本文關(guān)鍵詞：鄰位開環(huán)雙加氧酶TcID1介導(dǎo)朱砂葉螨對(duì)兩種殺螨劑抗性研究　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 朱砂葉螨 阿維菌素 甲氰菊酯 鄰位開環(huán)雙加氧酶 抗藥性

【摘要】：朱砂葉螨(Tetranychus cinnabarinus)在我國(guó)分布廣泛,主要危害棉花、蔬菜等農(nóng)作物。目前我國(guó)對(duì)于農(nóng)業(yè)害螨的防治主要以化學(xué)防治為主,其中阿維菌素和甲氰菊酯是使用十分廣泛的殺螨劑。這些殺螨劑的大量使用以及葉螨自身的生理特性,導(dǎo)致我國(guó)絕大多數(shù)地區(qū)的朱砂葉螨已經(jīng)對(duì)阿維菌素和甲氰菊酯產(chǎn)生了抗藥性。鄰位開環(huán)雙加氧酶(Introdial ring-cleavage dioxygenases,ID-RCDs)是一類能夠催化鄰苯二酚類底物氧化分解的雙加氧酶,主要存在于微生物如真菌、細(xì)菌中,其主要功能是降解帶有芳香環(huán)的化合物。朱砂葉螨的基因芯片檢測(cè)結(jié)果顯示,相對(duì)于敏感品系(Susceptible strain,SS),ID-RCDs基因在阿維菌素抗性品系(Abametin resistant strain,AbR)和甲氰菊酯抗性品系(Fenpropathrin reisitant strain,FeR)中均上調(diào)表達(dá)。因此,本論文在此基礎(chǔ)上,重點(diǎn)研究了ID-RCDs與朱砂葉螨對(duì)阿維菌素和甲氰菊酯抗性的關(guān)系并取得了以下研究結(jié)果:1、朱砂葉螨ID-RCDs酶活性測(cè)定。對(duì)朱砂葉螨ID-RCDs酶進(jìn)行粗酶活性測(cè)定,該酶在AbR及FeR中的活性均顯著高于SS品系,其中AbR品系酶活性是SS品系的1.63倍,FeR品系酶活性是SS品系的2.0倍。2、朱砂葉螨TcID1基因克隆及序列分析�；蚩寺〗Y(jié)果顯示,朱砂葉螨TcID1基因全長(zhǎng)的開放閱讀框長(zhǎng)度為774bp,編碼257個(gè)氨基酸,蛋白分子量為29.4kD,等電點(diǎn)為5.31。經(jīng)Signal IP 4.1在線預(yù)測(cè)發(fā)現(xiàn)TcID1的翻譯氨基酸含有23個(gè)氨基酸的信號(hào)肽,可能屬于分泌蛋白。對(duì)朱砂葉螨TcID1氨基酸序列進(jìn)行分析,發(fā)現(xiàn)TcID1編碼的蛋白具有鄰位開環(huán)雙加氧酶特有的Fe3+結(jié)合位點(diǎn):2個(gè)His和2個(gè)Tyr。與二斑葉螨、細(xì)菌、真菌等同源基因進(jìn)行氨基酸序列相似性和進(jìn)化分析,結(jié)果顯示:朱砂葉螨TcID1基因與二斑葉螨基因tetur06g00460的相似度最高,氨基酸一致性為98%;與伊氏葉螨AFY99039.1氨基酸一致性為66%;而與真菌或細(xì)菌ID-RCDs相關(guān)的氨基酸一致性均低于40%。系統(tǒng)發(fā)育樹結(jié)果顯示,相對(duì)于細(xì)菌,朱砂葉螨TcID1基因與真菌類ID-RCDs具有更高的同源性。3、TcID1基因在不同螨態(tài)、不同品系、藥劑脅迫下的mRNA表達(dá)模式。qPCR結(jié)果顯示:在朱砂葉螨敏感品系的不同螨態(tài)中,TcID1基因在幼螨、若螨及3日齡雌成螨中均呈高表達(dá),并且在若螨中表達(dá)量最高,其次為幼螨、成螨、卵;在不同品系之間,相對(duì)于敏感品系,AbR及FeR品系均呈高表達(dá),這也驗(yàn)證了基因芯片中的相關(guān)結(jié)果;在阿維菌素(藥劑在各品系中的LC30劑量)脅迫6h、12h及24h后,TcID1基因在SS品系表現(xiàn)為先增加后降低的表達(dá)模式;在AbR品系表現(xiàn)為先降低后增加的表達(dá)模式;甲氰菊酯脅迫6h、12h及24h后,TcID1基因的表達(dá)模式同阿維菌素脅迫結(jié)果類似。4、RNAi鑒定TcID1基因在朱砂葉螨對(duì)殺螨劑抗性的功能。采用葉碟法間接飼喂TcID1-dsRNA后,SS品系TcID1基因表達(dá)量下降60.7%,ID-RCDs酶活性降低38%,對(duì)阿維菌素的敏感性顯著增加,表現(xiàn)為死亡率顯著升高,分別為44%(阿維菌素LC30處理)和38%(阿維菌素LC50處理);而對(duì)甲氰菊酯的敏感性無(wú)顯著變化;飼喂TcID1-dsRNA后,AbR品系TcID1基因表達(dá)量下降67.5%,ID-RCDs酶活性降低44%,對(duì)阿維菌素的敏感性顯著增加,表現(xiàn)為死亡率顯著升高,分別為19.5%(阿維菌素LC30處理)和17%(阿維菌素LC50處理);飼喂TcID1-dsRNA后,FeR品系TcID1基因表達(dá)量下降47.7%,ID-RCDs酶活性降低40%,而對(duì)甲氰菊酯的敏感性無(wú)顯著變化。5、原核表達(dá)TcID1基因。以pColdⅡ?yàn)楸磉_(dá)載體,構(gòu)建去除信號(hào)肽的TcID1重組表達(dá)質(zhì)粒,并測(cè)序驗(yàn)證。將測(cè)序正確的重組質(zhì)粒TcID1-pColdⅡ轉(zhuǎn)化至大腸桿菌感受態(tài)細(xì)胞中,經(jīng)IPTG誘導(dǎo)表達(dá),SDS-PAGE電泳檢測(cè)發(fā)現(xiàn)該基因一部分以可溶性表達(dá),一部分以包涵體形式表達(dá);經(jīng)過鎳親和層析柱純化,Western Blot檢測(cè)表明TcID1基因在大腸桿菌中成功表達(dá)。以原兒茶酸為底物進(jìn)行蛋白活性測(cè)定,結(jié)果顯示重組蛋白比活力為0.1346±0.0090 nmol/μg.pro/min,Km為14.2±4.6 mM,Vmax為0.1163±0.0153nmol/μg.pro/min。6、TcID1異源表達(dá)產(chǎn)物與殺螨劑的作用模式。藥劑對(duì)重組蛋白酶活影響的測(cè)定結(jié)果顯示,使用0.001mmol/L~2mmol/L的阿維菌素及甲氰菊酯均對(duì)重組蛋白酶活無(wú)影響;借助高效液相色譜檢測(cè)TcID1重組蛋白對(duì)殺螨劑的代謝試驗(yàn),結(jié)果表明TcID1重組蛋白不能代謝阿維菌素。
