【作者】 姜晓静；

【导师】 韩召军；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 二化螟Chilo suppressalis是水稻的重要害虫之一,由于它钻蛀危害水稻茎秆,因而给化学防治带来一定的难度。采用的杀虫剂通常是渗透性强、具有一定的内吸作用、可传导,而且环境相容性好,价格合理农民可以承受的药剂品种。三唑磷最初以其优异的防治效果以及合理的价格成为继六六六、杀虫脒、杀虫单之后防治二化螟的主要替代药剂。但最近频频出现三唑磷对二化螟防治效果下降的报道,而且本实验室的前期工作也证实田间二化螟对三唑磷产生了显著的抗药性,并分析了抗药性的生化机理,发现三唑磷抗性二化螟除解毒酶活力加强外,其乙酰胆碱酯酶的敏感性也显著降低。在此基础上,本研究阐明了二化螟对三唑磷靶标抗性的分子机理,确定了靶标的抗性突变位点,进而建立了抗药性分子监测技术,为抗性二化螟治理和新药剂开发提供了新技术和新思路。1.二化螟乙酰胆碱酯酶基因的克隆和序列分析利用cDNA末端快速扩增技术(RACE)获取了二化螟AChE1基因全长,共2521 bp,其中5’-UTR为235 bp,3’-UTR为204 bp。由cDNA序列推导出来的氨基酸序列包括17个氨基酸的信号肽和677个氨基酸的成熟蛋白,与二化螟AChE2的相似性仅为47%,但与GenBank中其它鳞翅目昆虫的AChE1氨基酸序列的相似性较高,在75%～90%之间；而二化螟AChE2氨基酸序列与小菜蛾、苹果蠹蛾、棉铃虫、烟夜蛾以及家蚕的AChE2序列也具有很高的相似性,均大于90%以上。所推导的二化螟AChE1氨基酸序列具有AChE基因的特征性氨基酸残基,其中包括：(1)形成催化三联体的三个氨基酸残基Ser313、Glu439. His553;(2)活性部位的Ser313存在于所有胆碱酯酶中均存在的FGESAG序列中；(3)形成亚基内二硫键的6个保守氨基酸Cys181-Cys208,Cys 367-Cys 380,Cys515-Cys637,以及亚基间的保守氨基酸Cys660；(4)胆碱结合亚部位W198；(5)酰基口袋W346、F402、F443；(6)氧负离子孔G232、G233、A314。因此推断所获得的cDNA序列为二化螟AChEl的cDNA序列。2.二化螟抗三唑磷品系乙酰胆碱酯酶的点突变分析在室内用三唑磷对2003年采自浙江省苍南县的二化螟进行连续6代筛选,与2006年6月采自江苏连云港的赣榆地区的二化螟敏感品系比较,抗性水平达到1172倍。对敏感和抗性个体的AChE1、AChE2全长序列分别进行克隆和对比分析,结果发现所有三唑磷抗性二化螟个体的AChE1,在对应于电鳐Torpedocalifornica的AChE氧负离子孔A201这一功能位点上,发生了固定的点突变(A314S)。Ala与Ser的侧链分别是-CH3和-CH2OH,因此我们推测可能是该侧链的变化改变了相邻的催化三亚基中Ser的构象,最终影响了AChE1与抑制剂间的相互作用。另外,在抗性棉蚜和小菜蛾体内也发现了同样的点突变。由此分析认为AChE1的A314S突变很可能是导致二化螟靶标敏感性下降的功能突变位点。3.不同二化螟地理种群对三唑磷靶标抗性的分子检测通过抗性和敏感品系的二化螟AChE1的核酸序列比较发现,抗性相关突变A314S中从鸟嘌呤到胸腺嘧啶的碱基颠换(G／T),导致突变型基因组上的MspA1Ⅰ酶切位点发生变化。根据这个特点,本研究设计了基于RFLP-PCR的分子检测技术进行突变型等位基因的快速检测。结果显示,敏感纯合子SS基因组DNA的PCR产物被MspA1Ⅰ酶切为534bp和224bp两个片段；杂合子RS基因组DNA的PCR产物被MspA1Ⅰ酶切处理后,电泳检测到758bp、534bp和224bp三个片段；而抗性纯合子RR基因组DNA的PCR产物不能被MspA1Ⅰ酶切,电泳检测只有758bp一个片段。2007年、2008年利用该技术分别检测了7个不同田间地理种群的抗性水平,结果发现检测的抗性等位基因频率与已报道的有机磷杀虫剂抗性相符。该结果不仅创建了灵敏准确的二化螟靶标抗性分子检测技术,同时反证了该突变位点就是抗性功能突变位点。4.二化螟两个乙酰胆碱酯酶基因的相对表达量及体外真核表达利用实时定量PCR技术比较了野生型AChE1和AChE2两个基因在二化螟中的mRNA表达水平,发现在四龄幼虫中AChE1基因的表达量是AChE2的4.8倍。进一步构建了野生型AChE1和AChE2基因的供体质粒pFastBacl-AChE1和pFastBacl-AChE2;利用定点突变技术将突变A314S引入pFastBacl-AChE1构建成pFastBacl-AChE1Mu;将构建好的质粒pFastBacl-AChE1、pFastBacl-AChE2以及pFastBacl-AChE1Mu转到DH10BAC中,利用昆虫杆状病毒表达体系用重组病毒感染Tn细胞进行体外酶蛋白表达,收集细胞进行SDS-PAGE检测和蛋白杂交实验以及酶活测定。结果发现,虽然SDS-PAGE结果和蛋白杂交实验均证实目标蛋白在Tn细胞中正确表达,但酶活测定结果显示,体外表达的二化螟AChE1、AChElMu没有活性,AChE2的活性仅为1.954OD／min／mg。其原因可能是目前采用的系统不适宜表达二化螟AChE,二化螟2个AChE的生理毒理学特性尚有待利用其他表达体系进行进一步研究。综上所述,本研究的创新点包括以下三点：1.确定了二化螟有两个乙酰胆碱酯酶基因,为以后的AChEs的功能研究以及毒理学研究奠定了基础。2.发现了二化螟对三唑磷的靶标抗性突变位点,从分子水平上解释二化螟抗性表型产生的分子机理。3.建立了二化螟对三唑磷靶标抗性分子检测技术,为抗性二化螟治理和新药剂开发提供了新技术和新思路。更多还原

【Abstract】 Rice stem borer, Chilo suppressalis (Walker), is one of the most important insect pests in Asia. Because most of its life-cycle is spent inside rice stems, this pest is difficult to control. Only insecticides that combine good penetration or systemicity, low toxicity to fish, and affordability to farmers are appropriate for stem borer control. In the past, hexachlorocyolohexane (BHC), chlordimeform and monosultap were used extensively in China. However, the first two have been banned for ecotoxicological reasons, and monosultap has lost its effectiveness due to resistance. The organophosphate triazophos initially proved an