褐色橘蚜乙酰胆碱酯酶基因的分子特性及功能分析

发布时间：2018-05-06 19:03

  本文选题：褐色橘蚜 + 乙酰胆碱酯酶　； 参考：《西南大学》2017年硕士论文

【摘要】：本学位论文以橘园重要害虫褐色橘蚜Aphis(Toxoptera)citricidus(Kirkaldy)为研究对象,基于其转录组数据,利用生物信息学分析方法筛选得到2条乙酰胆碱酯酶基因序列,结合实时荧光定量PCR技术解析这2个基因在褐色橘蚜不同体段及不同发育阶段的表达模式;最后,分别利用RNAi技术以及真核表达技术从基因沉默及过表达两个方面深入解析这2个乙酰胆碱酯酶基因的分子特性和功能,以期为研究褐色橘蚜抗性发生的分子机理提供基础数据。主要研究结果如下:1褐色橘蚜乙酰胆碱酯酶Tcace1和Tcace2基因序列及系统发育分析基于本课题组测序获得的褐色橘蚜转录组数据,利用生物信息学分析方法,鉴定得到与昆虫乙酰胆碱酯酶同源的2条乙酰胆碱酯酶基因的序列信息。利用PCR技术克隆获得了褐色橘蚜Tcace1(KP723526)和Tcace2(KP723527)的cDNA全长序列。功能区域预测分析发现由这2条基因序列编码的蛋白质为乙酰胆碱酯酶(AChE,EC3.1.1.7),均具备包括酰基口袋、催化三联体、氧离子洞、胆碱结合位点、形成三个分子内二硫键的6个半胱氨酸残基和许多芳香族残基在内的乙酰胆碱酯酶典型的保守结构域,表明其具有乙酰胆碱酯酶保守位点,并可能有催化水解活性。通过推导的氨基酸序列的系统发育分析,发现TcAChE1和TcAChE2分属两支。此外,这2个乙酰胆碱酯酶与豌豆长管蚜Acyrthosiphon pisum及棉蚜Aphis gossypii乙酰胆碱酯酶同源性最高。2褐色橘蚜Tcace1和Tcace2基因时空表达模式分析采用实时荧光定量PCR技术对Tcace1和Tcace2在褐色橘蚜不同发育阶段及不同体段的表达特性进行分析。结果显示,Tcace1和Tcace2在褐色橘蚜的若虫期以及成虫期均有表达,表达量从若虫期到成虫期呈现逐渐上升的趋势,且在无翅成蚜的表达量高于有翅成蚜;两基因在褐色橘蚜头、胸、腹皆有表达,且在头部高表达。3基于RNAi技术的褐色橘蚜Tcace1和Tcace2基因的功能鉴定使用设计的饲喂装置通过饲喂法将Tcace1和Tcace2的dsRNA液导入褐色橘蚜体内,利用qPCR技术检测褐色橘蚜取食dsRNA不同时间后的沉默效率,发现在48 h后目的基因的沉默效果明显,Tcace1和Tcace2的沉默效率分别达到55%和50%,且不存在相互干扰及脱靶效应,沉默效率与对照相比存在显著差异(P0.05)。且在48 h内,干扰Tcace1基因后褐色橘蚜的死亡率显著上升。使用LC10-LC20的马拉硫磷及西维因分别处理饲喂dsRNA 48 h后的褐色橘蚜24 h,结果发现处理组试虫对药剂的敏感性显著上升(P0.05)。其中,马拉硫磷处理Tcace1干扰后的试虫死亡率达到74%,处理Tcace2干扰的试虫死亡率为52%,且二者存在显著差异(P0.05);西维因处理Tcace1干扰后的试虫的死亡率达到84%,处理Tcace2干扰的试虫死亡率为59%,且二者存在显著差异(P0.05)。进一步检测饲喂2个基因的dsRNA 48 h后存活褐色橘蚜的乙酰胆碱酯酶活性发现,发现其活性均发生了显著变化(P0.05):与对照相比,干扰Tcace1后其活性为61%,干扰Tcace2后其活性为73%。综上结果表明,Tcace1和Tcace2在褐色橘蚜对有机磷和氨基甲酸酯类杀虫剂敏感性中可能具有重要作用,且Tcace1较Tcace2发挥的作用更大。4褐色橘蚜Tcace1和Tcace2基因异源表达及活性分析采用Bac-to-Bac昆虫杆状病毒表达系统,在昆虫细胞Sf9中表达出褐色橘蚜Tcace1和Tcace2的重组蛋白。以乙酰胆碱酯酶的3种典型底物乙酰硫代胆碱碘化物acetylthiocholine iodide(ATCHI)、丁酰硫代胆碱碘化物butyrylthiocholine iodide(BTCHI)及丙炔基硫代胆碱碘化物propinylthiocholine iodide(PTCHI)对表达产物进行酶活性测定发现,表达TcAChE1和TcAChE2蛋白的Sf9细胞裂解液的酶活性显著增高,且对底物的亲和性更高,酶促反应速率更快,说明Tcace1和Tcace2在Sf9细胞中得到成功表达,且获得较高活性的可溶性蛋白。进一步统计分析发现,重组蛋白TcAChE1比TcAChE2对底物更具亲和性。比较分析重组蛋白对10种抑制剂的双分子速率常数发现,抑制剂对TcAChE1的双分子速率大于TcAChE2,也即TcAChE1对抑制剂更加敏感。这些结果证实TcAChE1和TcAChE2在褐色橘蚜神经突触内水解乙酰胆碱的重要生理功能,更为重要的是进一步说明TcAChE1是褐色橘蚜介导杀虫剂不敏感性的主要形式。综上所述,本学位论文研究综合利用生化毒理学、生物信息学和分子生物学等学科知识和技术手段从褐色橘蚜鉴定出介导其神经冲动及在对杀虫剂不敏感性中起重要作用的2个乙酰胆碱酯酶基因,全面解析了这2个乙酰胆碱酯酶基因的序列特征、系统进化关系、时空表达模式,并且综合运用RNAi扰和真核表达两种方法,分别从基因沉默和基因过表达两个层面对它们的功能进行了分析。研究结果为深度挖掘乙酰胆碱酯酶基因在昆虫体内的重要生理功能奠定了基础。
[Abstract]:This dissertation is based on the Aphis (Toxoptera) citricidus (Kirkaldy) of orange orchard, an important pest of orange garden. Based on its transcriptional data, 2 acetylcholinesterase gene sequences are screened by bioinformatics analysis, and the 2 genes in different body segments and different development of the brown orange aphids are analyzed by real time fluorescence quantitative PCR technology. In the end, the molecular properties and functions of the 2 acetylcholinesterase genes were analyzed by RNAi technology and eukaryotic expression technology from two aspects of gene silencing and overexpression, in order to provide basic data for the molecular mechanism of the resistance of brown orange aphids. The main results are as follows: 1 brown orange aphid acetyl The sequence and phylogenetic analysis of cholinesterase Tcace1 and Tcace2 gene and phylogenetic analysis were based on the data of the brown orange aphid transcriptional group obtained by the project group, and the sequence information of the 2 acetylcholinesterase gene homologous to the insect acetylcholinesterase was identified by bioinformatics analysis method. The brown orange aphid Tcace1 (KP7) was cloned by PCR technology. 