【作者】 邱高辉；

【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， 2007， 博士

【摘要】 麦长管蚜Sitobion avenae(Fabricius)是小麦的重要害虫之一,由于麦长管蚜对许多常规杀虫剂都产生了抗药性,而吡虫啉则因其对麦蚜的良好防效而成为目前防治麦蚜的当家农药品种。为了了解田间麦长管蚜对吡虫啉抗性的发生动态、延缓麦蚜对吡虫啉抗性的发展,延长吡虫啉在麦田的使用寿命,本文通过田间抗药性调查、室内抗性筛选以及适合度的研究、抗性品系的交互抗性研究,系统分析了麦长管蚜对吡虫啉产生抗性的风险;利用室内筛选的抗性品系和敏感品系试虫为材料,分析了抗性产生的生化机制;利用分子生物学技术克隆了麦长管蚜的烟碱型乙酰胆碱受体基因,取得了多项具有重要理论和实际意义的成果。一、麦长管蚜对吡虫啉的抗药性调查为了了解我国各麦区麦长管蚜对吡虫啉抗性的发生发展情况,连续两年调查了江苏南京、建湖、泗洪、山东德州、河南周口、甘肃兰州、天水等地麦长管蚜对吡虫啉和另一种常用于防治麦蚜的杀虫剂抗蚜威的敏感性。结果发现,不同地区麦长管蚜对吡虫啉的敏感性差异较大,江苏建湖和南京地区已经产生了一定的抗性(6.29-11.31倍),但其他地区仅表现为敏感性降低,尚没有产生明显的抗性。不同地区麦长管蚜对抗蚜威的敏感性差异较小,但抗蚜威的毒力显著小于吡虫啉,与早期报道的敏感基线相比发现,不同地区的麦长管蚜对抗蚜威已经产生了大约20-40倍的抗性。另外通过不同地区麦长管蚜年度间的敏感性比较发现,麦长管蚜对两种药剂的敏感性存在逐渐下降的趋势。综合分析认为,各麦区的麦长管蚜对抗蚜威已经产生了中等水平抗性,对吡虫啉虽然还处于敏感阶段,但在局部地区已经开始产生抗性,目前尚处于抗性发展的初期阶段。由于吡虫啉已成为防治麦蚜危害的当家农药品种,且目前没有更好的替代药剂,为了掌握麦长管蚜对吡虫啉的抗性发生发展动态、延缓抗性的发展、保证麦长管蚜的持续有效的治理,研究其抗性发生规律,制定有效的抗性治理策略是十分必要的。二、抗吡虫啉麦长管蚜品系的选育及适合度的研究采用点滴法在室内用吡虫啉对麦长管蚜进行连续32代的抗性筛选,获得了抗性倍数为25.22倍的室内品系。筛选早期抗性的增长速率相对缓慢,筛选到第18代后抗性快速上升,最终呈现“S”型增长的总体趋势。通过构建种群生命表,观察比较了吡虫啉抗性品系和敏感品系麦长管蚜的生长发育和繁殖特征,就抗性对种群适合度的影响进行了研究。结果表明,抗性品系表现出相对适合度下降。用种群数量趋势指数和内禀增长率来确定抗性品系的相对适合度发现,抗性品系的相对适合度为敏感品系的0.623和0.846。由此认为,麦长管蚜具有对吡虫啉产生抗性的风险,而抗性品系适合度较低是田间麦长管蚜对吡虫啉抗性发展缓慢的重要原因之一。三、麦长管蚜抗吡虫啉品系的交互抗性本研究测定了吡虫啉抗性筛选前后,麦长管蚜对不同杀虫剂的敏感性变化,以了解抗吡虫啉麦长管蚜对其他药剂的交互抗性。结果发现,筛选品系对吡虫啉的抗性上升了9.10倍,同时对啶虫脒的抗性也上升了4.11倍,表明啶虫脒和吡虫啉之间存在较明显得交互抗性,而筛选品系对抗蚜威、灭多威、氧化乐果、久效磷、溴氰菊酯等常用药剂的抗性没有明显的上升,说明这5种药剂与吡虫啉之间没有明显的交互抗性。四、麦长管蚜对吡虫啉的抗性生化机制分析本文通过室内筛选获得了麦长管蚜对吡虫啉的抗性品系(25.22倍),并利用增效试验和解毒酶活力测定,对其抗性机理进行了研究。增效试验结果表明:顺丁烯二酸二乙酯在抗、感品系中对吡虫啉都没有明显的增效作用,但氧化胡椒基丁醚和磷酸三苯酯在两个品系均有增效作用,而且在抗性品系中的增效比(3.49和2.62)显著大于敏感品系(1.59和1.29)。解毒酶活力测定发现,抗、感品系的谷胱甘肽S-转移酶比活力没有显著差异,但抗性品系中羧酸酯酶的比活力明显高于敏感品系(1.47倍)。综合分析认为多功能氧化酶和羧酸酯酶活力增强在麦长管蚜对吡虫啉的抗性中起重要作用。五、麦长管蚜烟碱型乙酰胆碱受体基因的克隆昆虫烟碱型乙酰胆碱受体(nAChR)是以吡虫啉为代表的新烟碱类杀虫剂的作用靶标。本研究采用RT-PCR技术,利用简并引物从麦长管蚜Sitobion avenae(Fabricius)中克隆出6个烟碱型乙酰胆碱受体基因的cDNA片段,其中5个为α型,1个为β型。同源性分析表明,6个麦长管蚜nAChR基因片段与其他昆虫相应基因序列相同区域具有较高的相似性,特别是与其他同翅目昆虫的相似性更高,达到95%以上,个别片段的相似性甚至达到100%。根据已克隆出的5个nAChRα型cDNA片段设计特异引物,结合快速扩增cDNA末端(RACE)技术,成功克隆了亚基的全长,分别命名为Saα1、Saα2、Saα3、Saα4和Saα5(GenBank登录号分别为:EF363700、EF363701、EF211977、EF363702、EF363703)。Saα1基因长1674bp,编码496个氨基酸(5’端不完整);Saα2全长2516bp,编码595个氨基酸;Saα3全长1763bp,编码537个氨基酸;Saα4全长1763bp;编码532个氨基酸;Saα5长度为1974bp,编码549个氨基酸。在Saα3中存在少见的Poly(A)序列;Saα5在5’端配基结合区有三种选择性剪接形式,分别造成碱基的缺失、插入和替换。序列分析发现,这些基因均具有nAChR基因家族的典型特征,并与昆虫烟碱型乙酰胆碱受体的相应亚基具有很高的同源性。该研究为进一步利用基因表达技术研究昆虫nAChR的天然亚基组成,以及分析麦长管蚜对新烟碱类杀虫剂的靶标抗性奠定了基础。本研究通过抗性调查,明确了我国麦长管蚜对吡虫啉的抗性现状。通过室内抗性筛选,成功获得了麦长管蚜抗吡虫啉品系,并证明麦长管蚜对吡虫啉具有产生抗性的风险,通过生物适合度的研究发现了田间麦长管蚜对吡虫啉抗性发展缓慢地原因。通过抗性机制分析明确了麦长管蚜对吡虫啉地重要抗性机理。这些研究成果对制定科学的抗性治理策略,有效地进行抗性治理,延长吡虫啉地田间使用寿命,保证麦蚜的持续有效治理,均具有重要的实践意义。利用分子生物学技术成功克隆了5个麦长管蚜烟碱型乙酰胆碱受体的α亚基基因,并发现了基因中的Poly(A)序列和选择性剪接现象,为进一步利用基因表达技术研究昆虫nAChR的天然亚基组成,以及分析麦长管蚜对新烟碱类杀虫剂的靶标抗性奠定了基础。更多还原

【Abstract】 Sitobion avenae（Fabricius） is one of the most important pests on wheat.Because of the resistance of S.avenae to conventionally used insecticide in the field,imidacloprid has become the key insecticide used for control of this pest.So,study on imidacloprid risistance is very important for sustainable control of S.avenae.In this paper,the resistance survey,resistance selection,relative fitness analysis,and cross-resistance test were all carded out to evaluate the risk for S.avenae to develop resistance to imidacloprid.With selected resistant strain and susceptible strain,the biochemical mechanisms for