BdGSTe8介导桔小实蝇对马拉硫磷抗性的分子机理研究
发布时间：2018-07-31 13:07
【摘要】：桔小实蝇Bactrocera dorsalis(Hendel)是热带和亚热带地区严重危害农业生产的重要害虫,化学杀虫剂的使用是目前最主要的防治方法,由于长期不合理的使用杀虫剂,桔小实蝇对于多种常用的杀虫剂产生了不同程度的抗性,明确其抗性机理对于合理的防治策略的提出以及更有效的杀虫剂的开发具有重要的意义。谷胱甘肽硫转移酶(GSTs)是否参与桔小实蝇对有机磷杀虫剂的解毒代谢从而介导抗性目前尚不明确。据此,本学位论文通过桔小实蝇转录组数据库筛选获得1个GSTs基因BdGSTe8,克隆获得开放阅读框并进行序列分析的基础上,分析该基因在桔小实蝇体内的表达分布特点和在马拉硫磷抗性和敏感品系中的表达差异;采用RNAi技术解析该基因在桔小实蝇马拉硫磷抗性中所起的作用;最后通过原核表达以及药剂代谢测定等实验明确该基因在代谢马拉硫磷过程中的作用机理。本研究旨在为全面揭示桔小实蝇代谢抗性分子机理提供基础。主要研究结果如下:1桔小实蝇BdGSTe8的筛选和鉴定采用RT-PCR技术全面解析基因的序列信息,结果显示该基因长度为666 bp,共编码221个氨基酸,系统发育分析表明目的基因属于epsilon家族,命名为BdGSTe8。通过软件和NCBI网站对其预测,结果显示BdGSTe8具有GSTs家族特有的保守位点:包括N端的6个GSH结合位点(G-site)和C端的10个底物结合位点(H-site)。其中昆虫delta和epsilon家族GSTs特有的Ser12高度保守,暗示是有活性的GSTs。通过对桔小实蝇抗性和敏感品系在cDNA水平和基因组水平扩增得到的序列进行分析,结果表明扩增得到了2条具有多态性的BdGSTe8。其中敏感品系仅存在BdGSTe8-A,抗性品系同时存在BdGSTe8-A和BdGSTe8-B一对等位基因。BdGSTe8-A和BdGSTe8-B核苷酸序列一致性为95%,氨基酸序列一致性为91%,其N端相对保守而C端具有多态性,暗示参与的生理功能可能存在差异。RT-qPCR分析BdGSTe8在桔小实蝇体内的分布情况,结果显示其在桔小实蝇的各个发育阶段都有所表达,在成虫期的表达水平随着日龄的增加表达量也不断上升,至成虫第7 d时表达量最高。在桔小实蝇各个组织和体段中也均有所表达,其中在马氏管表达量最高,而在生殖器官中表达量最低,说明BdGSTe8主要是在成虫体内解毒代谢器官中发挥着重要的作用。采用RT-qPCR技术分析其在抗性敏感中表达量差异,结果显示BdGSTe8在桔小实蝇抗性品系中通过转录因子增强使其表达量上调。上述结果暗示BdGSTe8可能参与介导桔小实蝇对马拉硫磷抗性的产生。2基于RNAi技术的BdGSTe8功能分析采用RNAi技术抑制BdGSTe8在抗性和敏感品系桔小实蝇体内的表达量,通过检测基因特异性沉默效率并进行马拉硫磷生物测定,从而深入简析此基因在介导桔小实蝇对马拉硫磷抗性形成中的作用。结果显示注射dsBdGSTe8后,桔小实蝇成虫的BdGSTe8转录水平显著下调,且在注射24 h后沉默效果最好。进一步对沉默目的基因后的桔小实蝇抗性和敏感品系试虫进行马拉硫磷敏感性测定,结果表明药剂处理24 h后,敏感品系试虫的死亡率与对照组相比无显著性差异,均在40%左右,但抗性品系试虫死亡率显著上升至58.4%。由于抗性品系BdGSTe8过表达,抑制其在抗性品系中的表达严重影响了桔小实蝇对马拉硫磷的解毒代谢,说明BdGSTe8参与介导桔小实蝇对马拉硫磷抗性的发展。3基于原核表达技术的离体BdGSTe8代谢能力解析采用原核表达和离体药剂代谢测定等技术在体外进一步明确两种BdGSTe8介导抗性的差异。SDS-PAGE结果显示在25 kD处有条带,与软件预测的条带大小基本一致;GSTs比活力测定结果显示重组蛋白BdGSTe8具有GSTs活性,说明获得了具有蛋白活性的BdGSTe8。细胞生物测定结果显示,表达有BdGSTe8-B的细胞相比于表达有BdGSTe8-A的细胞存活率更高,加入GSTs专一性抑制剂(DEM)后表明造成这种差异的原因是外源GSTs的表达,说明两种多态性的解毒能力存在差异。采用HPLC技术进一步对重组蛋白代谢能力进行测定,结果表明BdGSTe8-A处理在出峰时间为5 min的峰面积与对照相比无显著性变化,其它位置的出峰时间和峰面积也无显著性变化,说明BdGSTe8-A不能直接代谢马拉硫磷。BdGSTe8-B处理在5 min的峰面积与对照相比显著减少,并且在2 min左右出现一个新峰,可能为代谢出的一种新物质。进一步采用液质联用仪对该物质进行测定分析,发现确为一种新的代谢产物。采用定点突变的方法分析BdGSTe8-A和Bd GSTe8-B代谢马拉硫磷能力差异的原因,结果显示V128A是最重要的差异位点,说明抗性的产生有可能是这个位点的差异造成的。
[Abstract]:Bactrocera dorsalis (Hendel) is an important pest of agricultural production in tropical and subtropical areas. The use of chemical insecticides is the most important control method at present. Due to the long-term irrational use of insecticides, it has produced different degrees of resistance to a variety of commonly used insecticides and clear its resistance mechanism. It is of great significance to put forward a reasonable control strategy and to develop more effective insecticides. It is not clear whether the glutathione thiotransferase (GSTs) is involved in the detoxification of organophosphorus insecticides by the orange fly, and 1 GSTs is obtained through the screening of the transcriptional database of the orange fly. On the basis of an open reading frame and sequence analysis, the gene BdGSTe8 was cloned to analyze the expression distribution characteristics of the gene and the difference in the expression of malathion resistance and sensitive strain in the malathion resistance. The role of the gene in malathion resistance of the fruit fly was analyzed by RNAi. Finally, the expression of the gene was expressed in the prokaryotic expression. The main research results are as follows: 1 the screening and identification of the BdGSTe8 of citricus CITRIS was analyzed and identified by RT-PCR technique. The length of the gene is 666 BP, encoding a total of 221 amino acids. Phylogenetic analysis shows that the target gene belongs to the epsilon family, which is named BdGSTe8. through the software and NCBI website. The results show that BdGSTe8 has a specific conservative site of the GSTs family, including 6 GSH junction