桔小实蝇GSTs生化及分子毒理学特性研究
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摘要
桔小实蝇Bactrocera dorsalis (Hendel)隶属双翅目Diptera,实蝇科Tephritidae,果实蝇属Bactrocera Macquar,是一种重要的危险性果蔬害虫。鉴于桔小实蝇在生产上的经济重要性,近年来有关桔小实蝇种群遗传结构、引诱剂研发、化学防治等方面的研究日益增多。然而,长期持续、不合理的使用化学杀虫剂导致其产生了严重的抗药性。目前,桔小实蝇抗性机理方面的研究主要集中于靶标酶-乙酰胆碱酯酶的生化及分子生物学特性,而对于解毒酶系与桔小实蝇抗性发展的关系知之甚少。谷胱甘肽S-转移酶(gluthione S-transferase, GSTs, EC2.5.1.18)是一类多功能超基因家族酶。GSTs作为重要的解毒代谢酶,主要功能是催化一些内源性或外来有害物质的亲电子基团与还原型谷胱甘肽的巯基结合,增加其疏水性使其易于排出体外,从而达到解毒的目的。目前已知昆虫特有的GSTs Delta和Epsilon家族成员参与了昆虫对有机磷类、有机氯类和拟除虫菊酯类杀虫剂抗性的形成。
本学位论文以桔小实蝇为研究对象,瞄准昆虫适应逆境胁迫的机理这一研究热点,系统开展了桔小实蝇GSTs生化毒理学特性、GSTs基因的全长序列克隆、转录表达模式及异源表达研究。研究结果将有助于阐明桔小实蝇对杀虫剂抗性产生和发展的分子毒理学机理,从而在实践中建立基于GSTs的自然种群抗性的分子诊断技术。旨在为桔小实蝇的可持续治理提供理论依据,同时丰富和发展昆虫抗性机理研究的科学理论和技术体系。通过近三年的研究,取得的主要研究结果如下：
1桔小实蝇GSTs的纯化及生化毒理学特性
1.1桔小实蝇不同种群GSTs的纯化及生化毒理学特性
采用Glutathione Sepharose4B亲和层析法对桔小实蝇4个地理种群(东莞、广州、海南和云南)的GSTs进行了分离纯化和生化毒理学特性解析。结果表明,桔小实蝇4个种群GSTs纯化后的比活力相似,但广州种群的GSTs纯化回收率最低(31.54%)。聚丙酰胺凝胶电泳显示4个种群GSTs均只有一条大小为23kDa的条带。以CDNB为底物时,东莞和海南种群GSTs的Km值显著地高于广州和云南种群；海南种群GSTs的Vmax值在4个种群间最高。4个种群的GSTs最适反应温度均是37℃,最适pH值为7.5。离体抑制作用测定结果表明,利尿酸、四溴磺酚钠、马来酸二乙酯、双硫仑、姜黄素和高效氯氰菊酯均对桔小实蝇GSTs有很高的抑制率,而马拉硫磷和阿维菌素对桔小实蝇4个种群GSTs的抑制率均未到达50%。结果暗示GSTs可能介导桔小实蝇对高效氯氰菊酯的代谢抗性。
1.2桔小实蝇不同发育阶段GSTs的纯化及生化毒理学特性
采用Glutathione Sepharose4B亲和层析法对桔小实蝇不同发育阶段(三龄幼虫、蛹、成虫)GSTs进行了分离纯化和生化毒理学特性解析。结果表明,洗脱得到的桔小实蝇GSTs活性主要集中在洗脱收集到的3-5管。桔小实蝇成虫GSTs的总活性和比活力分别是370.76nmo1.min-1和16.91nmo1.min-1.μg-1,且显著高于三龄幼虫和蛹期。SDS-PAGE结果显示,桔小实蝇3个发育阶段的GSTs纯化后均获得单一条带,且分子量大小均为23kDa。无论以CDNB或GSH为底物时,桔小实蝇幼虫期GSTs的Km值均显著低于蛹和成虫期,而3个发育阶段间GSTs的Vmax无显著性差异。桔小实蝇3个发育阶段的GSTs最适反应温度均是37℃,最适pH值为7.5。利尿酸(1.15μM)和马来酸二乙酯(0.60μM)对桔小实蝇成虫期GSTs的I50。显著地低于其余两个发育阶段；四溴磺酚钠对桔小实蝇不同发育阶段GSTs的I50无显著差异；双硫仑对蛹和成虫期GSTs的I50均显著地高于三龄幼虫,而蛹和成虫间无显著差异；姜黄素对桔小实蝇成虫GSTs的I50(101.78μM)显著地高于三龄幼虫和蛹期；高效氯氰菊酯对蛹和成虫期GSTs的I50(0.90和0.78mM)显著地高于幼虫阶段；但马拉硫磷和阿维菌素对桔小实蝇3个发育阶段的GSTs的抑制率均未到达50%。结果暗示桔小实蝇幼虫期对杀虫剂高效氯氰菊酯相对更为敏感。
2桔小实蝇GSTs基因的克隆与序列分析
基于桔小实蝇转录组数据,利用RACE技术,从桔小实蝇体内分离克隆了18个GSTs基因的cDNA全长序列。通过序列分析,确定了这18个GSTs基因的开放阅读框,并推导了其编码的氨基酸序列。进一步利用Protparam等生物信息学软件分析了推导蛋白质的理化性质。依据其在细胞中的定位,将这18个GSTs基因分为胞质型和微粒体型两大类,分别包含17个和1个基因。根据黑腹果蝇、冈比亚按蚊、埃及伊蚊GSTs基因构建系统发育树,将其中的4个基因归类于Delta家族,8个基因归类于Epsilon家族,2个基因归类于Omega家族,另分别有1个基因归类于Theta和Zeta家族,而余下的1个基因则不能划分在已知的类型中,暂将其归为未分类家族Unclassified。这些胞质GSTs基因根据命名法则命名并在GenBank上登录,其名称和登录号分别为：BdGSTdl (JQ690090)、BdGSTd2(JN003593)、BdGSTd5(JN792452)、BdGSTd6(JN792453)、BdGSTe1(JN003588)、 BdGSTe2(JN003589)、BdGSTe3(JQ690091)、BdGSTe4(JN003590)、BdGSTe5(JN003591)、BdGSTe6(JQ690092)、BdGSTe7(JN003592)、BdGSTe9(JQ690093)、 BdGSTol (JQ690094)、BdGSTo2(JQ690095)、BdGSTt1(JQ690096)、BdGSTz2(JQ690097)和BdGSTu1(JN003587)。其中,Delta家族基因间的氨基酸同源性介于47.6-57.9%,Epsilon家族基因间的氨基酸的同源性范围为31.8-54.9%,Omega家族基因的氨基酸同源性高达85.4%,而在任意的非同一家族之间,氨基酸序列同源性仅在12.4-30%之间,但BdGSTu1与BdGSTe9的氨基酸特异性为35.9%。此外对这些GSTs基因进行分子特性分析,明确了酶促结合位点和维持酶活性构象的关键氨基酸残基。通过克隆并验证获得桔小实蝇微粒体GSTs基因1个,命名为BdGSTml, GenBank登录号JQ690098。BdGSTm1基因与黑腹果蝇DmGSTm-NP524696的氨基酸序列一致性高达91%。桔小实蝇和其它双翅目昆虫微粒体GSTs的多重比对分析发现,桔小实蝇微粒体GSTs基因中含有昆虫微粒体GSTs中特有的结构域D-P-X-V-E-R-V-R-R-A-H-X-N-D-X-E-N-I-L-P,且昆虫微粒体GSTs的氨基酸序列较短,通常在150个氨基酸左右。
3桔小实蝇GSTs基因的表达模式解析
3.1桔小实蝇GSTs基因在不同发育阶段的表达模式
在成功建立了桔小实蝇18个GSTs基因的qPCR反应体系的基础上,以桔小实蝇a-Tubulin基因作为内参基因,对这18个GSTs基因在不同发育阶段(卵、龄、二龄、三龄幼虫、蛹和成虫)mRNA表达水平进行了相对定量分析。结果发现,桔小实蝇卵期仅BdGSTel基因过量表达,暗示该基因可能与保幼激素和蜕皮激素的调控存在关联；桔小实蝇6个GSTs基因(BdGSTd2、BdGSTd5、BdGSTe5、 BdGSTe9、BdGSTt1和BdGSTz2)在幼虫期的相对表达量高于成虫期,暗示参与了幼虫期的代谢活动；桔小实蝇BdGSTe2、BdGSTe3、BdGSTe4和BdGSTe6基因随着桔小实蝇的生长发育,表达量增加上调,这表明在桔小实蝇个体发育过程中解毒代谢能力也随之增强。
3.2桔小实蝇GSTs基因在不同组织部位的表达模式
以a-Tubulin基因为内参基因,对18个GSTs基因在桔小实蝇中肠、脂肪体和马氏管3个组织中的mRNA表达水平进行了相对定量分析。结果表明,桔小实蝇BdGSTd5、BdGSTe3和BdGSTe9基因在中肠中的表达量最高,而且这3个GSTs基因都归属于昆虫特有的家族,暗示这些基因可能在外源化合物解毒代谢过程中起着重要的功能作用。其它GSTs基因在中肠不表达,可能在其他组织中起着类似的功能作用。桔小实蝇BdGSTd6、BdGSTe4、BdGSTe6和BdGSTz2基因在脂肪体中相对表达量最高,表明这些基因可能参与解毒代谢外源化合物或保护机体免受氧化应激。桔小实蝇BdGSTd1、BdGSTe1、BdGSTe2、BdGSTe5、BdGSTe7和BdGSTul基因在马氏管中有较高的表达量。GSTs基因在桔小实蝇马氏管中的具体作用还不清楚,推测可能和参与增强马氏管的排泄作用有关。
3.3桔小实蝇GSTs基因在3种杀虫剂诱导后的表达模式
利用有机磷类杀虫剂马拉硫磷、生物源杀虫剂阿维菌素及拟除虫菊酯类杀虫剂高效氯氰菊酯对桔小实蝇进行不同时间和剂量诱导,进而应用qPCR技术解析桔小实蝇18个GSTs基因在药剂诱导后的表达模式。结果表明,马拉硫磷对桔小实蝇诱导的时间效应中,5个GSTs基因(BdGSTd6、BdGSTe4、BdGSTe5、BdGSTe6和BdGSTm1)诱导12h后相对表达量最高,2个GSTs基因(BdGSTd2和BdGSTo1)诱导24h后相对表达量达最高峰,而另外8个GSTs基因(BdGSTd1、BdGSTe1、 BdGSTe2、BdGSTe3、BdGSTo2、BdGSTtl、BdGSTz2和BdGSTul)相对表达量的最高峰延迟到诱导后48h,说明这些GSTs基因可能均参与马拉硫磷在桔小实蝇体内的代谢过程。马拉硫磷亚致死剂量(LD10)处理桔小实蝇24h后,10个GSTs基因(BdGSTd1、BdGSTd5、BdGSTd6、BdGSTe2、BdGSTe4、BdGSTe5、BdGSTe6、 BdGSTo2、BdGSTt1和BdGSTul)相对表达量最高,表明这些基因可能介导桔小实蝇对马拉硫磷的代谢抗性。
阿维菌素对桔小实蝇诱导的时间效应中,2个GSTs基因(BdGSTe6和BdGSTm1)诱导12h后相对表达量最高,6个GSTs基因(BdGSTd1、BdGSTd2、 BdGSTd5、BdGSTd6、BdGSTe4和BdGSTe9)诱导24h后相对表达量达最高峰,阿维菌素致死中剂量(LD50)诱导桔小实蝇24h后,7个GSTs基因(BdGSTd1、 BdGSTd5、BdGSTd6、BdGSTe4、BdGSTe5、BdGSTe6和BdGSTml)相对表达量达到最高峰,暗示这些GSTs基因可能均参与阿维菌素在桔小实蝇体内的解毒代谢作用。
