干扰TcRXR2对朱砂叶螨卵期的致死效果分析

Analysis of the lethal effect of RNA interfering with TcRXR2 on the egg stage of Tetranychus cinnabarinus

【目的】基于RNA干扰(RNA interference,RNAi)技术筛选朱砂叶螨Tetranychus cinnabarinus卵孵化过程中的关键基因,为开发靶向朱砂叶螨卵期的RNAi控制技术奠定基础。【方法】通过基因克隆、序列分析和qPCR分析,筛选出在朱砂叶螨卵期特异性表达的基因(类视黄醇X受体2,RetinoidX receptor 2,TcRXR2),并应用RNAi,表型观察分析干扰TcRXR2基因表达后,对朱砂叶螨各个发育过程的影响。【结果】克隆获得了TcRXR2的全长序列,开放阅读框长度为1293 bp,可编码氨基酸为430 aa,具有核受体的DNA结合域(DNA-binding domain,DBD)和配体结合域(Ligand binding domain,LBD)。TcRXR2在第二若螨阶段高表达,在其他阶段无显著性差异(P>0.05),但是进一步检测其在不同日龄卵中的表达发现,TcRXR2在卵后期表达量显著提高(P<0.05)。在连续饲喂ds TcRXR2的条件下,朱砂叶螨幼螨仍能成功发育为成螨,使用点滴ds TcRXR2的方式处理卵则可显著影响卵的孵化(P<0.05),具有致死效应。这些结果表明,TcRXR2基因是影响朱砂叶螨卵孵化的特异性RNAi靶点。【结论】研究找到了一个对朱砂叶螨卵期具有致死效应RNAi靶点,为利用RNAi技术防治朱砂叶螨奠定了基础。

[Aim]To utilize RNA interference(RNAi)technology to identify crucial genes involved in the hatching process of Tetranychus cinnabarinus,and thereby lay the groundwork for the development of targeted RNAi control techniques specifically aimed at the egg stage of this pest.[Methods]Genes specifically expressed during the egg stage of T.cinnabarinus were screened using gene cloning,sequence analysis,and qPCR.One,the retinoid X receptor 2(TcRXR2),was identified and subjected to RNAi.Phenotypic observations were conducted to analyze the impact of RNAi on TcRXR2 gene expression in various developmental stages of T.cinnabarinus.[Results]The full-length sequence of TcRXR2 was successfully cloned,with an open reading frame of 1293 bp,encoding 430 amino acids.It features a nuclear receptor DNA-binding domain(DBD)and a ligand-binding domain(LBD).TcRXR2 was highly expressed during the second nymph stage,with no significant differences in expression in other stages(P<0.05).However,further examination of its expression during egg development revealed that its expression significantly increased as development progressed.Nymphs that were continuously fed dsTcRXR2 were still able to successfully develop into mites.However,the treatment of eggs with dots of dsTcRXR2 significantly affected hatching,demonstrating a lethal effect on embryos.These results suggest that the TcRXR2 gene is a specific RNAi target that influences the hatching of T.cinnabarinus eggs.[Conclusion]A specific RNAi target with lethal effects on the eggs of T.cinnabarinus,was successfully identified,thereby providing a foundation for utilizing RNAi technology to control this pest.