朱砂叶螨β-COP和Sro基因鉴定及其沉默致死效果

Identification of β-COP and Sro of Tetranychus cinnabarinus and Their Lethal Effects After Silencing

【目的】明确朱砂叶螨(Tetranychus cinnabarinus)β-COP和Sro两条基因分子生物学信息,并基于优化的RNAi体系评价它们的致死效应,为筛选适用于RNAi防控的靶基因打下基础。【方法】首先克隆目的基因的全长,并通过序列的同源比对、保守区域及蛋白结构预测以及系统进化树的构建明确其分子生物学信息;其次通过减少ds RNA降解因素、并适时补充ds RNA等方法改进朱砂叶螨的RNAi体系,从而延长干扰时间。利用定量PCR技术检测特定时间点的沉默效率,评价优化RNAi体系后的沉默效果;最后在沉默目的基因β-COP和Sro后,检测朱砂叶螨在各特定时间点的死亡率,评价目的基因在RNAi下的致死效果,并观察相应的致死表型。【结果】β-COP开放阅读框长度为2 688 bp,编码895 aa,属于"WD40 superfamily"及"Coatomer_WDAD superfamily",包含了"WD 40"和"Coatomar_WDAD"的保守区域。Sro的开放阅读框长度为1 119 bp,编码372 aa,具有典型的短链脱氢酶所特有的两个特征,即与NADP+biding结合位点基序"TGxxx Gx"和"Yxxx K"及其上游的天冬氨酸(Asn)、丝氨酸(Ser)活性位点。优化后的RNAi体系可在96 h内能稳定保持50%左右的沉默效率,使用该方法干扰朱砂叶螨β-COP和Sro两条致死基因48 h后试验组与对照组的死亡率均有显著性差异,用β-COP的ds RNA片段干扰雌成螨108 h后,死亡率达57.4%;用Sro的ds RNA片段干扰若螨96 h后,死亡率达28.8%。死亡个体均具有明显的表型,β-COP试验组死亡表型为4对足蜷缩在体侧;Sro试验组则在静伏期死亡或在蜕皮过程中无法蜕皮而死亡。【结论】优化的朱砂叶螨RNAi技术有稳定持续的沉默效果。β-COP和Sro与RNAi技术相结合,验证了这两条目的基因的沉默可对朱砂叶螨产生一定的致死效应,表明此类基因与技术加成的模式极具开发和利用的潜力,为今后基因功能验证和筛选以及开发以RNAi技术为基础的朱砂叶螨防控方法提供了依据。

【Objective】 The objective of this study is to clarify the molecular information of Sro and β-COP genes of Tetranychus cinnabarinus, using the optimized RNAi system to study the lethal effects of these two genes on the T. cinnabarinus, and to provide a basic information to identify target genes, which are useful in the application of RNAi control. 【Method】 Firstly, full sequences of the two genes were identified and the bioinformatics were analyzed by homologous alignment of the sequence, prediction of the conserved region, protein structure and construction of the phylogenetic tree. Secondly, the RNAi system was improved by reducing the factors that will degrade the ds RNA, timely replenishment of ds RNA and other methods, and the gene silencing effect of the new RNAi system was evaluated by quantitative PCR technique. Finally, based on the optimized RNAi system, the expressions of β-COP and Sro genes were silenced to evaluate the lethal effects, and the lethal phenotypes were observed. 【Result】The length of open reading frame of β-COP is 2 688 bp, encoding a 895 aa protein, which belonging to ＂WD40 superfamily＂ and ＂Coatomer_WDAD superfamily＂, containing the conserved regions of ＂WD 40＂ and ＂Coatomar_WDAD＂. The length of open reading frame of Sro is 1 119 bp, encoding a 372 aa protein. It has two characteristics of typical short chain dehydrogenase： one is binding sequence of NADP＋biding binding sequence ＂TGxxx Gx＂, the other is ＂Yxxx K＂ Motif and its upstream aspartic acid（Asn）, serine（Ser） active sites. The optimized RNAi could maintain the silencing efficiency of about 50% after 96 h. Based on this system, the expressions of β-COP and Sro were silenced and the death rate showed significant differences between experimental group and control group after 48 h. β-COP ds RNA caused 57.4% mortality rates at 108 h post-treatment, and Sro ds RNA caused 28.8% death rates at 96 h post-treatment. In addition, the phenotype of dead individuals in the β-COP group showed that the four pairs of feet were curled up on the body side and died, and in the Sro group, the mites dead during the molting process. 【Conclusion】The optimized RNAi technique has stable and continuous silencing effect on the T. cinnabarinus. The combination of β-COP and Sro genes functional verification with RNAi technique showed that the silencing of the two target genes can produce a certain lethal effect on the mite. It also shows that the combination of gene and technology has great potential for development and utilization, which can lay a basis for the future functional verification, screening gene and developing the RNAi technology to control the T. cinnabarinus.