高效氯氰菊酯对桔小实蝇诱导的时间效应中,3个GSTs基因(BdGSTe2、 BdGSTe5和BdGSTe6)诱导12h后相对表达量最高,而另外6个GSTs基因(BdGSTe4、BdGSTe9、BdGSTo2、BdGSTt1、BdGSTu1和BdGSTml)相对表达量的最高峰延迟到诱导后36h,说明这些GSTs基因可能均参与高效氯氰菊酯在桔小实蝇体内的代谢过程。而高效氯氰菊酯亚致死剂量(LD10)诱导桔小实蝇后,BdGSTd1、BdGSTe4、BdGSTe7和BdGSTtl基因表达上调,暗示桔小实蝇GSTs基因可能参与高效氯氰菊酯的解毒代谢过程。
3.4桔小实蝇GSTs基因在不同品系中的表达模式
采用定量PCR技术,以a-Tubulin基因为内参基因,解析克隆获得的桔小实蝇18个GSTs基因在实验室选育并保存的桔小实蝇马拉硫磷和高效氯氰菊酯抗性品系及敏感品系中的表达模式。结果表明,在马拉硫磷抗性品系中,BdGSTd5、 BdGSTe9和BdGSTz2基因的相对表达量显著高于敏感品系,而BdGSTe1、BdGSTe2和BdGSTtl在抗性品系中的相对表达量显著低于敏感品系。暗示这些GSTs基因可能介导了桔小实蝇对马拉硫磷抗性的形成。桔小实蝇高效氯氰菊酯抗性品系中,8个GSTs基因(BdGSTd6、BdGSTe1、BdGSTe2、BdGSTe5、BdGSTe6、BdGSTe7、 BdGSTtl和BdGSTm1)在抗性品系中的相对表达量显著高于敏感品系,暗示这些基因可能参与了桔小实蝇对高效氯氰菊酯的解毒代谢作用。
4桔小实蝇BdGSTe5基因的原核表达
采用NdeI和HindⅢ双酶切位点及DNA重组技术构建了桔小实蝇BdGSTe5基因基于BdGSTe5-pET28a (+)的原核表达质粒,经IPTG诱导后,SDS-PAGE电泳发现含有重组质粒BdGSTe5-pET28a(+)的细菌中在27kDa下方有一条特异性的蛋白质条带。根据蛋白质分子量估算,表达的融合蛋白质分子量大约为26kDa,由于6×His-tag标签及其侧翼序列片段大约占1kDa,因此该特异性条带分子量与预期表达的融合蛋白大小相一致。采用Ni柱和DEAE柱依次对BdGSTe5-pET28a(+)重组蛋白进行纯化,经SDS-PAGE验证,在分子量为27kDa处获得单一条带。
综上所述,通过亲和层析法分离纯化GSTs并比较研究纯化产物的动力学和毒理学特性；应用高通量测序及RACE技术克隆桔小实蝇GSTs基因的全长序列,结合qPCR技术解析其mRNA在桔小实蝇不同发育阶段、不同组织部位和不同品系中的表达模式,鉴定了参与该虫抗性的GSTs基因。研究结果将促进对桔小实蝇GSTs基因生理功能的认识,为桔小实蝇抗性相关GSTs基因的鉴定提供理论依据,并对阐释GSTs介导桔小实蝇代谢抗性的分子生物学机制具有重要的理论意义。
The oriental fruit fly, Bactrocera dorsalis (Hendel)(Diptera:Tephritidae), is a severe pest of fruit and vegetable pests. Based on its significantly economic importance, researches on population genetic structure, pheromone products, and chemical control were conducted in recent years, while B. dorsalis has generated heavy resistance to insecticides due to long-term and unreasonable using chemical control. Efforts to control this pest often result in development of insecticide resistance. It has been shown to develop resistance and cross-resistance to various different classes of insecticides, including organophosphates, pyrethroids, and carbamates. Until now, the mechanism of insecticides resistance of B. dorsalis has focused on target enzyme:the biochemical and molecular characterization of acetylcholinesterase. However, very little is known about the characteristics of detoxification enzymes of oriental fruit fly, despite their significance in the metabolic of xenobiotics and insecticide resistance. Glutathione S-transferases (GSTs, EC2.5.1.18) are a diverse super family of enzymes with a range of catalytic functions, including cellular protection from reactive oxygen species, reductive maintenance of thiolated proteins, prostaglandin synthesis, and glutathione conjugation of endogenous and exogenous ligands. The GSTs from Delta and Epsilon family are insect specific, which have important roles in phase Ⅱ detoxification of several chemical insecticides classes, including organophosphates, chlorinated hydrocarbons, and pyrethroids.
The current study was aimed at the insect adaption mechanisms environment stress. We comprehensicely studied the purification, biochemical and toxicological characterization of GSTs, molecular cloning and sequences analysis of GSTs genes, GSTs genes expression profiling, and prokaryotic expression of GSTs genes. The results would help to clarify the molecular toxicology mechanism of to molecular toxicology of B. dorsalis to insecticide resistance, which could also act as a molecular diagnostic technique to monitor the resistant level of field populations. Our results will provide a theoretical basis for the sustainable management of B. dorsalis, and enrich and develop the scientific theoretical study system of insect pests resistance to insecticides. The main results are as follows:
1. Purification, biochemical and toxicological characterization of GSTs from B. dorsalis
1.1Purification, biochemical and toxicological characterization of GSTs from four field populations of B. dorsalis
In this study, GSTs were purified from four field populations of B. dorsalis with different insecticide susceptibilities by glutathione-agarose affinity chromatography. The populations were collected from Dongguan (DG) and Guangzhou (GZ) of Guangdong Province, Haikou of Hainan province (HN), and Kunming of Yunnan province (YN), China. Differences in GST characteristics among the four populations were studied using purified enzyme samples through comparative SDS-PAGE, kinetic, and inhibition experiments. The specific activities of the purified enzymes were similar, but the purification yield of the GZ population (31.54%) was the lowest. SDS-PAGE analysis showed only one band at approximately23kDa for these four populations. Kinetic analyses showed that the affinities of the purified GSTs from the GZ and YN populations for1-chloro-2.4-dinitrobenzene (CDNB) were much higher than those of GSTs from the other two populations, while the HN population had the highest catalytic capability in terms of Vmax value. The optimum temperature for CDNB conjugation was37℃, and the optimum pH was7.5in all four populations. Inhibition kinetics showed that ethacrynic acid, diethyl maleate, tetraethylthiuram disulfide, curcumin, bromosulfalein, and beta-cypermethrin had excellent inhibitory effects on GSTs in the four populations of B. dorsalis, but the low inhibitory effects of malathion and avermectin did not differ between populations. These results suggest that GSTs may have a role in detoxification of (3-cypermethrin in B. dorsalis.
