【作者】 张琪；

【导师】 戴武；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2016， 硕士

【摘要】 谷胱甘肽S-转移酶(Glutathione S-transferases,GSTs,EC 2.5.1.18)是一类广泛分布于动植物、微生物等有机体内的多功能酶。GSTs主要功能是代谢生物体内的有毒物质,从而避免或降低机体受到损伤,与昆虫的抗药性相关。韭菜迟眼蕈蚊(Bradysia odoriphaga Yang et Zhang)是韭菜等百合科蔬菜上的重要害虫,主要以幼虫聚集在韭菜地下鳞茎处进行取食危害。目前,生产上主要采用化学农药灌根进行防治,造成韭菜农药残留超标,食物中毒事件频发。由于化学农药的不合理使用导致该虫的抗药性增强,给防治工作带来困难。本研究对韭菜迟眼蕈蚊谷胱甘肽S-转移酶进行克隆、表达并初步分析其功能,有助于了解韭菜迟眼蕈蚊抗性形成的分子机制,为农业生产中韭菜迟眼蕈蚊的防治提供理论依据。本研究主要结果如下:1.韭菜迟眼蕈蚊谷胱甘肽S-转移酶基因克隆与生物信息学分析利用RT-PCR和RACE技术克隆获得两个韭菜迟眼蕈蚊谷胱甘肽S-转移酶基因,即BoGSTd1和BoGSTd2。BoGSTd1开放阅读框为636bp,编码211个氨基酸,其等电点和蛋白分子量大小分别为5.94和23.72kDa。BoGSTd2开放阅读框为642bp,编码213个氨基酸,其等电点和蛋白分子量大小分别为5.12和23.78kDa。通过序列对比以及系统发育分析可知,两个基因均为Delta家族的GSTs基因。两者均无信号肽和跨膜区并且两个基因组DNA均不包含内含子。BoGSTd1和BoGSTd2氨基酸序列相似性为55.61%。2.韭菜迟眼蕈蚊BoGSTd1和BoGSTd2时空表达利用Real-time PCR技术测定不同发育时期和不同组织BoGSTd1和BoGSTd2的表达量。结果显示,韭菜迟眼蕈蚊BoGSTd1和BoGSTd2在其发育的各个时期均有表达,BoGSTd1在卵期表达量最高,三、四龄幼虫表达量次之,一、二龄幼虫和蛹期表达量相对较少。BoGSTd2则在卵期表达量最高,其余各个时期表达量差异不显著。BoGSTd1和BoGSTd2在不同组织中表达量存在显著差异。BoGSTd1和BoGSTd2在成虫体内的分布较为相似,在腹部表达量最高,头部和胸部次之,其他组织中表达量很少甚至不表达。3.韭菜迟眼蕈蚊谷胱甘肽S-转移酶原核表达及活性测定利用原核表达系统在大肠杆菌BL21(DE3)中表达并纯化获得大小约为25kDa的可溶性体外重组蛋白BoGSTd1和BoGSTd2。用BCA蛋白定量试剂盒测定其浓度,结果显示,BoGSTd1和BoGSTd2体外重组蛋白的浓度分别为37.29 mg/ml和21.25 mg/ml。SDS-PAGE结果表明,两个纯化后的重组蛋白具有较高的纯度。以CDNB和GSH为底物,测定其活性,结果表明两个重组蛋白均能催化GSH与CDNB发生共轭反应,BoGSTd1最大反应速率Vmax为1062±60.84μmol/mg/min,米氏常数Km为0.35±0.05 mM,BoGSTd2动力学常数Vmax和Km分别为290.4±11.42μmol/mg/min和0.24±0.02 mM。GSTs在不同的pH值和温度条件下表现出的活性也存在差异,BoGSTd1和BoGSTd2都表现出随缓冲液pH值的升高活性先升高随后降低的趋势。BoGSTd1在pH约为8.0-8.5时活性最高,而BoGSTd2在缓冲液pH约为7时活性最高。体外重组蛋白BoGSTd1和BoGSTd2在40℃以下时,均表现出较高的活性。4.杀虫剂对酶活性的影响测定了三种杀虫剂对两个重组蛋白活性的影响。结果显示,浓度为0.25 mM的甲基毒死蜱、高效氯氟氰菊酯、西维因处理后,BoGSTd1和Bo GSTd2活性均显著下降。其中甲基毒死蜱对酶活性的抑制作用最为明显,BoGSTd1和BoGSTd2残留酶活仅为11.7%和5.44%。测定了上述药剂对BoGSTd1和BoGSTd2酶活性的抑制中浓度(IC50)。结果显示,随着药剂浓度的增加,BoGSTd1酶活性逐渐降低。甲基毒死蜱、高效氯氟氰菊酯和西维因对BoGSTd1的IC50分别为0.19 mM、0.21m M和0.22 mM。随着药剂浓度的增加,BoGSTd2活性先逐渐增大然后减小直至完全被抑制。当甲基毒死蜱、高效氯氟氰菊酯和西维因浓度分别达到0.25 mM、0.3125 mM和0.3125 mM时,BoGSTd2活性被完全抑制。谷胱甘肽S-转移酶抑制剂马来酸二乙酯显著地抑制了BoGSTd1和BoGSTd2活性,随着抑制剂浓度的升高蛋白活性逐渐降低,马来酸二乙酯对重组蛋白BoGSTd1和BoGSTd2的抑制中浓IC50分别为0.041 mM和0.038 mM。更多还原