23526) and the full length cDNA sequence of Tcace2 (KP723527). Functional region prediction analysis found that the proteins encoded by the 2 gene sequences are acetylcholinesterase (AChE, EC3.1.1.7), all contain acyl pockets, catalyze three interbody, oxygen ion hole, choline binding site, form 6 cysteine residues of two sulfur bonds in three molecules and many aromatic compounds. The typical conserved domain of acetylcholinesterase, including the residue of the family, indicates that it has the conserved site of acetylcholinesterase and may have catalytic hydrolysis activity. By phylogenetic analysis of the deduced amino acid sequence, two branches of TcAChE1 and TcAChE2 are found. In addition, the 2 acetylcholinesterase and pea aphid Acyrthosiphon Pisum and Analysis of spatio-temporal expression patterns of the Tcace1 and Tcace2 genes of the highest homology of Aphis gossypii acetylcholinesterase in the Aphis gossypii (Aphis gossypii) and.2 brown orange aphid, the expression characteristics of Tcace1 and Tcace2 in different developmental stages and different segments of the brown orange aphid were analyzed by real-time quantitative quantitative PCR technique. The results showed that Tcace1 and Tcace2 were in the nymph stage of brown orange aphid. The expression amount increased from the nymph to the adult stage, and the expression amount was higher than that of the aphis aphis, and the two gene was expressed in the brown orange aphid head, the chest and the abdomen, and the functional identification of the brown orange aphid Tcace1 and Tcace2 gene based on the RNAi technique was highly expressed in the head. The dsRNA solution of Tcace1 and Tcace2 was introduced into brown orange aphid by feeding, and qPCR technique was used to detect the silencing efficiency of the brown orange aphid after dsRNA for different time. It was found that the silence effect of the target gene was obvious after 48 h. The silence efficiency of Tcace1 and Tcace2 was 55% and 50% respectively, and there was no mutual interference and miss effect and silence effect. There was a significant difference in the rate compared with the control (P0.05). And in 48 h, the death rate of brown orange aphid was significantly increased after the interference of Tcace1 gene. The brown orange aphid after the use of LC10-LC20 was treated with 24 h of the brown orange aphid after the 48 h of dsRNA, and the results showed that the sensitivity of the insect pest in the treatment group increased significantly (P0.05). The mortality rate of the test worm after Tcace1 interference was 74%, the mortality rate of the test worms treated with Tcace2 interference was 52%, and the two were significantly different (P0.05); the mortality of the test