sites (G-site) at the N end and 10 substrate binding sites at the C end (H-si) TE). Among them, the insect Delta and the epsilon family GSTs are highly conserved, suggesting that the active GSTs. is analyzed by the sequence of the resistance of the flies and the sequence of the susceptible strains at cDNA level and genomic level. The results show that 2 polymorphic BdGSTe8. of them have been amplified by the amplification of only BdGSTe8-A. The consistency of.BdGSTe8-A and BdGSTe8-B nucleotide sequences of the one peer-to-peer gene of BdGSTe8-A and BdGSTe8-B was 95%, the consistency of the amino acid sequence was 91%, the N end was relatively conservative while the C end was polymorphic, suggesting the possible existence of the physiological function of the participation of BdGSTe8 in the distribution of the.BdGSTe8-A in the orange fly. It was expressed in every stage of the growth of the fruit fly, and the expression level of the adult period increased with the increase of day age. The expression was highest when the adult was seventh D. The expression was also expressed in every tissue and body segment of the adult. The expression of the martensitic tube was the highest, but the expression in the reproductive organs was the lowest. BdGSTe8 mainly plays an important role in the detoxification of metabolic organs in the adult. RT-qPCR technique is used to analyze the difference of expression in resistance sensitivity. The results show that BdGSTe8 is enhanced by the enhancement of transcription factors in the resistant strain of the fruit fly. The results suggest that BdGSTe8 may be involved in mediating orange fly to horses. The production of.2 based on the BdGSTe8 function analysis of RNAi technology, the expression of BdGSTe8 in the resistance and sensitive strain of C. orange was inhibited by RNAi technique, and the gene specific silencing efficiency was detected and the biometric determination of malathion was carried out, thus the gene was deeply analyzed to mediate the formation of malathion resistance in the malathion. The results showed that after the injection of dsBdGSTe8, the BdGSTe8 transcriptional level of the adult of the adult was significantly down, and the silencing effect was best after the injection of 24 h. The sensitivity of the resistance and the sensitive strain of the susceptible strain of the insect after the silencing of the target gene was further measured. The results showed that the susceptible strain was killed after 24 h treatment. There was no significant difference in the death rate compared with the control group, all at about 40%, but the death rate of the resistant strain increased significantly to 58.4%. due to the overexpression of the resistant strain BdGSTe8, and the inhibition of its expression in the resistant strain affected the detoxification of malathion by the fruit fly, indicating that BdGSTe8 was involved in mediating resistance to malathion. Development of.3 based on prokaryotic expression technology in vitro BdGSTe8 metabolism ability analysis using prokaryotic expression and isolation of drug metabolism assay in vitro to further clarify the difference between two kinds of BdGSTe8 mediated resistance.SDS-PAGE results show that there is a strip at 25 kD, which is basically the same as the band size predicted by the software; GSTs specific activity determination results show that The recombinant protein BdGSTe8 has the activity of GSTs, indicating that the biometric results of BdGSTe8. cells with protein activity showed that the cells expressing BdGSTe8-B had a higher survival rate than those expressing BdGSTe8-A, and after adding the GSTs specificity inhibitor (DEM), the reason for this difference was the