imidacloprid resistance were analyzed,and some genes for nicotinic acetylcholine receptors （nAChR） were cloned.Valuable breakthroughs have been achieved.1.Field survey for imidacloprid resistance in S.avenaeThe toxicity of imidacloprid and pirimicarb（another effective insecticide usually used to control wheat aphid） to field populations of S.avenae had been tested with the insects collected from Nanjing,Jianhu,Sihong,Dezhou,Zhoukou,Lanzhou and Tianshui in 2005 and 2006.Results showed that the sensitivity of S.avenae to imidacloprid varied obviously with the geographic populations.The aphids collected from Jianhu and Nanjiang had developed low level of resistance（RR was 11.31 and 6.29,respectively）.But the aphids from other areas were still susceptible to imidacloprid（RR ranged 1.99-3.85）.However, pirimicarb resistance varied not so much among different populations.The survey results showed that pirimicarb was much less toxic to S.avenae than imidacloprid.Comparing with the baseline data reported previously,S.avenae in different areas were found to be moderate resistant（RR:20-40）.Furthermore,the data of different years showed that the resistance of S.avenae to imidacloprid and pirimicarb were increasing slowly.In conclusion,imidacloprid resistance was still in its early stage of development.Most field populations of S.avenae were still susceptible to imidacloprid,but a few populations had really developed resistance to this insecticides.Thus,it is necessary to study the development of imidecloprid resistance and complement reasonable resistance management.2.Imidacloprid resistance selected in S.avenae and the fitnessAn imidacloprid-resistant stain（25.22-fold） of S.avenae was developed by continuous selection for 32 generations with imidaclopfid in laboratory.During the selection, imidaclopfid resistance was found with a phased increase.From the 1^st to 18^th generation, the resistance developed very slowly.The rapid increase occurred from 18^th to 24^th generation,and leaving 24^th to 32^nd as a resistance developing plate.There should be a risk of resistance in S.avenae after long-time extensive use of imidaclopfid in the field.The effects of resistance on the fitness of S.avenae were evaluated in term of developmental and reproductive characteristics by constructing and comparing the life tables of imidaclopfid-resistant and susceptible strains.The results revealed that imidacloprid-resistant stain had no developmental disadvantage but obvious reproductive disadvantage.The fitness of irnidacloprid-resistant strain was determining by population number tendency index（I） and intrinsic rate of increase（r_m） with susceptible strain as standard.The relative fitness value for the imidalopfid-resistant strain was calculated to be 0.623 and 0.846 respectively.So,it was concluded that imidacloprid resistance in S.avenae costs lots of fitness,which could be one of the main factors for the slow development of imidacloprid resistance in the field population of S.avenae.3.Cross-resistance of the imidacloprid-resistant strain of S.avenaeThe cross-resistance of the imidacloprid-resistant strain of S.avenae to six conventionally used insecticides in the field was measure.It was found that selection with imidaclopfid could make a obvious increase in the resistance of S.avenae to imidaclopfid and acetamiprid（9.10 and 4.11 times,respectively）,which means positive cross-resistance between imidacloprid and acetamiprid.However,no distinct cross-resistance to carbamate （pirimicarb and methomyl）,organphosphates（omethoate and monocrotophos） and pyrethroid （deltaraethrin） was found. 