Characteristics Of AChE Expression In Two Nilaparvata Lugens Biotypes

Nilaparvata lugens,the brown planthopper（BPH）feeds on the phloem of rice through stab-sucking mouthparts,which not only interferes with the transport of rice assimilation materials but also induces and spreads rice diseases due to its excreta.Outbreak of the pest seriously affects the normal growth and development of rice and causes a large amount of grain yield reduction.It is one of the important pests of rice.The current major control strategy is to spray pesticides,but overuse of pesticides will cause environmental pollution and develoment of pest resistance.There is a co-evolution between brown planthopper and rice.Different biotypes of brown planthopper harboring different virulence genes,can adapt to rice varieties with different resistant levels.Acetylcholinesterase（AChE）is an important target for insecticides and can also promote apoptosis of the midgut^[1,2].Therefore,it is significant to study the epigenetic relationship among brown planthopper with different biotypes.In this work,characteristics of AChE expression in two Nilaparvata lugens with different biotypes were studied as follows:（1）The chromosomal walking technique was used to clone anⅠbiotype AChE upstream flanking DNA sequence with 1919 bp in size.Novel promoter element analysis showed that there were several binding sites（elements）binding to resistance-related transcription factors such as MYB,WRKY,and ARE.),indicating that the promoter may be related to stress.An expression vector was constructed based on the skeleton vector p GL3-AChE,to detect the expression level of the promoter by using double luciferase assay.The results demonstrated that the fluorescence intensity was directly proportional to the rice resistance,implying the AChE promoter is induced by host rice resistance.（2）The methylation-specific PCR（MS-PCR）technology was used to analyze the methylation degree of the AChE gene promoter region between the biotypeⅠand biotypeⅡ.The results showed that the promoter region in the biotypeⅠwas more highly methylated than in biotypeⅡ.Bisulfite sequencing（BSP）assay revealed that the methylation standard of the promoter region in biotype I（24.09%）was higher than in biotype II（7.27%）.RT-PCR showed that the expression level of AChE in biotypeⅡwas higher than in biotypeⅠ.All the data suggests that the high methylation level of the promoter region is a possible reason for the low expression level of AChE in the brown planthopper.（3）A Cas RNP complex vector was constructed based on CRISPR/Cas9 technology.The sgRNA-AChE construct was confirmed by digestion in vitro,and the expressed sgRNA exhibited a specific site-directed digestion of AChE fragment.Subsequently,a sgRNA-AChE expression vector was constructed and used for microinjecting 4th instar nymph of BPH.Knockout of AChE reduced the survival rate of the brown planthopper by 22.22%.RT-PCR showed that the expression level of AChE gradually decreased by 1.26 fold a day later and0.94 fold 2 days later.The injected insects displayed various abnormal biotypes such as missing wings or mutilated wings.The expression levels of wing-controling genes like ln R1,Ubx,and Apa were elevated,however,the expression of ln R2 gradually decreased.（4）An expression vector pET32a-AChE was successfully constructed,and AChE protein was expressed and purified.The optimal expression was performed under 16℃for 16 h by using 1 m M IPTG as inducer.This experiment laied a foundation for determining the crystal structure of AChE.The prediction of the secondary structure showed that 32%of the amino acid residues formedα-helix,10%of the amino acids formedβ-sheets and 2%were TM sheets.The tertiary structure was predicted as that this protein was 65%identical to that of Drosophila and 44%to human AChE2.