excellent replacement for monosultap, but recently its effectiveness has declined. A previous publication confirmed resistance to triazophos in C. suppressalis from some locations in China, and suggested reduced sensitivity of acetylcholinesterase-the target site of organophosphates-to be the primary resistance mechanism. In this paper, we reported a mutation in Ch-AChEl consistently associated with resistance, and an assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. The results were summarized as follows:1. Cloning and sequence analysis of AChEl from C. suppressalisThe Rapid Amplification of cDNA Ends (RACE) procedure was employed to obtain full-length sequence of AChEl from C. suppressalis. The result showed that it is 2521 bp in full-length. The 5’and 3’untranslated regions (UTR) were 235 bp and 204 bp, respectively. The complete amino acid sequence deduced from the cDNA consisted of 17 residues for the putative signal peptide and 677 residues for the mature protein. The sequence of this gene has the features shared by members of AChE family. Compared with AChE in Torpedo californica, the functional motifs were highly conserved:the catalytic triads (S313, E439 and H553 in Ch-AChE1, and S266, E395 and H509 in Ch-AChE2), the characteristic’FGESAG’ motif surrounding the active serine,3 intra-chain disulfide bridges (C181-C208, C367-380 and C515-C637 in Ch-AChEl, and C115-C143, C320-C335 and C471-C590 in Ch-AChE2), a anionic choline-binding site (W198 in Ch-AChE1 and W133 in Ch-AChE2), acyl pocket residues (W346, F402 and F443 in Ch-AChE1, and W299, F358 and F399 in Ch-AChE2) that accommodate the acyl moiety of the active site, and the oxyanion hole (G232, G233 and A314 in Ch-AChEl, and G179, G180 and A267 in Ch-AChE2) that helps to stabilize the tetrahedral molecule during catalysis. The C-termimal Cys residues that contributed to an intermolecular dimerization were Cys660 and Cys608, respectively. Following these Cys residues, the protein has amino acid tails enriched in hydrophobic residues as predicted using ProtScale. Thus, it is proved to be the AChE gene. Otherwise, this gene shares only 47%similarity with previously reported AChE2 in this pest, but has a high degree of homology to AChEls from other Lepidopetra species. All these results firmly established that the amplified cDNA fragment is the sequence of AChE 1 gene in rice stem borer. 2. Mutation in AChEl associated with triazophos resistance in the rice stem borerOne strain of the rice stem borer was selected in the lab by exposure to increasing concentrations of triazophos. Insecticide bioassays showed that the resistant strain (Tp-R) exhibited a 1172-fold resistance ratio to the insecticide, compared to a susceptible strain (Gy-S). Previous work suggested that insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer. To verify the hypothesis, we compared the full-length AChEs cDNA sequences from five monocrotophos-resistant and five susceptible rice stem borer individuals. Sequence analysis found an amino acid mutation A314S in Ch-AChEl (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This