4.Mechanism for imidacloprid resistance in Sitobion avenaeA strain（about 25.22 fold） of Sitobion avenae resistant to irnidacloprid was developed in the laboratory.Synergism experiments and detoxification enzyme analysis were carried out to reveal the resistance mechanisms.The results showed that piperonyl butoxide and triphenyl phosphite had significant synergism on imidaeloprid in both the resistant and susceptible strains,but diethyl maleate had not.The synergism ratio of piperonyl butoxide and triphenyl phosphite in the resistant strain were much higher（3.49-fold and 2.62-fold, respectively） than in the susceptible strain（1.59-fold and 1.29-fold,respectively）.The resistant strain showed higher activity of carboxylesterases（about 1.53-fold） than the susceptible strain,but the activity of gluathione S-transferases was similar.These results indicated that the enhancement of the activity of carboxylesterase and Mixed function oxidase should contribute to the imidaeloprid-resistance in this S.avenae strain.5.Cloning of nAChR genes from S.avenaeNicotinic aeetylcholine receptor（nAChR） is one of the most important insecticide targets.With general primers designed for nAChR genes,RT-PCR was carded out and 6 cDNA fragment of nAChR genes were cloned from S.avenae.Of which,five had the typical characteristics of nAChR alpha subunits and one belonged to beta subunits.Further analysis demonstrated that this six fragments had high amino acid sequence identity with other insect nAChR subunits previously reported,especially those of Homoptera.Based on the five fragments of nAChRαsubunits,general primers were designed and five different genes were successfully cloned to their full-length by using the RACE strategy.They were named Saα1,Saα2,Saaα,Saα4 and Saα5,and their GenBank access number were EF363700,EF363701,EF211977,EF363702 and EF363703,respectively. These five genes had all the characteristics of nAChR gene family and were high homology with other nAChR genes from insects.This work facilitates the further studies on natural subunit composition of nAChR and the molecular mechanisms for target resistance to new nicotinic insecticides in this pest.Additionally,an unusual adenosine rich region were found in Saα3 and alternative splicing in Saα5.There were three forms of alternative splicing in N-coding regain in Saα5, causing absence,insert and change in the amino acid sequence.The role of these alternative splicing need further research.In conclusion,studies had demonstrated the imidaeloprid-resistance statue in S.avenae, the risk to develop resistance to imidacloprid,the reasons of the slow development of imidacloprid resistance in the field,and the main mechanisms for imieacloprid resistance. This work is significant in the management of S.avenae resistance.On the other hand,five nAChR subunits genes cloned from S.avenae facilitates the further studies on natural subunit composition of nAChR and the molecular mechanisms for target resistance to new nicotinic insecticides in this pest.更多还原