【作者】 卢文才；

【导师】 何林；

【作者基本信息】 西南大学 ， 农药学， 2009， 硕士

【摘要】 朱砂叶螨（Tetranychus cinnabarinus）在我国分布广泛,是严重为害棉花、蔬菜等多种作物而又难于防治的一种害螨。该螨繁殖能力强,产卵力高,世代周期短,在短时间里能够迅速形成一定数量的种群,并且容易产生抗药性。本文通过比较朱砂叶螨敏感品系（SS）、阿维菌素抗性品系（AbR）和高温饲养品系（HR）γ-氨基丁酸（γ-aminobutyric acid,GABA）及γ-氨基丁酸受体（γ-aminobutyric acid receptor,GABAR）含量差异,从生物化学和分子水平研究朱砂叶螨适应阿维菌素、高温逆境胁迫与其体内GABA和GABAR的相关关系,旨在阐明对阿维菌素产生抗性的朱砂叶螨在高温下具有适合度优势的机理,为延长阿维菌素的使用寿命,有效治理朱砂叶螨对阿维菌素的抗性提供理论依据。本研究得到了国家自然科学基金（30600059）的资助,获得以下主要研究结果。一、建立了朱砂叶螨GABA含量检测的高效液相色谱（HPLC）法,以异硫氰酸苯酯（PITC）为衍生剂,进行柱前衍生反应,采用C₁₈进行分离,紫外检测器测定衍生物的吸光量。GABA标准液和样品液得到了良好的洗脱和分离,GABA在柱上的保留时间是4.29min,添加回收率在98.53%与100.60%之间。二、采用HPLC检测朱砂叶螨体内GABA含量,朱砂叶螨AbR品系体内GABA含量显著高于SS品系（1.39倍,P＜0.05）:用SS品系LC₉₅剂量的阿维菌素（0.055 mg/L）汰选SS获得耐药性个体（SS-A）,其GABA含量明显高于SS品系（为SS的1.41倍）,耐药性个体繁殖的下一代（SS-AF₁）,在未接触任何药剂情况下体内GABA含量高于SS品系（1.52倍）:用SS品系LC₅₀剂量的阿维菌素（0.018 mg/L）分别处理朱砂叶螨SS和AR品系,SS品系药后8h GABA含量迅速提高（提高了52%）,而AbR品系变化不大;HR品系体内GABA含量仅为SS品系的73.1%,差异显著（P＜0.05）;40℃热激1h后,HR和SS品系个体GABA含量均显著提高（分别升高了27.2%和20.6%）。我们初步推测朱砂叶螨通过调节GABA含量来适应阿维菌素和高温的胁迫。三、通过对朱砂叶螨行为学观察,发现凡是体内GABA含量较高的朱砂叶螨品系或个体,其爬行速度都比较缓慢:AbR、从SS品系汰选的抗性个体SS-A及其下一代SS-AF₁个体相对于SS品系均表现为行动迟缓（平均爬行速度分别为SS品系个体的62.3%、70.8%和76.4%）;经药剂诱导8h后,SS品系个体活动速度明显变慢（为诱导前71%）,而抗性个体活动速度变化不显著;HR品系和SS品系活动速度相当,无显著差异;热应激后,SS和HR品系活动速度均变得缓慢（爬行速度分别为热激前的74.2%和70.1%）。体内GABA含量较高的朱砂叶螨的活动都受到不同程度抑制,从而间接验证GABA是无脊椎动物的抑制性神经递质。四、分子克隆获得一条GABA受体α亚基全长序列,Genbank登录号为EU362111,命名Rdl^TC。开放阅读框（ORF）为1127bp,编码376个氨基酸残基,其中包括了由21个氨基酸组成的信号肽。该基因编码的氨基酸序列理论分子量为41.1kDa,等电点（pI）为9.25;5’端有27bp的非编码区,3’端有555bp的非编码区和polyA尾,第1681处有AATAAA加尾信号;与其他昆虫、螨同源性搜索比较,发现与二班叶螨Tetranychus urticae、黑腹果蝇Drosophila melanogaster GABA受体α亚基同源性分别高达72%和71%。生物信息学分析表明,推导的Rdl^Tc结构也包含典型的GABA受体亚基的四个跨膜结构域,且第三结构域至第四结构域之间有一个明显的亲水区;Rdl^Tc氨基酸序列中存在高度保守的糖基化位点(Rdl^Tc氨基酸序列92N和133N)与磷酸化位点(Rdl^Tc氨基酸序列187Y,341S,346Y,362Y),另外还含有可能在螨类中是特有的糖基化点(60Nin Rdl^Tc)与磷酸化点(48S and 339S in Rdl^Tc);构建分子进化树结果显示,朱砂叶螨和二斑叶螨GABAR基因在GABAR类群中最为原始,在进化树里独立于其他物种的GABAR。采用同样的方法得到了室内选育的朱砂叶螨AbR品系GABAR的基因序列,对SS品系和AbR品系核苷酸及推导的氨基酸序列进行对位排列分析,结果显示,两品系核苷酸与氨基酸序列没有差异。五、建立了基于SYBR Green I染料技术的real-time PCR方法,以β-actin基因作为内参基因进行校正,运用实时定量PCR检测SS品系、AbR品系GABA受体mRNA表达量。检测结果为AbR品系的相对表达量显著低于SS品系的表达量（仅为SS品系的14%,P＜0.05）,用SS品系LC₅₀剂量的阿维菌素对朱砂叶螨AbR品系、SS品系诱导8h后,AbR品系GABA受体的表达量基本没有变化,而SS品系表达量显著降低（降为2.4%）,HR品系GABAR表达量显著低于SS品系（前者是后者的54.7%）;热激后,SS品系表达量显著下降（降低了56.6%）,而HR品系表达量有所降低,但无显著差异。朱砂叶螨GABA受体mRNA表达下调介导了其对阿维菌素和高温胁迫的适应性。更多还原

【Abstract】 The carmine spider mite,Tetranychus cinnabarinus（Boisduval）,one of the most important pests on cotton,vegetables and other crops,is widely distributed in China.Because of its high fecundity and short generation time,the mite can form rapidly population of a certain number of stocks in a short period of time,develop resistance easily and thus it is difficult to prevent and control this mite.The thesis was designed to detect and compare the differences of GABA and GABAR content among susceptible strain（SS）,abmectin-resistance（AbR） and high temperature resistance