alanine residue is located in the characteristic’FGESAG’motif surrounding the active serine, and lies in the oxyanion hole that contributes to stabilizing the tetrahedral molecule during catatysis. The alanine to serine substitution changes the side group from-CH3 to-CH2OH, which is believed to alter the conformation of the adjacent serine of the catalytic triad and to affect the interaction between AChE and both substrates and inhibitors. The mutation A314S in Ch-AChEl is likely to be responsible for the AChE insensitivity to triazophos.3. Frequency of the mutation in field populations with different resistanceThe mutation GCG to TCG in Ch-acel removes an MspAl I restriction site from the wild type allele. An RFLP-PCR assay was therefore designed using primers able to amplify specifically a 758-bp fragment encompassing the mutation site in Ch-ace1 from genomic DNA, and by checking the digestion of the resulting fragment with MspAl I. The PCR product amplified from the wild type allele was completely cut into two pieces (534 bp and 224 bp long). That amplified from the heterozygote was partially cut and presented as three bands (758 bp,534 bp and 224 bp long), whereas that amplified from the mutated allele remained intact.With this method, individual larvae of C. suppressalis collected from different field sites were analysed, and results showed a strong correlation between mutation frequencies and levels of triazophos resistance. The RFLP-PCR assay developed to diagnose the A314S mutation in field populations provides a potentially valuable way of monitoring resistance and investigating the relative frequencies of resistance genotypes. It overcomes many of the difficulties encountered with time-consuming bioassays against C. suppressalis, and offers the prospect of a rapid and high throughput kit to assist with choosing insecticides and combating resistance in this species.4. Eukaryotic expression and transcript comparison in vivo of AChEs from rice stem borerReal-time PCR was performed to compare the transcript levels of Ch-AChEl and Ch-AChE2 from individual fourth-instar larva. The results showed that the transcript level of Ch-AChE1 was higher than Ch-AChE2’s.Based on cloned AChEs from rice stem borer, the insect Bac-to-Bac expression system was selected to construct the eukaryotic expression plasmids of wild AChEl and AChE2. Meanwhile, plasmids of wild AChEl was changed into plasmids of mutant AChE1 by site-directed mutagenesis. The constructed donor plasmids of pFastBac1-AChE1、pFastBac 1-AChE2 and pFastBac 1-AChE 1 Mu were transfected into the DH10BAC, and then select the positive clone and extract the Bacmid, using Bacmid to transfect the Tn cells obtain the recombine virus, then amplificate the first generation virus and infect the Tn cells to express the target protein.All of the three recombinant proteins showed the obvious band through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. But the wild recombinant AChE1 and mutant showed no enzyme activity, the recombinant AChE2 showed enzyme activity.更多还原