拟环纹豹蛛乙酰胆碱酯酶功能验证与新乙酰胆碱酯酶基因克隆
发布时间：2018-10-04 18:27
【摘要】：乙酰胆碱酶(acetylcholinesterase,AChE)是催化乙酰胆碱(acetylcholine,ACh)的丝氨酸水解酶,主要分布于胆碱能神经末梢突触间隙,多集中在运动神经突触后膜的褶皱中,其主要作用是终止胆碱能神经冲动、保证信号分子正常传导,在神经传导中担任重要的角色。乙酰胆碱酯酶是有机磷类和氨基甲酸酯类杀虫剂的主要作用靶标,杀虫剂使乙酰胆碱酯酶活性部位磷酰化或者氨基甲酰酯化从而抑制乙酰胆碱酯酶的活性,导致乙酰胆碱在突触处积累,引起突触后膜上的乙酰胆碱受体过度兴奋,破坏了神经信号正常传导,产生中毒症状,最终造成昆虫死亡。拟环纹豹蛛Pardosa pseudoannulata分布于亚洲大部分稻田之中,属于广谱性捕食性天敌,主要捕食对象为褐飞虱,其体型大,种群数量多,空间生态位宽,捕食能力强,在稻田害虫防治中发挥着重要作用。如今,生态环境的恶化使得人们越发意识到环境保护的重要性,如何更好地开发利用自然天敌控制害虫已成为当今的研究热点。目前国内外的研究大多集中于杀虫剂对天敌生物毒性等方面,鲜有关于其对害虫和天敌药剂选择性机理的研究报道,本研究通过RNAi技术验证了拟环纹豹蛛AChE1和AChE2对杀虫剂敏感性的差异。同时,在本实验室拟环纹豹蛛转录组测序中曾发现多个注释为AChE的基因片段,它们与其它物种的AChE具有很高的相似度,这表明拟环纹豹蛛中可能含有多个不同的ace基因,本文成功克隆了拟环纹豹蛛4个新的乙酰胆碱酯酶基因,通过与部分昆虫的乙酰胆碱酯酶氨基酸序列对比,分析有机磷和氨基甲酸酯类杀虫剂在蜘蛛与昆虫中的选择毒力。一、利用RNAi验证拟环纹豹蛛AChE1与AChE2的功能在本实验室前期研究中,克隆了拟环纹豹蛛AChE1(PpAChE1)和AChE2(PpAChE2)基因,并分析了外源重组酶的动力学特征与杀虫剂敏感性。为了进一步验证PpAChE1与PpAChE2在杀虫剂敏感性中的差异,本章建立了一种适用于拟环纹豹蛛的RNAi方法,通过显微注射法将dsRNA导入拟环纹豹蛛体内,注射位置为腹侧接近性腺处,50nL为最佳注射体积,60h为RNAi效率最佳时间点。RNAi结果显示,沉默PpAChE1和PpAChE2编码基因,均导致有机磷杀虫剂敏感性变化,说明两者可能都是有机磷杀虫剂的作用靶标,但PpAChE1是主要的靶标酶。此外,沉默PpAChE2几乎不改变蜘蛛对氨基甲酸酯类杀虫剂的敏感性,沉默PpAChE1显著改变敏感性,说明氨基甲酸酯杀虫剂的靶标酶为PpAChE1,而PpAChE2可能不是其靶标酶。由此可见,比较两个乙酰胆碱酯酶,PpAChE1是有机磷杀虫剂和氨基甲酸酯杀虫剂的主要靶标。二、拟环纹豹蛛4个新乙酰胆碱酯酶基因的克隆与序列分析利用本实验室拟环纹豹蛛转录组数据,选择与蛛形纲和昆虫纲乙酰胆碱酯酶相似性最高的4个unigene,在PCR验证序列的基础上,采用RACE技术克隆获得4个可能的乙酰胆碱酯酶基因全长。这4个可能的乙酰胆碱酯酶基因编码的氨基酸具有乙酰胆碱酯酶的典型特征,包括氧阴离子洞,胆碱结合位点,催化三联体,六个半胱氨酸形成三个链内二硫键,覆盖丝氨酸Ser200活性位点在内的FGESA保守序列等关键位点。但是,个别推到的氨基酸序列存在保守性位点上的区别,重要胆碱结合位点Trp84被Tyr替代,催化三联体Glu327被Asp取代,AChE的典型特征序列“FGESAG”在这4个基因中也发生轻微的氨基酸变化,此外14个芳香氨基酸中保守的数目也不尽相同。说明本研究克隆的4个基因可能均为拟环纹豹蛛的AChE基因,但需要进一步功能验证。结合系统进化树以及同源性对比,分别将克隆得到的4个乙酰胆碱酯酶基因命名为 Pp-ace3、Pp-ace4、Pp-ace5、Pp-ace6。将拟环纹豹蛛4个可能的乙酰胆碱酯酶与昆虫乙酰胆碱酯酶序列对比分析后发现,一些重要功能位点上的氨基酸与昆虫存在差异,这些关键氨基酸差异性可能是有机磷和氨基甲酸酯类杀虫剂在蜘蛛与昆虫之间出现选择毒力的重要机制,但需要进一步的后续研究。
[Abstract]:The sericin (ACh) is a serine hydrolase of catalytic hydrogenation (ACh), which is mainly distributed in the synaptic cleft of cholinergic neurons, and is concentrated in the folds of postsynaptic membrane of motor nerve. The main function is to terminate the cholinergic nerve impulses. To ensure the normal conduction of signal molecules and play an important role in nerve conduction. Esterase is the main target of the organophosphorus and carbamate insecticides, and the insecticide is esterified so as to inhibit the esterases from being esterified so as to inhibit the activity of esterases and lead to accumulation in the synapses. It causes hyperstimulation of the proton acceptor on the postsynaptic membrane, destroys the normal conduction of the nerve signal, generates the poisoning symptom, and finally causes the insect to die. Pdsa ptuboannulata is distributed in most of the rice fields in Asia, and belongs to predatory predation natural enemies. The main predator-prey is brown fly lice. Its population is large, the population is large, the space ecological position is wide, the predation ability is strong, and plays an important role in the prevention and treatment of pests in paddy fields. Nowadays, the deterioration of the ecological environment makes people more aware of the importance of environmental protection, and how to develop and utilize natural enemies to control pests has become a hot spot in today's research. At present, most of the researches at home and abroad have focused on the toxicity of insecticides to natural enemies. Few studies have been made on the selective mechanism of pesticide on pests and natural enemies. At the same time, in this lab, we have found a number of gene fragments annotated as cyclin E, which are similar to that of other species, suggesting that there may be a number of different ace genes in the Pantula spider. Four new Esterase genes were cloned successfully in this paper, and the selection virulence of organophosphorus and carbamate insecticides in spiders and insects was analyzed by comparing the amino acid sequences of esterase amino acids with some insects. 