【作者】 李如美；

【导师】 张友军；

【作者基本信息】 湖南农业大学 ， 农药学， 2013， 博士

【摘要】 近年来,烟粉虱Bemisia tabaci (Gennadius)已经成为国内一种重要的农业害虫。由于杀虫剂大量的使用,烟粉虱对很多药剂都产生了抗性,加大了防治难度。噻虫嗪作为新烟碱类药剂的典型代表,是目前防治烟粉虱较好的药剂。为了延长噻虫嗪的田间使用寿命,延缓烟粉虱抗性发展,本研究利用现代生物技术,系统地研究了烟粉虱(B型)对噻虫嗪的代谢抗性机制。本研究首次筛选获得了适合定量检测烟粉虱代谢酶系基因表达的内参基因,率先克隆了烟粉虱体内细胞色素P450CYP6DZ7基因,并阐明了抗性与敏感种群CYP6DZ7基因表达差异、在不同发育阶段和不同组织内的表达特性,最后通过RNAi技术和原核表达技术,初步揭示了CYP6DZ7基因在烟粉虱对噻虫嗪抗性中的作用。相关研究将为烟粉虱的抗性预防与治理提供重要的理论依据。主要研究内容及结论如下：1烟粉虱的内参基因的筛选本文选用了15个候选内参基因,应用实时荧光定量PCR对在生物和非生物处理下的烟粉虱的内参基因进行评估,应用软件geNorm和Normfinder进行分析。结果显示,烟粉虱体内至少有2个内参基因在各种实验处理下可以稳定表达。值得注意的是,不同生物处理条件下(包括不同寄主植物、植物病毒、烟粉虱不同发育阶段、烟粉虱组织差异以及不同的生物型),RPL29是最稳定的内参基因。相比而言,在不同非生物条件下(光周期、温度和杀虫剂敏感性等),EFlα、PPIA、NADH、SDHA和HSP40基因能稳定表达。2烟粉虱细胞色素P450CYP6DZ7基因的克隆及序列分析应用cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)克隆P450基因CYP6DZ7全长序列,结果显示该基因开发阅读框架为1668bp,预测编码555个氨基酸残基。由P450命名委员会的David Nelson命名为CYP6DZ7。与20个昆虫CYP6基因比对以及BLAST分析,结果表明噻虫嗪抗性和敏感品系氨基酸序列是一致的。3烟粉虱抗性、敏感品系CYP6DZ7基因表达量的分析通过荧光定量PCR检测CYP6DZ7的表达量,发现噻虫嗪抗性品系TH-2000中该基因的表达量是敏感品系TH-S的9倍,TH-R品系中的表达量是TH-S中的7倍。结果显示药剂噻虫嗪处理条件下CYP6DZ7基因表达量上调,表明该基因被噻虫嗪明显的诱导。4烟粉虱细胞色素P450基因CYP6DZ7在不同发育阶段和不同组织内的表达量的分析通过荧光定量PCR测定了该基因各个发育龄期和组织的表达量,表明该基因在各个发育阶段的表达量是一致的,其中卵期是最低的,但是与若虫和成虫并未形成显著差异。组织间的定量结果表明腹部中该基因的表达量最高,胸部是最低的,差异达到4倍。5烟粉虱在药剂处理下,CYP6DZ7基因表达量的变化为了验证本研究的假设,检测不同时间段2000mg,/L的噻虫嗪处理的TH-R品系中CYP6DZ7基因表达量,同时处理TH-S,结果表明该基因的表达量与噻虫嗪抗性密切相关。6利用RNAi技术揭示CYP6DZ7基因在抗噻虫嗪中的作用通过RNA干扰(RNAi)沉默TH-R中的CYP6DZ7基因以进一步研究其功能。结果表明双链RNA注射72h后,CYP6DZ7基因的表达量降低>600%,表明该基因能够通过RNAi技术成功敲除。生物测定结果表明进行干扰以后的抗性品系具有更高的死亡率。总而言之,研究结果表明基因CYP6DZ7烟粉虱的噻虫嗪抗性中起到重要作用。7CYP6DZ7在E.Coli中的原核表达本研究利用PET表达系统重组质粒,并成功在大肠杆菌细胞中表达CYP6DZ7蛋白。通过SDS-PAGE电泳分析,在63kDa处发现一条特异性条带,表明目的基因得到成功表达,为下一步烟粉虱抗性机制研究和抗性治理奠定了基础。更多还原

