二化螟抗药性监测及大螟鱼尼丁受体基因克隆与表达分析

发布时间：2018-07-29 13:31

【摘要】：水稻二化螟[Chilo suppressalis(Walker)]和大螟[Sesamia inferens(Walker)]是水稻上的重要害虫,广泛分布于我国主要水稻种植区。二化螟的为害程度较大螟重,大螟通常在稻田周边零星发生。长期以来,水稻螟虫的防治主要依靠化学药剂,其中主要包括有机磷类杀虫剂毒死蜱和三唑磷、大环内酯抗生素类杀虫剂阿维菌素和甲维盐、沙蚕毒素类杀虫剂杀虫单及新型双酰胺类杀虫剂氯虫苯甲酰胺和氟苯虫酰胺,目前双酰胺类杀虫剂是田间防治螟虫的主要药剂。但是由于化学药剂的长期不合理使用,二化螟对这些药剂均产生了一定的抗药性。本研究进行了田间二化螟种群对上述七种药剂的抗药性监测,以期能够及时了解上述药剂的抗性发展情况。同时还进行了二化螟对双酰胺类杀虫剂交互抗性的研究,旨在延长该类药剂的使用寿命,使其在二化螟的防治中发挥更好的作用。为了明确大螟对氯虫苯甲酰胺和氟苯虫酰胺的活性,本研究测定了三个田间大螟种群对上述两种药剂的敏感性。昆虫鱼尼丁受体是双酰胺类杀虫剂的主要靶标,鉴于目前还没有关于大螟对双酰胺类杀虫剂作用机制的研究,因此本研究克隆了大螟鱼尼丁受体基因cDNA的全长序列,并对其不同组织及不同发育阶段鱼尼丁受体基因表达水平的差异进行研究,以期为未来大螟对双酰胺类杀虫剂抗药性分子机制的研究提供依据。1.二化螟对七种药剂的抗药性监测本研究分别采用点滴法和稻苗浸渍法于2013-2015年测定了我国10省30地59个二化螟田间种群对氯虫苯甲酰胺、氟苯虫酰胺甲维盐、阿维菌素、毒死蜱、三唑磷及杀虫单七种药剂的抗药性。结果显示:田间二化螟种群对这七种药剂均存在明显的地理差异,抗性高的种群基本都来自浙江省、江西省及湖南省,江苏省、山东省及辽宁省的二化螟种群相对敏感,其中辽宁地区的二化螟种群对上述七种药剂均处于敏感阶段。在2013-2015年,已经发现在2015年浙江苍南二化螟种群对氟苯虫酰胺已产生了138.4倍的高水平抗性,同时已有21个种群对该药剂产生了低到中等水平抗性(5.5-85.1倍);浙江余姚、苍南、象山及江西南城二化螟种群对氯虫苯甲酰胺已达到中等水平抗性(RR=10.9-77.6倍),并且有10个种群对其也产生了低水平抗性(5.7-8.9倍)。从连续监测的数据可以发现,浙江余姚、象山及江西南城二化螟种群对双酰胺类杀虫剂一直处于较高水平的抗性,而江苏仪征、邗江及安徽徽州3个种群一直处于相对敏感的阶段。同时为了明确氯虫苯甲酰胺原药和制剂的抗性水平是否存在差异,我们比较了浙江余姚和象山两个抗性种群对97.3%氯虫苯甲酰胺原药和20%氯虫苯甲酰胺悬浮剂的抗性倍数,发现两个二化螟种群对20%氯虫苯甲酰胺悬浮剂的抗性均高于其原药。2014-2015年,我国大部分二化螟田间种群对甲维盐、阿维菌素及杀虫单仍处于敏感至敏感水平下降阶段,分别占其监测种群总数的78.2%、76.9%及88.9%,而浙江象山、金华、湖南东安及江西上高、南城种群对甲维盐为低到中等水平抗性(5.6-22.5倍);浙江苍南、余姚、江西上高、南城及湖南东安、攸县对阿维菌素表现为低到中等水平抗性(5.5-15.6倍);只有湖南东安、安徽徽州及山东临沂三个种群对杀虫单产生了低到中等水平抗性(6.5-16.1倍);浙江苍南、象山及江西上高等七个种群对毒死蜱产生了中等水平抗性(11.2-67.6倍);而对于三唑磷,湖南东安和浙江金华对其仍为高水平抗性(RR=216.8和209.9倍),浙江象山、苍南及江西上高南城等七个种群对其也达到中等抗性水平。2.二化螟对双酰胺类杀虫剂的交互抗性为了明确二化螟对双酰胺类杀虫剂溴氰虫酰胺、四氯虫酰胺及氯氟氰虫酰胺与氯虫苯甲酰胺和氟苯虫酰胺间是否存在交互抗性,我们以浙江余姚和象山抗性种群为试材,进行了其对三种新双酰胺类杀虫剂的交互抗性研究,结果显示,对氟苯虫酰胺和氯虫苯甲酰胺均产生了中到高水平抗性(27.6-133.6)的浙江象山、余姚二化螟种群,对溴氰虫酰胺、四氯虫酰胺和氯氟氰虫酰胺也产生了中到高水平抗性(30.3-127.0倍),则表明上述三种新双酰胺类杀虫剂与氯虫苯甲酰胺和氟苯虫酰胺之间存在交互抗性。3.水稻大螟对氯虫苯甲酰胺和氟苯虫酰胺的敏感性测定本研究采用稻苗浸渍法对于2014年采自江苏南京、通州及浙江余杭的大螟田间种群对氯虫苯甲酰胺和氟苯虫酰胺两种双酰胺类杀虫剂的敏感性进行了测定,结果显示:这两种药剂对大螟的毒力差异显著,大螟对氟苯虫酰胺比氯虫苯甲酰胺的敏感性高。但是不同地区大螟种群对同一种药剂的敏感性差异不明显。4.大螟鱼尼丁受体基因克隆与表达分析利用RT-PCR和RACE的方法,克隆得到大螟鱼尼丁受体的cDNA全长序列,命名为SiRyR,该序列全长15428bp,开放阅读框15357 bp,编码5118个氨基酸。大螟RyR氨基酸序列与甜菜夜蛾和棉铃虫RyR的相似性最高,分别为97%和96%,与线虫、人和大鼠的相似性较低(43%-50%);此外通过构建进化树,同样发现大螟RyR与棉铃虫和甜菜夜蛾RyR的亲缘关系最近。同时对其蛋白质结构预测发现存在七种蛋白质结构域,包括跨膜区结构域、MIR结构域、RIH结构域、RIH-associated结构域、SPRY区域、RyR结构域和离子通道蛋白家族自身螺旋结构。此外,通过荧光定量PCR对大螟鱼尼丁受体基因在不同发育阶段及不同组织部位中的表达情况进行研究,发现鱼尼丁受体在大螟整个生活史及不同组织部位中鱼尼丁受体基因均有表达,且在雄成虫和四龄幼虫的胸部表达量最高。
[Abstract]:[Chilo suppressalis (Walker) of rice borer and [Sesamia inferens (Walker) of rice borer] is an important pest on rice. It is widely distributed in the main rice planting areas in China. The borer of the rice borer is heavy and the borer is usually scattered around the paddy field. It includes organophosphorus insecticides and three azophos, macrolide antibiotic insecticides and methylene salts, insecticide insecticides, new diamide insecticides, chloropetamide and florfenicides. At present, diamide insecticides are the main insecticides in the field prevention and control of the borers. For a long period of irrational use, the borer produced certain resistance to these drugs. This study conducted the resistance monitoring of the Seven Insecticides in the field, in order to understand the resistance development of the above agents in time. At the same time, the