strains（HR） of T.cinnabarinus,and discussed the biochemistry and molecular resistance mechanism of T.cinnabarinus to abamectin and heat stresses,in order to reveal the thorough mechanism of superior fitness of abamectin-resistant strains under high temperature condition,and to provide a theoretical basis for Integrated Resistance Management（IRM）.This work was supported by grants from the National Natural Science Foundation of China（No.30600059）,and the main results were as the following.1.The method for measuring the content of GABA,using Phenylisothiocyanate（PITC） as pre-column derivatization reagent with high-performance liquid chromatographic（HPLC） UV detection,was established.The method exhibited good retention time（4.29min） and peak area reproducibility for GABA-PITC.The recoveries of GABA were among the the range of 98.53%-100.60%.2.The GABA content in T.cinnabarinus was measured by HPLC,the content in AbR strain of T. cinnabarinus was significantly higher than that in SS strain of T.cinnabarinus（1.39 fold,P＜0.05）, and the GABA content in abamectin-resistance individuals selected from SS with abamectin LC95 of SS（SS-A） and SS-AF₁（the F1 generation of SS-A） strains were also significantly higher than that in SS（1.41-and 1.52-fold,respectively）.The significantly higher expression level was observed in the SS strain 8 hours later of being treated with abamectin（0.018 mg/L） when compared that with no treatment（the content of GABA increased 52%）,whereas the expression level in AbR strain did not change significantly after being treated with abamectin.The GABA content of HR strain was significantly lower than that of SS（about 73%of that in SS） and the content of GABA in SS and HR were significantly risen an hour later of 40℃heat-shock（increased 27.2%and 20.6%, respectively）.We speculate the changes of GABA content is involved in the resistance to abamectin and high temperature in T.cinnabarinus.3.A comparative study on crawling speed of T.cinnabarinus with the high and normal GABA content was carried on to verify the inhibitory function of GABA.We found that the crawling speeds of AR、SS-A and SS-AF₁ were significantly slower than that of SS（about 62.3%,70.8%and 76.4% of the speed of SS,respeetively）.The significantly slower speed was also observed in the SS 8 hours later of being treated with abamectin when compared that with no treatment,however the speed in AbR strain did not change significantly after being treated with abamectin.There was no significance difference between the speeds of SS and HR strains,whereas their movement slowed down obviously after being treated with high temperature（40℃）.4.We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from T.cinnabarinus,through rapid amplification of cDNA ends（RACE） and polymerase chain reaction using primers based on the 5’ and 3’ ends of the mRNA(Genbank