BdGSTe8介導(dǎo)桔小實(shí)蠅對(duì)馬拉硫磷抗性的分子機(jī)理研究
發(fā)布時(shí)間：2018-07-31 13:07
【摘要】：桔小實(shí)蠅Bactrocera dorsalis(Hendel)是熱帶和亞熱帶地區(qū)嚴(yán)重危害農(nóng)業(yè)生產(chǎn)的重要害蟲(chóng),化學(xué)殺蟲(chóng)劑的使用是目前最主要的防治方法,由于長(zhǎng)期不合理的使用殺蟲(chóng)劑,桔小實(shí)蠅對(duì)于多種常用的殺蟲(chóng)劑產(chǎn)生了不同程度的抗性,明確其抗性機(jī)理對(duì)于合理的防治策略的提出以及更有效的殺蟲(chóng)劑的開(kāi)發(fā)具有重要的意義。谷胱甘肽硫轉(zhuǎn)移酶(GSTs)是否參與桔小實(shí)蠅對(duì)有機(jī)磷殺蟲(chóng)劑的解毒代謝從而介導(dǎo)抗性目前尚不明確。據(jù)此,本學(xué)位論文通過(guò)桔小實(shí)蠅轉(zhuǎn)錄組數(shù)據(jù)庫(kù)篩選獲得1個(gè)GSTs基因BdGSTe8,克隆獲得開(kāi)放閱讀框并進(jìn)行序列分析的基礎(chǔ)上,分析該基因在桔小實(shí)蠅體內(nèi)的表達(dá)分布特點(diǎn)和在馬拉硫磷抗性和敏感品系中的表達(dá)差異;采用RNAi技術(shù)解析該基因在桔小實(shí)蠅馬拉硫磷抗性中所起的作用;最后通過(guò)原核表達(dá)以及藥劑代謝測(cè)定等實(shí)驗(yàn)明確該基因在代謝馬拉硫磷過(guò)程中的作用機(jī)理。本研究旨在為全面揭示桔小實(shí)蠅代謝抗性分子機(jī)理提供基礎(chǔ)。主要研究結(jié)果如下:1桔小實(shí)蠅BdGSTe8的篩選和鑒定采用RT-PCR技術(shù)全面解析基因的序列信息,結(jié)果顯示該基因長(zhǎng)度為666 bp,共編碼221個(gè)氨基酸,系統(tǒng)發(fā)育分析表明目的基因?qū)儆趀psilon家族,命名為BdGSTe8。通過(guò)軟件和NCBI網(wǎng)站對(duì)其預(yù)測(cè),結(jié)果顯示BdGSTe8具有GSTs家族特有的保守位點(diǎn):包括N端的6個(gè)GSH結(jié)合位點(diǎn)(G-site)和C端的10個(gè)底物結(jié)合位點(diǎn)(H-site)。其中昆蟲(chóng)delta和epsilon家族GSTs特有的Ser12高度保守,暗示是有活性的GSTs。通過(guò)對(duì)桔小實(shí)蠅抗性和敏感品系在cDNA水平和基因組水平擴(kuò)增得到的序列進(jìn)行分析,結(jié)果表明擴(kuò)增得到了2條具有多態(tài)性的BdGSTe8。其中敏感品系僅存在BdGSTe8-A,抗性品系同時(shí)存在BdGSTe8-A和BdGSTe8-B一對(duì)等位基因。BdGSTe8-A和BdGSTe8-B核苷酸序列一致性為95%,氨基酸序列一致性為91%,其N(xiāo)端相對(duì)保守而C端具有多態(tài)性,暗示參與的生理功能可能存在差異。RT-qPCR分析BdGSTe8在桔小實(shí)蠅體內(nèi)的分布情況,結(jié)果顯示其在桔小實(shí)蠅的各個(gè)發(fā)育階段都有所表達(dá),在成蟲(chóng)期的表達(dá)水平隨著日齡的增加表達(dá)量也不斷上升,至成蟲(chóng)第7 d時(shí)表達(dá)量最高。在桔小實(shí)蠅各個(gè)組織和體段中也均有所表達(dá),其中在馬氏管表達(dá)量最高,而在生殖器官中表達(dá)量最低,說(shuō)明BdGSTe8主要是在成蟲(chóng)體內(nèi)解毒代謝器官中發(fā)揮著重要的作用。采用RT-qPCR技術(shù)分析其在抗性敏感中表達(dá)量差異,結(jié)果顯示BdGSTe8在桔小實(shí)蠅抗性品系中通過(guò)轉(zhuǎn)錄因子增強(qiáng)使其表達(dá)量上調(diào)。