study on the cross resistance to the diacylamine insecticides was also carried out. The service life of this kind of agent makes it play a better role in the prevention and control of the borer. In order to clarify the activity of borer to chloroacetamide and fluoranamide, this study has determined the sensitivity of the three field borer population to the above two insecticides. There is no study on the mechanism of the action of the borer on the double amide insecticides. Therefore, this study cloned the full-length sequence of the cDNA gene of the neidine receptor gene, and studied the difference in the expression level of the gene expression in the different tissues and different developmental stages, in order to be the molecular mechanism of the resistance to the double amide insecticides in the future borer. The study was based on the resistance monitoring of the Seven Insecticides by the.1. borer. This study was used to determine the resistance of the field population of 59 Chilo suppressalis, 30 in 10 provinces, to seven kinds of insecticides, including benzamide, abamectin, chlorpyrifos, three zazophos and Seven Insecticides in 2013-2015 years in 10 provinces of China. The results showed that there were obvious geographical differences between the Seven Insecticides in the field. The population of high resistance was basically from Zhejiang, Jiangxi and Hunan, Jiangsu Province, Shandong province and Liaoning province were relatively sensitive, and the population of borer suppressalis in Liaoning area was at the sensitive stage to the above seven agents. In 2013-2015 In 2015, it was found that 138.4 times the high level of resistance to fluoroacetamide in the Cangnan borer population in Zhejiang in 2015, 21 populations had produced low to medium level resistance (5.5-85.1 times) to the drug, and the two of the population in Yuyao, Cangnan, Xiangshan and Jiangxi, South City of Jiangxi, had reached a medium level resistance to chloroacetamide (RR =10.9-77.6 times), and 10 populations also produced low level resistance (5.7-8.9 times) to it. From continuous monitoring data, it was found that the population of borer in Yuyao, Xiangshan and Jiangxi, Zhejiang, has been highly resistant to the double amide insecticides, while Jiangsu Yizheng, Hanjiang and Anhui Huizhou have been relatively sensitive. At the same time, in order to determine whether there were differences in the resistance level of the original and preparation of chloroacetamide, we compared the resistance multiplier of two resistant populations in Yuyao and Xiangshan of Zhejiang to the benzamide original and 20% chloroacetamide suspending agent of 97.3% chloroacetamide, and found that the resistance of the two borer species to the 20% chloroacetylene benzamide suspending agent was all. Higher than its original drug.2014-2015, most of the field population of borer in China is still in the stage of sensitive to sensitive decline, which is 78.2%, 76.9% and 88.9% of the total number of monitoring populations, while Xiangshan in Zhejiang, Jinhua, Hunan Dongan and Jiangxi are high, and Nancheng population is low to medium level. Sex (5.6-22.5 times); Zhejiang Cangnan, Yuyao, Jiangxi high, South City