發(fā)布時(shí)間：2018-05-06 19:03

  本文選題：褐色橘蚜 + 乙酰膽堿酯酶�。� 參考：《西南大學(xué)》2017年碩士論文

【摘要】：本學(xué)位論文以橘園重要害蟲(chóng)褐色橘蚜Aphis(Toxoptera)citricidus(Kirkaldy)為研究對(duì)象,基于其轉(zhuǎn)錄組數(shù)據(jù),利用生物信息學(xué)分析方法篩選得到2條乙酰膽堿酯酶基因序列,結(jié)合實(shí)時(shí)熒光定量PCR技術(shù)解析這2個(gè)基因在褐色橘蚜不同體段及不同發(fā)育階段的表達(dá)模式;最后,分別利用RNAi技術(shù)以及真核表達(dá)技術(shù)從基因沉默及過(guò)表達(dá)兩個(gè)方面深入解析這2個(gè)乙酰膽堿酯酶基因的分子特性和功能,以期為研究褐色橘蚜抗性發(fā)生的分子機(jī)理提供基礎(chǔ)數(shù)據(jù)。主要研究結(jié)果如下:1褐色橘蚜乙酰膽堿酯酶Tcace1和Tcace2基因序列及系統(tǒng)發(fā)育分析基于本課題組測(cè)序獲得的褐色橘蚜轉(zhuǎn)錄組數(shù)據(jù),利用生物信息學(xué)分析方法,鑒定得到與昆蟲(chóng)乙酰膽堿酯酶同源的2條乙酰膽堿酯酶基因的序列信息。利用PCR技術(shù)克隆獲得了褐色橘蚜Tcace1(KP723526)和Tcace2(KP723527)的cDNA全長(zhǎng)序列。功能區(qū)域預(yù)測(cè)分析發(fā)現(xiàn)由這2條基因序列編碼的蛋白質(zhì)為乙酰膽堿酯酶(AChE,EC3.1.1.7),均具備包括�；诖�、催化三聯(lián)體、氧離子洞、膽堿結(jié)合位點(diǎn)、形成三個(gè)分子內(nèi)二硫鍵的6個(gè)半胱氨酸殘基和許多芳香族殘基在內(nèi)的乙酰膽堿酯酶典型的保守結(jié)構(gòu)域,表明其具有乙酰膽堿酯酶保守位點(diǎn),并可能有催化水解活性。通過(guò)推導(dǎo)的氨基酸序列的系統(tǒng)發(fā)育分析,發(fā)現(xiàn)TcAChE1和TcAChE2分屬兩支。此外,這2個(gè)乙酰膽堿酯酶與豌豆長(zhǎng)管蚜Acyrthosiphon pisum及棉蚜Aphis gossypii乙酰膽堿酯酶同源性最高。2褐色橘蚜Tcace1和Tcace2基因時(shí)空表達(dá)模式分析采用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)Tcace1和Tcace2在褐色橘蚜不同發(fā)育階段及不同體段的表達(dá)特性進(jìn)行分析。結(jié)果顯示,Tcace1和Tcace2在褐色橘蚜的若蟲(chóng)期以及成蟲(chóng)期均有表達(dá),表達(dá)量從若蟲(chóng)期到成蟲(chóng)期呈現(xiàn)逐漸上升的趨勢(shì),且在無(wú)翅成蚜的表達(dá)量高于有翅成蚜;兩基因在褐色橘蚜頭、胸、腹皆有表達(dá),且在頭部高表達(dá)。3基于RNAi技術(shù)的褐色橘蚜Tcace1和Tcace2基因的功能鑒定使用設(shè)計(jì)的飼喂裝置通過(guò)飼喂法將Tcace1和Tcace2的dsRNA液導(dǎo)入褐色橘蚜體內(nèi),利用qPCR技術(shù)檢測(cè)褐色橘蚜取食dsRNA不同時(shí)間后的沉默效率,發(fā)現(xiàn)在48 h后目的基因的沉默效果明顯,Tcace1和Tcace2的沉默效率分別達(dá)到55%和50%,且不存在相互干擾及脫靶效應(yīng),沉默效率與對(duì)照相比存在顯著差異(P0.05)。且在48 h內(nèi),干擾Tcace1基因后褐色橘蚜的死亡率顯著上升。