【Abstract】 Glutathione S-transferases(GSTs, EC 2.5.1.18), a superfamily of multifunctional enzymes, have been found widely exsisting in plants, animals and microorganisms. GSTs play a crucial role in metabolizing xenobiotics and endogenous compounds to protect organisms from damage of the toxins and are also closely related to insecticide resistance. Bradysia odoriphaga is the major pest of Chinese chives, which mainly feeds on the roots and stems of Chinese chives. The primary management practice for controlling B. odoriphaga is the application of synthetic insecticides. However, excessive use of chemical insecticides causes pollution in the environment and leaves high residues on marketed Chinese chives which affects the human health. Furthermore, it is difficult to control because of increasing resistance. In this study, the glutathione S-transferases Bo GSTd1 and Bo GSTd2 in B. odoriphaga were cloned, and the expression levels of these genes in different developmental stages and tissues were analyzed by qPCR. Their functions were preliminarily analyzed to understand the mechanisms of resistance. The results are summarized as follows: 1. Cloning and sequence analysis of glutathione S-transferase genes in B. odoriphagaBy RT-PCR and RACE techniques,two glutathione S-transferase genes,BoGSTd1 and BoGSTd2 were cloned in B. odoriphaga. BoGSTd1 contained an open reading frame of 636 bp that encodes a protein of 211 amino acids with a predicted molecular weight of 23.72 kDa and with a theoretical isoelectric point of 5.94. BoGSTd2 has an open reading frame of 642 bp that encodes a protein of 213 amino acids with a predicted molecular weight of 23.78 kDa and with a theoretical isoelectric point of 5.12. The absence of a signal peptide and transmembrane domain in BoGSTd1 and BoGSTd2 secondary structures suggests that both of them are cytosolic GST proteins. By sequence alignment and phylogenetic analysis, the results indicate that BoGSTd1 and BoGSTd2 belong to Delta class. There was no intron in the BoGSTd1 and BoGSTd2 genes. Amino acids sequences of the two GSTs share 55.61% identity. 2. The spatiotemporal expression pattern of BoGSTd1 and BoGSTd2The expression patterns in different developmental stages and tissues of BoGSTd1 and BoGSTd2 were measured by quantitative real-time PCR. The results showed that both BoGSTd1 and BoGSTd2 were expressed in all developmental stages. BoGSTd1 was mainly expressed in the egg, followed by the 3rd- and 4th-instar larvae stage, but was weakly expressed in the 1st- and 2nd-instar larvae stage and the pupal stage. BoGSTd2 was mainly expressed in the egg, but expression of BoGSTd2 was weakly with no significant differences in other stages. Both BoGSTd1 and BoGSTd2 were mainly expressed in the abdomen, followed by the head and thorax, and were weakly expressed in legs and wings, and there is almost no expression of BoGSTd1 and BoGSTd2 in the antenna. 3. Prokaryotic expression and catalytic activity analysis of BoGSTd1 and BoGSTd2The BoGSTd1 and BoGSTd2 were expressed in E. coli BL21(DE3). The target recombinant proteins were purified through Ni2+-NTA agarose and two ~ 25 kDa soluble recombinant proteins were obtained. The concentrations of the purified proteins were 37.29mg/ml and 21.25mg/ml, respectively. Kinetic parameters of recombinant BoGSTd1 and BoGSTd2 were measured using CDNB as substrate. The results show that the Vmax and Km of BoGSTd1 were 1062μmol/min/mg and 0.35 mM, respectively. The Vmax and Km of BoGSTd2 were 290.4 μmol/min/mg and 0.24 mM, respectively. The activities of both BoGSTd1 and BoGSTd1 first increased and then declined with increasing pH. The optimal reaction pH range of the BoGSTd1 is 8.0- 8.5; the optimal reaction pH of BoGSTd2 is about 7.0. When the temperature is below 40℃, both BoGSTd1 and BoGSTd2 show high activities. 4. Effect of insecticides and inhibitor on BoGSTs activitiesIn vitro the effects of insecticides on inhibition of GSTs activities were measured. The results indicated that the activities of the enzymes were obviously inhibited in the precence of 0.25 mM chlorpyrifos-methyl, lambda-cyhalothrin and carbaryl. After treatment chlorpyrifos-methyl, the remaining enzyme activities of BoGSTd1 and BoGSTd2 were 11.7% and 5.44% as compared with the control, respectively. The activity of BoGSTd1 reduced with the increasing concentration of insecticides. The IC50 values were 0.19 mM, 0.21 mM and 0.22 mM for chlorpyrifos-methyl, lambda-cyhalothrin and carbaryl, respectively. The activity of the BoGSTd2 protein was increased at low concentrations of insecticides. With the increase of insecticide concentration, the activity of BoGSTd2 reached a peak and eventually started to decline. The activity of BoGSTd2 was inhibited totally in the presence of 0.25 mM chlorpyrifos-methyl, 0.3125 mM lambda-cyhalothrin or 0.3125 mM carbaryl. The values of IC50 of the Diethyl maleate(an inhibitor of GSTs) for BoGSTd1 and BoGSTd2 were 0.041 mM and 0.038 mM.更多还原