昆虫乙酰胆碱受体β亚基毒理学特性研究与新外源表达平台的建立
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摘要
基因的外源表达是研究基因功能的一种重要方法。通过普通分子生物学技术和基因组测序技术,目前已经从很多种昆虫中鉴定了烟碱型乙酰胆碱受体亚基的基因,但是在外源表达时并不能表达出有功能的受体。
本文用分子生物学方法和电生理学方法,对昆虫的烟碱型乙酰胆碱受体(nAChRs)p亚基进行了克隆,并对几种昆虫的烟碱型乙酰胆碱受体(nAChRs) β亚基重要功能环和环上氨基酸变化进行了毒理学特性分析,以期探明几种昆虫nAChRs β亚基的结构与功能,进一步探索新烟碱类杀虫剂选择作用的分子机制。由于昆虫单亚基表达或少数几个亚基共表达均不能在外源表达系统中表达出功能受体(一般认为是p亚基的问题),说明需要更多的烟碱型乙酰胆碱受体亚基共表达,或者需要与一些附属蛋白共表达,这样就要求在外源表达系统中转染或者注射更多的受体mRNA,需要提高外源细胞的接受量。根据非洲爪蟾卵母细胞表达外源受体的特点,考虑到中华大蟾蜍卵母细胞的体积优势,本文在开发中华大蟾蜍卵母细胞表达系统方面开展了一些探索。成功组建了电生理实验室,掌握了中华大蟾蜍的室内饲养方法、室内饲养对中华大蟾蜍生殖的影响因素和人工条件下获得中华大蟾蜍成熟卵母细胞的方法,最后对中华大蟾蜍成熟卵母细胞内源受体进行了分析,对可表达的外源蛋白进行研究,取得了部分有价值的进展和成果。
一、昆虫nAChRs β亚基D、E、F环上氨基酸对新烟碱类杀虫剂选择性影响
本文对模式昆虫果蝇和农业昆虫桃蚜的1nAChRs β1亚基D、E、F环及环上氨基酸变化对乙酰胆碱和吡虫啉的影响进行了双电极电压钳检测。检测结果发现,把模式昆虫果蝇和农业昆虫桃蚜的nAChRs β1亚基D、E、F环单独或整体引入到大鼠的p2亚基中与褐飞虱a1亚基共表达,与野生型(褐飞虱a1亚基和大鼠的p2亚基,Nlal-β2)相比,电生理检测发现构建的嵌合体对新烟碱类杀虫剂吡虫啉的敏感性上升,而对乙酰胆碱的影响基本上没有变化。同时把发生在loopE S131Y(R)、D133N和loopF T191W、P192K氨基酸变化引入到大鼠p2亚基中与褐飞虱a1亚基共表达,与野生型Nlαl-β2相比,电生理检测发现对新烟碱类杀虫剂吡虫啉的敏感性上升,而对乙酰胆碱的影响基本上没有变化。电生理记录结果显示昆虫nAChRs p亚基D、E、F环上氨基酸对新烟碱类杀虫剂选择性有重要的影响。
二、褐飞虱nAChRs β1亚基的克隆和A-to-I RNA编辑位点的发现
采用简并引物PCR和RACE技术首次从水稻害虫褐飞虱中克隆了具有典型结构烟碱型乙酰胆碱受体β亚基,同源性比对发现此基因具有烟碱型乙酰胆碱受体亚基的典型特征,如位于胞外区氨基端与配基结合密切相关的保守氨基酸残基形成的环1oopD-F、由13个氨基酸残基隔开的含有两个二硫键的半胱氨酸环、4个保守的跨膜片段、以及TM3和TM4之间可变的胞内区等。因此,根据系统进化关系,将这个基因命名为N1β1。同时发现在这个β亚基的氨基端有6个A-to-I RNA编辑位点,其中四个引起了氨基酸变化,位点2和位点5分别位于loopD和loopE上,引起了氨基酸天冬酰胺(N)到天冬氨酸(D)变化。在不同的褐飞虱品系中发现,位点2在敏感品系中发生频率较高,而位点5在抗性品系中发生频率较高,其他几个位点在两种褐飞虱品系中发生频率是没有变化的。
三、A-to-I RNA编辑在褐飞虱中对新烟碱类杀虫剂敏感性的作用
把在褐飞虱烟碱型乙酰胆碱受体β1亚基loopD和loopE上发现的RNA编辑引起的氨基酸天冬酰胺(N)到天冬氨酸(D)变化引入到大鼠的β2亚基中,构建成突变体β2N73D、β2loopD-N73D以及β2N73D、β2loopD-N73D与褐飞虱α1亚基共表达,与野生型即褐飞虱α1亚基和大鼠的β2亚基(N1α1-β2和N1α1-β2loopD)相比,电生理检测发现,发生在D环上的RNA编辑引起的N73D变化,降低了激动剂乙酰胆碱(ACh)和吡虫啉(IMI)的敏感性,但是对乙酰胆碱的影响大于吡虫啉；而发生在E环上的RNA编辑引起的N133D变化仅对吡虫啉的敏感性有影响,对乙酰胆碱的影响是没有变化的。由此认为,发生在褐飞虱烟碱型乙酰胆碱受体β1亚基loopD和loopE上RNA编辑引起的氨基酸天冬酰胺(N)到天冬氨酸(D)变化,对新烟碱类杀虫剂的敏感性是有关的,但是不同环上氨基酸的变化起的作用不同。
