丝氨酸蛋白酶与朱砂叶螨抗性相关关系的研究

发布时间：2018-03-08 16:05

本文选题：朱砂叶螨　切入点：丝氨酸蛋白酶　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)是农业生产中一种危害严重、分布广泛的害螨,在世界各地均有分布。在我国华南、西北、西南、湖北等地方都有发生,其寄主植物多达1000多种,其中经济作物有100多种,因其具有繁殖能力强、世代周期短、活动范围小、近亲交配率高等特点,以及几年来杀虫(螨)剂的大量、持续和不科学使用,导致抗药性问题尤为严重。丝氨酸蛋白酶作为昆虫体内一类重要的水解酶,在维持昆虫机体正常生命活动中扮演着重要的角色,它参与了昆虫机体消化、胚胎发育、组织重建、细胞分化、血管形成、血液凝固、免疫反应、激素活化等多种生理生化过程,它是否也可能与朱砂叶螨的抗性存在着相关关系,为探究两者之间存在的相关关系,我们设计了相关研究内容:在室内筛选出的甲氰菊酯抗性品系(FeR)、阿维菌素抗性品系(AbR)朱砂叶螨以及敏感品系(SS)朱砂叶螨的基础上,研究丝氨酸蛋白酶与朱砂叶螨抗性之间存在的相关关系,并初步对其机制进行初步探究,主要的研究内容和结果如下:1.对丝氨酸蛋白酶基因(SP-900)进行核苷酸序列全长克隆,获得一条长为897 bp碱基序列,并且顺利翻译成含有298个氨基酸的序列,序列分析确定其为一条丝氨酸蛋白酶基因,并且通过进化树构建分析结果表明它亦是一条类胰蛋白酶基因。2.采用q-PCR技术检测目的基因SP-900在SS、FeR、AbR三个品系中表达情况,利用数据分析软件SPSS22.0采用独立样本T检验的分析方法分析抗性品系与SS品系表达量的差异性,结果显示FeR、AbR品系目的基因SP-900的表达量均高于SS品系,分别是SS品系的2.45、5.54倍。对三个品系朱砂叶螨经甲氰菊酯或阿维菊素诱导后6h、12h、24h SP-900基因的表达量情况也进行了检测,结果显示,SS品系经甲氰菊酯诱导后12h SP-900基因表达量上调至3.05倍,6h、24h表达量没有上调,FeR品系经甲氰菊酯诱导后6h、12h SP-900基因表达量分别上调至2.77、2.65倍;SS品系经阿维菌素诱导后12h SP-900基因表达量上调至2.42倍,6h、24h表达量没有上调,AbR品系经阿维菌素诱导后12h SP-900基因表达量上调至2.25倍,6h、24h表达量没有上调。上述研究结果表明在目前的抗性水平,朱砂叶螨抗性的增加可能与丝氨酸蛋白酶基因表达量的上调有关,并且在药剂诱导下,SP-900基因的表达量会上升,表明SP-900可能参与了朱砂叶螨在应对药剂带来的不利影响时的某些生理生化反应。3.SS、FeR、AbR三个品系朱砂叶螨丝氨酸蛋白酶粗酶活性测定结果为:SS、FeR、AbR三个品系朱砂叶螨丝氨酸蛋白酶粗酶活力分别为3.0024nmol/μg/min、5.2634 nmol/μg/min和6.2774 nmol/μg/min,FeR、AbR两个抗性品系的粗酶活力分别是SS品系的1.75、2.09倍,具有显著性差异。采用药膜法对三个品系朱砂叶螨进行丝氨酸蛋白酶抑制剂处理,这种处理方法能够抑制朱砂叶螨体内丝氨酸蛋白酶粗酶活性,处理后SS、FeR、AbR三个品系的粗酶活性分别下降了38.4%、24.8%和21.9%,三个品系朱砂叶螨丝氨酸蛋白酶活性被抑制后,其对甲氰菊酯和阿维菌素的敏感性上升。表明朱砂叶螨丝氨酸蛋白酶活性下降,对药剂的敏感性上升。4.采用叶碟饲喂法对SS、FeR、AbR三个品系朱砂叶螨进行RNAi处理,SP-900基因的表达量分别被抑制了49.19%、51.74%和53.69%;粗酶活性分别下降了28.6%、18.2%和23.5%;SS品系对甲氰菊酯、阿维菌素的LC50分别下降了174ppm、0.089ppm,FeR品系对甲氰菊酯的LC50下降了14234ppm,AbR品系对阿维菌素的LC50下降了0.211ppm。RNAi结果显示,叶碟饲喂法能够较好的抑制朱砂叶螨基因SP-900的表达,基因SP-900表达量下降,酶活降低,药剂敏感性上升,。5.丝氨酸蛋白酶与甲氰菊酯或阿维菌素结合实验的结果显示,丝氨酸蛋白酶没有与药剂结合,表明其没有直接参与朱砂叶螨对甲氰菊酯和阿维菌素的分解代谢活动;利用HPLC测定SS、FeR、AbR三个品系朱砂叶螨ATP含量,结果显示三个品系之间ATP含量没有表现出差异性。可能是因为FeR、AbR两个抗性品系的解毒代谢活动强于SS品系,ATP需求增加,但是两个抗性产生了适合度代价,导致其其它的生命活动减弱(生殖力下降),ATP需求量下降,从而在ATP总量上没有表现出差异性。
[Abstract]:Tetranychuscinnabarinus (Tetranychus cinnabarinus) is a kind of agricultural production in the serious, widespread pest mites, distributed throughout the world. In China, Southern China, northwest, southwest, Hubei and other places have occurred, the host plant as many as 1000 kinds, including 100 kinds of economic crops, because of its strong ability of reproduction the scope of activities, short generation cycle, small, high rate of inbreeding, and several years insect (mite) agent for large, continuous and scientific use, lead to drug resistance problem is especially serious. As a serine protease in insects is an important class of enzymes, play a critical role in maintaining the body's normal life activity in insects it is involved in the insect body, digestion, embryonic development, tissue remodeling, cell differentiation, angiogenesis, blood coagulation, immune response, hormone activation and other physiological and biochemical processes, it is impossible and tetranychuscinnabarinus resistant There is correlation between the relationship between the inquiry between the two, we designed the related research contents: fenpropathrin resistant strains were screened in laboratory (FeR), abamectin (AbR) tetranychuscinnabarinus and sensitive strains (SS) based on tetranychuscinnabarinus, correlation the research between serine protease and tetranychuscinnabarinus resistant, and the preliminary mechanism of the preliminary inquiry, the main research contents and results are as follows: 1. the serine protease gene (SP-900) full-length nucleotide sequence, have a length of 897 BP nucleotide sequence, and successfully translated into a sequence containing 298 amino acids. Sequence analysis identified it as a serine protease gene, and through the construction of phylogenetic tree analysis showed that it is also a kind of trypsin gene.2. q-PCR was used to detect the gene SP-900 in SS, FeR, AbR three