使用LC10-LC20的馬拉硫磷及西維因分別處理飼喂dsRNA 48 h后的褐色橘蚜24 h,結(jié)果發(fā)現(xiàn)處理組試蟲(chóng)對(duì)藥劑的敏感性顯著上升(P0.05)。其中,馬拉硫磷處理Tcace1干擾后的試蟲(chóng)死亡率達(dá)到74%,處理Tcace2干擾的試蟲(chóng)死亡率為52%,且二者存在顯著差異(P0.05);西維因處理Tcace1干擾后的試蟲(chóng)的死亡率達(dá)到84%,處理Tcace2干擾的試蟲(chóng)死亡率為59%,且二者存在顯著差異(P0.05)。進(jìn)一步檢測(cè)飼喂2個(gè)基因的dsRNA 48 h后存活褐色橘蚜的乙酰膽堿酯酶活性發(fā)現(xiàn),發(fā)現(xiàn)其活性均發(fā)生了顯著變化(P0.05):與對(duì)照相比,干擾Tcace1后其活性為61%,干擾Tcace2后其活性為73%。綜上結(jié)果表明,Tcace1和Tcace2在褐色橘蚜對(duì)有機(jī)磷和氨基甲酸酯類(lèi)殺蟲(chóng)劑敏感性中可能具有重要作用,且Tcace1較Tcace2發(fā)揮的作用更大。4褐色橘蚜Tcace1和Tcace2基因異源表達(dá)及活性分析采用Bac-to-Bac昆蟲(chóng)桿狀病毒表達(dá)系統(tǒng),在昆蟲(chóng)細(xì)胞Sf9中表達(dá)出褐色橘蚜Tcace1和Tcace2的重組蛋白。以乙酰膽堿酯酶的3種典型底物乙酰硫代膽堿碘化物acetylthiocholine iodide(ATCHI)、丁酰硫代膽堿碘化物butyrylthiocholine iodide(BTCHI)及丙炔基硫代膽堿碘化物propinylthiocholine iodide(PTCHI)對(duì)表達(dá)產(chǎn)物進(jìn)行酶活性測(cè)定發(fā)現(xiàn),表達(dá)TcAChE1和TcAChE2蛋白的Sf9細(xì)胞裂解液的酶活性顯著增高,且對(duì)底物的親和性更高,酶促反應(yīng)速率更快,說(shuō)明Tcace1和Tcace2在Sf9細(xì)胞中得到成功表達(dá),且獲得較高活性的可溶性蛋白。進(jìn)一步統(tǒng)計(jì)分析發(fā)現(xiàn),重組蛋白TcAChE1比TcAChE2對(duì)底物更具親和性。比較分析重組蛋白對(duì)10種抑制劑的雙分子速率常數(shù)發(fā)現(xiàn),抑制劑對(duì)TcAChE1的雙分子速率大于TcAChE2,也即TcAChE1對(duì)抑制劑更加敏感。這些結(jié)果證實(shí)TcAChE1和TcAChE2在褐色橘蚜神經(jīng)突觸內(nèi)水解乙酰膽堿的重要生理功能,更為重要的是進(jìn)一步說(shuō)明TcAChE1是褐色橘蚜介導(dǎo)殺蟲(chóng)劑不敏感性的主要形式。綜上所述,本學(xué)位論文研究綜合利用生化毒理學(xué)、生物信息學(xué)和分子生物學(xué)等學(xué)科知識(shí)和技術(shù)手段從褐色橘蚜鑒定出介導(dǎo)其神經(jīng)沖動(dòng)及在對(duì)殺蟲(chóng)劑不敏感性中起重要作用的2個(gè)乙酰膽堿酯酶基因,全面解析了這2個(gè)乙酰膽堿酯酶基因的序列特征、系統(tǒng)進(jìn)化關(guān)系、時(shí)空表達(dá)模式,并且綜合運(yùn)用RNAi擾和真核表達(dá)兩種方法,分別從基因沉默和基因過(guò)表達(dá)兩個(gè)層面對(duì)它們的功能進(jìn)行了分析。研究結(jié)果為深度挖掘乙酰膽堿酯酶基因在昆蟲(chóng)體內(nèi)的重要生理功能奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S436.66

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