1.2Purification, biochemical and toxicological characterization of GSTs from three development stages of B. dorsalis
In this study, GSTs were purified and characterized from three development stages (third larva, pupa, adult) of B. dorsalis by glutathione sepharose4B affinity chromatography. The chromatography elutions are showed the highest specific activity of GSTs between third to fifth fraction. The both total activity and specific activity of adult showed higher values than those of third larva and pupa, with370.76nmol.min-1and16.91nmol.min-1.μg-1, respectively. SDS-PAGE analysis showed only one band at approximately23kDa for these three development stages. In both CDNB or GSH as substrate, the GSTs Km value of larval stage were significantly lower than those in the pupa and adult stages, while there were no significant difference among three development stages of GSTs Vmax. The optimum temperature for CDNB conjugation was37℃, and the optimum pH was7.5in all three development stages. The I50values of ethacrynic acid (1.15μM) and diethyl maleate (0.60μM) against the adult stage were significantly lower than those of the other stages. The I50value of bromosulfalein did not differ significantly among the three development stages. The I50value of disulfiram among the pupa and adult stages were significantly higher than that of third larva, while there were no significant difference between pupa and adult stages. The I50value of curcumin (101.78μM) against the adult stage was significantly higher than those of the other stages. The I50values of β-cypermethrin against the pupa (0.90mM) and adult (0.78mM) stages were significantly higher than that of larva stages. The low inhibitory effects of malathion and avermectin did not differ between development stages. The results suggested that B. dorsalis was more sensitive to the insecticide β-cypermethrin in the larva stage than other two stages.
2. Molecular cloning and sequences analysis of GST genes from B. dorsalis
Based on the transcriptome data,18novel GST genes were cloned from B. dorsalis using the RT-PCR and RACE techniques. Molecular characterizations of the putative proteins have been predicted by Protparam software. According the location with the cell, these18GSTs genes were classified into17cytosolic and1microsomal. Phylogenetic analysis of these17cytosolic GSTs deduced from their cDNAs revealed16GSTs that belong to five different cytosolic classes, including4in delta,8in epsilon,2in omega,1in theta, and1in zeta, based on their sequence similarities to other insect GSTs, particularly those from Drosophila melanogaster, Anopheles gambiae, and Aedes aegypti. The remaining one GST was unclassified due to the lack of homologies to the currently known class. The nomenclature of these B. dorsalis GSTs cDNA and their deduced amino acid sequences have been deposited in Genbank with the following accession numbers:BdGSTdl (JQ690090), BdGSTd2(JN003593), BdGSTd5(JN792452), BdGSTd6(JN792453), BdGSTel (JN003588), BdGSTe2(JN003589), BdGSTe3(JQ690091), BdGSTe4(JN003590), BdGSTe5(N003591), BdGSTe6(JQ690092), BdGSTe7(JN003592), BdGSTe9(JQ690093), BdGSTol (JQ690094), BdGSTo2(JQ690095), BdGSTtl (JQ690096), BdGSTz2(JQ690097), and BdGSTul (JN003587). The percentages of deduced amino acid identities were47.6-57.9%among the four delta GSTs,31.8-54.9%among the eight epsilon GSTs,85.4%between the two omega GSTs, but only12.4to30%among different classes of the GSTs in the oriental fruit fly GSTs. Furthermore, several conserved amino acid residues which represent the catalytic pocket and binding site were analyzed by sequence alignment. Meanwhile, the full-length cDNA of microsomal GST gene BdGSTml was identified and cloned from B. dorsalis using RACE technique. The Genbank accession number was JQ690098. In addition, BdGSTm1was closely related to DmGSTm-NP524696with91%amino acid sequence identity. The deduced amino acid sequence included one insect typical motif of the microsomal GSTs family, was D-P-X-V-E-R-V-R-R-A-H-X-N-D-X-E-N-I-L-P, based on their sequence similarities to other diptera insect microsomal GSTs. At the same time, insect microsomal GSTs amino acid sequences were shorter, usually only about150amino acids.
