Screening And Functional Study Of Cytochrome P450 Genes Associated With Resistance To Imidacloprid In Bemisia Tabaci

Bemisia tabaci is a notorious pest insect of agriculture and horticulture worldwide.Intensive chemical insecticides were used to control the damage caused by B.tabaci and inevitably led to the appearance of insecticide-resistant populations.Imidacloprid is widely used for the control of B.tabaci which belongs to neonicotinoids.Previous studies have demonstrated that overexpression of cytochrome P450 genes is one of the most important resistance mechanisms to neonicotinoid insecticides of B.tabaci.The elucidation of resistance mechanism is the prerequisite for the insect resistance management,and the early resistance detection can provide scientific guidance for the effective control of B.tabaci.At present,B.tabaci from Xinjiang has developed resistance to commonly used neonicotinoid insecticides,but the resistance mechanism has not been well known In this paper,we firstly investigated the resistance levels to imidacloprid of B.tabaci from south and north of Xinjiang.Overexpressed P450 genes involved in imidacloprid resistance were then screened by semi-quantitative PCR and further verified by quantitative PCR-The candidate genes were successfully expressed in Sf9 and metabolism of imidacloprid in the cells was analyzed.Our results not only lay a foundation for exploring the molecular detection technology of resistance to imidacloprid,but also provided the scientific basis for making the res:istance managements strategies of B.tabaci.The main results of this study are summarized as follows:1.Resistance to imidacloprid and thiamethogam of Bemisia tabaci field populations from XinjiangThe resistance level to imidacloprid and thiamethoxam was investigated in five field populations of Bemisia tabaci from Turpan(Seed farm and vegetable production base),Shache,Hotan and Yining in northern and southern Xinjiang using the leaf dipping method.The results showed that the four populations except for Yining had developed different level resistance to the imidacloprid,compared to susceptible strain Turpan2,Hotan and Shache populations had low to moderate level resistance to imidacloprid respectively with 6.74,8.49 and 18.18-fold of RR.All field populations tested showed susceptible to thiamethoxam.2.Screening of valid reference genes for gene expression studies by quantitative real-time PCR in different resistant populations of Bemisia tabaciIn order to acquire the accurate results of the expression levels of target genes in different resistant Bemisia tabaci populations by quantitative real-time PCR(qPCR),selection of the suitable reference genes is necessary.Five candidate reference genes,including β-actin,HSP20,HSP90,y-tubulin and RPL29,were chosen from B.tabaci transcriptome data.The expression of the five candidate reference genes was estimated by qPCR in different imidacloprid-resistant populations of B.tabaci.The stability of the five genes was investigated using geNorm and NormFinder.Based on the results of geNorm analysis,expression of HSP20 and HSP90 was the most stable,while HSP20 and RPL29 were the most stable reference genes according to NormFinder analysis.HSP20 was selected as the suitable reference gene for qPCR in different resistant populations of B.tabaci.The work is contributable to the future gene expression studies in different resistant and field populations of B.tabaci.3 The expression analysis of cytochrome P450 Genes from Bemisia tabaci in XinjiangTo elucidate resistance mechanism of B.tabaci from Xinjiang to imidacloprid,the expressions of P450 genes from different Bemisia tabaci populations were analyzed by semi-quantitative and quantitative real-time fluorescence quantitative PCR.The results showed that expression of nine P450 genes in the field resistant populations was higher than that in the susceptible strain,including CYP4G68,CYP6CM1,CYP6CX4,CYP4G68,CYP415A1,CYP6DV4,CYP402C2,CYP6DV6,CYP6DW3 and CYP416A1.The results obtained from real-time quantitative PCR further confirmed that CYP6CM1 and CYP4G68 were overexpressed in imidacloprid-resistant field populations,which suggested the two genes involved in imiacloprid resistance mechanism of B.tabaci from Xinjiang4.Functional expression of CYP6CM1 and CYP4G68 in Sf9 and metabolism to imidacloprid of the two gene recombinantsCYP6CM1 and CYP4G68 were cloned into pFastBacHTA vector and expressed in Sf9 cells.The metabolic efficiency of the two recombinants against substrate p-nitro anisole was examined in order to verify that the recombinant enzyme is catalytically active.The results showed that the activity of recombinant CYP6CM1 and CYP4G68 was 17.2 and 16.9-fold higher than that in EGFP respectively,suggesting that the recombinant enzymes were functional In addition,analysis of imidacloprid metabolism by liquid chromatography-mass spectrometry(LC-MS)showed the two recombinant proteins could metabolize imidacloprid,with metabolite being desnitro-olefin imidacloprid.These results suggested that the overexpression of CYP6CM1 and CYP4G68 is one of important mechanisms of resistance to imidacloprid ofB.tabci from Xirgiang.