【作者】 任晓霞；

【导师】 王荫长； 韩召军；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2002， 博士

【摘要】 本论文以棉铃虫为研究对象，系统地研究了棉铃虫有机磷抗性的机制，其重点是对乙酰胆碱酯酶（AChE）生物化学及分子生物学的研究。 用久效磷对采集于山东聊城抗性地区的棉铃虫进行抗性品系的选育。在16代期间经过10代的室内选育，抗性倍数达到211.88倍，为选育前的6.67倍。同时发现筛选后对氰戊菊酯、氯氰菊酯和灭多威的抗性分别下降了8.13、7.44和13.36倍，对甲基对硫磷和辛硫磷的抗性基本保持不变。这说明久效磷与这5种被试药剂之间没有明显的交互抗性。从久效磷、马拉硫磷、辛硫磷、甲基对硫磷、甲胺磷、灭多威、巴沙和氯氰菊酯8种杀虫剂对棉铃虫XJ-S品系和LC-R品系的毒力测定可以看出，LC-R品系对久效磷的抗性倍数为211.9倍，属高水平抗性，而对其它7种药剂均为低水平抗性或耐药力增加。 通过构建种群生命表，观察比较了棉铃虫对久效磷的抗性品系和敏感性品系一系列生长发育和繁殖特征。结果表明，两品系的适合度差异显著。相对于敏感品系，抗性品系生长发育上表现为幼虫期和蛹期延长，蛹重减轻，化蛹率和羽化率降低；繁殖上表现为交配率、产卵量和卵孵化率降低。用种群数量趋势指数（I）来确定两个品系的相对适合度，抗性品系对敏感品系的相对适合度仅为0.14。本文认为这是生产上棉铃虫对有机磷的抗性发展较慢的原因之一。 本研究通过生化方法对抗、感棉铃虫体内AChE进行了研究。研究表明两个品系AChE的动力学参数存在着显著差异。以粗提酶液进行研究，发现久效磷对抗性棉铃虫体内AChE的抑制中浓度(I₅₀)是敏感品系的3.18倍。纯化后，抗、感品系的AChE敏感性之间仍存在1.66倍的差异。酶液的纯化使抗、感品系AChE的I₅₀分别下降6.69和3.49倍，说明粗提酶液中存在AChE保护因子，且这种保护在抗性品系中更有效。此外，本文还对两品系的酯酶活力进行了测定，发现两个品系的酯酶活力存在着显著的差异。由此认为AChE敏感性降低与另外一些因子共同组成了棉铃虫对久效磷的抗性机制。 在比较已发表昆虫的AChE基因氨基酸序列的基础上，我们根据保守序列设计合成了简并引物，利用反转录多聚酶链式反应（RT-PCR）技术扩增了棉铃虫AChE的cDNA片段。所获得的281bp的棉铃虫cDNA片段由94个氨基酸残基组成，通过与GenBank 棉铃虫从eljcoverna arm枉era（nUbner）对有机磷杀虫剂靶标抗性机制的研究中其它几种昆虫AChE基因序列的同源性比较，发现所得到的序列与已知几种昆虫和动物的北M氨基酸序列具有很高的同源性，由此推断所获得的片段序列是棉铃虫ACbB基因的 片段序列。 利用 CDNA末端快速扩增枝术（RACE）获取了新疆棉铃虫敏感品系 AChE的 CDNA序列，由。mA序列推导出来的氨基酸序列包括32个氨基酸的信号肽和615个氨基酸的成熟蛋白，分子量为69，059 KDa。5’端非翻译区为315ba，3’端非翻译区为324hp。通过与 GenBank 中黑尾叶蝉N勾劝灯。ttix cj。ctjcops、马铃薯甲虫Lop ttno拈 rSS WcCmLCmL切ea is、斯氏按④砌山汕打es S拈ph＊DS／和黑腹果蝇Nosophjla。打。Ogss比。4种昆虫 AChE基困序列的同源性比较，发现彼此均具有较高的同源性，其同源性分别为70儿68儿60％和57巩 并且，所推导的氨基酸序列具有从M基困的特征性氨基酸残基，其中包括形成催化三联体的三个氨基酸残基钻r，Gin和His以及形咸亚基间H硫键的 6个保守的Cys。由此推断所获得的CDNA序列为棉铃虫AChE基因的CDNA序列。 通过比较棉铃虫久效磷抗性品系和敏感品系 AChE的全长 CDNA序列，发现抗性品系才对于敏感品系存在9个点突变，分另为Arg一4一GIn，y8117一Met，ASp一170－＋01y，GIfl－217－－＋Aflg，Ilfl－433－＋mf，AS少435－－＋Gly，MSt叫68－－＋VSI，们S－硼7、灯S和Ala刁85—Thr。其中，Ala刁85—Thr出现于所有的测试抗性棉铃虫个体中，而敏感个体中却不存在这一突变，说明这一点突变可能导致了棉铃虫AChE的不敏感性，从而导致了棉铃虫对久效磷的抗性。 本研究还对棉铃虫辛硫磷抗性品系的AChE基因进行了克隆和序列测定。通过比较抗性品系和敏感品系AChE的全长CDNA序列，发现杭性品系相对于敏感品系存在6个点突变，分别为Gly－9、Gly，GIU－128—Gly，LyS－291－－＋GIS，ASP－415－＋Gly，Ala－555一Val和Ala－585叶Thr。其中，点突变Ala－585－＋Thr出现于所有的坝试抗性棉铃虫个体中，而敏感个体中却不存在这一突变，说明这一点突变可能导致了棉铃虫AChE的不敏感性，从而导致了棉铃虫对辛硫磷的抗性。 本研究还从棉铃虫抗性品系和敏感品系中克隆TAChE基因组mA片段。序列比较表明，抗性品系和敏感品系之间存在着内含子序列上的差异。这些DNA序列上的信息在排除地理种群的多态性之后可为棉铃虫抗药性的分子检测提供序列依据。 本研究选用了敏感性不同的几个田间品系，采用点滴法和浸渍法，室内测定了丙涣磷对辛硫磷的增效作用。结果表明：门)所有配比都具有一定的增效作用；随着辛梆的比例增大，共毒系数的变化呈抛物线状，以中间比例（1：4和1：5）的共毒系更多还原