3. GST genes expression profiles from B. dorsalis
3.1Developmental stages expression profiles of GST genes
The developmental stages expression profiles of18GST genes from B. dorsalis were analyzed using a-Tubulin as an internal control gene. The developmental stages included egg, three different larval instars (first-, second-, and third), pupa, and adult. The results showed that the relative expression level of BdGSTel was the only overexpressed gene in the egg developmental stage, which suggested that it might be associated with regulation of juvenile hormone and ecdysone in egg. Higher expression levels of six GST genes (BdGSTd2, BdGSTd5, BdGSTe5, BdGSTe9, BdGSTtl, and BdGSTz2) were observed at larval stages compared with adulthood, implies it may play an important metabolic role in the larval stages. The expression level of four Epsilon GST genes (BdGSTe2, BdGSTe3, BdGSTe4, and BdGSTe6) were increased during the development of fruit fly, which indicates that the detoxification capacity also increase in the ontogeny of B. dorsalis.
3.2Expression profiles of GST genes among different tissues
The relative quantitative of mRNA expression levels of18GST genes in three tissues including midgut, fat body and Malpighian tubules were analyzed using a-Tubulin as reference gene. In the midgut tissue, the expression of three insect-specific family GST genes (BdGSTd5, BdGSTe3, and BdGSTe9) were the highest, suggesting that these genes may play an important role in the detoxification of exogenous compounds. Other GST genes were not expressed in the midgut, may play a similar functional roles in other tissues. Four GST genes(BdGSTd6, BdGSTe4, BdGSTe6, and BdGSTz2) in the fat body tissue were relatively the highest expression level, indicating that these genes may be involved in the detoxification of exogenous compounds or protect the body from oxidative stress. The relative mRNA expression of BdGSTdl, BdGSTel, BdGSTe2, BdGSTe5, BdGSTe7, and BdGSTu1were higher in the Malpighian tubules tissue than the other tissues. The specific role of GSTs in Malpighian tubules is still not clear, but it could be involved in the enhanced excretion of Malpighian tubules.
3.3Expression profiles of GST genes induced by three insecticides
To determine the induction responses of GST genes, three insecticides malathion, avermectin, and β-cypermethrin were chosen in this study. Based on the bioassay, the flies were induced by the insecticides in two different regimes:time treatment (12,24,36, and48h) and dose treatment (LD10, LD50, and LD80). qPCR with a-Tubulin as the reference gene was used to determine the relative expression quantity of GST genes. Five GST genes (BdGSTd6, BdGSTe4, BdGSTe5, BdGSTe6, and BdGSTm1) transcripts showed the highest after12h malathion induction; BdGSTd2and BdGSTo1were reached the highest after24h; and other eight GST genes(BdGSTd1, BdGSTel, BdGSTe2, BdGSTe3, BdGSTo2, BdGSTtl, BdGSTz2, and BdGSTu1) reached the peak48h after malathion induction. The relative expression quantities of ten GST genes (BdGSTd1, BdGSTd5, BdGSTd6, BdGSTe2, BdGSTe4, BdGSTe5, BdGSTe6, BdGSTo2, BdGSTtl, and BdGSTu1) were increased significantly after exposure to sub-lethal dose (LD10) malathion for24h. These GST genes may participate in the metabolic processes to malathion of B. dorsalis.
In the avermectin induction, the expression of BdGSTe6and BdGSTml were significantly enhanced after12h, meanwhile these six GST genes(BdGSTd1, BdGSTd2, BdGSTd5, BdGSTd6, BdGSTe4, and BdGSTe9) transcripts were reached the peak24h later, seven GST genes(BdGSTd2, BdGSTd5, BdGSTd6, BdGSTe4, BdGSTe5, BdGSTe6, and BdGSTml) were the highest after induce to lethal dose (LD50) avermectin for24h. The results suggest that these GST genes may have a role in detoxification of avermectin in B. dorsalis.
Similar to the above insecticides, after the12h β-cypermethrin induction, the expression of three GST genes(BdGSTe2, BdGSTe5, and BdGSTe6) were significant higher; meanwhile these six GST genes (BdGSTe4, BdGSTe9, BdGSTo2, BdGSTtl, BdGSTu1, and BdGSTm1) expression were reached the peak36h later, four GST genes (BdGSTd1, BdGSTe4, BdGSTe7, and BdGSTtl) were up regulation after induce to lethal dose (LD10) P-cypermethrin for24h, suggesting that these GST gene may have a role in detoxification of β-cypermethrin in B. dorsalis.
3.4Expression profiles of GST genes among different strains
In order to verify whether GSTs are over-expressed in resistant fruit flies, qPCR with a-Tubulin as the reference gene was employed to determine the relative expression quantity of18GST genes in three different strains of B. dorsalis. As indicated by qPCR, three GST genes (BdGSTd5, BdGSTe9, and BdGSTz2) transcripts in malathion resistant strain were significantly higher than susceptible strain, while the expression of BdGSTel, BdGSTe2, and BdGSTtl were significantly lower than susceptible strain, which implied that these GST genes were possibly involved in B. dorsalis malathion resistance. The relative expression levels of these eight GST genes (BdGSTd6, BdGSTel, BdGSTe2, BdGSTe5, BdGSTe6, BdGSTe7, BdGSTtl, and BdGSTm1) were significantly higher in β-cypermethrin resistant strain than susceptible strain, suggesting that these GST gene may have a role in detoxification of β-cypermethrin in B. dorsalis.
4. Heterologous expression of BdGSTe5in Escherichia coli
Using the double digestion of Ndel and HindⅢ restriction enzymes and the DNA recombination technology, the expression vector for BdGSTe5was constructed based on pET28a (+) vector. After IPTG induction to E. coli, SDS-PAGE analysis showed only one specific band at approximately27kDa for the recombinant plasmid BdGSTe5-pET28a (+). According to the protein molecular weight, the expression of fusion protein molecular weight was about26kDa. Because of6x His-tag and flanking fragment accounting for1kDa, therefore, the specific band was consistent with the expected size of fusion protein. BdGSTe5-pET28a (+) recombinant protein was purified and confirmed by Ni and DEAE column.
