邻位开环双加氧酶TcID1介导朱砂叶螨对两种杀螨剂抗性研究

发布时间：2018-01-16 22:06

本文关键词：邻位开环双加氧酶TcID1介导朱砂叶螨对两种杀螨剂抗性研究　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：朱砂叶螨(Tetranychus cinnabarinus)在我国分布广泛,主要危害棉花、蔬菜等农作物。目前我国对于农业害螨的防治主要以化学防治为主,其中阿维菌素和甲氰菊酯是使用十分广泛的杀螨剂。这些杀螨剂的大量使用以及叶螨自身的生理特性,导致我国绝大多数地区的朱砂叶螨已经对阿维菌素和甲氰菊酯产生了抗药性。邻位开环双加氧酶(Introdial ring-cleavage dioxygenases,ID-RCDs)是一类能够催化邻苯二酚类底物氧化分解的双加氧酶,主要存在于微生物如真菌、细菌中,其主要功能是降解带有芳香环的化合物。朱砂叶螨的基因芯片检测结果显示,相对于敏感品系(Susceptible strain,SS),ID-RCDs基因在阿维菌素抗性品系(Abametin resistant strain,AbR)和甲氰菊酯抗性品系(Fenpropathrin reisitant strain,FeR)中均上调表达。因此,本论文在此基础上,重点研究了ID-RCDs与朱砂叶螨对阿维菌素和甲氰菊酯抗性的关系并取得了以下研究结果:1、朱砂叶螨ID-RCDs酶活性测定。对朱砂叶螨ID-RCDs酶进行粗酶活性测定,该酶在AbR及FeR中的活性均显著高于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、朱砂叶螨TcID1基因克隆及序列分析。基因克隆结果显示,朱砂叶螨TcID1基因全长的开放阅读框长度为774bp,编码257个氨基酸,蛋白分子量为29.4kD,等电点为5.31。经Signal IP 4.1在线预测发现TcID1的翻译氨基酸含有23个氨基酸的信号肽,可能属于分泌蛋白。对朱砂叶螨TcID1氨基酸序列进行分析,发现TcID1编码的蛋白具有邻位开环双加氧酶特有的Fe3+结合位点:2个His和2个Tyr。与二斑叶螨、细菌、真菌等同源基因进行氨基酸序列相似性和进化分析,结果显示:朱砂叶螨TcID1基因与二斑叶螨基因tetur06g00460的相似度最高,氨基酸一致性为98%;与伊氏叶螨AFY99039.1氨基酸一致性为66%;而与真菌或细菌ID-RCDs相关的氨基酸一致性均低于40%。系统发育树结果显示,相对于细菌,朱砂叶螨TcID1基因与真菌类ID-RCDs具有更高的同源性。3、TcID1基因在不同螨态、不同品系、药剂胁迫下的mRNA表达模式。qPCR结果显示:在朱砂叶螨敏感品系的不同螨态中,TcID1基因在幼螨、若螨及3日龄雌成螨中均呈高表达,并且在若螨中表达量最高,其次为幼螨、成螨、卵;在不同品系之间,相对于敏感品系,AbR及FeR品系均呈高表达,这也验证了基因芯片中的相关结果;在阿维菌素(药剂在各品系中的LC30剂量)胁迫6h、12h及24h后,TcID1基因在SS品系表现为先增加后降低的表达模式;在AbR品系表现为先降低后增加的表达模式;甲氰菊酯胁迫6h、12h及24h后,TcID1基因的表达模式同阿维菌素胁迫结果类似。4、RNAi鉴定TcID1基因在朱砂叶螨对杀螨剂抗性的功能。采用叶碟法间接饲喂TcID1-dsRNA后,SS品系TcID1基因表达量下降60.7%,ID-RCDs酶活性降低38%,对阿维菌素的敏感性显著增加,表现为死亡率显著升高,分别为44%(阿维菌素LC30处理)和38%(阿维菌素LC50处理);而对甲氰菊酯的敏感性无显著变化;饲喂TcID1-dsRNA后,AbR品系TcID1基因表达量下降67.5%,ID-RCDs酶活性降低44%,对阿维菌素的敏感性显著增加,表现为死亡率显著升高,分别为19.5%(阿维菌素LC30处理)和17%(阿维菌素LC50处理);饲喂TcID1-dsRNA后,FeR品系TcID1基因表达量下降47.7%,ID-RCDs酶活性降低40%,而对甲氰菊酯的敏感性无显著变化。5、原核表达TcID1基因。以pColdⅡ为表达载体,构建去除信号肽的TcID1重组表达质粒,并测序验证。将测序正确的重组质粒TcID1-pColdⅡ转化至大肠杆菌感受态细胞中,经IPTG诱导表达,SDS-PAGE电泳检测发现该基因一部分以可溶性表达,一部分以包涵体形式表达;经过镍亲和层析柱纯化,Western Blot检测表明TcID1基因在大肠杆菌中成功表达。以原儿茶酸为底物进行蛋白活性测定,结果显示重组蛋白比活力为0.1346±0.0090 nmol/μg.pro/min,Km为14.2±4.6 mM,Vmax为0.1163±0.0153nmol/μg.pro/min。6、TcID1异源表达产物与杀螨剂的作用模式。药剂对重组蛋白酶活影响的测定结果显示,使用0.001mmol/L~2mmol/L的阿维菌素及甲氰菊酯均对重组蛋白酶活无影响;借助高效液相色谱检测TcID1重组蛋白对杀螨剂的代谢试验,结果表明TcID1重组蛋白不能代谢阿维菌素。
[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.

【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】：S482.52;S433.7

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