worms after the interference of Tcace1 was 84%, the mortality rate of the Tcace2 interference was 59%, and the two were significantly different (P0.05). Further tests were fed on the DS of 2 genes. The activity of acetylcholinesterase in the surviving brown orange aphid after RNA 48 h found that the activity had significant changes (P0.05): compared with the control, the activity of interfering Tcace1 was 61%. After interference Tcace2, its activity was 73%. synthesis, and Tcace1 and Tcace2 were possible in the sensitivity of the brown orange aphid to organophosphorus and carbamate insecticides. It plays an important role, and Tcace1 plays a greater role than Tcace2 in.4 brown orange aphid Tcace1 and Tcace2 gene expression and activity analysis using Bac-to-Bac insect baculovirus expression system. The recombinant protein of brown orange aphid Tcace1 and Tcace2 is expressed in insect cell Sf9. The 3 typical substrates of acetylcholinesterase are acetylcholine thiocholine. The enzyme activity of acetylthiocholine iodide (ATCHI), butyacylthiocholine iodide butyrylthiocholine iodide (BTCHI) and propargyl thiocholine iodide propinylthiocholine iodide (PTCHI) showed that the enzyme activity of the Sf9 cell lysate expressing TcAChE1 and TcAChE2 protein was significantly higher, and the substrate was observed. Higher affinity and faster enzyme reaction rate showed that Tcace1 and Tcace2 were successfully expressed in Sf9 cells and obtained high active soluble proteins. Further statistical analysis found that the recombinant protein TcAChE1 was more compatible with the substrate than TcAChE2. Comparative analysis of the double molecular rate constant of the recombinant protein to the 10 inhibitors was found, and the inhibitor was found to be a inhibitor. The bimolecular rate of TcAChE1 is greater than TcAChE2 and TcAChE1 is more sensitive to inhibitors. These results confirm the important physiological function of TcAChE1 and TcAChE2 in the hydrolysis of acetylcholine in the synapses of brown orange aphids. More importantly, it is important to further explain that TcAChE1 is the main form of the insensitivity of the brown orange aphid to mediate the insecticide. The dissertation studies 2 acetylcholinesterase genes, which mediate their nerve impulse and play an important role in insecticide insensitivity, by using biochemical toxicology, bioinformatics and molecular biology to identify the 2 acetylcholinesterase genes which mediate their nerve impulse and play an important role in the insensitivity to insecticides, and the sequence characteristics of the 2 acetylcholinesterase genes are fully analyzed. The evolution relationship, the spatio-temporal expression pattern, and the comprehensive use of two methods of RNAi disturbance and eukaryotic expression, were analyzed from two layers of gene silencing and gene overexpression, respectively. The results laid the foundation for deep mining of the important physiological functions of acetylcholinesterase gene in insects.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.66

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