In summary, the purification, biochemical and toxicological characterization of GSTs of B. dorsalis were studied by glutathione-agarose affinity chromatography; based on the transcriptome data,18novel GST genes were cloned from B. dorsalis using RACE technique; GST genes expression profiles were studied using qPCR technique from B. dorsalis including developmental stages, tissues, insecticides inductions, and different strains, furthermore, the expression vector for BdGSTe5was constructed and expressed. These results will not only provide great insights into exploringthe functions of the oriental fruit fly GSTs system in development and physiology, but also provide evidence for clarifying the adaptive mechanisms of oriental fruit fly to environment. Meanwhile, the results would also enrich and develope the scientific theoretical study system for the resistance mechanism of other insect pests.
本学位论文以桔小实蝇为研究对象,瞄准昆虫适应逆境胁迫的机理这一研究热点,系统开展了桔小实蝇GSTs生化毒理学特性、GSTs基因的全长序列克隆、转录表达模式及异源表达研究。研究结果将有助于阐明桔小实蝇对杀虫剂抗性产生和发展的分子毒理学机理,从而在实践中建立基于GSTs的自然种群抗性的分子诊断技术。旨在为桔小实蝇的可持续治理提供理论依据,同时丰富和发展昆虫抗性机理研究的科学理论和技术体系。通过近三年的研究,取得的主要研究结果如下：
1桔小实蝇GSTs的纯化及生化毒理学特性
1.1桔小实蝇不同种群GSTs的纯化及生化毒理学特性
采用Glutathione Sepharose4B亲和层析法对桔小实蝇4个地理种群(东莞、广州、海南和云南)的GSTs进行了分离纯化和生化毒理学特性解析。结果表明,桔小实蝇4个种群GSTs纯化后的比活力相似,但广州种群的GSTs纯化回收率最低(31.54%)。聚丙酰胺凝胶电泳显示4个种群GSTs均只有一条大小为23kDa的条带。以CDNB为底物时,东莞和海南种群GSTs的Km值显著地高于广州和云南种群；海南种群GSTs的Vmax值在4个种群间最高。4个种群的GSTs最适反应温度均是37℃,最适pH值为7.5。离体抑制作用测定结果表明,利尿酸、四溴磺酚钠、马来酸二乙酯、双硫仑、姜黄素和高效氯氰菊酯均对桔小实蝇GSTs有很高的抑制率,而马拉硫磷和阿维菌素对桔小实蝇4个种群GSTs的抑制率均未到达50%。结果暗示GSTs可能介导桔小实蝇对高效氯氰菊酯的代谢抗性。
1.2桔小实蝇不同发育阶段GSTs的纯化及生化毒理学特性
采用Glutathione Sepharose4B亲和层析法对桔小实蝇不同发育阶段(三龄幼虫、蛹、成虫)GSTs进行了分离纯化和生化毒理学特性解析。结果表明,洗脱得到的桔小实蝇GSTs活性主要集中在洗脱收集到的3-5管。桔小实蝇成虫GSTs的总活性和比活力分别是370.76nmo1.min-1和16.91nmo1.min-1.μg-1,且显著高于三龄幼虫和蛹期。SDS-PAGE结果显示,桔小实蝇3个发育阶段的GSTs纯化后均获得单一条带,且分子量大小均为23kDa。无论以CDNB或GSH为底物时,桔小实蝇幼虫期GSTs的Km值均显著低于蛹和成虫期,而3个发育阶段间GSTs的Vmax无显著性差异。桔小实蝇3个发育阶段的GSTs最适反应温度均是37℃,最适pH值为7.5。利尿酸(1.15μM)和马来酸二乙酯(0.60μM)对桔小实蝇成虫期GSTs的I50。显著地低于其余两个发育阶段；四溴磺酚钠对桔小实蝇不同发育阶段GSTs的I50无显著差异；双硫仑对蛹和成虫期GSTs的I50均显著地高于三龄幼虫,而蛹和成虫间无显著差异；姜黄素对桔小实蝇成虫GSTs的I50(101.78μM)显著地高于三龄幼虫和蛹期；高效氯氰菊酯对蛹和成虫期GSTs的I50(0.90和0.78mM)显著地高于幼虫阶段；但马拉硫磷和阿维菌素对桔小实蝇3个发育阶段的GSTs的抑制率均未到达50%。结果暗示桔小实蝇幼虫期对杀虫剂高效氯氰菊酯相对更为敏感。
2桔小实蝇GSTs基因的克隆与序列分析
基于桔小实蝇转录组数据,利用RACE技术,从桔小实蝇体内分离克隆了18个GSTs基因的cDNA全长序列。通过序列分析,确定了这18个GSTs基因的开放阅读框,并推导了其编码的氨基酸序列。进一步利用Protparam等生物信息学软件分析了推导蛋白质的理化性质。依据其在细胞中的定位,将这18个GSTs基因分为胞质型和微粒体型两大类,分别包含17个和1个基因。根据黑腹果蝇、冈比亚按蚊、埃及伊蚊GSTs基因构建系统发育树,将其中的4个基因归类于Delta家族,8个基因归类于Epsilon家族,2个基因归类于Omega家族,另分别有1个基因归类于Theta和Zeta家族,而余下的1个基因则不能划分在已知的类型中,暂将其归为未分类家族Unclassified。这些胞质GSTs基因根据命名法则命名并在GenBank上登录,其名称和登录号分别为：BdGSTdl (JQ690090)、BdGSTd2(JN003593)、BdGSTd5(JN792452)、BdGSTd6(JN792453)、BdGSTe1(JN003588)、 BdGSTe2(JN003589)、BdGSTe3(JQ690091)、BdGSTe4(JN003590)、BdGSTe5(JN003591)、BdGSTe6(JQ690092)、BdGSTe7(JN003592)、BdGSTe9(JQ690093)、 BdGSTol (JQ690094)、BdGSTo2(JQ690095)、BdGSTt1(JQ690096)、BdGSTz2(JQ690097)和BdGSTu1(JN003587)。其中,Delta家族基因间的氨基酸同源性介于47.6-57.9%,Epsilon家族基因间的氨基酸的同源性范围为31.8-54.9%,Omega家族基因的氨基酸同源性高达85.4%,而在任意的非同一家族之间,氨基酸序列同源性仅在12.4-30%之间,但BdGSTu1与BdGSTe9的氨基酸特异性为35.9%。此外对这些GSTs基因进行分子特性分析,明确了酶促结合位点和维持酶活性构象的关键氨基酸残基。通过克隆并验证获得桔小实蝇微粒体GSTs基因1个,命名为BdGSTml, GenBank登录号JQ690098。BdGSTm1基因与黑腹果蝇DmGSTm-NP524696的氨基酸序列一致性高达91%。桔小实蝇和其它双翅目昆虫微粒体GSTs的多重比对分析发现,桔小实蝇微粒体GSTs基因中含有昆虫微粒体GSTs中特有的结构域D-P-X-V-E-R-V-R-R-A-H-X-N-D-X-E-N-I-L-P,且昆虫微粒体GSTs的氨基酸序列较短,通常在150个氨基酸左右。
3桔小实蝇GSTs基因的表达模式解析
3.1桔小实蝇GSTs基因在不同发育阶段的表达模式
在成功建立了桔小实蝇18个GSTs基因的qPCR反应体系的基础上,以桔小实蝇a-Tubulin基因作为内参基因,对这18个GSTs基因在不同发育阶段(卵、龄、二龄、三龄幼虫、蛹和成虫)mRNA表达水平进行了相对定量分析。结果发现,桔小实蝇卵期仅BdGSTel基因过量表达,暗示该基因可能与保幼激素和蜕皮激素的调控存在关联；桔小实蝇6个GSTs基因(BdGSTd2、BdGSTd5、BdGSTe5、 BdGSTe9、BdGSTt1和BdGSTz2)在幼虫期的相对表达量高于成虫期,暗示参与了幼虫期的代谢活动；桔小实蝇BdGSTe2、BdGSTe3、BdGSTe4和BdGSTe6基因随着桔小实蝇的生长发育,表达量增加上调,这表明在桔小实蝇个体发育过程中解毒代谢能力也随之增强。
3.2桔小实蝇GSTs基因在不同组织部位的表达模式