EU362111 named as Rdl^Tc).The complete amino acid sequence of Rdl^Tc deduced from the cDNA is 1730 residues with the putative signal peptide consisted of 21 residues with a theoretical molecular weight 41.1 kDa and an isoelectric point（pI） of 9.25.The 5’ and 3’ untranslated regions（UTR） consist of 28 and 555 bp, and there is a 16 bp poly A tail in 3’untranslated region.The predicted GABA receptor protein shared 72 and 71%identity with other GABA receptors proteins isolated from Tetranychus urticae and Drosophila mlanogaster,at amino acid level,respectively.Analysis of the amino acid sequence of Rdl^Tc indicated it contains a putative polydenylation signal AATAAA,highly conserved dicysteine-loop（Cys-loop） and four transmembrane domains（TM1,TM2,TM3,and TM4）.Online software（http://www.cbs.dtu.dk/serviees/NetNGlyc,http://www.cbs.dtu.dk/serviees/NetPhos） was used to analysis the N-glycosylation sites and phosphorylation sites.The highly conserved N-glycosylation sties(N92 and N133 in Rdl^Tc) and highly conserved phosphrylation sites(187Y, 341S,346Y and 362Y in Rdl^Tc) are all occurred in other insect and mite species.While N-glycosylation sties(60N in Rdl^Tc) and phosphrylation sites(48S and 339S in Rdl^Tc) are mainly confined in mites,which suggesting these motifs are species-specific.The phylogenetic tree shows that acarus is well separated from insect.Among acarus,the Tetranychidae and Ixodidae are well separated.Among insect,Diptera,Hymenoptera,Homoptera and Lepidoptera are well separated from each other,but under order level,family can’t be well separated,such as H.virescens,B.mori, P.xylostella,and S.exigua in Lepidoptera.Therefore,our results suggested that the sequences of GABAR are well separated on order level and might be used to construct phylogenetic tree on this level.we also compared the cDNA sequences of the Rdl^Tc between SS and AbR strain of T. cinnabarinus and found that there was no mutation in Rdl^Tc in abamectin-resistant strain seleted in laboratory.5.The mRNA expression level of Rdl^Tc was detected by the method of SYBR Green I dye-based technology with theβ-actin as reference gene.The expression level of the GABA receptor gene in AbR strain was significant lower than that in the SS strain.The significant lower expression level was observed in SS strain 8 hours later after being treated with abamectin when compared that with no treatment,whereas the expression level in AbR strain did not change significantly after being treated with abamectin.The expression level of the GABAR in HR was significant lower than that in SS.The significant lower expression level was also observed in SS after heat shock,however there was almost no change after being treated with high temperature in HR.The low expression level of GABA receptor is involved in the resinstance to abamectin and high temperature in T.cinnabarinus.更多还原