The expression of strains, using data analysis software SPSS22.0 analysis using independent samples T test analysis of expression difference of resistant strains and SS strains, the results showed that FeR, expression of SP-900 gene of AbR strain was higher than that of the SS strain, which is 2.45,5.54 times of the SS strain of three. Strains of tetranychuscinnabarinus by fenpropathrin or acitretin pyrethrins after induction of 6h, 12h, 24h expression of SP-900 gene was detected, the results showed that the SS strain from fenpropathrin 12h after the induction of SP-900 gene expression was increased to 3.05 times, 6h, 24h expression did not increase, FeR the fenpropathrin after induction of 6h, 12h SP-900 expression were up to 2.77,2.65 times; SS strain by Avermectin 12h after the induction of SP-900 gene expression was increased to 2.42 times, 6h, 24h expression was not up-regulated AbR expression induced by strains of 12h SP-900 gene of avermectin quantity To 2.25 times, 6h, 24h expression was not up-regulated. These results demonstrated that the resistance level at present, tetranychuscinnabarinus resistance may be associated with increased serine protease gene expression amount increases, and in the chemical induction, the expression level of SP-900 gene will rise, suggest that SP-900 may be some physiological and biochemical reactions involved in.3.SS the adverse effects of tetranychuscinnabarinus agents to deal with the FeR, AbR of three strains of crude enzyme activity of tetranychuscinnabarinus serine protease determination results: SS, FeR, AbR in three strains of crude enzyme tetranychuscinnabarinus serine protease activity were 3.0024nmol/ g/min, 5.2634 nmol/ g/min and 6.2774 nmol/ g/min FeR, AbR, crude enzyme activity of two resistant strains were 1.75,2.09 times SS lines, with a significant difference. To tetranychuscinnabarinus three strains of serine protease inhibitor treatment by membrane method, this processing method Method can inhibit the crude enzyme activity in tetranychuscinnabarinus serine protease, SS after treatment, FeR, AbR crude enzyme activity of three strains were decreased by 38.4%, 24.8% and 21.9%, three strains of tetranychuscinnabarinus serine protease activity was inhibited, increased its sensitivity to fenpropathrin and avermectin showed a decrease. Tetranychuscinnabarinus serine protease activity and sensitivity to fungicides increased.4. by using leaf disc feeding method of SS, FeR, AbR of three strains of Tetranychus cinnabarinus were treated with RNAi, the expression level of SP-900 gene was inhibited by 49.19%, 51.74% and 53.69%; the crude enzyme activity decreased by 28.6%, 18.2% and 23.5%; SS Department of fenpropathrin, abamectin and LC50 were decreased by 174ppm, 0.089ppm, LC50 to fenpropathrin FeR strain decreased by 14234ppm and AbR strains of avermectin LC50 decreased by 0.211ppm.RNAi results show that the method can better suppress the Ye Diesi fed The expression of tetranychuscinnabarinus gene SP-900, SP-900 gene expression decreased, enzyme activity decreased, drug sensitivity increased,.5. serine protease and fenpropathrin and avermectin binding experiments showed that serine protease with no medicament combination, that is not directly involved in the catabolic activity of Tetranychus cinnabarinus to fenpropathrin and avermectin; determination SS, by HPLC FeR AbR, three strains of tetranychuscinnabarinus ATP content, showed that three strains of ATP content showed no difference. It may be because FeR, AbR of the two resistant strains of detoxification activity is strong in the SS strain, ATP demand increases, but the two resistance for the degree of price, the other life activities (reduced fertility decline), ATP demand dropped, resulting in the total ATP showed no difference.

【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】：S433.7

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