【作者】 丁中；

【导师】 高必达； 彭德良；

【作者基本信息】 湖南农业大学 ， 植物病理， 2007， 博士

【摘要】 乙酰胆碱酯酶（acetylcholinesterase,AChE）是有机磷和氨基甲酸酯类杀虫剂作用的主要靶标部位,它们通过抑制AChE对乙酰胆碱的水解作用,干扰和最终阻断突触传导。有机磷和氨基甲酸酯类杀虫剂的广泛使用已经导致许多害虫产生了明显的抗药性。近年来,在我国一些地区采用涕灭威防治甘薯茎线虫（Ditylenchus destructor）,其效果呈现下降的趋势。为此,本文以甘薯茎线虫为研究对象,采集了河北省不同种群,进行了毒力生测、乙酰胆碱酯酶活力测定、甘薯茎线虫乙酰胆碱酯酶三个基因cDNA全长的克隆、抗药性种群的汰选,以及利用实时荧光定量PCR检测了不同种群各乙酰胆碱酯酶基因的相对表达水平,明确了当前甘薯茎线虫对乙酰胆碱酯酶抑制剂的抗性水平,探讨了抗性机理,证实了甘薯茎线虫抗药性的产生与乙酰胆碱酯酶的过量表达相关。具体研究结果如下:1、采用浸渍法和沙柱法对河北省昌黎、涿州、卢龙和抚宁四地的甘薯茎线虫进行了涕灭威、灭线磷、阿维菌素的敏感性测定。浸渍法结果表明,昌黎甘薯茎线虫种群对涕灭威、灭线磷的LC₅₀分别为891.73mg/L、743.82mg/L,其它三地涿州、卢龙和抚宁甘薯茎线虫种群对涕灭威、灭线磷的敏感性均有所降低,与昌黎种群相比较,其对涕灭威的抗性倍数分别为4.0、3.5和3.7倍,对灭线磷的抗性倍数分别为3.8、2.1和1.6倍;阿维菌素在400mg/L以下时,各种群的死亡率均在45%以下,为600 mg/L,时其死亡率陡增,均为90%以上,且无显著性差异。沙柱法结果表明,5mg/L的涕灭威和50mg/L的灭线磷对线虫的活动能力具有很好的抑制作用,50mg/L的阿维菌素抑制作用则相对较低。2、在对不同温度和盐浓度的适应性测定发现,昌黎种群较其它三地种群对46℃以下的高温有较高的耐受力;抚宁种群对盐具有较高的耐受力,卢龙和昌黎两种群线虫对盐的耐受力较低。不同的地理生态环境使线虫具不同的特殊生态适应性。3、用改进的Ellman方法测定了河北省昌黎、涿州、卢龙和抚宁四个不同种群甘薯茎线虫的乙酰胆碱酯酶的生化特性及其对抑制剂涕灭威和毒扁豆碱的敏感性差异。结果表明:甘薯茎线虫AChE至少存在两种类型,一种类型对毒扁豆碱的IC₅₀值小于10^-6mol/L,另一种类型大于10^-4mol/L。当抑制剂毒扁豆碱浓度为10^-6～10^-10mol/L、涕灭威浓度为10^-5～10^-10mol/L时,甘薯茎线虫昌黎种群AChE的敏感性抑制率明显高于涿州、抚宁、卢龙三地的甘薯茎线虫种群的AChE的抑制率。各地种群AChE对抑制剂敏感性差异是由于甘薯茎线虫AChE的多态性和各分子型综合性改变的结果。4、利用RT-PCR结合RACE技术首次克隆了甘薯茎线虫乙酰胆碱酯酶基因cDNA全序列:Dd-ace-1、Dd-ace-2和Dd-ace-3,提交GenBank登录号分别是EF583059、EF583058和EF583057。其长度分别是2196bp、2425bp和2517bp。这三个ace基因5’端均存在反式剪接引导序列SL1。其中Dd-ace-1编码635个氨基酸,Dd-ace-2编码734个氨基酸,Dd-ace-3编码611个氨基酸。克隆的三个基因都具有乙酰胆碱酯酶的特征:催化三联体、胆碱结合位点、3对分子内二硫键桥和保守的芳香族氨基酸残基等。聚类分析显示,Dd-ACE-1、Dd-ACE-2、Dd-ACE-3分别与其它线虫乙酰胆碱酯酶ACE-1、ACE-2、ACE-3同属一个支系。5、采用实时荧光定量PCR技术比较了河北省昌黎、涿州、卢龙和抚宁四地的甘薯茎线虫AChE基因Dd-ace-1、Dd-ace-2和Dd-ace-3的mRNA表达水平的差异。涿州、卢龙和抚宁三地种群的ace-1、ace-2和ace-3在转录水平上不同程度的高于昌黎种群。涿州、卢龙和抚宁三地种群AChE敏感性降低与ace-1、ace-2、ace-3多基因的过量表达相关。6、采用沙柱法对敏感种群进行涕灭威、灭线磷和阿维菌素抗药性汰选,进一步证实了涕灭威、灭线磷能显著提高甘薯茎线虫ace-1、ace-2、ace-3基因mRNA的表达水平,但这种量的变化不足以显著提高汰选种群的抗药性,但可以观察到不同药剂汰选种群在含药剂或不含药剂的沙柱中的运动情况有所变化。更多还原

