摘要
乙酰胆碱酯酶(acetylcholinestrase，AChE，EC3.1.1.7)，是一种丝氨酸水解酶，主要存在于神经元和神经肌肉接头处。它的主要功能是快速水解神经递质乙酰胆碱(acetylcholine，ACh)，从而终止ACh对突触后膜的兴奋作用，保证神经信号在生物体内正常传递，因此，在胆碱能神经传导过程中起着至关重要的作用。AChE是神经毒剂、有机磷和氨基甲酸酯类杀虫剂的作用靶标。杀虫剂通过和AChE结合，降低AChE活性，引起ACh在突触间隙的过量积累，导致昆虫的抽搐甚至死亡。此外，AChE还与细胞凋亡有关。
为了研究AChE的结构与功能之间的相互关系，本文以家蚕为材料，作了如下研究：比较研究了辛硫磷农药诱导前后的两种ace（ace1和ace2）的转录水平；利用原核表达系统表达了ace1的编码区，并成功制备了兔多克隆抗体，检测了辛硫磷农药诱导后的脑组织中的ace1蛋白的表达水平；对AChE1关键位点进行突变，利用杆状病毒表达系统进行表达，测定了表达蛋白的生化属性以及抑制动力学属性；采用高通量测序技术测定了家蚕脑组织中的各基因转录水平，并以定量PCR检测了AChE调控相关基因在辛硫磷农药诱导后的转录水平。主要研究结果如下：
1家蚕添食辛硫磷前后两种ace的转录特征
采用了实时荧光定量PCR（Real-time quantitative Polymerase Chain Reaction）方法，研究了ace1和ace2基因在家蚕5龄幼虫各组织（中肠、丝腺、脂肪体、马氏管、血液、精巢、卵巢、脑）中的转录水平，以及在辛硫磷(4.0μg/mL)添食后的转录特征。结果表明，在正常的家蚕中，ace1在各组织中表达量差距很大，在脑组织中高量表达，在其他所有组织中只有微弱的表达，表达量仅为脑组织表达量的0.21%-3.68%。而ace2在各组织中的表达量差距不大，在中肠、丝腺和血淋巴中相对表达量较低，在脂肪体和卵巢中相对表达量较高。对比两种ace的转录水平发现，ace1在各组织中的表达量比ace2的表达量高，平均值为ace2表达量的13.50倍，脑组织中的ace1的表达量为ace2的95.97倍。在有机磷诱导24h后，各组织ace1表达水平都有所提高，其中脑组织中ace1基因表达增加了1.81倍；大部分组织的ace2表达水平都有所提高，精巢和血淋巴中上升倍数最大，分别为3.48和4.22倍，脑组织中的表达量为诱导前的1.12倍。检测了辛硫磷诱导48h，脑组织中ace1和ace2的转录水平，结果表明，ace1和ace2的表达趋势一致，在24h时呈增量表达，48h时表达量最低，72h表达量升高。Ace1的变化倍数大于ace2，农药诱导后的ace2在各组织中的表达也相对稳定。证明ace1是编码主要的AChE基因，与辛硫磷的关系更为密切，为研究两种AChE在家蚕生理过程中的作用机理打下了基础。
2家蚕Ace1的克隆及抗体制备
为了进一步研究ace1的功能，本研究将ace1的编码区克隆到表达载体pET28a上，转化到大肠杆菌BL21感受态细胞中，经0.1mmol/L IPTG诱导进行原核表达。SDS-PAGE电泳分析表明，转化了ace1基因重组载体的BL21菌，在约76kDa位置出现了一条特异性条带，而在对照组空BL21菌和转化了空载体pET28a(+)的菌在同样位置没有此特异性条带，说明ace1在大肠杆菌中得到正确表达。Western blottting分析结果表明，已经成功地表达出了带有6×His标签的融合蛋白。将表达的蛋白纯化后免疫家兔，成功制备了兔多克隆抗体，抗体效价为1:512000，为深入研究AChE1在家蚕组织中的表达，以及以后的功能研究打下了基础。
3家蚕辛硫磷诱导前后AChE1在脑组织中的表达水平
我们用制备的兔多克隆抗体检测AChE1在中毒前后的家蚕脑组织的表达水平。结果表明AChE1在诱导24h后表达量增加1.5倍，而48h呈下降趋势，72h后蛋白表达水平重新上升。证明AChE1在家蚕的辛硫磷抗性方面起着重要作用，和ace1的定量分析结果一致。
4家蚕ace1的定点突变及真核表达
为了研究家蚕ace1特征位点的突变与杀虫剂抗性之间的关系，我们采用定点突变方法，对保守位点处的3个氨基酸进行了突变：Ala (GCG)303Ser (TCG)，Gly (GGA)329Ala (GCA)，Leu(TCT)554Ser (TTC)。利用杆状病毒表达系统对野生型ace1（wace1）和突变型ace1（mace1）进行真核表达。首先将野生型和突变型的AChE基因（wace1和mace1）进行亚克隆到转移载体pFastBacTMHT B中，然后转化Ac DH10Bac E coli大肠杆菌菌株，得到转移质粒bacmid，最后转染sf9细胞，得到重组蛋白。测序结果表明，已成功突变了目标碱基。SDS-PAGE和Western blotting方法检测到了特异性表达的目的蛋白，分子量大小约76kDa，说明目的蛋白得到了正确表达，为ace1基因的结构和功能研究打下了基础。
5表达产物的纯化及生化属性测定
