关键词: 松材线虫, 乙酰胆碱酯酶, 石杉碱甲, 抑制剂, 苦参碱, RNA干扰

Abstract:

[Objective]Bursaphelenchus xylophilus(nematode), the causative agent of pine wilt disease, has devastated pine forests for a long time. A safe and efficient technique is needed urgently to control the nematode and prevent the pine wilt disease. The previous studies indicated that the matrine is not suitable for preventing the pine wilt disease although it is an environmentally friendly biocontrol agent. To develop low toxicity and effecient nematicides, the gene, function and protein inhibition of acetylcholinesterase 1 in pine wood nematode B.xylophilus, was studied.[Method]The B.xylophilus populations used in this study were collected from Guangdong Province in 2007. The host is masson pine (Pinus massoniana).The nematodes were separated by the Behrman funnel method, cultured at 25 ℃ without light and fed on Botrytis cinerea moss. The gene, function and protein inhibition of acetylcholinesterase 1, was studied. The full-length of BxACE 1 gene was cloned and the sequence was analysed. The BxACE 1 gene function was silenced by the method of RNAi and inhibited by proteins inhibitor to study the insecticidal effect of matrine.In the meantime, Q-PCR was used to identify RNAi effects. [Result]A total of 2 145 bp full-length gene was obtained by splicing 5' and 3' sequence, named BxACE 1. Homologous sequences of multiple sequence alignment showed that the nematode ACE evolution speed was consistent with nematodes. Protein 3D structure analysis showed that BxACE 1 was 13 β-fold surrounded by 14 α-helix. In view of the fact that the more hydrogen bonds in combination with the more stable, the huperzine A was selected as a more efficient inhibitor of acetylcholinesterase 1 protein by docking technique. Huperzine A itself had low toxicity to nematodes and the common aplication of RNAi and huperzine A had still low toxicity to nematodes, but the nematodes survivals in 24 h, 48 h and 72 h after RNAi treatment and inhibitor (treated nematodes with 0.03% matrine + 1 μmol ·L^-1 Huperzine) treatment were not significantly different with each other indicating that Huperzine A and RNAi BxACE 1 genes had similar treatment effects to nematodes.[Conclusion and other]The results indicated that the nematicidal activity of matrine was improved after the BxACE 1 was silenced by the method of RNAi or the BxACE1 was inhibited. RNAi technology easily proved that blocking BxACE 1 gene function could improve the nematicidal effect of matrine, while dsRNA could degrade easily, which limited the extensive application of the technique in production practice. Huperzine A, screened with docking technique,was able to combine with BxACE1 stabily. Appraisal of inhibitory effects of inhibitors showed that huperzine A improved the nematicidal effect of matrine, which had the development potential as bio-control auxiliary agents, could be used as an alternative of gene silencing technology that inhibited acetylcholinesterase 1 gene of nematodes. Therefore, this study further put forward that huperzine A, the acetylcholinesterase inhibitor, used to block BxACE 1 gene function could improve the effects of botanical nematicidal like matrine and so on. Through inhibition of ACE, ACEIs made the acetylcholine accumulate in the synaptic clearance, and hence made the effect time of acetylcholine longer. ACEIs caused the choline levels to increase acutely in a short period of time by inhibiting nematodes acetylcholinesterase activity, which resulted in the poisoning death of nematodes. The representative pesticides class are organophosphorus compounds and carbamate compounds, most of which were non-reversible ACEIs. Huperzine A is a half terpene alkaloid derived from Huperziaceae plants, Huperzia serrata, and is a kind of high efficient, high selective, and reversible ACEIs. In this study, huperzine A was introduced to B.xylophilus, which was a new attempt to apply the botanical source medicament.

Key words: Bursaphelenchus xylophilus, acetylcholinesterase, huperzine A, inhibitor, matrine, RNAi