expression of exogenous GSTs, indicating two kinds of more. HPLC technique was used to determine the metabolic ability of recombinant protein. The results showed that the peak area of BdGSTe8-A treatment at the peak time of 5 min had no significant change, and there was no significant change in the peak time and peak area of other positions, indicating that BdGSTe8-A could not directly metabolize the Mala sulphur. The peak area of P.BdGSTe8-B treatment at 5 min was significantly reduced compared with the control, and a new peak appeared at about 2 min, which might be a new metabolite. Further, the liquid chromatograph was used to determine the substance, and it was found to be a new metabolite. The point mutation method was used to analyze the BdGSTe8-A and Bd GSTe8-B generation. The results showed that V128A was the most important difference site, suggesting that the resistance was probably caused by the difference of this site.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433
本文编号：2155671
[Abstract]:Bactrocera dorsalis (Hendel) is an important pest of agricultural production in tropical and subtropical areas. The use of chemical insecticides is the most important control method at present. Due to the long-term irrational use of insecticides, it has produced different degrees of resistance to a variety of commonly used insecticides and clear its resistance mechanism. It is of great significance to put forward a reasonable control strategy and to develop more effective insecticides. It is not clear whether the glutathione thiotransferase (GSTs) is involved in the detoxification of organophosphorus insecticides by the orange fly, and 1 GSTs is obtained through the screening of the transcriptional database of the orange fly. On the basis of an open reading frame and sequence analysis, the gene BdGSTe8 was cloned to analyze the expression distribution characteristics of the gene and the difference in the expression of malathion resistance and sensitive strain in the malathion resistance. The role of the gene in malathion resistance of the fruit fly was analyzed by RNAi. Finally, the expression of the gene was expressed in the prokaryotic expression. The main research results are as follows: 1 the screening and identification of the BdGSTe8 of citricus CITRIS was analyzed and identified by RT-PCR technique. The length of the gene is 666 BP, encoding a total of 221 amino acids. Phylogenetic analysis shows that the target gene belongs to the epsilon family, which is named BdGSTe8. through the software and NCBI website. The results show that BdGSTe8 has a specific conservative site of the GSTs family, including 6 GSH junction sites (G-site) at the N end and 10 substrate binding sites at the C end (H-si) TE). Among them, the insect Delta and the epsilon family GSTs are highly conserved, suggesting that the active GSTs. is analyzed by the sequence of the resistance of the flies and the sequence of the susceptible strains at cDNA level and genomic level. The results show that 2 polymorphic BdGSTe8. of them have been amplified by the amplification of only BdGSTe8-A. The consistency of.BdGSTe8-A and BdGSTe8-B nucleotide sequences of the one peer-to-peer gene of BdGSTe8-A and BdGSTe8-B was 95%, the consistency of the amino acid sequence was 91%, the N end was relatively conservative while the C end was polymorphic, suggesting the possible existence of the physiological function of the participation of BdGSTe8 in the distribution of the.BdGSTe8-A in the orange fly. It was expressed in every stage of the growth of the fruit fly, and the expression level of the adult period increased with the increase of day age. The expression was highest when the adult was seventh