摘要
【目的】为提高苦参碱的杀虫效果,对松材线虫的药剂防治靶标乙酰胆碱酯酶1基因(Bx ACE1)进行研究。【方法】松材线虫采集自广东省内马尾松,贝曼漏斗法分离,灰葡萄孢菌苔上25℃避光培养扩繁线虫。克隆Bx ACE1基因全长并进行序列分析,通过基因沉默和蛋白抑制2种技术阻断该基因功能,研究苦参碱的杀虫效果,并应用Q-PCR技术鉴定RNAi效果。【结果】5'和3'序列拼接得到基因全长2 145 bp,命名为Bx ACE1。同源序列多序列比对表明线虫ACE的进化速度与线虫进化速度一致。蛋白质三维结构分析表明,Bx ACE1由14个α螺旋包围着13个β折叠,鉴于氢键数量越多结合越稳定,利用docking技术筛选出石杉碱甲是松材线虫乙酰胆碱酯酶1蛋白的较佳抑制剂。RNAi明显增加了苦参碱的杀虫效果,石杉碱甲本身对线虫毒性较小,共同施用RNAi和石杉碱甲对线虫毒性仍然较小,但RNAi处理组线虫与Inhibitor(0.03%苦参碱+1μmol·L-1石杉碱甲处理线虫)处理组线虫在24,48及72 h存活率差异不显著,表明石杉碱甲处理线虫和Bx ACE1基因RNAi处理效果相近。【结论与其他】通过基因沉默和蛋白抑制2种技术阻断Bx ACE1功能均可以提高苦参碱的杀虫效果,RNAi技术能够简单易行地证明若阻断Bx ACE1功能可以提苦参碱的杀虫效果,但dsRNA容易降解等因素限制了该技术在生产实践中的广泛应用。石杉碱甲可以与Bx ACE1稳定结合,鉴定表明石杉碱甲可以提高苦参碱的杀虫效果,具有生防药剂辅剂的开发潜力,可替代基因沉默技术实现抑制Bx ACE1的目的。因此,通过乙酰胆碱酯酶抑制剂石杉碱甲可阻断Bx ACE1的功能,进而达到提高苦参碱等植物源药剂杀虫效果的目的。ACEIs通过抑制线虫体内的乙酰胆碱酯酶的活性,使线虫体内的胆碱水平短时间内大量提高,从而导致其中毒死亡。该类具有代表性的杀虫剂是有机磷类化合物和氨基甲酸酯类化合物,大多为非可逆型ACEIs。石杉碱甲是来源于石杉科植物蛇足石杉的一种半萜生物碱,是一种高效、高选择性、可逆性的ACEIs。本研究将石杉碱甲引入松材线虫研究,是对该植物源药剂的一个新尝试。
【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 ofBx ACE1 gene was cloned and the sequence was analysed. The Bx ACE1 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 Bx ACE1.Homologous sequences of multiple sequence alignment showed that the nematode ACE evolution speed was consistent with nematodes. Protein 3D structure analysis showed that Bx ACE1 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- 1Huperzine）treatment were not significantly different with each other indicating that Huperzine A and RNAi Bx ACE1 genes had similar treatment effects to nematodes.【Conclusion and other】The results indicated that the nematicidal activity of matrine was improved after the Bx ACE1 was silenced by the method of RNAi or the Bx ACE1 was inhibited. RNAi technology easily proved that blocking Bx ACE1 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 Bx ACE1 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 Bx ACE1 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.
出处
《林业科学》
EI
CAS
CSCD
北大核心
2015年第1期66-73,共8页
Scientia Silvae Sinicae
基金
国家林业公益性行业科研专项"重大森林病虫灾害防控技术的关键理论基础"(201204501)
黑龙江省自然科学基金项目项目(ZD201404)
中央高校基本科研业务费专项资金项目(DL13BA01)