以a-Tubulin基因为内参基因,对18个GSTs基因在桔小实蝇中肠、脂肪体和马氏管3个组织中的mRNA表达水平进行了相对定量分析。结果表明,桔小实蝇BdGSTd5、BdGSTe3和BdGSTe9基因在中肠中的表达量最高,而且这3个GSTs基因都归属于昆虫特有的家族,暗示这些基因可能在外源化合物解毒代谢过程中起着重要的功能作用。其它GSTs基因在中肠不表达,可能在其他组织中起着类似的功能作用。桔小实蝇BdGSTd6、BdGSTe4、BdGSTe6和BdGSTz2基因在脂肪体中相对表达量最高,表明这些基因可能参与解毒代谢外源化合物或保护机体免受氧化应激。桔小实蝇BdGSTd1、BdGSTe1、BdGSTe2、BdGSTe5、BdGSTe7和BdGSTul基因在马氏管中有较高的表达量。GSTs基因在桔小实蝇马氏管中的具体作用还不清楚,推测可能和参与增强马氏管的排泄作用有关。
3.3桔小实蝇GSTs基因在3种杀虫剂诱导后的表达模式
利用有机磷类杀虫剂马拉硫磷、生物源杀虫剂阿维菌素及拟除虫菊酯类杀虫剂高效氯氰菊酯对桔小实蝇进行不同时间和剂量诱导,进而应用qPCR技术解析桔小实蝇18个GSTs基因在药剂诱导后的表达模式。结果表明,马拉硫磷对桔小实蝇诱导的时间效应中,5个GSTs基因(BdGSTd6、BdGSTe4、BdGSTe5、BdGSTe6和BdGSTm1)诱导12h后相对表达量最高,2个GSTs基因(BdGSTd2和BdGSTo1)诱导24h后相对表达量达最高峰,而另外8个GSTs基因(BdGSTd1、BdGSTe1、 BdGSTe2、BdGSTe3、BdGSTo2、BdGSTtl、BdGSTz2和BdGSTul)相对表达量的最高峰延迟到诱导后48h,说明这些GSTs基因可能均参与马拉硫磷在桔小实蝇体内的代谢过程。马拉硫磷亚致死剂量(LD10)处理桔小实蝇24h后,10个GSTs基因(BdGSTd1、BdGSTd5、BdGSTd6、BdGSTe2、BdGSTe4、BdGSTe5、BdGSTe6、 BdGSTo2、BdGSTt1和BdGSTul)相对表达量最高,表明这些基因可能介导桔小实蝇对马拉硫磷的代谢抗性。
阿维菌素对桔小实蝇诱导的时间效应中,2个GSTs基因(BdGSTe6和BdGSTm1)诱导12h后相对表达量最高,6个GSTs基因(BdGSTd1、BdGSTd2、 BdGSTd5、BdGSTd6、BdGSTe4和BdGSTe9)诱导24h后相对表达量达最高峰,阿维菌素致死中剂量(LD50)诱导桔小实蝇24h后,7个GSTs基因(BdGSTd1、 BdGSTd5、BdGSTd6、BdGSTe4、BdGSTe5、BdGSTe6和BdGSTml)相对表达量达到最高峰,暗示这些GSTs基因可能均参与阿维菌素在桔小实蝇体内的解毒代谢作用。
高效氯氰菊酯对桔小实蝇诱导的时间效应中,3个GSTs基因(BdGSTe2、 BdGSTe5和BdGSTe6)诱导12h后相对表达量最高,而另外6个GSTs基因(BdGSTe4、BdGSTe9、BdGSTo2、BdGSTt1、BdGSTu1和BdGSTml)相对表达量的最高峰延迟到诱导后36h,说明这些GSTs基因可能均参与高效氯氰菊酯在桔小实蝇体内的代谢过程。而高效氯氰菊酯亚致死剂量(LD10)诱导桔小实蝇后,BdGSTd1、BdGSTe4、BdGSTe7和BdGSTtl基因表达上调,暗示桔小实蝇GSTs基因可能参与高效氯氰菊酯的解毒代谢过程。
3.4桔小实蝇GSTs基因在不同品系中的表达模式
采用定量PCR技术,以a-Tubulin基因为内参基因,解析克隆获得的桔小实蝇18个GSTs基因在实验室选育并保存的桔小实蝇马拉硫磷和高效氯氰菊酯抗性品系及敏感品系中的表达模式。结果表明,在马拉硫磷抗性品系中,BdGSTd5、 BdGSTe9和BdGSTz2基因的相对表达量显著高于敏感品系,而BdGSTe1、BdGSTe2和BdGSTtl在抗性品系中的相对表达量显著低于敏感品系。暗示这些GSTs基因可能介导了桔小实蝇对马拉硫磷抗性的形成。桔小实蝇高效氯氰菊酯抗性品系中,8个GSTs基因(BdGSTd6、BdGSTe1、BdGSTe2、BdGSTe5、BdGSTe6、BdGSTe7、 BdGSTtl和BdGSTm1)在抗性品系中的相对表达量显著高于敏感品系,暗示这些基因可能参与了桔小实蝇对高效氯氰菊酯的解毒代谢作用。
4桔小实蝇BdGSTe5基因的原核表达
采用NdeI和HindⅢ双酶切位点及DNA重组技术构建了桔小实蝇BdGSTe5基因基于BdGSTe5-pET28a (+)的原核表达质粒,经IPTG诱导后,SDS-PAGE电泳发现含有重组质粒BdGSTe5-pET28a(+)的细菌中在27kDa下方有一条特异性的蛋白质条带。根据蛋白质分子量估算,表达的融合蛋白质分子量大约为26kDa,由于6×His-tag标签及其侧翼序列片段大约占1kDa,因此该特异性条带分子量与预期表达的融合蛋白大小相一致。采用Ni柱和DEAE柱依次对BdGSTe5-pET28a(+)重组蛋白进行纯化,经SDS-PAGE验证,在分子量为27kDa处获得单一条带。
综上所述,通过亲和层析法分离纯化GSTs并比较研究纯化产物的动力学和毒理学特性；应用高通量测序及RACE技术克隆桔小实蝇GSTs基因的全长序列,结合qPCR技术解析其mRNA在桔小实蝇不同发育阶段、不同组织部位和不同品系中的表达模式,鉴定了参与该虫抗性的GSTs基因。研究结果将促进对桔小实蝇GSTs基因生理功能的认识,为桔小实蝇抗性相关GSTs基因的鉴定提供理论依据,并对阐释GSTs介导桔小实蝇代谢抗性的分子生物学机制具有重要的理论意义。
The oriental fruit fly, Bactrocera dorsalis (Hendel)(Diptera:Tephritidae), is a severe pest of fruit and vegetable pests. Based on its significantly economic importance, researches on population genetic structure, pheromone products, and chemical control were conducted in recent years, while B. dorsalis has generated heavy resistance to insecticides due to long-term and unreasonable using chemical control. Efforts to control this pest often result in development of insecticide resistance. It has been shown to develop resistance and cross-resistance to various different classes of insecticides, including organophosphates, pyrethroids, and carbamates. Until now, the mechanism of insecticides resistance of B. dorsalis has focused on target enzyme:the biochemical and molecular characterization of acetylcholinesterase. However, very little is known about the characteristics of detoxification enzymes of oriental fruit fly, despite their significance in the metabolic of xenobiotics and insecticide resistance. Glutathione S-transferases (GSTs, EC2.5.1.18) are a diverse super family of enzymes with a range of catalytic functions, including cellular protection from reactive oxygen species, reductive maintenance of thiolated proteins, prostaglandin synthesis, and glutathione conjugation of endogenous and exogenous ligands. The GSTs from Delta and Epsilon family are insect specific, which have important roles in phase Ⅱ detoxification of several chemical insecticides classes, including organophosphates, chlorinated hydrocarbons, and pyrethroids.