and Dongan of Hunan, Youxian showed low to middle level resistance (5.5-15.6 times) to abamectin; only Hunan Dongan, Anhui Huizhou and Shandong Linyi produced low to medium level resistance (6.5-16.1 times) to the insecticidal list (6.5-16.1 times); The population had moderate resistance to chlorpyrifos (11.2-67.6 times), while three azophos, Dongan in Dongan and Jinhua in Zhejiang, Hunan, were still highly resistant (RR=216.8 and 209.9 times), Xiangshan in Zhejiang, Cangnan and the upper south city of Jiangxi, which also reached the moderate resistance to the cross resistance of the.2.. In order to determine whether there was a cross resistance between the double amide insecticide cyanamide, four chloroacetamide and chlorofluoramide and chlorfenamidamide and flubenzamidamide, we used the resistant population of Yuyao and Xiangshan in Zhejiang as test materials to study the cross resistance to three new diamide insecticides. Both fluoroacetamide and chloroacetanilide produced medium to high level resistance (27.6-133.6) in Xiangshan, Zhejiang, Yuyao borer population, which also produced medium to high level resistance (30.3-127.0 times) to bromo cyanamide, four chloramidamides and chlorofluoride amides, indicating the above three new diamide insecticides and chloroacetanilide and fluorobenzamide. The sensitivity of rice seedling borer to chloroacetanilide and florfenicide in.3. rice borer was detected by the method of rice seedling impregnation, and the sensitivity of two diamides of benzamide and florfenicides in Nanjing, Tongzhou and Yuhang, Zhejiang, in 2014, was measured by the rice seedling impregnation method. The results showed that the virulence difference between the two agents was significant, and the sensitivity of the borer to the chloroacetamide was higher than that of the chloroacetamide. However, the difference in sensitivity to the same insecticide was not obvious in different regions of the borer. The cloning and expression of the gene of.4. was cloned by RT-PCR and RACE. The full length sequence of cDNA, named SiRyR, is full length 15428bp, open reading frame 15357 BP, encoding 5118 amino acids. The RyR amino acid sequence of borer has the highest similarity with the beet armyworm and Helicoverpa armigera RyR, 97% and 96%, respectively, with the nematode, human and rat (43%-50%). In addition, the same hair is made by constructing the evolutionary tree. The relationship between RyR and RyR of Helicoverpa armigera and beet armyworm and Spodoptera Spodoptera was recent. At the same time, seven protein domains were found to be predicted for protein structure, including transmembrane domain, MIR domain, RIH domain, RIH-associated domain, SPRY region, RyR domain and ionic channel protein family spiral structure. The expression of PCR receptor gene in different developmental stages and different tissue parts of the fish was studied. It was found that the fish neidine receptor was expressed in the whole life history and different tissue parts of the borer, and the highest expression in the chest part of the male and four instar larvae was found.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.112.1

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