由于未纯化的粗酶液可能影响抑制剂和底物与酶的相互作用，我们利用Ni-NTA纯化系统对杆状病毒表达系统表达的野生型AChE1（wAChE1）和突变型AChE1（mAChE1）进行了纯化。并用AChE专一性抑制剂毒扁豆碱以及辛硫磷农药测定了其对表达产物的抑制作用。结果表明，经过纯化，我们得到了较为单一的目的蛋白，符合酶活测定的要求。对纯化产物进行生物学测定发现两种表达蛋白的酶促动力学属性没有明显差异，野生型的AChE1的Km为0.0305mM，突变型的AChE1的Km为0.0284mM，两种蛋白的Vmax值分别为0.731和0.743μM min1mg1。但是对专一性抑制剂的敏感性有所区别，突变后的AChE1对专一性抑制剂毒扁豆碱和辛硫磷农药的敏感性更低，为AChE1蛋白关键位点的突变与抗药性关系提供了理论基础，也为培育家蚕抗农药新品种和开发选择性杀虫剂提供了新的思路。
6高通量测序研究辛硫磷诱导前后家蚕脑组织基因转录水平
采用高通量测序技术，研究经辛硫磷（4.0μg/mL）诱导48h后家蚕脑组织基因表达变化，从氧化代谢、核酸及氨基酸代谢等方面分析辛硫磷添毒对家蚕脑组织的影响。采用DGE方法对对照组（CK）、添毒组（OP）两个文库进行测序，分别产生3588285，3379576个clean tag。Clean tag种类分别为67679，59278个。与基因组比对后，比对上的tag种类分别为12173和10116，分别占总clean tag种类的17.99%和17.07%。筛选家蚕添毒后脑组织中差异表达基因共315个，其中上调基因74个，下调基因241个。其中ace1的表达呈下调趋势，与我们的定量检测结果一致。线粒体相关氧化磷酸化基因上调表达，DNA损伤修复、碱基合成及代谢相关基因下调表达。透射电镜结果表明，辛硫磷诱导后细胞出现线粒体肿胀，呈空泡状、染色质结块等细胞凋亡症状，证明了辛硫磷中毒对线粒体产生损伤，对核内染色体也产生影响。为我们进一步研究家蚕及其他鳞翅目昆虫代谢及有机磷杀虫剂的分子机制打下基础。
7辛硫磷诱导后AChE调控相关基因的转录特征
为了研究家蚕ace1在辛硫磷农药诱导后表达的调控作用，我们测定了辛硫磷农药诱导后AChE调控相关的5个基因（cJun-N-terminal kinase、activating transcription factor2、calpain、calcineurinA和calcineurinB）在家蚕脑组织中的转录水平。结果表明，cJun-N-terminal kinase、activating transcription factor2在辛硫磷农药诱导不同时间后，表达量都随着时间的推移呈上升状态，48h时cJun-N-terminal kinase增加最为明显。atf2表达量在72h时增加倍数最多。calpain、calcineurinA和calcineurinB三个基因呈先上升后下降的趋势。证明家蚕在农药诱导后，AChE调控相关的基因表达与AChE的表达存在时空差异，具有24h的时间差，但是具体机制还有待深入研究。
本研究首次从基因转录水平和蛋白表达水平证明辛硫磷诱导后家蚕ace1承担重要的角色，借鉴鳞翅目抗药性害虫AChE的突变位点，对家蚕的ace1进行了定点突变，发现突变后对专一性抑制剂的敏感性下降，为转基因培育抗药性家蚕新品种提供了重要的育种素材。同时，对辛硫磷诱导后家蚕脑组织的基因表达谱进行了研究，分析了农药代谢相关基因的转录特征，并对ace1转录调控相关基因的转录特征进行了分析，为研究家蚕ace1的表达调控机制打下了基础。
Acetylcholinesterase (AChE, EC3.1.1.7) is a kind of serine hydrolytic enzyme, located at thejunction place of neurons and neuromuscular. Acetylcholinesterase can maintain the normaltransmission of neural impulses in synaptic clefts through catalyzing the hydrolysis of neurotransmitteracetylcholine. AChE is a target enzyme for organophosphorus and carbamate pesticides. Thesepesticides can bind to AChE and reduce its activity, leading to massive accumulation of postsynapticmembrane acetylcholine, continuous stimulation, biological convulsion, and insect death. Meanwhile,AChE is related to cell apoptosis.