四、中华大蟾蜍生物学特性研究
本文研究了中华大蟾蜍的室内饲养方法、室内饲养对中华大蟾蜍卵母细胞成熟的影响因素和人工条件下获得成熟卵母细胞的方法。研究发现,中华大蟾蜍在环境温度<15℃时,处于冬眠期,这时室内饲养只要保持足够的湿度和通风即可,而环境温度>15℃时,中华大蟾蜍开始取食,每周在清晨或黄昏喂食2～3次活体黄粉虫,每周换水3～4次,保证中华大蟾蜍的排泄物及时清除,室内饲养的适宜温度是18～22℃。长期生长在高温或昏暗环境下的中华大蟾蜍是不能产生成熟卵母细胞的。中华大蟾蜍必须经过足够时间的低温处理,体内一种被称为“冬眠因子”的生长调控因子才能发挥作用,从而引发生发泡的破裂,使中华大蟾蜍卵母细胞成熟。4～6℃环境中处理一周至一个月时间的中华大蟾蜍,注射4000IU/千克绒毛膜促性腺激素(CG)或8个/千克脑垂体(PG),经过3～5天,可产生成熟的卵母细胞。
五、中华大蟾蜍卵母细胞内源性离子通道的表达分析
对中华大蟾蜍卵母细胞内源性基因表达进行了研究。实验用双电极电压钳技术在具有滤泡膜和无滤泡膜的中华大蟾蜍卵母细胞上记录由乙酰胆碱(Ach)、γ-氨基丁酸(GABA).甘氨酸(Gly)激活的配基门控离子受体超家族受体引起的电流,并与非洲爪蟾卵母细胞和先前在中华大蟾蜍卵母细胞中的表达进行了比较。结果显示,在具有滤泡膜的中华大蟾蜍卵母细胞上,乙酰胆碱、γ-氨基丁酸、甘氨酸受体均有不同程度的表达,表达对1mM乙酰胆碱、γ-氨基丁酸、甘氨酸引起的电流值分别是87.1±4.9nA、45.4±13.8nA、36.6±22.0nA；而无滤泡膜的中华大蟾蜍卵母细胞上,对乙酰胆碱、γ-氨基丁酸、甘氨酸的刺激检测不到任何电流信号。本工作的一个目的是探讨两种蟾蜍卵母细胞内源性神经递质受体和离子通道的表达,探索中华大蟾蜍卵母细胞作为外源表达工具的可能性。
六、中华大蟾蜍卵母细胞表达外源通道的研究
本章主要是对中华大蟾蜍卵母细胞的外源表达进行了研究。分别将美洲大蠊神经索和秀丽隐杆线虫的mRNA注射到中华大蟾蜍的卵母细胞中,利用双电极电压钳检测发现,在去除滤泡膜的中华大蟾蜍卵母细胞上记录到由乙酰胆碱(Ach)、γ-氨基丁酸(GABA)刺激引起的电流信号。在注射美洲大蠊神经索mRNA的卵母细胞上,1mM/L乙酰胆碱、γ-氨基丁酸引起的电流值分别是98.1±4.9nA、329.5±6.3nA；在注射秀丽隐杆线虫的mRNA的卵母细胞上,1mM/L乙酰胆碱、γ-氨基丁酸引起的电流值分别是84.1±13.8nA、89.5±7.9nA。作为隐性对照,在未注射mRNA的去除滤泡膜的卵母细胞上,没有检测到任何由乙酰胆碱、γ-氨基丁酸刺激引起的电流信号。结果显示,注射了外源mRNA的去除滤泡膜的中华大蟾蜍卵母细胞上,乙酰胆碱受体和γ-氨基丁酸受体均有表达。本工作的一个目的是探讨中华大蟾蜍卵母细胞作为外源性神经递质受体和离子通道表达系统的可行性。根据得到的结果认为中华大蟾蜍卵母细胞也可以作为一个表达工具,对配基门控离子受体超家族受体基因的外源表达研究。
Exogenous expression is an important method to study protein function. Many insect nicotinic acetylcholine receptor (nAChR) subunits have been identified and characterized by molecular cloning and genome sequencing. However, in the case of insects such as Drosophila with all subunits identified, considerable problems remain in identification of native subunit composition and even in generating functional insect nAChRs in heterologous expression systems.
