二化螟抗藥性監(jiān)測及大螟魚尼丁受體基因克隆與表達分析
發(fā)布時間：2018-07-29 13:31
【摘要】：水稻二化螟[Chilo suppressalis(Walker)]和大螟[Sesamia inferens(Walker)]是水稻上的重要害蟲,廣泛分布于我國主要水稻種植區(qū)。二化螟的為害程度較大螟重,大螟通常在稻田周邊零星發(fā)生。長期以來,水稻螟蟲的防治主要依靠化學藥劑,其中主要包括有機磷類殺蟲劑毒死蜱和三唑磷、大環(huán)內酯抗生素類殺蟲劑阿維菌素和甲維鹽、沙蠶毒素類殺蟲劑殺蟲單及新型雙酰胺類殺蟲劑氯蟲苯甲酰胺和氟苯蟲酰胺,目前雙酰胺類殺蟲劑是田間防治螟蟲的主要藥劑。但是由于化學藥劑的長期不合理使用,二化螟對這些藥劑均產生了一定的抗藥性。本研究進行了田間二化螟種群對上述七種藥劑的抗藥性監(jiān)測,以期能夠及時了解上述藥劑的抗性發(fā)展情況。同時還進行了二化螟對雙酰胺類殺蟲劑交互抗性的研究,旨在延長該類藥劑的使用壽命,使其在二化螟的防治中發(fā)揮更好的作用。為了明確大螟對氯蟲苯甲酰胺和氟苯蟲酰胺的活性,本研究測定了三個田間大螟種群對上述兩種藥劑的敏感性。昆蟲魚尼丁受體是雙酰胺類殺蟲劑的主要靶標,鑒于目前還沒有關于大螟對雙酰胺類殺蟲劑作用機制的研究,因此本研究克隆了大螟魚尼丁受體基因cDNA的全長序列,并對其不同組織及不同發(fā)育階段魚尼丁受體基因表達水平的差異進行研究,以期為未來大螟對雙酰胺類殺蟲劑抗藥性分子機制的研究提供依據。1.二化螟對七種藥劑的抗藥性監(jiān)測本研究分別采用點滴法和稻苗浸漬法于2013-2015年測定了我國10省30地59個二化螟田間種群對氯蟲苯甲酰胺、氟苯蟲酰胺甲維鹽、阿維菌素、毒死蜱、三唑磷及殺蟲單七種藥劑的抗藥性。結果顯示:田間二化螟種群對這七種藥劑均存在明顯的地理差異,抗性高的種群基本都來自浙江省、江西省及湖南省,江蘇省、山東省及遼寧省的二化螟種群相對敏感,其中遼寧地區(qū)的二化螟種群對上述七種藥劑均處于敏感階段。在2013-2015年,已經發(fā)現(xiàn)在2015年浙江蒼南二化螟種群對氟苯蟲酰胺已產生了138.4倍的高水平抗性,同時已有21個種群對該藥劑產生了低到中等水平抗性(5.5-85.1倍);浙江余姚、蒼南、象山及江西南城二化螟種群對氯蟲苯甲酰胺已達到中等水平抗性(RR=10.9-77.6倍),并且有10個種群對其也產生了低水平抗性(5.7-8.9倍)。從連續(xù)監(jiān)測的數據可以發(fā)現(xiàn),浙江余姚、象山及江西南城二化螟種群對雙酰胺類殺蟲劑一直處于較高水平的抗性,而江蘇儀征、邗江及安徽徽州3個種群一直處于相對敏感的階段。同時為了明確氯蟲苯甲酰胺原藥和制劑的抗性水平是否存在差異,我們比較了浙江余姚和象山兩個抗性種群對97.3%氯蟲苯甲酰胺原藥和20%氯蟲苯甲酰胺懸浮劑的抗性倍數,發(fā)現(xiàn)兩個二化螟種群對20%氯蟲苯甲酰胺懸浮劑的抗性均高于其原藥。