To study the relationship between structure and function of Bombyx mori AChE, two ace’stranscriptional level of two types of AChE was compared after induced by the pesticide, phoxim.Furthermore, the ORF of ace1was expressed by prokaryotic expression system, and rabbit polyclonalantibody was successfully prepared. AChE1protein expression level in brain tissue after phoximstimulation was detected by Western blot. After that, a mutant (mace1) for three amino acids usingsite-directed mutagenesis was generated. The Baculovirus expression system was used for theexpression of the wild type ace1(wace1) and mace1. The expression products were purified todetermine the AChE activity and the inhibitory effects of physostigmine and phoxim. We used theDGE(Digital Gene Expression Tag Profiling) to measure the transcription level of the ace genes andtheir regulation related genes in the brain of B. mori by quantitative PCR. The main results are asfollows:
1Transcriptional characteristics of ace in B. mori induced by phoxim.
The Real-time qPCR method was used to analyze ace1, and ace2transcriptional level in the B.mori larvae tissues (midgut, silk gland, fat body, Malpighian tubules, hemolymph, testis, ovary, andbrain) and the transcription characteristics by the phoxim induced (4.0μg/mL). The results showed thatace1was highly expressed in brain tissue, and had weak expression in other tissues,with the levels ofonly0.21%-3.68%to normal brain tissue. In normal tissues, there was a relatively lower expressionlevels of ace2gene in the midgut, silk gland, and hemolymph, but a relative higher expression level inthe fat body and the ovarian. Comparing the two ace transcriptional levels, we found that, ace1 expression in various tissues were higher than ace2. The average expression level of ace1is13.50timeshigher to ace2. Specifically, the expression level of ace1was95.97times higher to ace2in the braintissue. Quantitative analysis of the ace expression level at different time points after induced by phoximin brain tissue showed that, ace1and ace2expressions appeared in the same trend: decreased firstly andthen increased. The changes of ace1were greater than ace2. Pesticide-induced ace2expression invarious tissues was relatively stable. These results indicated that ace1was the major AChE gene, and itwas more sensitive to phoxim. These studies lay the foundation for two aces in B. mori physiologicalprocesses.
2Cloning and antibody preparation of ace1
In order to further study ace1function, ace1ORF was cloned into the expression vector pET28a，and then the vector was transformed into the competent cells of E. coli BL21. The recombinant proteinwas prokaryotic expressed by0.1mmol/L IPTG induced. SDS-PAGE electrophoresis analysis showedthat the transformed ace1recombinant vector in E. coli BL21had a specific band at approximately76kDa size, while in the control groups (BL21bacteria and transformed with the vector pET28a) did nothave this specific bands at the same position. Western blottting analysis showed that6×His tag fusionprotein has been successfully expressed. These results illustrated that the ace1was correctly expressedin E. coli. Then a rabbit polyclonal antibody was successfully prepared by immunizing rabbits with thepurified protein. The titer of antibody was1:512000. These results lay the foundation for the futurefunctional studies.
3The expression level of AChE1in B. mori exposed to phoxim.
In order to further study the protein expression levels of ace1, we detected the expression levels ofthe AChE1in B. mori brain induced by organophosphorus pesticide, Phoxim. By western blot assay, theresults proved that AChE1expression induced for24h was increased to1.5fold, expression at48hshowed a reduced trend, and protein expression levels at72h rised again. These results are consistentwith the result by qPCR. It indicated that AChE1played an important role in the B. mori phoximresistance.