Nicotinic acetylcholine receptors (nAChRs) β subunits in insects were cloned by molecular techniques. To investigate the structure and function of insect nAChR β subunits and the possible mechanisms of neonicotinoids selectivity, the pharmcological properties of functional loops (D, E and F) and amino acids within loops of insect nicotinic acetylcholine receptor β subunits have been researched and analysised by electrophysiological techniques. Even when all insect a and β subunits were combined to generate a functional receptors, all tries are not successful. Previous studies showed that some proteins other than nAChRs themselves might play important roles in insect nAChRs formation, such as the chaperone, regulator and modulator, which indicates that more proteins should be expressed together to generate a functional insect nAChRs. To express more proteins together in frog oocytes, such as Xenopus oocytes, more cRNA or cDNA should be injected at the same time, which requires bigger capacities of oocytes. In this thesis, we are tyring to develop a new tool for exogenous expression of insect nAChRs and their accessary proteins, with big capacities. We have successfully set up the electrophysiology methods for Bufo bufo gargarizans (Chinese big toad) oocytes, including the indoor feeding methods, the factors affecting the reproduction of the big toad, the methods to obtain mature oocytes under artificial conditions, the injection of cRNA and electrophsiological recording. The endogenous expression of membrane channels in the big toad mature oocytes we tested and the exogenous expression of nAChRs were recorded.
1. Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor P subunits influenced neonicotinoid selectivity
To investigate the mechanism of acetylcholine and neonicotinoid selectivity, we have examined the effects of altering insect-specific loops D, E and F and amino acids within loops D, E and F of insect nicotinic acetylcholine receptor with two-electrode voltage clamp.The results indicated that the introduction of the insect-specific loops D, E and F, singly or together, into rat02subunit resulted in a leftward shift of the imidacloprid dose-response curves for hybrid nAChRs containing insect and mammalian subunits Nlal-β2(Nlal from the brown planthopper Nilaparvata lugens and02from rat) chimeras, reflecting decreases in EC50, compared to wildtype nAChRs Nlal-β2. By contrast, the influences on ACh potency were minimal or negligible. S131Y (R) and D133N in loop E and T191W and P192K in loop F were found to contribute to the neonicotinoid selectivity of insect-specific loops E and F, reflecting in the significant leftward shifts in EC50to imidacloprid, but the influences on ACh potency were minimal, compared to wildtype nAChRs Nlal-β2. These results indicated the insect-specific loops D, E and F each play important roles in neonicotinoids selectivity.
2. Molecular cloning nicotinic acetylcholine receptor β1subunit from brown planthopper and A-to-I RNA editing in this subunit
We obtained the full-length cDNAs encoding nicotinic acetylcholine receptor P subunit in the Nilaparvata lugens, a major rice pest, by RT-PCR and RACE technology. The gene contain nicotinic acetylcholine receptor β subunit signature motifs, such as an extracellular N-terminal region with conserved residues within loops D-F which are involved in ligand binding, the cys-loop consisting of two disulphide bond-forming cysteines separated by13amino acid residues, four well-conserved transmembrane regions (TM1-4) and a variable intracellular region between TM3and TM4, and was denoted as N.lugens β(N1β1). Six A-to-I RNA editing sites were found in N1β1N-terminal domain, Among the six editing sites, four caused amino acid changes, in which the site2(E2) and site5(E5) caused an N to D change in loop D (N73D) and loop E (N133D) respectively. E2frequency was high in Sus (susceptible) strain and E5frequency was high in Res (resistant) strain. The frequency of the other sites is no change in the two different strains of N. lugens.