[Abstract]:Tetranychuscinnabarinus (Tetranychus cinnabarinus) is widely distributed in China, the main harm of cotton, vegetables and other crops. At present in our country for the control of agricultural pest mites mainly based on chemical control, which is the use of fenpropathrin and avermectin acaricide widely. These acaricides used and physiological characteristics of leaf mite itself the result of the vast majority of areas in our country have tetranychuscinnabarinus to abamectin and fenpropathrin resistant. Ortho ring opening dioxygenase (Introdial ring-cleavage, dioxygenases, ID-RCDs) is a kind of dioxygenase pyrocatechol catalyzed substrate oxidation and decomposition, mainly in microorganisms such as fungi, bacteria its main function is to degrade, compounds with aromatic rings. The detection results of gene chip tetranychuscinnabarinus showed that compared with susceptible strains (Susceptible, strain, SS) ID-RCDs gene in Abamectin Hormone resistant strains (Abametin resistant, strain, AbR) and fenpropathrin resistant strains (Fenpropathrin, reisitant strain, FeR) expression were raised. Therefore, this paper on the basis of this, we focus on ID-RCDs and tetranychuscinnabarinus of Abamectin and fenpropathrin resistance, and achieved the following results: 1 ID-RCDs, Tetranychus cinnabarinus. Enzyme activity on t.cinnabarinus ID-RCDs enzyme determination of crude enzyme activity, the enzyme activity in AbR and FeR were significantly higher than those in the SS strain, the enzyme activity of AbR strain was 1.63 times of the SS strain, enzyme activity of FeR strain was 2 times of.2 SS strain cloning of TcID1 gene, cinnabar mites and gene cloning sequence analysis. Results showed that the full-length Zhu Shaye mite TcID1 gene ORF length of 774bp, encoding 257 amino acids, the molecular weight of the protein was 29.4kD, isoelectric point was 5.31. by Signal IP 4.1 is a translation of the TcID1 online prediction of ammonia Amino acid signal peptide containing 23 amino acids, may belong to secretory protein. To tetranychuscinnabarinus TcID1 amino acid sequence analysis, found that TcID1 encoding protein has an ortho ring opening specific dioxygenase Fe3+ binding sites: 2 His and 2 Tyr. and two spotted spider mites, bacteria, fungi and other similar homologous genes analysis and evolution of the amino acid sequence showed that tetranychuscinnabarinus TcID1 gene and two spotted spider mite tetur06g00460 gene had the highest similarity, amino acid consistency was 98%; and an mite AFY99039.1 amino acid identity was 66%; with fungi or bacteria ID-RCDs related amino acid consistency was lower than the 40%. phylogenetic tree shows. Compared with bacteria, fungi and Zhu Shaye mite TcID1 gene ID-RCDs has higher homology.3, TcID1 gene in different developmental states, different strains, chemical stress mRNA expression pattern of.QPCR showed that in cinnabar Different states of mite mite susceptible strains, TcID1 gene in larvae, nymphs and 3 day old female