2014-2015年,我國大部分二化螟田間種群對甲維鹽、阿維菌素及殺蟲單仍處于敏感至敏感水平下降階段,分別占其監(jiān)測種群總數的78.2%、76.9%及88.9%,而浙江象山、金華、湖南東安及江西上高、南城種群對甲維鹽為低到中等水平抗性(5.6-22.5倍);浙江蒼南、余姚、江西上高、南城及湖南東安、攸縣對阿維菌素表現(xiàn)為低到中等水平抗性(5.5-15.6倍);只有湖南東安、安徽徽州及山東臨沂三個種群對殺蟲單產生了低到中等水平抗性(6.5-16.1倍);浙江蒼南、象山及江西上高等七個種群對毒死蜱產生了中等水平抗性(11.2-67.6倍);而對于三唑磷,湖南東安和浙江金華對其仍為高水平抗性(RR=216.8和209.9倍),浙江象山、蒼南及江西上高南城等七個種群對其也達到中等抗性水平。2.二化螟對雙酰胺類殺蟲劑的交互抗性為了明確二化螟對雙酰胺類殺蟲劑溴氰蟲酰胺、四氯蟲酰胺及氯氟氰蟲酰胺與氯蟲苯甲酰胺和氟苯蟲酰胺間是否存在交互抗性,我們以浙江余姚和象山抗性種群為試材,進行了其對三種新雙酰胺類殺蟲劑的交互抗性研究,結果顯示,對氟苯蟲酰胺和氯蟲苯甲酰胺均產生了中到高水平抗性(27.6-133.6)的浙江象山、余姚二化螟種群,對溴氰蟲酰胺、四氯蟲酰胺和氯氟氰蟲酰胺也產生了中到高水平抗性(30.3-127.0倍),則表明上述三種新雙酰胺類殺蟲劑與氯蟲苯甲酰胺和氟苯蟲酰胺之間存在交互抗性。3.水稻大螟對氯蟲苯甲酰胺和氟苯蟲酰胺的敏感性測定本研究采用稻苗浸漬法對于2014年采自江蘇南京、通州及浙江余杭的大螟田間種群對氯蟲苯甲酰胺和氟苯蟲酰胺兩種雙酰胺類殺蟲劑的敏感性進行了測定,結果顯示:這兩種藥劑對大螟的毒力差異顯著,大螟對氟苯蟲酰胺比氯蟲苯甲酰胺的敏感性高。但是不同地區(qū)大螟種群對同一種藥劑的敏感性差異不明顯。4.大螟魚尼丁受體基因克隆與表達分析利用RT-PCR和RACE的方法,克隆得到大螟魚尼丁受體的cDNA全長序列,命名為SiRyR,該序列全長15428bp,開放閱讀框15357 bp,編碼5118個氨基酸。大螟RyR氨基酸序列與甜菜夜蛾和棉鈴蟲RyR的相似性最高,分別為97%和96%,與線蟲、人和大鼠的相似性較低(43%-50%);此外通過構建進化樹,同樣發(fā)現(xiàn)大螟RyR與棉鈴蟲和甜菜夜蛾RyR的親緣關系最近。同時對其蛋白質結構預測發(fā)現(xiàn)存在七種蛋白質結構域,包括跨膜區(qū)結構域、MIR結構域、RIH結構域、RIH-associated結構域、SPRY區(qū)域、RyR結構域和離子通道蛋白家族自身螺旋結構。此外,通過熒光定量PCR對大螟魚尼丁受體基因在不同發(fā)育階段及不同組織部位中的表達情況進行研究,發(fā)現(xiàn)魚尼丁受體在大螟整個生活史及不同組織部位中魚尼丁受體基因均有表達,且在雄成蟲和四齡幼蟲的胸部表達量最高。
[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.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S435.112.1
本文編號：2152829
[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.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S435.112.1
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