D. The expression was also expressed in every tissue and body segment of the adult. The expression of the martensitic tube was the highest, but the expression in the reproductive organs was the lowest. BdGSTe8 mainly plays an important role in the detoxification of metabolic organs in the adult. RT-qPCR technique is used to analyze the difference of expression in resistance sensitivity. The results show that BdGSTe8 is enhanced by the enhancement of transcription factors in the resistant strain of the fruit fly. The results suggest that BdGSTe8 may be involved in mediating orange fly to horses. The production of.2 based on the BdGSTe8 function analysis of RNAi technology, the expression of BdGSTe8 in the resistance and sensitive strain of C. orange was inhibited by RNAi technique, and the gene specific silencing efficiency was detected and the biometric determination of malathion was carried out, thus the gene was deeply analyzed to mediate the formation of malathion resistance in the malathion. The results showed that after the injection of dsBdGSTe8, the BdGSTe8 transcriptional level of the adult of the adult was significantly down, and the silencing effect was best after the injection of 24 h. The sensitivity of the resistance and the sensitive strain of the susceptible strain of the insect after the silencing of the target gene was further measured. The results showed that the susceptible strain was killed after 24 h treatment. There was no significant difference in the death rate compared with the control group, all at about 40%, but the death rate of the resistant strain increased significantly to 58.4%. due to the overexpression of the resistant strain BdGSTe8, and the inhibition of its expression in the resistant strain affected the detoxification of malathion by the fruit fly, indicating that BdGSTe8 was involved in mediating resistance to malathion. Development of.3 based on prokaryotic expression technology in vitro BdGSTe8 metabolism ability analysis using prokaryotic expression and isolation of drug metabolism assay in vitro to further clarify the difference between two kinds of BdGSTe8 mediated resistance.SDS-PAGE results show that there is a strip at 25 kD, which is basically the same as the band size predicted by the software; GSTs specific activity determination results show that The recombinant protein BdGSTe8 has the activity of GSTs, indicating that the biometric results of BdGSTe8. cells with protein activity showed that the cells expressing BdGSTe8-B had a higher survival rate than those expressing BdGSTe8-A, and after adding the GSTs specificity inhibitor (DEM), the reason for this difference was the expression of exogenous GSTs, indicating two kinds of more. HPLC technique was used to determine the metabolic ability of recombinant protein. The results showed that the peak area of BdGSTe8-A treatment at the peak time of 5 min had no significant change, and there was no significant change in the peak time and peak area of other positions, indicating that BdGSTe8-A could not directly metabolize the Mala sulphur. The peak area of P.BdGSTe8-B treatment at 5 min was significantly reduced compared with the control, and a new peak appeared at about 2 min, which might be a new metabolite. Further, the liquid chromatograph was used to determine the substance, and it was found to be a new metabolite. The point mutation method was used to analyze the BdGSTe8-A and Bd GSTe8-B generation. The results showed that V128A was the most important difference site, suggesting that the resistance was probably caused by the difference of this site.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433
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