The Detoxification Function Of Glutathione S-transferase Genes In Locusta Migratoria On Insecticides

Locusta migratoria is one of the important agricultural pests worldwide. Flood, drought and locust plague were the three biggest agricultural disasters in Chinese history, locust plague brought great influence to agricultural production[5]. Organophosphate insecticides have been used extensively to control the locusta, which makes an increasing resistance for the L. migratoria and has a serious impact on the agro-ecological ecological environment, the insecticides resistance may be related to the metabolic detoxification of glutathione S-transferase (GST). In order to carrying out effective management strategies of locust, glutathione S-transferases of L. migratoria were studied in this thesis, The main contents were as follows:1. The function of glutathione S-transferases in L. migratoriaAmong the10LmGSTs, five representative genes (LmGSTs3, LmGSTs5, LmGSTd1, LmGSTt1, and LmGSTul) were chosen for RNAi. The second instar-nymph of locusts were injected3μg representative LmGSTs dsRNA or deionized water (control), respectively. The bioassay was conducted after24h dsRNA injection,3υL drop of pesticides (chlorpyrifos, deltamethrin, malathion, carbaryl or DDT) was topically applied for each nymph. The nymph mortalities after carbaryl treatment increased38.7%,28.2%, and12.0%after LmGSTs3, LmGSTs5, and LmGSTul were silenced, respectively. The nymph mortalities after malathion treatment increased from13.9%to40%after LmGSTs5was silenced, meanwhile the nymph mortality after chlorpyrifos treatment increased from39.4%to57.5%after LmGSTul was silenced. Neither deltamethrin nor DDT showed significant changes in nymph mortalities after all the five LmGSTs were silenced. These results indicated that the different LmGSTs genes were involved in different insecticides detoxification in the L. migratoria. As validated by RNAi followed by insecticides bioassay, sigma LmGST played a significant role in conferring carbaryl detoxification, and some of sigma LmGST also involved in malathion detoxification. The unclassified LmGST played significant roles in conferring carbaryl and chlorpyrifos detoxification.2.The effects of different insecticides on glutathione S-transferases in L. migratoriaThree kinds of pesticides (chlorpyrifos, malathion or carbaryl) were topically applied to each L. migratoria nymph. The activities of GST were detecte after24h treatment. The mRNA levels of10LmGSTs were analysed by Real-time PCR. The protein expression of four representative LmGST were detected by Western bolt. The results showed that the DCNB and pNBC activities were decreased after chlorpyrifos exposure, while the CDNB activity did not change. Malathion decreased GST activities by all the three kinds of substrates And carbaryl decresed GST activities except for pNBC activity. The mRNA expression of LmGSTd1was elevated, while other LmGSTs were decreased by chlorpyrifos, except for LmGSTsl. The results of four LmGST protein expression were in accordance with that of mRNA levels. LmGSTs2mRNA levels were decreased, while those of LmGSTd1and LmGSTs7, as well as LmGSTd1protein expression, were increased after malathion or carbaryl exposure. The activities and expression of some LmGST were decreased, which might due to the oxidative damage in vivo. It is suggested that LmGSTdl, LmGSTul, and LmGSTs7might play potential roles in resistance of L. migratoria.3.The research on the expression of L. migratoria glutathione S-transferase enzyme in different tissuesTissue-specific expression patterns of four L. migratoria GST genes (LmGSTd1, LmGSTs5, LmGSTt1and LmGSTul) were determined in nine different issues, including foregut, midgut, gastric caecum, hindgut, Malpighian tubules, fatbodies, muscles, spermaries and ovaries by using Western Blot. This thesis indicated that LmGSTd1was expressed in all tissues examined and showed significantly higher expressions in Malpighian tubules, fatbodies and muscles. LmGSTs5was expressed in all tissues examined except foregut and showed significantly higher expressions in Malpighian tubules. LmGSTt1and LmGSTu1were expressed in midgut, gastric caecum, hindgut, Malpighian tubules and fatbodies with highest expressions in the Malpighian tubules. These results suggested that GSTs protein had higher expression levels in the gastrointestinal tract, Malpighian tubules, and fatbodies of L. migratoria, which was constant to our previous conclusion.This thesis focused on the functions of glutathione S-transferase genes in L. migratoria and the effects of insecticides in metabolic detoxification on glutathione S-transferase in L. migratoria, which are expected to provide the basic information on develop effective control of this important insect pest.