The current study was aimed at the insect adaption mechanisms environment stress. We comprehensicely studied the purification, biochemical and toxicological characterization of GSTs, molecular cloning and sequences analysis of GSTs genes, GSTs genes expression profiling, and prokaryotic expression of GSTs genes. The results would help to clarify the molecular toxicology mechanism of to molecular toxicology of B. dorsalis to insecticide resistance, which could also act as a molecular diagnostic technique to monitor the resistant level of field populations. Our results will provide a theoretical basis for the sustainable management of B. dorsalis, and enrich and develop the scientific theoretical study system of insect pests resistance to insecticides. The main results are as follows:
1. Purification, biochemical and toxicological characterization of GSTs from B. dorsalis
1.1Purification, biochemical and toxicological characterization of GSTs from four field populations of B. dorsalis
In this study, GSTs were purified from four field populations of B. dorsalis with different insecticide susceptibilities by glutathione-agarose affinity chromatography. The populations were collected from Dongguan (DG) and Guangzhou (GZ) of Guangdong Province, Haikou of Hainan province (HN), and Kunming of Yunnan province (YN), China. Differences in GST characteristics among the four populations were studied using purified enzyme samples through comparative SDS-PAGE, kinetic, and inhibition experiments. The specific activities of the purified enzymes were similar, but the purification yield of the GZ population (31.54%) was the lowest. SDS-PAGE analysis showed only one band at approximately23kDa for these four populations. Kinetic analyses showed that the affinities of the purified GSTs from the GZ and YN populations for1-chloro-2.4-dinitrobenzene (CDNB) were much higher than those of GSTs from the other two populations, while the HN population had the highest catalytic capability in terms of Vmax value. The optimum temperature for CDNB conjugation was37℃, and the optimum pH was7.5in all four populations. Inhibition kinetics showed that ethacrynic acid, diethyl maleate, tetraethylthiuram disulfide, curcumin, bromosulfalein, and beta-cypermethrin had excellent inhibitory effects on GSTs in the four populations of B. dorsalis, but the low inhibitory effects of malathion and avermectin did not differ between populations. These results suggest that GSTs may have a role in detoxification of (3-cypermethrin in B. dorsalis.
1.2Purification, biochemical and toxicological characterization of GSTs from three development stages of B. dorsalis
In this study, GSTs were purified and characterized from three development stages (third larva, pupa, adult) of B. dorsalis by glutathione sepharose4B affinity chromatography. The chromatography elutions are showed the highest specific activity of GSTs between third to fifth fraction. The both total activity and specific activity of adult showed higher values than those of third larva and pupa, with370.76nmol.min-1and16.91nmol.min-1.μg-1, respectively. SDS-PAGE analysis showed only one band at approximately23kDa for these three development stages. In both CDNB or GSH as substrate, the GSTs Km value of larval stage were significantly lower than those in the pupa and adult stages, while there were no significant difference among three development stages of GSTs Vmax. The optimum temperature for CDNB conjugation was37℃, and the optimum pH was7.5in all three development stages. The I50values of ethacrynic acid (1.15μM) and diethyl maleate (0.60μM) against the adult stage were significantly lower than those of the other stages. The I50value of bromosulfalein did not differ significantly among the three development stages. The I50value of disulfiram among the pupa and adult stages were significantly higher than that of third larva, while there were no significant difference between pupa and adult stages. The I50value of curcumin (101.78μM) against the adult stage was significantly higher than those of the other stages. The I50values of β-cypermethrin against the pupa (0.90mM) and adult (0.78mM) stages were significantly higher than that of larva stages. The low inhibitory effects of malathion and avermectin did not differ between development stages. The results suggested that B. dorsalis was more sensitive to the insecticide β-cypermethrin in the larva stage than other two stages.
2. Molecular cloning and sequences analysis of GST genes from B. dorsalis
Based on the transcriptome data,18novel GST genes were cloned from B. dorsalis using the RT-PCR and RACE techniques. Molecular characterizations of the putative proteins have been predicted by Protparam software. According the location with the cell, these18GSTs genes were classified into17cytosolic and1microsomal. Phylogenetic analysis of these17cytosolic GSTs deduced from their cDNAs revealed16GSTs that belong to five different cytosolic classes, including4in delta,8in epsilon,2in omega,1in theta, and1in zeta, based on their sequence similarities to other insect GSTs, particularly those from Drosophila melanogaster, Anopheles gambiae, and Aedes aegypti. The remaining one GST was unclassified due to the lack of homologies to the currently known class. The nomenclature of these B. dorsalis GSTs cDNA and their deduced amino acid sequences have been deposited in Genbank with the following accession numbers:BdGSTdl (JQ690090), BdGSTd2(JN003593), BdGSTd5(JN792452), BdGSTd6(JN792453), BdGSTel (JN003588), BdGSTe2(JN003589), BdGSTe3(JQ690091), BdGSTe4(JN003590), BdGSTe5(N003591), BdGSTe6(JQ690092), BdGSTe7(JN003592), BdGSTe9(JQ690093), BdGSTol (JQ690094), BdGSTo2(JQ690095), BdGSTtl (JQ690096), BdGSTz2(JQ690097), and BdGSTul (JN003587). The percentages of deduced amino acid identities were47.6-57.9%among the four delta GSTs,31.8-54.9%among the eight epsilon GSTs,85.4%between the two omega GSTs, but only12.4to30%among different classes of the GSTs in the oriental fruit fly GSTs. Furthermore, several conserved amino acid residues which represent the catalytic pocket and binding site were analyzed by sequence alignment. Meanwhile, the full-length cDNA of microsomal GST gene BdGSTml was identified and cloned from B. dorsalis using RACE technique. The Genbank accession number was JQ690098. In addition, BdGSTm1was closely related to DmGSTm-NP524696with91%amino acid sequence identity. The deduced amino acid sequence included one insect typical motif of the microsomal GSTs family, was D-P-X-V-E-R-V-R-R-A-H-X-N-D-X-E-N-I-L-P, based on their sequence similarities to other diptera insect microsomal GSTs. At the same time, insect microsomal GSTs amino acid sequences were shorter, usually only about150amino acids.