3. The possible roles of A-to-I RNA editings in N1β1subunit in neonicotinoid sensitivity
To evaluate the influence of N73D in loop D and N133D in loop E by A-to-I RNA editing on neonicotinoid selectivity, loop D or loop E of N1β1was introduced into rat β2of N1αl-βnAChRs to construct the hybrid Nlal-β2LoopD or Nlal-β2LoopE nAChRs, and then N73D or N133D mutation was introduced into β2LoopD or β2LoopE to construct mutant Nlal-p2LoopD-N73D or Nlα1-p2LoopE-N133D. By expressing in Xenopus oocytes, N73D mutation in loop D significantly reduced the agonist potency of both ACh and imidacloprid, and the influence on ACh was more significant than on imidacloprid. In contrast, N133D mutation in loop E only showed significant effects on imidacloprid potency, and the influences on ACh potency were minimal or negligible. These results indicated, although E2and E5in loop D and E both caused an N to D change in important loops, their roles in imidacloprid sensitivity might be different.
4. The biological characteristics of the big toad
In this section, the indoor feeding methods for the big toad, the factors affecting the reproduction of the big toad and the methods to obtain mature oocytes under artificial conditions were analyzed. When the environmental temperature was below15℃, the big toad is in a hibernation period, and adequate humidity and ventilation should be provided to keep such hibernation. When the environmental temperature was above15℃, the big toad start feeding Tenebrio molitor2or3times in a week at the early morning or dusk, and the water should be changed3to4times a week to ensure that the excrement of the big toad was immediately removed. The appropriate temperature indoors is18～22℃. When kept in in high temperature or under a dim environment, the Chinese big toad can not produce mature oocytes. The sufficient treatment by the low temperature was necessary for the oocyte maturation. After treatment by the low temerature of4～6℃for one week to one month and the injection of4000IU/kg CG (Chorionic gonadotropin) or8/kg PG (pituitary gland), the big toad toad could produce mature oocytes in3to5days.
5. The endogenous expression of membrane channel proteins in the big toad oocytes
The endogenous expression of membrane channel proteins in the big toad oocytes was analysised by two-electrode voltage-clamp recording, in oocytes with or without follicular. The receptors for acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glycine (Gly) were tested, compared to the expression in Xenopus laevis oocytes and earlier findings in the big toad oocytes. The results showed that acetylcholine, gamma-aminobutyric acid and glycine could cause the obvious currents in the big toad oocytes with follicular membrane, with the current in response to1mM ACh, GABA and Gly in oocytes with follicular membrane was87.1±4.9nA,45.4±13.8nA and36.6±22.0nA. However, in the big toad oocytes without follicular membrane, no currents were detected when the application of ACh, GABA and Gly. The results indicated there was not the endogenous expression of the receptors for such chemcials in the big toad oocytes.
6. The exogenous expression of membrane channel proteins in the big toad oocytes
The mRNA purified from American cockroach nerve cord and Caenorhabditis elegans was injected into oocytes of the big toad. The currents in oocytes without follicular membrane evoked by the application of ACh and GABA were recoded using the two-electrode voltage-clamp, compared to the expression in Xenopus laevis and earlier findings in the big toad oocytes. The obvious currents were observed in the big toad oocytes with the injection of exogenous mRNA when application of ACh and GABA. The current in response to1mM ACh and GABA in oocytes injected with American cockroach nerve cord mRNA was98.1±4.9nA and329.5±6.3nA. The current in response to1mM ACh and GABA in oocytes injected with Caenorhabditis elegans mRNA was84.1±13.8nA and89.5±7.9nA. Because there was not any currents were evoked by ACh and GABA in the big toad oocytes without any injection, the results showed the suscessful expression of exogenous ACh receptors and GABA receptors in the big toad oocytes. In future, more receptors and channels were tested in the big toad oocytes. According to the results at present, we could conclude, at least, that the big toad oocytes can be used as an expression tool for exogenous expression of ACh and GABA receptors.