【Abstract】 In recent years, Bemisia tabaci (Gennadius) B biotype has been one of the most destructive invasive pests in many cropping systems worldwide. This whitefly species has developed high resistance to several insecticides commonly used in the world. Thiamethoxam is an important insecticide which has been widely used for the control of this pest. So, the resistance mechanism of the pest to thiamethoxam is a key for sustainable control of this pest. In this study, the resistance mechanisms of B. tabaci B biotype were analyzed using molecular biology method. The main conclusions were listed as follows:1. Selection of reference gene in B. tabaciIn this study,15candidate reference genes in B. tabaci were evaluated under a diverse set of biotic and abiotic conditions using two Excel-based algorithms geNorm and Normfinder. In summary, at least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions, respectively. Specifically, for biotic conditions including host plant, acquisition of plant virus, developmental stage, tissue, and whitefly biotype, ribosomal protein L29was the most stable reference gene. In contrast, the expression of elongation factor1alpha, peptidylprolyl isomeraseA, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein40were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility.2. Cloning and analysis of CYP6DZ7gene in B. tabaciThe full sequence of the mRNA was obtained by RACE (rapid amplification of cDNA ends). CYP6DZ7has a1668bp open reading frame (ORF) with a putative protein size of555amino acid. Full gene sequences were named by Dr. David Nelson. The putative full-length protein sequences of CYP6DZ7were compared between the resistant and susceptible strains, and were also compared to the CYP6protein sequences in20insect species identified by BLASTX. This study found that protein sequence of CYP6DZ7in the resistant and susceptible strains were identical.3. Relative expression analysis of CYP6DZ7in TH-S, TH-R and TH-2000strain of B.tabaciRT-qPCR analyses showed that the expression of CYP6DZ7in TH-2000strain was approximately9-fold higher than that in TH-S strain and was approximately7-fold higher than that in TH-R strain. This result showed that the expression increased when TH-R strain were treated with thiamethoxam, suggesting the CYP6DZ7gene may play an important role in the thiamethoxam resistance of the TH-R strain.4. Relative expression analysis of CYP6DZ7in developmental stages and tissues in B.tabaciThe expression of CYP6DZ7gene in different developmental stages of B. tabaci was examined by RT-qPCR. The result indicated that CYP6DZ7was consistently expressed in all life stages. The expression level of CYP6DZ7was the lowest in eggs but not significantly different from that in nymph or adult. The expression of CYP6DZ7gene was high in abdomen whereas low in the chest. The tissue-dependent expression pattern was validated by RT-qPCR. The expression of CYP6DZ7in abdomen was4-fold higher than that in the chest.5. Differential expression of CYP6DZ7gene after treating with thiamethoxamTo further test our hypothesis, we quantified the expression level of CYP6DZ7of different time course after2000mg/L thiamethoxam treatment and with different treating concentrations in TH-R. At the same time, we quantified the expression level of CYP6DZ7with different treating concentrations in TH-S. The results indicated that the expression of CYP6DZ7gene correlated with thiamethoxam resistance in whiteflies.6. RNA interference (RNAi) methods were used to explore the role of P450genes in B.tabaciThe expression of CYP6DZ7gene in the TH-R strain was partially silenced by RNAi with the double-stranded RNA (dsRNA), which further confirmed the function of CYP6DZ7gene. The results showed that the gene expression of CYP6DZ7decreased>60%by72hr after CYP6DZ7dsRNA injection, which indicated that this gene was successfully knocked down by RNAi. The bioassay tests showed a significantly higher mortalities for the dsRNA-injected adults compared to the control adults injected with buffer. Taken together, our results indicated that CYP6DZ7gene plays an important role in thiamethoxam resistance of the whitefly.7. Expression of CYP6DZ7in E. coli cellsThis study constructed recombinant plasmid using PET expression system, which successfully expressed in E. coli cells. The sresult showed that a63kDa protein was examined and confirmed by SDS-PAGE. Our work laid a foundation for the next resistance mechanism study and resistance management.更多还原