1. Using RNAi, we cloned the gene of PpHEE1 and PpPEE2 in the early stage of our lab, and analyzed the dynamics and pesticide sensitivity of the exogenous recombinase. In order to further verify the difference between PpHE1 and PpHE2 in pesticide sensitivity, this chapter establishes an RNAi method suitable for the PpHE1 and PpHE2 in pesticide sensitivity. 50nL is the optimal injection volume, 60h is the best time point for RNAi efficiency. The RNAi results showed that silencing of PpHEE1 and PpHEE2 encoded genes resulted in sensitive changes in organophosphate insecticides, suggesting that both could be the target of organophosphorus pesticide action, but PpHEE1 was the primary target enzyme. In addition, silence PpHEE2 hardly changes the susceptibility of spiders to carbamate insecticides, silence PpHE1 significantly altered sensitivity, suggesting that the target enzyme of the carbamate insecticide is PpHE1, and PpHEE2 may not be its target enzyme. It can be seen that the two esterase and PpPEE1 are the main targets of organophosphate insecticides and carbamate insecticides. (2) cloning and sequence analysis of the four new esterase genes of the spider spider spider, utilizing the data of the loop-shaped leopard spiders in the laboratory, selecting 4 unigenes with the highest similarity to the spider and the insect class, and on the basis of the PCR verification sequence, Four possible full-length esterase genes were cloned by RACE technique. These four possible amino acid esterase gene-coded amino acids have typical characteristics of esterase, including oxygen anion holes, choline binding sites, catalytic triplets, hexines forming three chains of intra-chain disulfide bonds, Key sites such as the FGESA conserved sequence covering serine Ser200 active sites. However, there is a difference in the conservative sites of the individual extrapolated amino acid sequences, the important choline binding site Trp84 is replaced by Tyr, the catalytic triplets Glu327 is replaced by Asp, and the typical characteristic sequence 鈥淔GESAG鈥，
本文编号：2251421
[Abstract]:The sericin (ACh) is a serine hydrolase of catalytic hydrogenation (ACh), which is mainly distributed in the synaptic cleft of cholinergic neurons, and is concentrated in the folds of postsynaptic membrane of motor nerve. The main function is to terminate the cholinergic nerve impulses. To ensure the normal conduction of signal molecules and play an important role in nerve conduction. Esterase is the main target of the organophosphorus and carbamate insecticides, and the insecticide is esterified so as to inhibit the esterases from being esterified so as to inhibit the activity of esterases and lead to accumulation in the synapses. It causes hyperstimulation of the proton acceptor on the postsynaptic membrane, destroys the normal conduction of the nerve signal, generates the poisoning symptom, and finally causes the insect to die. Pdsa ptuboannulata