本文用分子生物学方法和电生理学方法,对昆虫的烟碱型乙酰胆碱受体(nAChRs)p亚基进行了克隆,并对几种昆虫的烟碱型乙酰胆碱受体(nAChRs) β亚基重要功能环和环上氨基酸变化进行了毒理学特性分析,以期探明几种昆虫nAChRs β亚基的结构与功能,进一步探索新烟碱类杀虫剂选择作用的分子机制。由于昆虫单亚基表达或少数几个亚基共表达均不能在外源表达系统中表达出功能受体(一般认为是p亚基的问题),说明需要更多的烟碱型乙酰胆碱受体亚基共表达,或者需要与一些附属蛋白共表达,这样就要求在外源表达系统中转染或者注射更多的受体mRNA,需要提高外源细胞的接受量。根据非洲爪蟾卵母细胞表达外源受体的特点,考虑到中华大蟾蜍卵母细胞的体积优势,本文在开发中华大蟾蜍卵母细胞表达系统方面开展了一些探索。成功组建了电生理实验室,掌握了中华大蟾蜍的室内饲养方法、室内饲养对中华大蟾蜍生殖的影响因素和人工条件下获得中华大蟾蜍成熟卵母细胞的方法,最后对中华大蟾蜍成熟卵母细胞内源受体进行了分析,对可表达的外源蛋白进行研究,取得了部分有价值的进展和成果。
一、昆虫nAChRs β亚基D、E、F环上氨基酸对新烟碱类杀虫剂选择性影响
本文对模式昆虫果蝇和农业昆虫桃蚜的1nAChRs β1亚基D、E、F环及环上氨基酸变化对乙酰胆碱和吡虫啉的影响进行了双电极电压钳检测。检测结果发现,把模式昆虫果蝇和农业昆虫桃蚜的nAChRs β1亚基D、E、F环单独或整体引入到大鼠的p2亚基中与褐飞虱a1亚基共表达,与野生型(褐飞虱a1亚基和大鼠的p2亚基,Nlal-β2)相比,电生理检测发现构建的嵌合体对新烟碱类杀虫剂吡虫啉的敏感性上升,而对乙酰胆碱的影响基本上没有变化。同时把发生在loopE S131Y(R)、D133N和loopF T191W、P192K氨基酸变化引入到大鼠p2亚基中与褐飞虱a1亚基共表达,与野生型Nlαl-β2相比,电生理检测发现对新烟碱类杀虫剂吡虫啉的敏感性上升,而对乙酰胆碱的影响基本上没有变化。电生理记录结果显示昆虫nAChRs p亚基D、E、F环上氨基酸对新烟碱类杀虫剂选择性有重要的影响。
二、褐飞虱nAChRs β1亚基的克隆和A-to-I RNA编辑位点的发现
采用简并引物PCR和RACE技术首次从水稻害虫褐飞虱中克隆了具有典型结构烟碱型乙酰胆碱受体β亚基,同源性比对发现此基因具有烟碱型乙酰胆碱受体亚基的典型特征,如位于胞外区氨基端与配基结合密切相关的保守氨基酸残基形成的环1oopD-F、由13个氨基酸残基隔开的含有两个二硫键的半胱氨酸环、4个保守的跨膜片段、以及TM3和TM4之间可变的胞内区等。因此,根据系统进化关系,将这个基因命名为N1β1。同时发现在这个β亚基的氨基端有6个A-to-I RNA编辑位点,其中四个引起了氨基酸变化,位点2和位点5分别位于loopD和loopE上,引起了氨基酸天冬酰胺(N)到天冬氨酸(D)变化。在不同的褐飞虱品系中发现,位点2在敏感品系中发生频率较高,而位点5在抗性品系中发生频率较高,其他几个位点在两种褐飞虱品系中发生频率是没有变化的。
三、A-to-I RNA编辑在褐飞虱中对新烟碱类杀虫剂敏感性的作用
把在褐飞虱烟碱型乙酰胆碱受体β1亚基loopD和loopE上发现的RNA编辑引起的氨基酸天冬酰胺(N)到天冬氨酸(D)变化引入到大鼠的β2亚基中,构建成突变体β2N73D、β2loopD-N73D以及β2N73D、β2loopD-N73D与褐飞虱α1亚基共表达,与野生型即褐飞虱α1亚基和大鼠的β2亚基(N1α1-β2和N1α1-β2loopD)相比,电生理检测发现,发生在D环上的RNA编辑引起的N73D变化,降低了激动剂乙酰胆碱(ACh)和吡虫啉(IMI)的敏感性,但是对乙酰胆碱的影响大于吡虫啉；而发生在E环上的RNA编辑引起的N133D变化仅对吡虫啉的敏感性有影响,对乙酰胆碱的影响是没有变化的。由此认为,发生在褐飞虱烟碱型乙酰胆碱受体β1亚基loopD和loopE上RNA编辑引起的氨基酸天冬酰胺(N)到天冬氨酸(D)变化,对新烟碱类杀虫剂的敏感性是有关的,但是不同环上氨基酸的变化起的作用不同。