4Site-Directed Mutagenesis of Bm-ace1and eukaryotic expression of the AChE1
To study the relationship between mutations in ace1feature sites and insecticide resistance of the B.mori, by site-directed mutagenesis method, a mutant of three amino acids at the conserved sites in theAChE was generated, with mutation sites of Ala (GCG)303Ser (TCG), Gly (GGA)329Ala (GCA), Leu(TCT)554Ser (TTC). The Baculovirus expression system was used for the eukaryotic expression of thewild type ace1(wace1) and the mutant ace1(mace1). First, the gene s (wace1and mace1) weresubcloned into the transfer vector pFastBacTMHT B, and then transformed into the Ac DH10Bac E coli strain to give the transfer plasmid Bacmid, finally transfected Sf9cells to produce the recombinantprotein. The sequences were confirmed. The result laid the foundation for the structure and function ofgenes ace1. SDS-PAGE and Western blotting were used to detect the targeting proteins with expectedsize of about76kDa. These results showed that target protein was correctly expressed.
5Expression product purification and biochemical properties determination.
We purified the expression products of wild-type the AChE (wAChE) and mutation of AChE(mAChE) in the baculovirus expression system using Ni-NTA purification system. The products wereused for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. Nosignificant differences were observed in the overall activity of the wild type and mutant AChEs. TheKms of wAChE and mAChE were0.0305mM and0.0284mM, respectively. The Vmax values were0.731and0.743μM min1mg1, respectively. However, the remaining activity of wAChE wassignificantly lower than that of mAChE after physostigmine and phoxim inhibition. These resultsindicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine andphoxim, which laid the foundation for future in vivo studies onAChE’s roles in pesticide resistance.
6Using DGE technology to detect gene expression in brain tissue induced by phoxim.
We used DGE technology to study the changes of gene expression in brain tissue induced byphoxim (4.0μg/mL)48h. We analyzed influence of phoxim pesticide on the B. mori brain tissue fromthe nerve conduction, apoptosis and so on. The method of DGE was used to sequence control group(CK), add poison group (OP) two library, which produced3588285and379576clean tags,respectively. The type of clean tags were67679, and59278, respectively. Genomic alignment showedthat previous tag types were12173and10116, respectively, countting for17.99%and17.07%of thetotal clean tag types. There were315differentially expressed genes, which including74up-regulatedgenes, and241down-regulated genes. Expression of ace1showed a down trend. There was aup-regulation in mitochondrial oxidative phosphorylation related gene expression, and adown-regulation in DNA damage repair, base synthesis and metabolism related genes. Transmissionelectron microscopy (sem) results showed that phoxim caused mitochondria swelling, after induction invacuolated, chromatin agglomeration. This lay the foundation for our further study of B. mori and otherlepidoptera insect metabolism, and molecular mechanism of organophosphorus pesticide.
7The AChE regulation related gene transcription characteristics by phoxim induced
In order to study relationship between phoxim and Bm-ace1, we used q RT-PCR method todetermine the transcription level of the five genes related to AChE regulation in B. mori brain afterinducted by phoxim: cJun-N-terminal kinase, activating transcription factor2, calpain protein,calcineurin A and calcineurin B. The results showed that the expression of cJun-N-terminal kinase and activating transcription factor2rised with the passage of time after induced by phoxim. cJun-N-terminal kinase showed an obvious increase at the48h. Atf2expression levels increased fastest at72h.Calpain protein, calcineurin A and calcineurin B expressions increased firstly and then decreased. Theseresults provided a reference for the elaboration of the mechanism of apoptosis, insecticide choice andinsecticidal mechanism’s clarifies.
This study proved an important role of ace1phoxim after induction from the gene transcriptionlevel and protein expression levels for the first time. Referring to the mutations of Lepidopteradrug-resistant pests AChE, ace1was mutated by site-directed mutagenesis. We found a decline in thesensitivity of the product of mutation to specific inhibitors. These results provided an importantbreeding material for genetically modified to cultivate the new varieties resistant of B. mori. Meanwhile,the gene expression profile of the B. mori brain tissue were studied after phoxim induction.Thetranscription characteristics of pesticide metabolism related gene were analyzed. The transcriptionalcharacteristics of Ace1transcription-regulated genes were analyzed. These studies lay the foundation forthe regulation mechanisms of the B. mori ace1expression.
引文
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