上述結(jié)果暗示BdGSTe8可能參與介導(dǎo)桔小實(shí)蠅對(duì)馬拉硫磷抗性的產(chǎn)生。2基于RNAi技術(shù)的BdGSTe8功能分析采用RNAi技術(shù)抑制BdGSTe8在抗性和敏感品系桔小實(shí)蠅體內(nèi)的表達(dá)量,通過(guò)檢測(cè)基因特異性沉默效率并進(jìn)行馬拉硫磷生物測(cè)定,從而深入簡(jiǎn)析此基因在介導(dǎo)桔小實(shí)蠅對(duì)馬拉硫磷抗性形成中的作用。結(jié)果顯示注射dsBdGSTe8后,桔小實(shí)蠅成蟲(chóng)的BdGSTe8轉(zhuǎn)錄水平顯著下調(diào),且在注射24 h后沉默效果最好。進(jìn)一步對(duì)沉默目的基因后的桔小實(shí)蠅抗性和敏感品系試蟲(chóng)進(jìn)行馬拉硫磷敏感性測(cè)定,結(jié)果表明藥劑處理24 h后,敏感品系試蟲(chóng)的死亡率與對(duì)照組相比無(wú)顯著性差異,均在40%左右,但抗性品系試蟲(chóng)死亡率顯著上升至58.4%。由于抗性品系BdGSTe8過(guò)表達(dá),抑制其在抗性品系中的表達(dá)嚴(yán)重影響了桔小實(shí)蠅對(duì)馬拉硫磷的解毒代謝,說(shuō)明BdGSTe8參與介導(dǎo)桔小實(shí)蠅對(duì)馬拉硫磷抗性的發(fā)展。3基于原核表達(dá)技術(shù)的離體BdGSTe8代謝能力解析采用原核表達(dá)和離體藥劑代謝測(cè)定等技術(shù)在體外進(jìn)一步明確兩種BdGSTe8介導(dǎo)抗性的差異。SDS-PAGE結(jié)果顯示在25 kD處有條帶,與軟件預(yù)測(cè)的條帶大小基本一致;GSTs比活力測(cè)定結(jié)果顯示重組蛋白BdGSTe8具有GSTs活性,說(shuō)明獲得了具有蛋白活性的BdGSTe8。細(xì)胞生物測(cè)定結(jié)果顯示,表達(dá)有BdGSTe8-B的細(xì)胞相比于表達(dá)有BdGSTe8-A的細(xì)胞存活率更高,加入GSTs專(zhuān)一性抑制劑(DEM)后表明造成這種差異的原因是外源GSTs的表達(dá),說(shuō)明兩種多態(tài)性的解毒能力存在差異。采用HPLC技術(shù)進(jìn)一步對(duì)重組蛋白代謝能力進(jìn)行測(cè)定,結(jié)果表明BdGSTe8-A處理在出峰時(shí)間為5 min的峰面積與對(duì)照相比無(wú)顯著性變化,其它位置的出峰時(shí)間和峰面積也無(wú)顯著性變化,說(shuō)明BdGSTe8-A不能直接代謝馬拉硫磷。BdGSTe8-B處理在5 min的峰面積與對(duì)照相比顯著減少,并且在2 min左右出現(xiàn)一個(gè)新峰,可能為代謝出的一種新物質(zhì)。進(jìn)一步采用液質(zhì)聯(lián)用儀對(duì)該物質(zhì)進(jìn)行測(cè)定分析,發(fā)現(xiàn)確為一種新的代謝產(chǎn)物。采用定點(diǎn)突變的方法分析BdGSTe8-A和Bd GSTe8-B代謝馬拉硫磷能力差異的原因,結(jié)果顯示V128A是最重要的差異位點(diǎn),說(shuō)明抗性的產(chǎn)生有可能是這個(gè)位點(diǎn)的差異造成的。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S433
本文編號(hào)：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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S433
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