四、中华大蟾蜍生物学特性研究
本文研究了中华大蟾蜍的室内饲养方法、室内饲养对中华大蟾蜍卵母细胞成熟的影响因素和人工条件下获得成熟卵母细胞的方法。研究发现,中华大蟾蜍在环境温度<15℃时,处于冬眠期,这时室内饲养只要保持足够的湿度和通风即可,而环境温度>15℃时,中华大蟾蜍开始取食,每周在清晨或黄昏喂食2～3次活体黄粉虫,每周换水3～4次,保证中华大蟾蜍的排泄物及时清除,室内饲养的适宜温度是18～22℃。长期生长在高温或昏暗环境下的中华大蟾蜍是不能产生成熟卵母细胞的。中华大蟾蜍必须经过足够时间的低温处理,体内一种被称为“冬眠因子”的生长调控因子才能发挥作用,从而引发生发泡的破裂,使中华大蟾蜍卵母细胞成熟。4～6℃环境中处理一周至一个月时间的中华大蟾蜍,注射4000IU/千克绒毛膜促性腺激素(CG)或8个/千克脑垂体(PG),经过3～5天,可产生成熟的卵母细胞。
五、中华大蟾蜍卵母细胞内源性离子通道的表达分析
对中华大蟾蜍卵母细胞内源性基因表达进行了研究。实验用双电极电压钳技术在具有滤泡膜和无滤泡膜的中华大蟾蜍卵母细胞上记录由乙酰胆碱(Ach)、γ-氨基丁酸(GABA).甘氨酸(Gly)激活的配基门控离子受体超家族受体引起的电流,并与非洲爪蟾卵母细胞和先前在中华大蟾蜍卵母细胞中的表达进行了比较。结果显示,在具有滤泡膜的中华大蟾蜍卵母细胞上,乙酰胆碱、γ-氨基丁酸、甘氨酸受体均有不同程度的表达,表达对1mM乙酰胆碱、γ-氨基丁酸、甘氨酸引起的电流值分别是87.1±4.9nA、45.4±13.8nA、36.6±22.0nA；而无滤泡膜的中华大蟾蜍卵母细胞上,对乙酰胆碱、γ-氨基丁酸、甘氨酸的刺激检测不到任何电流信号。本工作的一个目的是探讨两种蟾蜍卵母细胞内源性神经递质受体和离子通道的表达,探索中华大蟾蜍卵母细胞作为外源表达工具的可能性。
六、中华大蟾蜍卵母细胞表达外源通道的研究
本章主要是对中华大蟾蜍卵母细胞的外源表达进行了研究。分别将美洲大蠊神经索和秀丽隐杆线虫的mRNA注射到中华大蟾蜍的卵母细胞中,利用双电极电压钳检测发现,在去除滤泡膜的中华大蟾蜍卵母细胞上记录到由乙酰胆碱(Ach)、γ-氨基丁酸(GABA)刺激引起的电流信号。在注射美洲大蠊神经索mRNA的卵母细胞上,1mM/L乙酰胆碱、γ-氨基丁酸引起的电流值分别是98.1±4.9nA、329.5±6.3nA；在注射秀丽隐杆线虫的mRNA的卵母细胞上,1mM/L乙酰胆碱、γ-氨基丁酸引起的电流值分别是84.1±13.8nA、89.5±7.9nA。作为隐性对照,在未注射mRNA的去除滤泡膜的卵母细胞上,没有检测到任何由乙酰胆碱、γ-氨基丁酸刺激引起的电流信号。结果显示,注射了外源mRNA的去除滤泡膜的中华大蟾蜍卵母细胞上,乙酰胆碱受体和γ-氨基丁酸受体均有表达。本工作的一个目的是探讨中华大蟾蜍卵母细胞作为外源性神经递质受体和离子通道表达系统的可行性。根据得到的结果认为中华大蟾蜍卵母细胞也可以作为一个表达工具,对配基门控离子受体超家族受体基因的外源表达研究。
Exogenous expression is an important method to study protein function. Many insect nicotinic acetylcholine receptor (nAChR) subunits have been identified and characterized by molecular cloning and genome sequencing. However, in the case of insects such as Drosophila with all subunits identified, considerable problems remain in identification of native subunit composition and even in generating functional insect nAChRs in heterologous expression systems.
