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【目的】探讨脱氧鬼臼毒素(deoxyopodophyllotoxin,DOP)对美洲大蠊Periplaneta americana神经系统乙酰胆碱受体信号通路的影响。【方法】从美洲大蠊头部克隆乙酰胆碱受体信号通路上的关键信号分子nAChRα6亚基、CaM和CaMKⅡ的部分mRNA,并测定其序列。应用荧光定量PCR技术分别观察注射不同浓度(10,45,80,115和150μg/μL)DOP 24和48 h后上述3种基因表达水平的变化。【结果】测序结果显示,克隆出的美洲大蠊nAChR基因部分序列(539 bp)与赤拟谷盗Tribolium castaneum nAChRα6亚基基因的核苷酸序列一致性为84%;美洲大蠊CaM基因(435 bp)与雕叶蝉Graphocephala atropunctata CaM基因的核苷酸序列一致性为85%;美洲大蠊CaMKⅡ基因(513 bp)与黑腹果蝇Drosophila melanogaster CaMKⅡ基因的核苷酸一致性为77%。实时定量荧光PCR实验表明:DOP处理48 h后对美洲大蠊nAChRα6亚基、CaM和CaMKII基因表达水平大体表现出低剂量激活,高剂量抑制的特点。45~80μg/μL DOP浓度范围内3种基因表达水平达到高峰,80~150μg/μL浓度范围内表现为抑制作用,基因表达水平呈下降趋势。【结论】DOP需要在美洲大蠊体内蓄积一定时间才有明显的作用,能与nAChR结合引起CaM-CaMKⅡ级联反应,使3种基因的表达在低浓度组上调、高浓度组被抑制,进而对美洲大蠊产生潜在的毒杀作用。
【Objective】 To investigate the effect of deoxyopodophyllotoxin (DOP) on acetylcholine receptor signal pathway in Periplaneta americana nervous system of American cockroach. 【Method】 Some mRNAs of the key signal molecules nAChRα6 subunit, CaM and CaMKⅡ on the acetylcholine receptor signal pathway were cloned from the head of American cockroach and their sequences were determined. Fluorescent quantitative PCR was used to observe the changes of the expression levels of the above three genes after DOP 24 of different concentrations (10, 45, 80, 115 and 150 μg / μL) for 24 and 48 h, respectively. 【Result】 The results of sequencing showed that the nucleotide sequence of the cloned nAChR gene of 539 bp was 84% identical to the Tribolium castaneum nAChRα6 subunit gene. The CaM gene of American cockroach (435 bp) was 85% identical to that of Graphocephala atropunctata CaM gene. The nucleotide identity of CaMKⅡ gene (513 bp) and Drosophila melanogaster CaMKⅡ gene of Periplaneta americana was 77%. Real-time quantitative PCR experiments showed that the expression of nAChRα6 subunit, CaM and CaMKII genes in the cockroach JAC showed broadly low-dose activation and high-dose inhibition 48 h after DOP treatment. The expression levels of the three genes reached the peak in the concentration range of 45 ~ 80μg / μL DOP, and inhibited in the concentration range of 80 ~ 150μg / μL, the gene expression level showed a downward trend. 【Conclusion】 DOP needs to accumulate in the body of American cockroach for a certain period of time to have obvious effect. It can combine with nAChR to cause cascade of CaM-CaMKⅡ, up-regulate the expression of three genes in low concentration group and inhibit the high concentration group Possibly toxic to Periplaneta americana.