3. GST genes expression profiles from B. dorsalis
3.1Developmental stages expression profiles of GST genes
The developmental stages expression profiles of18GST genes from B. dorsalis were analyzed using a-Tubulin as an internal control gene. The developmental stages included egg, three different larval instars (first-, second-, and third), pupa, and adult. The results showed that the relative expression level of BdGSTel was the only overexpressed gene in the egg developmental stage, which suggested that it might be associated with regulation of juvenile hormone and ecdysone in egg. Higher expression levels of six GST genes (BdGSTd2, BdGSTd5, BdGSTe5, BdGSTe9, BdGSTtl, and BdGSTz2) were observed at larval stages compared with adulthood, implies it may play an important metabolic role in the larval stages. The expression level of four Epsilon GST genes (BdGSTe2, BdGSTe3, BdGSTe4, and BdGSTe6) were increased during the development of fruit fly, which indicates that the detoxification capacity also increase in the ontogeny of B. dorsalis.
3.2Expression profiles of GST genes among different tissues
The relative quantitative of mRNA expression levels of18GST genes in three tissues including midgut, fat body and Malpighian tubules were analyzed using a-Tubulin as reference gene. In the midgut tissue, the expression of three insect-specific family GST genes (BdGSTd5, BdGSTe3, and BdGSTe9) were the highest, suggesting that these genes may play an important role in the detoxification of exogenous compounds. Other GST genes were not expressed in the midgut, may play a similar functional roles in other tissues. Four GST genes(BdGSTd6, BdGSTe4, BdGSTe6, and BdGSTz2) in the fat body tissue were relatively the highest expression level, indicating that these genes may be involved in the detoxification of exogenous compounds or protect the body from oxidative stress. The relative mRNA expression of BdGSTdl, BdGSTel, BdGSTe2, BdGSTe5, BdGSTe7, and BdGSTu1were higher in the Malpighian tubules tissue than the other tissues. The specific role of GSTs in Malpighian tubules is still not clear, but it could be involved in the enhanced excretion of Malpighian tubules.
3.3Expression profiles of GST genes induced by three insecticides
To determine the induction responses of GST genes, three insecticides malathion, avermectin, and β-cypermethrin were chosen in this study. Based on the bioassay, the flies were induced by the insecticides in two different regimes:time treatment (12,24,36, and48h) and dose treatment (LD10, LD50, and LD80). qPCR with a-Tubulin as the reference gene was used to determine the relative expression quantity of GST genes. Five GST genes (BdGSTd6, BdGSTe4, BdGSTe5, BdGSTe6, and BdGSTm1) transcripts showed the highest after12h malathion induction; BdGSTd2and BdGSTo1were reached the highest after24h; and other eight GST genes(BdGSTd1, BdGSTel, BdGSTe2, BdGSTe3, BdGSTo2, BdGSTtl, BdGSTz2, and BdGSTu1) reached the peak48h after malathion induction. The relative expression quantities of ten GST genes (BdGSTd1, BdGSTd5, BdGSTd6, BdGSTe2, BdGSTe4, BdGSTe5, BdGSTe6, BdGSTo2, BdGSTtl, and BdGSTu1) were increased significantly after exposure to sub-lethal dose (LD10) malathion for24h. These GST genes may participate in the metabolic processes to malathion of B. dorsalis.
In the avermectin induction, the expression of BdGSTe6and BdGSTml were significantly enhanced after12h, meanwhile these six GST genes(BdGSTd1, BdGSTd2, BdGSTd5, BdGSTd6, BdGSTe4, and BdGSTe9) transcripts were reached the peak24h later, seven GST genes(BdGSTd2, BdGSTd5, BdGSTd6, BdGSTe4, BdGSTe5, BdGSTe6, and BdGSTml) were the highest after induce to lethal dose (LD50) avermectin for24h. The results suggest that these GST genes may have a role in detoxification of avermectin in B. dorsalis.
Similar to the above insecticides, after the12h β-cypermethrin induction, the expression of three GST genes(BdGSTe2, BdGSTe5, and BdGSTe6) were significant higher; meanwhile these six GST genes (BdGSTe4, BdGSTe9, BdGSTo2, BdGSTtl, BdGSTu1, and BdGSTm1) expression were reached the peak36h later, four GST genes (BdGSTd1, BdGSTe4, BdGSTe7, and BdGSTtl) were up regulation after induce to lethal dose (LD10) P-cypermethrin for24h, suggesting that these GST gene may have a role in detoxification of β-cypermethrin in B. dorsalis.
3.4Expression profiles of GST genes among different strains
In order to verify whether GSTs are over-expressed in resistant fruit flies, qPCR with a-Tubulin as the reference gene was employed to determine the relative expression quantity of18GST genes in three different strains of B. dorsalis. As indicated by qPCR, three GST genes (BdGSTd5, BdGSTe9, and BdGSTz2) transcripts in malathion resistant strain were significantly higher than susceptible strain, while the expression of BdGSTel, BdGSTe2, and BdGSTtl were significantly lower than susceptible strain, which implied that these GST genes were possibly involved in B. dorsalis malathion resistance. The relative expression levels of these eight GST genes (BdGSTd6, BdGSTel, BdGSTe2, BdGSTe5, BdGSTe6, BdGSTe7, BdGSTtl, and BdGSTm1) were significantly higher in β-cypermethrin resistant strain than susceptible strain, suggesting that these GST gene may have a role in detoxification of β-cypermethrin in B. dorsalis.
4. Heterologous expression of BdGSTe5in Escherichia coli
Using the double digestion of Ndel and HindⅢ restriction enzymes and the DNA recombination technology, the expression vector for BdGSTe5was constructed based on pET28a (+) vector. After IPTG induction to E. coli, SDS-PAGE analysis showed only one specific band at approximately27kDa for the recombinant plasmid BdGSTe5-pET28a (+). According to the protein molecular weight, the expression of fusion protein molecular weight was about26kDa. Because of6x His-tag and flanking fragment accounting for1kDa, therefore, the specific band was consistent with the expected size of fusion protein. BdGSTe5-pET28a (+) recombinant protein was purified and confirmed by Ni and DEAE column.
In summary, the purification, biochemical and toxicological characterization of GSTs of B. dorsalis were studied by glutathione-agarose affinity chromatography; based on the transcriptome data,18novel GST genes were cloned from B. dorsalis using RACE technique; GST genes expression profiles were studied using qPCR technique from B. dorsalis including developmental stages, tissues, insecticides inductions, and different strains, furthermore, the expression vector for BdGSTe5was constructed and expressed. These results will not only provide great insights into exploringthe functions of the oriental fruit fly GSTs system in development and physiology, but also provide evidence for clarifying the adaptive mechanisms of oriental fruit fly to environment. Meanwhile, the results would also enrich and develope the scientific theoretical study system for the resistance mechanism of other insect pests.
引文
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