Nicotinic acetylcholine receptors (nAChRs) β subunits in insects were cloned by molecular techniques. To investigate the structure and function of insect nAChR β subunits and the possible mechanisms of neonicotinoids selectivity, the pharmcological properties of functional loops (D, E and F) and amino acids within loops of insect nicotinic acetylcholine receptor β subunits have been researched and analysised by electrophysiological techniques. Even when all insect a and β subunits were combined to generate a functional receptors, all tries are not successful. Previous studies showed that some proteins other than nAChRs themselves might play important roles in insect nAChRs formation, such as the chaperone, regulator and modulator, which indicates that more proteins should be expressed together to generate a functional insect nAChRs. To express more proteins together in frog oocytes, such as Xenopus oocytes, more cRNA or cDNA should be injected at the same time, which requires bigger capacities of oocytes. In this thesis, we are tyring to develop a new tool for exogenous expression of insect nAChRs and their accessary proteins, with big capacities. We have successfully set up the electrophysiology methods for Bufo bufo gargarizans (Chinese big toad) oocytes, including the indoor feeding methods, the factors affecting the reproduction of the big toad, the methods to obtain mature oocytes under artificial conditions, the injection of cRNA and electrophsiological recording. The endogenous expression of membrane channels in the big toad mature oocytes we tested and the exogenous expression of nAChRs were recorded.
1. Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor P subunits influenced neonicotinoid selectivity
To investigate the mechanism of acetylcholine and neonicotinoid selectivity, we have examined the effects of altering insect-specific loops D, E and F and amino acids within loops D, E and F of insect nicotinic acetylcholine receptor with two-electrode voltage clamp.The results indicated that the introduction of the insect-specific loops D, E and F, singly or together, into rat02subunit resulted in a leftward shift of the imidacloprid dose-response curves for hybrid nAChRs containing insect and mammalian subunits Nlal-β2(Nlal from the brown planthopper Nilaparvata lugens and02from rat) chimeras, reflecting decreases in EC50, compared to wildtype nAChRs Nlal-β2. By contrast, the influences on ACh potency were minimal or negligible. S131Y (R) and D133N in loop E and T191W and P192K in loop F were found to contribute to the neonicotinoid selectivity of insect-specific loops E and F, reflecting in the significant leftward shifts in EC50to imidacloprid, but the influences on ACh potency were minimal, compared to wildtype nAChRs Nlal-β2. These results indicated the insect-specific loops D, E and F each play important roles in neonicotinoids selectivity.
2. Molecular cloning nicotinic acetylcholine receptor β1subunit from brown planthopper and A-to-I RNA editing in this subunit
We obtained the full-length cDNAs encoding nicotinic acetylcholine receptor P subunit in the Nilaparvata lugens, a major rice pest, by RT-PCR and RACE technology. The gene contain nicotinic acetylcholine receptor β subunit signature motifs, such as an extracellular N-terminal region with conserved residues within loops D-F which are involved in ligand binding, the cys-loop consisting of two disulphide bond-forming cysteines separated by13amino acid residues, four well-conserved transmembrane regions (TM1-4) and a variable intracellular region between TM3and TM4, and was denoted as N.lugens β(N1β1). Six A-to-I RNA editing sites were found in N1β1N-terminal domain, Among the six editing sites, four caused amino acid changes, in which the site2(E2) and site5(E5) caused an N to D change in loop D (N73D) and loop E (N133D) respectively. E2frequency was high in Sus (susceptible) strain and E5frequency was high in Res (resistant) strain. The frequency of the other sites is no change in the two different strains of N. lugens.