adult mites showed high expression, and the nymphs of the highest expression level, followed by the young mites, mites, eggs; among different strains compared to the susceptible strain, high expression AbR and FeR strains showed, which also verified the results in gene chip; in avermectin (LC30 dose in different strains of 6h and 12h reagent) stress, 24h, expression of TcID1 gene in SS strain for the expression pattern of decrease after the first increase in performance; AbR strain for expression pattern the increase at first and then decreased; fenpropathrin stress 6h, 12h and 24h, the expression pattern of TcID1 gene with avermectin stress results similar to.4, RNAi identified the TcID1 gene in tetranychuscinnabarinus of acaricide resistance function. By using leaf disc method indirect feeding TcID1-dsRNA, SS strains TcID1 gene expression decreased by 60.7% the activity of ID-RCDs decreased to 38%. Avermectin sensitivity increased significantly, showed significantly increased mortality, respectively 44% and 38% (avermectin LC30) (avermectin LC50 treatment); and the sensitivity to fenpropathrin showed no significant changes; after feeding TcID1-dsRNA AbR strain TcID1 gene expression decreased 67.5%, ID-RCDs activity decreased by 44%, the sensitivity of abamectin significantly increased, showed significantly increased mortality, respectively (19.5% LC30 and 17% avermectin (LC50) abamectin treatment); after feeding TcID1-dsRNA FeR strain TcID1 gene expression decreased 47.7%, ID-RCDs activity decreased by 40%, while the sensitivity of fenpropathrin was no significant change in.5, prokaryotic expression of TcID1 gene expression vector. In pCold II, construction of the signal peptide removed TcID1 expression plasmid and sequencing. The recombinant plasmid was transformed into Escherichia coli TcID1-pCold competent cells, by IP TG induced expression, SDS-PAGE analysis revealed that part of the gene in soluble expression, part of the expression in the form of inclusion body; by nickel affinity chromatography, Western Blot assay showed that TcID1 gene was expressed in Escherichia coli. As protocatechuic acid protein assay substrate, results showed that the recombinant protein specific activity was 0.1346. 0.0090 nmol/ g.pro/min, Km 14.2 + 4.6 mM, Vmax 0.1163 + 0.0153nmol/ g.pro/min.6, expression of the mode of action of product and acaricide TcID1 heterologous. The testing results of effect of chemicals on the recombinant protease, using 0.001mmol/L~2mmol/L abamectin and fenpropathrin on recombinant protease activity had no effect; by means of high performance liquid chromatography detection of recombinant TcID1 protein on the metabolism of test acaricides, the results showed that TcID1 recombinant protein cannot metabolize abamectin.

【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
【分類號(hào)】：S482.52;S433.7

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