is distributed in most of the rice fields in Asia, and belongs to predatory predation natural enemies. The main predator-prey is brown fly lice. Its population is large, the population is large, the space ecological position is wide, the predation ability is strong, and plays an important role in the prevention and treatment of pests in paddy fields. Nowadays, the deterioration of the ecological environment makes people more aware of the importance of environmental protection, and how to develop and utilize natural enemies to control pests has become a hot spot in today's research. At present, most of the researches at home and abroad have focused on the toxicity of insecticides to natural enemies. Few studies have been made on the selective mechanism of pesticide on pests and natural enemies. At the same time, in this lab, we have found a number of gene fragments annotated as cyclin E, which are similar to that of other species, suggesting that there may be a number of different ace genes in the Pantula spider. Four new Esterase genes were cloned successfully in this paper, and the selection virulence of organophosphorus and carbamate insecticides in spiders and insects was analyzed by comparing the amino acid sequences of esterase amino acids with some insects. 1. Using RNAi, we cloned the gene of PpHEE1 and PpPEE2 in the early stage of our lab, and analyzed the dynamics and pesticide sensitivity of the exogenous recombinase. In order to further verify the difference between PpHE1 and PpHE2 in pesticide sensitivity, this chapter establishes an RNAi method suitable for the PpHE1 and PpHE2 in pesticide sensitivity. 50nL is the optimal injection volume, 60h is the best time point for RNAi efficiency. The RNAi results showed that silencing of PpHEE1 and PpHEE2 encoded genes resulted in sensitive changes in organophosphate insecticides, suggesting that both could be the target of organophosphorus pesticide action, but PpHEE1 was the primary target enzyme. In addition, silence PpHEE2 hardly changes the susceptibility of spiders to carbamate insecticides, silence PpHE1 significantly altered sensitivity, suggesting that the target enzyme of the carbamate insecticide is PpHE1, and PpHEE2 may not be its target enzyme. It can be seen that the two esterase and PpPEE1 are the main targets of organophosphate insecticides and carbamate insecticides. (2) cloning and sequence analysis of the four new esterase genes of the spider spider spider, utilizing the data of the loop-shaped leopard spiders in the laboratory, selecting 4 unigenes with the highest similarity to the spider and the insect class, and on the basis of the PCR verification sequence, Four possible full-length esterase genes were cloned by RACE technique. These four possible amino acid esterase gene-coded amino acids have typical characteristics of esterase, including oxygen anion holes, choline binding sites, catalytic triplets, hexines forming three chains of intra-chain disulfide bonds, Key sites such as the FGESA conserved sequence covering serine Ser200 active sites. However, there is a difference in the conservative sites of the individual extrapolated amino acid sequences, the important choline binding site Trp84 is replaced by Tyr, the catalytic triplets Glu327 is replaced by Asp, and the typical characteristic sequence 鈥淔GESAG鈥，
本文编号：2251421
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