【Abstract】 In this paper, we studied the organophosphorus resistance mechanism in cotton bollworm Helicoverpa armigera (Hubner), one of the serious cotton pests in the world. Our study especially put stress on the biochemical and molecular analysis of acetylcholinesterase (AChE, EC 3.1.1.7), which is the inhibition target enzyme of organophosphate and carbamate insecticides.Monocrotophos resistance was selected in laboratory with the field resistant cotton bollworm collected from Liaocheng County (LC-R), Shandong Province. After ten-generation selections with monocrotophos during sixteen generations, the resistance increased 6.67-fold and was as high as 211.88-fold when compared with a susceptible strain collected from Xinjiang (XJ-S). But the resistance of the selected strain to fenvalerate, cypermethrin and methomyl decreased by 8.13-, 7.44- and 13.36-fold, respectively, during the selection. Methylparathion and phoxim resistance was basically unchanged as compared with that of FQ. These indicated there was no significant cross-resistance between monocrotophos and the other 5 insecticides tested. Toxicity of 8 insecticides to resistant and susceptible H. armigera showed that the LC-R strain has developed high resistance to monocrotophos (RR=211.9) and low resistance or high insecticide-proof ability to the other 7 insecticides.By constructing life table of resistant strain and susceptible strain, the effects of monocrotophos resistance on fitness in the cotton bollworm were evaluated in terms of developmental and reproductive characteristics. The results indicated that the LC-R strain possessed developmental disadvantages including prolonged larva and pupa duration, lighter pupae weight, lower pupation and emergence rate, and reproductive disadvantages including lower copulation rate, fecundity and hatchability when compared with the XJ-S strain. Relative fitness was determined by population number tendency index (I). The LC-R strain was calculated to have a fitness value of 0.14relative to the XJ-S strain, which could be contributed to the slow development of organophosphate resistance in cotton bollworm in fields.Acetylcholinesterase (AChE) in the resistant and susceptible H. armigera was also studied. The results showed that AChE in resistant strain was much less sensitive to monocrotophos. The I50 value for resistant AChE was 3.18-fold higher than that for susceptible. Partial purification of AChE to remove esterase and some other proteins increased the sensitivity dramatically. The I50 reduced by 6.69-fold and 3.49-fold for resistant and susceptible strain, respectively. This indicated that there might be some factors protecting AChE, which was more powerful in resistant strain. Even so, there was still 1.66-fold difference in the I50 value of purified AChEs from resistant and susceptible strains. Kinetic parameters of AChE also showed significant difference between these two strains. Esterase activity was 5-fold higher in resistant larvae than in the susceptible strain. So, it is concluded that the decreased sensitivity of AChE together with some other factors in the resistant strain contributes to monocrotophos resistance.With the degenerate primers we have amplified a 281bp cDNA fragment of AChE gene in cotton bollworm by reverse transcription-polymerase chain reaction (RT-PCR) method using total RNA extracted from 4th larva as the template. The cDNA fragment was inserted into pGEM-T vector and then cloned. The deduced amino acid sequence of AChE was consisted of 94 residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with AChE gene from other published insects and animals. The acquired sequence had 84%, 79%, 74%, 70%, 70%, 72%, 68%, 61%, 55% and 57% of amino acid residues identical to those of Leptinotarsa decemlineata (L.d.), Nephotettix cincticeps (N.c.), Anopheles stephensi (A.s.), Aedes aegypti (A.a.), Lucilia cuprina (L.c.\ Drosophila melanogaster (D.m.), Musca domestica (M.d.), Meloidogyne incognita (M. i.), Torpedo californica更多还原