【Abstract】 Acetylcholinesterase （AChE, EC 3.1.1.7） catalyzes the hydrolysis of the neurotransmitter acetylcholine at cholinergic synapses to keep the normal transmission of nerve impulses. It is the target of the organophosphate and carbamate compounds. When AChE is inhibited, acetylcholine builds up in the synapse, causing hyperexcitation and then desensitization of the muscle receptors, resulting in paralysis. Intensive use of these pesticides over the past years has led to the development of resistance in many target species. The efficacy of nematicides on the control of Ditylenchus destructor showed the tendency of decrease in the northern areas of China in the recent years. This study difinitizes the level of resistance of D. destructor to aldicarb, ethoprophos and abamectin, provides the molecular evidence that over-expression of AChE and AChE genes was associated with the resistance in D. destructor, with the evidences from toxicity assay, determination of enzyme activity, induction of nematicides to D. destructor, and mRNA expression of AChE genes.1. Four populations of Ditylenchus destructor were collected from different areas of Hebei Province and their susceptibilities to 3 different types of nematicides were tested with contact assays and sand column methods in laboratory. The contact assays results showed that the Changli populations were more sensitive to aldicarb and ethoprophos, the values of LC₅₀ were 891.73 mg/L and 743.82 mg/L, respectively. The other populations had lower susceptibilities to aldicarb and ethoprophos. The sus- ceptibilities of the three populations had 3.5～4.0 fold difference to aldicarb, and 1.6～3.8 fold to ethoprophos. The susceptibilities of the four populations to abamectin were no significant difference. The results of sand column assays showed that the movement of nematode could be inhibited by 5mg/L aldicarb and 50mg/L ethoprophos. The inhibition of 50 mg/L abamectin on nematodes movement was lower than that of aldicarb and ethoprophos.2. Different populations had different tolerance to salt concentration and temperature. ChangLi population had rather high tolerance at 46℃than that of other population. Funing population survived better than those from other area in 3%～5% salt solution.3. The AChE properties of four populations of D. destructor and susceptibilities to inhibitors were investigated. AChE was extracted in a direct method and ultracentrifugetion in sucrose gradients. AChE activities were determined by the amended spectrophotometric method of Ellman. Effect of inhibitors on AChE hydrolysis was analyzed. The results showed that there are two major class AChEs in acetylcholine hydrolysis at least. One class of AChE is highly sensitive to eserine (IC₅₀<10^-6 mol/L) and other class is less sensitive to eserine (IC₅₀>10^-4 mol/L). Inhibition of AChE activity of Changli population was higher than that of Zhuozhou, Lulong and Funing populations at the concentration 10^-6-10^-10 mol/L of eserine and at 10^-6-10^-10 mol/L of aldicarb. This susceptibilities difference of AChE of different populations to inhibitors may be due to the polymorphism of AChE and the multiple changes of different molecular polymorphism.4. Three new cDNA, named Dd-ace-1 （EF583059）, Dd-ace-2 （EF583057） and Dd-ace-3 （EF583058） encoding acetylcholinesterases were isolated from D. destructor. The three full length cDNA, carrying the transspliced SL1 leader sequence, are 2196bp, 2425bp and 2517bp long with an open reading frame of 1908bp, 2205bp and 1836bp encoding 635, 734 and 611 amino acid residues, respectively. All of them shared of the characteristics of the AChE, the catalytic triads, the choline binding sit, 3 intra-chain disulfide bridges and aromatic residues lining the catalytic gorge. Phylogenetic analysis based on other nematodes and species AChEs showed that the deduced Dd-ACE-1, Dd-ACE-2 and Dd-ACE-3 formed a cluster with ACE-1s, ACE-2s and ACE-3s, respectively.5. Using the real-time quantitative PCR, the mRNA expression of three AChE genes in Funing, Lulong and Zhuozhou population was compared with those in Changli population. Expression level of AChE genes in Funing, Lulong and Zhuozhou population was higher than Changli population at different level. It provided the molecular evidence that over-expression of AChE genes was associated with decrease of susceptibilities of D. destructor to inhibitors.6. The induction of inhibitor to susceptive population provided stronger evidence that the level of AChE mRNA expression could be significantly enhanced after treated with sand column soaked with aldicarb and ethoprophos. But the susceptibilities of D. destructor to inhibitors could not be affected by the changes of expression of mRNA. The changing of movement percentage of different populations in sand column was observed.更多还原