朱砂叶螨β-COP和Sro基因鉴定及其沉默致死效果

doi:10.3864/j.issn.0578-1752.2017.18.009

Abstract

Abstract: 【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 dsRNA, timely replenishment of dsRNA 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 “TGxxxGx”, the other is “YxxxK” 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 dsRNA caused 57.4% mortality rates at 108 h post-treatment, and Sro dsRNA 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.

Key words: Tetranychus cinnabarinus, RNA interference technology, lethal effect, comprehensive prevention and green control

AO YiQianYun, SHEN GuangMao, WANG MengYao, LIU JiaLu, PAN Yu, HE Lin. Identification of β-COP and Sro of Tetranychus cinnabarinus and Their Lethal Effects After Silencing[J].Scientia Agricultura Sinica, 2017, 50(18): 3529-3539.

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

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