王越,张苏芳,徐瑶,方加兴,孔祥波,刘福,张真.美国白蛾几丁质酶细菌表达的RNA干扰载体构建及其介导的RNA干扰[J].林业科学研究,2019,32(2):1-8
美国白蛾几丁质酶细菌表达的RNA干扰载体构建及其介导的RNA干扰
Construction of the Expression Vector and RNAi Mediated by Bacteria Expressed dsRNA of Chitinase Gene from Hyphantria cunea
投稿时间:2017-11-20  修订日期:2018-02-05
DOI:10.13275/j.cnki.lykxyj.2019.02.001
中文关键词:  美国白蛾  HT115菌株  L4440  几丁质酶  RNA干扰
英文关键词:Hyphantria cunea  HT115 strain  L4440  chitinase  RNAi
基金项目:林业公益性行业科研专项(201504302);中国林科院森环森保所基本科研业务费专项资金(CAFRIFEEP 201406)
作者单位E-mail
王越 中国林业科学研究院森林生态环境与保护研究所, 北京 100091  
张苏芳 中国林业科学研究院森林生态环境与保护研究所, 北京 100091  
徐瑶 中国林业科学研究院森林生态环境与保护研究所, 北京 100091  
方加兴 中国林业科学研究院森林生态环境与保护研究所, 北京 100091  
孔祥波 中国林业科学研究院森林生态环境与保护研究所, 北京 100091  
刘福 中国林业科学研究院森林生态环境与保护研究所, 北京 100091  
张真 中国林业科学研究院森林生态环境与保护研究所, 北京 100091 zhangzhen@caf.ac.cn 
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中文摘要:
      [目的]探索细菌表达dsRNA介导的RNAi在美国白蛾中的可行性,为RNAi技术在美国白蛾等林业害虫上的应用提供依据。[方法]选择几丁质酶HcChi基因作为靶标,设计有效的干扰片段,构建到L4440干扰载体上,并转入HT115大肠杆菌菌株。IPTG诱导HT115表达HcChi的干扰片段,用菌液持续饲喂美国白蛾幼虫,观察幼虫生长情况,定量PCR检测HcChi的转录水平。[结果]构建了带有HcChi-L4440表达载体的HT115菌株,经IPTG诱导能够合成HcChi-dsRNA,浓缩菌液持续饲喂幼虫显著抑制HcChi的表达,相对表达量显著下降了76.7%~90.3%,幼虫生长发育迟缓,体重增长量与对照相比显著减小40.7%。[结论]成功构建HcChi RNA干扰载体,通过饲喂法在美国白蛾中获得RNAi效应。该体系首次在美国白蛾中建立,为该物种的基因功能研究和生物防治提供新思路。
英文摘要:
      [Objective] To explore the feasibility of dsRNA expressed by HT115-mediated RNAi in Hyphantria cunea.[Method] Hyphantria cunea chitinase gene (HcChi) was chosen as the target gene. An interference fragment was designed and inserted into the expression vector L4440, and then transformed into the Escherichia coli strain HT115. The H. cunea larvae were fed on bacteria solution which was induced by IPTG. The growth of larvae were observed, and the mRNA level of HcChi was detected using qPCR.[Result] The HT115 strain containing HcChi-L4440 expression vector can express HcChi-dsRNA by IPTG induction. After fed on bacteria solution, the expression level of HcChi in H. cunea decreased significantly by 76.7%-90.3%. The body weight growth decreased about 40.7% compared with the control.[Conclusion] The RNA interference vector was constructed successfully. The effect of RNAi was observed in H. cunea by feeding method. This is the first time that developing a method of RNAi based on bacteria expressed dsRNA in H. cunea, which provides a new idea on the study of gene function and biological control of H. cunea.
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