3. The possible roles of A-to-I RNA editings in N1β1subunit in neonicotinoid sensitivity
To evaluate the influence of N73D in loop D and N133D in loop E by A-to-I RNA editing on neonicotinoid selectivity, loop D or loop E of N1β1was introduced into rat β2of N1αl-βnAChRs to construct the hybrid Nlal-β2LoopD or Nlal-β2LoopE nAChRs, and then N73D or N133D mutation was introduced into β2LoopD or β2LoopE to construct mutant Nlal-p2LoopD-N73D or Nlα1-p2LoopE-N133D. By expressing in Xenopus oocytes, N73D mutation in loop D significantly reduced the agonist potency of both ACh and imidacloprid, and the influence on ACh was more significant than on imidacloprid. In contrast, N133D mutation in loop E only showed significant effects on imidacloprid potency, and the influences on ACh potency were minimal or negligible. These results indicated, although E2and E5in loop D and E both caused an N to D change in important loops, their roles in imidacloprid sensitivity might be different.
4. The biological characteristics of the big toad
In this section, the indoor feeding methods for the big toad, the factors affecting the reproduction of the big toad and the methods to obtain mature oocytes under artificial conditions were analyzed. When the environmental temperature was below15℃, the big toad is in a hibernation period, and adequate humidity and ventilation should be provided to keep such hibernation. When the environmental temperature was above15℃, the big toad start feeding Tenebrio molitor2or3times in a week at the early morning or dusk, and the water should be changed3to4times a week to ensure that the excrement of the big toad was immediately removed. The appropriate temperature indoors is18～22℃. When kept in in high temperature or under a dim environment, the Chinese big toad can not produce mature oocytes. The sufficient treatment by the low temperature was necessary for the oocyte maturation. After treatment by the low temerature of4～6℃for one week to one month and the injection of4000IU/kg CG (Chorionic gonadotropin) or8/kg PG (pituitary gland), the big toad toad could produce mature oocytes in3to5days.
5. The endogenous expression of membrane channel proteins in the big toad oocytes
The endogenous expression of membrane channel proteins in the big toad oocytes was analysised by two-electrode voltage-clamp recording, in oocytes with or without follicular. The receptors for acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glycine (Gly) were tested, compared to the expression in Xenopus laevis oocytes and earlier findings in the big toad oocytes. The results showed that acetylcholine, gamma-aminobutyric acid and glycine could cause the obvious currents in the big toad oocytes with follicular membrane, with the current in response to1mM ACh, GABA and Gly in oocytes with follicular membrane was87.1±4.9nA,45.4±13.8nA and36.6±22.0nA. However, in the big toad oocytes without follicular membrane, no currents were detected when the application of ACh, GABA and Gly. The results indicated there was not the endogenous expression of the receptors for such chemcials in the big toad oocytes.
6. The exogenous expression of membrane channel proteins in the big toad oocytes
The mRNA purified from American cockroach nerve cord and Caenorhabditis elegans was injected into oocytes of the big toad. The currents in oocytes without follicular membrane evoked by the application of ACh and GABA were recoded using the two-electrode voltage-clamp, compared to the expression in Xenopus laevis and earlier findings in the big toad oocytes. The obvious currents were observed in the big toad oocytes with the injection of exogenous mRNA when application of ACh and GABA. The current in response to1mM ACh and GABA in oocytes injected with American cockroach nerve cord mRNA was98.1±4.9nA and329.5±6.3nA. The current in response to1mM ACh and GABA in oocytes injected with Caenorhabditis elegans mRNA was84.1±13.8nA and89.5±7.9nA. Because there was not any currents were evoked by ACh and GABA in the big toad oocytes without any injection, the results showed the suscessful expression of exogenous ACh receptors and GABA receptors in the big toad oocytes. In future, more receptors and channels were tested in the big toad oocytes. According to the results at present, we could conclude, at least, that the big toad oocytes can be used as an expression tool for exogenous expression of ACh and GABA receptors.
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