【摘要】：乙酰膽堿酶(acetylcholinesterase,AChE)是催化乙酰膽堿(acetylcholine,ACh)的絲氨酸水解酶,主要分布于膽堿能神經(jīng)末梢突觸間隙,多集中在運(yùn)動(dòng)神經(jīng)突觸后膜的褶皺中,其主要作用是終止膽堿能神經(jīng)沖動(dòng)、保證信號(hào)分子正常傳導(dǎo),在神經(jīng)傳導(dǎo)中擔(dān)任重要的角色。乙酰膽堿酯酶是有機(jī)磷類和氨基甲酸酯類殺蟲劑的主要作用靶標(biāo),殺蟲劑使乙酰膽堿酯酶活性部位磷�；蛘甙被柞ｕセ瘡亩种埔阴Ｄ憠A酯酶的活性,導(dǎo)致乙酰膽堿在突觸處積累,引起突觸后膜上的乙酰膽堿受體過度興奮,破壞了神經(jīng)信號(hào)正常傳導(dǎo),產(chǎn)生中毒癥狀,最終造成昆蟲死亡。擬環(huán)紋豹蛛Pardosa pseudoannulata分布于亞洲大部分稻田之中,屬于廣譜性捕食性天敵,主要捕食對(duì)象為褐飛虱,其體型大,種群數(shù)量多,空間生態(tài)位寬,捕食能力強(qiáng),在稻田害蟲防治中發(fā)揮著重要作用。如今,生態(tài)環(huán)境的惡化使得人們?cè)桨l(fā)意識(shí)到環(huán)境保護(hù)的重要性,如何更好地開發(fā)利用自然天敵控制害蟲已成為當(dāng)今的研究熱點(diǎn)。目前國(guó)內(nèi)外的研究大多集中于殺蟲劑對(duì)天敵生物毒性等方面,鮮有關(guān)于其對(duì)害蟲和天敵藥劑選擇性機(jī)理的研究報(bào)道,本研究通過RNAi技術(shù)驗(yàn)證了擬環(huán)紋豹蛛AChE1和AChE2對(duì)殺蟲劑敏感性的差異。同時(shí),在本實(shí)驗(yàn)室擬環(huán)紋豹蛛轉(zhuǎn)錄組測(cè)序中曾發(fā)現(xiàn)多個(gè)注釋為AChE的基因片段,它們與其它物種的AChE具有很高的相似度,這表明擬環(huán)紋豹蛛中可能含有多個(gè)不同的ace基因,本文成功克隆了擬環(huán)紋豹蛛4個(gè)新的乙酰膽堿酯酶基因,通過與部分昆蟲的乙酰膽堿酯酶氨基酸序列對(duì)比,分析有機(jī)磷和氨基甲酸酯類殺蟲劑在蜘蛛與昆蟲中的選擇毒力。一、利用RNAi驗(yàn)證擬環(huán)紋豹蛛AChE1與AChE2的功能在本實(shí)驗(yàn)室前期研究中,克隆了擬環(huán)紋豹蛛AChE1(PpAChE1)和AChE2(PpAChE2)基因,并分析了外源重組酶的動(dòng)力學(xué)特征與殺蟲劑敏感性。為了進(jìn)一步驗(yàn)證PpAChE1與PpAChE2在殺蟲劑敏感性中的差異,本章建立了一種適用于擬環(huán)紋豹蛛的RNAi方法,通過顯微注射法將dsRNA導(dǎo)入擬環(huán)紋豹蛛體內(nèi),注射位置為腹側(cè)接近性腺處,50nL為最佳注射體積,60h為RNAi效率最佳時(shí)間點(diǎn)。RNAi結(jié)果顯示,沉默PpAChE1和PpAChE2編碼基因,均導(dǎo)致有機(jī)磷殺蟲劑敏感性變化,說明兩者可能都是有機(jī)磷殺蟲劑的作用靶標(biāo),但PpAChE1是主要的靶標(biāo)酶。此外,沉默PpAChE2幾乎不改變蜘蛛對(duì)氨基甲酸酯類殺蟲劑的敏感性,沉默PpAChE1顯著改變敏感性,說明氨基甲酸酯殺蟲劑的靶標(biāo)酶為PpAChE1,而PpAChE2可能不是其靶標(biāo)酶。由此可見,比較兩個(gè)乙酰膽堿酯酶,PpAChE1是有機(jī)磷殺蟲劑和氨基甲酸酯殺蟲劑的主要靶標(biāo)。二、擬環(huán)紋豹蛛4個(gè)新乙酰膽堿酯酶基因的克隆與序列分析利用本實(shí)驗(yàn)室擬環(huán)紋豹蛛轉(zhuǎn)錄組數(shù)據(jù),選擇與蛛形綱和昆蟲綱乙酰膽堿酯酶相似性最高的4個(gè)unigene,在PCR驗(yàn)證序列的基礎(chǔ)上,采用RACE技術(shù)克隆獲得4個(gè)可能的乙酰膽堿酯酶基因全長(zhǎng)。這4個(gè)可能的乙酰膽堿酯酶基因編碼的氨基酸具有乙酰膽堿酯酶的典型特征,包括氧陰離子洞,膽堿結(jié)合位點(diǎn),催化三聯(lián)體,六個(gè)半胱氨酸形成三個(gè)鏈內(nèi)二硫鍵,覆蓋絲氨酸Ser200活性位點(diǎn)在內(nèi)的FGESA保守序列等關(guān)鍵位點(diǎn)。但是,個(gè)別推到的氨基酸序列存在保守性位點(diǎn)上的區(qū)別,重要膽堿結(jié)合位點(diǎn)Trp84被Tyr替代,催化三聯(lián)體Glu327被Asp取代,AChE的典型特征序列“FGESAG”在這4個(gè)基因中也發(fā)生輕微的氨基酸變化,此外14個(gè)芳香氨基酸中保守的數(shù)目也不盡相同。說明本研究克隆的4個(gè)基因可能均為擬環(huán)紋豹蛛的AChE基因,但需要進(jìn)一步功能驗(yàn)證。結(jié)合系統(tǒng)進(jìn)化樹以及同源性對(duì)比,分別將克隆得到的4個(gè)乙酰膽堿酯酶基因命名為 Pp-ace3、Pp-ace4、Pp-ace5、Pp-ace6。將擬環(huán)紋豹蛛4個(gè)可能的乙酰膽堿酯酶與昆蟲乙酰膽堿酯酶序列對(duì)比分析后發(fā)現(xiàn),一些重要功能位點(diǎn)上的氨基酸與昆蟲存在差異,這些關(guān)鍵氨基酸差異性可能是有機(jī)磷和氨基甲酸酯類殺蟲劑在蜘蛛與昆蟲之間出現(xiàn)選擇毒力的重要機(jī)制,但需要進(jìn)一步的后續(xù)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，

本文編號(hào)：2251421