature of these C. tentans GSTs cDNA and their deduced amino acid sequences have been deposited in GenBank with the following accession numbers: FJ851365(CtGSTd1), FJ851366(CtGSTd2), FJ851367 (CtGSTs1), FJ851368(CtGSTs2), FJ851369(CtGSTs3), FJ851370(CtGSTs4), FJ851371(CtGSTu1), FJ851372 (CtGSTu2), FJ851373 (CtGSTu3), FJ851374(CtGSTu4) and FJ851375 (CtGSTo1) Furthermore, several conserved amino acid residues which represent the catalytic pocket and binding site were analysised by sequence alignment.3. Tissue-specific expression patterns of the 11 C. tentans GSTs genes were analyzed in each of six different tissues, including salivary glands, hemolymph, midgut, Malpighian tubules, fatbodies, and cuticle, by using semi-quantitative RT-PCR. Stage-specific expression patterns of C. tentans GSTs genes were determined in eggs, four different larval instars (1st, 2nd, 3rd and 4th), pupae and adults by using semi-quantitative RT-PCR. Such tissue- and stage- specific expression patterns may reflect specific roles and functions of the GSTs genes within different tissue and during the midge development.4. After treatment with alachlor for 72h at 10, 100, 1,000μg/L, GSTs activity of fourth-instar midges were reduced by 1.3-, 1.7-, and 2.2-fold, respectively, compared with the control when CDNB were used as a substrate. Similarly, alachlor at 10, 100, 1,000μg/L reduced the GSTs activity by 1.3-, 1.7-, and 1.8-fold, respectively, when DCNB was used as a substrate. Semi-quantitative RT-PCR results clearly showed that alachlor significantly increased the mRNA levels of the CtGSTd1, CtGSTs2 and CtGSTs3 genes in an alachlor concentration-dependent manner in the midges exposed to alachlor. These results suggested that alachlor can induce the expression of some genes in at least two classes of the GSTs gene family, including delta (CtGSTd1) and sigma (CtGSTs2, CtGSTs3). Real-time quantitative PCR results showed the exposure of forth-instar larvae to alachlor at 1,000μg/L for 72 h increased the CtGSTd1, CtGSTs2 and CtGSTs3 mRNA levels by 2.1-, 2.8- and 4.3-fold, respectively. Those GST genes can be used as biochemical marker for environment pollution monitoring.5. The Tripterygium wilfordii alkaloids showed a high toxicity to fourth-instar larvae of the aquatic midge with the LC50 values of 33.08,19.09 and 16.76μg/L at 24 , 48 and 72 h, respectively. After the treatments with T. wilfordii alkaloids at 5,10,15μg/L for 72h, GSTs activity of fourth-instar midges were reduced by 2.37-, 2.82-, and 3.77-fold, respectively, compared with the control when CDNB were used as a substrate. Similarly, the GSTs activity was reduced by 2.34-, 2.79-, and 3.73-fold, respectively, when DCNB was used as a substrate. Semi-quantitative RT-PCR results clearly showed that alachlor significantly increased the mRNA levels of the CtGSTu3 and CtGSTu4 genes in a T. wilfordii alkaloid concentration-dependent manner in the midges exposed to T. wilfordii alkaloids. These results indicated that T. wilfordii alkaloids were highly toxic to aquatic midges and can be potentially used to develop a mosquito control agent.6. The extracting techniques for the alkaloids from the root bark of T. wilfordii by using ethyl acetate were evaluated both at small laboratory experimental and pilot-plant scales. The optimal extraction conditions were obtained as follows: root bark powder 30 mesh, temperature 50-60oC, duration of extraction 8 h for first time and 4h for another 4 tiems. Applications of these extracting conditions can reduce the costs of production, decrease the energy consuming, and increase the alkaloid yields. This extraction technique appears to be feasiable and practicable.7. The alkaloids extracted from the root bark of T. wilfordii were formulated to micro-emulsifier (ME) by the way of combining researches in physical and chemical characters with laboratory bioassay and field trial. The solvent and emulsifier which suited 1.0% T. wilfordii alkaloids ME had been screened out and successfully developed. Its quality index met the standard of a commercial pesticide. The 1.0% T. wilfordii alkaloids ME possesses excellent insecticidal activities, and has a great potential for its development and application.