【作者】 李静；

【导师】 李建洪； 马康生；

【作者基本信息】 华中农业大学 ， 农业硕士（专业学位）， 2022， 硕士

【摘要】 棉蚜（Aphis gossypii Glover）属半翅目（Hemiptera）,蚜科（Aphidiae）,是一种世界性分布的重要害虫,主要通过吸食植物汁液和传播植物病害造成危害。目前,由于化学杀虫剂的大量长期使用,棉蚜已对多种杀虫剂产生了高水平抗药性。G蛋白偶联受体（G protein-coupled receptors,GPCRs）是一种重要的存在于细胞膜上可以感受各类信号的蛋白受体。GPCRs参与昆虫许多重要的生理过程,包括生长发育、繁殖、代谢和行为。GPCRs作为潜在新型杀虫剂靶标,有研究表明其通过对抗性相关P450基因表达的调控介导了致倦库蚊（Culex quinquefasciatus）对苄氯菊酯的抗药性。前期研究已证明细胞色素P450介导了棉蚜对氟啶虫胺腈的代谢抗性,但其上游调控通路仍未可知。基于此,本文以室内敏感品系和棉蚜对氟啶虫胺腈抗性品系为材料,通过生物信息学、生物化学和分子生物学等方法和技术,系统鉴定了棉蚜中具有典型7个跨膜结构域的GPCRs基因,分析了其在棉蚜不同品系、发育阶段、组织和药剂诱导下的表达谱,揭示了GPCRs调控棉蚜P450基因表达的分子抗性机制。主要结果如下:1.棉蚜基因组中具有典型7个跨膜结构域的GPCRs基因鉴定参考已发表的黑腹果蝇（Drosophila melanogaster）、赤拟谷盗（Tribolium castaneum）和豌豆蚜（Acyrthosiphon pisum）GPCRs基因,通过BLASTP搜索棉蚜基因组数据库,获得了120条候选基因。进一步,根据GPCRs基因鉴定标准,最终确定了88个棉蚜GPCRs基因,其中66个A家族成员,12个B家族成员,7个C家族成员,还有3个F家族成员。2.棉蚜GPCRs基因表达谱分析用LC₁₅（31.03 mg/L）和LC₅₀（157.68 mg/L）浓度的氟啶虫胺腈处理棉蚜,发现11个GPCRs基因被氟啶虫胺腈显著诱导。此外,采用实时荧光定量PCR分析了57个GPCRs基因在敏感品系（SS）和氟啶虫胺腈抗性（Sul R）品系中的相对表达量,发现共有34个GPCRs基因在Sul R中均显著上调表达。龄期和组织特异性表达分析发现18个GPCRs基因在成虫期的表达量均显著高于若虫期,且在有翅蚜和无翅蚜中呈现明显差异表达;12个、5个和2个GPCRs基因分别在头、表皮和中肠中高表达。3.GPCRs对棉蚜氟啶虫胺腈抗性相关P450基因表达的调控作用研究为了阐明GPCRs调控棉蚜P450基因表达的抗性分子机制,利用RNAi分别干扰Ago GPCR49和Ago GPCR54,沉默后棉蚜对氟啶虫胺腈的敏感性增加并显著抑制解毒酶P450的活力。干扰Ago GPCR49后,20个P450基因显著下调表达;而沉默Ago GPCR54后12个P450基因显著下调。CYP6DB1、CYP6CY18、CYP301A1、CYP6CY4、CYP6CY14、CYP4CJ3、CYP6A14、CYP4CK1和CYP306A1均在沉默Ago GPCR49或Ago GPCR54后显著下调表达,其中CYP6CY14表达量分别在沉默Ago GPCR49和Ago GPCR54显著下降79%和71%。以上研究为初步揭示GPCRs调控P450s介导棉蚜对氟啶虫胺腈代谢抗性的分子机制奠定了理论基础。更多还原

【Abstract】 The cotton aphid,Aphis gossypii Glover（Hemiptera:Aphididae）,is one of the most serious insect pests in the cotton field worldwide,which causes economic damage through direct feeding and indirectly virus transmission.So far,the control of cotton aphids in China is largely depended on chemical insecticides.Consequently,cotton aphids have evolved resistance to many types of insecticides.G protein-coupled receptors（GPCRs）are important protein receptors to exist on cell membranes and can sense various signals.They are involved in the regulation of many important physiological processes in insects,including growth,development,reproduction,metabolism,and behaviors.Studies have shown that GPCRs mediated the resistance of Culex quinquefasciatus to permethrin through regulating the expression of resistance-related P450 genes.It has been proved that cytochrome P450 mediates the main metabolic resistance mechanism of sulfoxaflor in cotton aphids.However,the upstream regulation pathway of GPCRs is unclear.In this study,a series of biological,biochemical,and molecular experiments were conducted on the susceptible strain and a sulfoxaflor-resistant strain.Therefore,A.gossypii GPCR genes with typical seven-transmembrane（7TM）domains were systematically identified,the expression pattern of A.gossypii GPCR genes were analyzed in different strains,developmental stages,tissues,and sulfoxaflor treatments,the molecular resistance mechanism of GPCRs regulating the P450 gene expression was clarified in the sulfoxaflor resistance in A.gossypii.The main results are as follows.1.Identification of A.gossypii GPCR genes with typical seven transmembrane domainsBased on previous studies,the GPCRs of Drosophila melanogaster,Tribolium castaneum,and Acyrthosiphon pisum were collected and used as references.By performing BLASTP searches in the genome database of A.gossypii,120 GPCR genes were found in candidate pools.According to the criteria,88 A.gossypii GPCR genes were identified,among which there are 66 family-in A members,12 family-in B members,7 family-in C members,and 3 family-in F members.2.Analysis of expressing pattern of A.gossypii GPCR genesThe expression of eleven GPCR genes was significantly induced by sulfoxaflor with concentrations of LC₁₅（31.03 mg/L）and LC₅₀（157.68 mg/L）.The expression levels of 57A.gossypii GPCR genes in the susceptible strain SS and sulfoxaflor resistant（Sul R）strain were performed by quantitative real-time PCR.The results showed that a total of 34 GPCR genes were up-regulated in the Sul R strain.Age-specific and tissue-specific expression analysis indicated that the expression levels of 18 GPCR genes in the adult stages were higher than those in other stages and were significantly differentially expressed in the wingless or winged aphids;twelve,five,and two GPCR genes were highly expressed in the head,epidermis,and midgut,respectively.3.Study on regulation of GPCRs to the expression of P450 gene related to A.gossypii resistance to sulfoxaflorIn order to elucidate the mechanism of GPCRs regulating the P450 gene expression,knocking down Ago GPCR49 or Ago GPCR54 by RNAi was performed,the susceptibility of A.gossypii to sulfoxaflor increased significantly and the relative activities of detoxification enzymes P450 decreased significantly.It was further found that 20 of P450genes were significantly down-regulated with Ago GPCR49 knocked down.After silencing Ago GPCR54,12 of P450 genes were significantly down-regulated.In addition,CYP6DB1,CYP6CY18,CYP301A1,CYP6CY4,CYP6CY14,CYP4CJ3,CYP6A14,CYP4CK1,and CYP306A1 were significantly decreased after feeding with ds Ago GPCR49 or ds Ago GPCR54,among which the expression level of CYP6CY14 was significantly reduced by 79%and 71%,respectively.These findings layed a theoretical foundation for preliminary mechanism of GPCRs in regulating P450 genes mediated the sulfoxaflor metabolic resistance in Aphis gossypii Glover.更多还原