文献类型：期刊文献

中文题名：中华紫胶虫FAD基因RNA干扰载体构建与功能初步分析

英文题名：Construction of RNA Interference Vector and Function Analysis of FAD Gene in Kerria chinensis

作者：王伟伟[1] 凌晓霏[1] 陆沁[1] 柳鹏飞[1] 张金稳[1] 陈航[1,2]

第一作者：王伟伟

机构：[1]中国林业科学研究院资源昆虫研究所,云南昆明650224;[2]国家林业与草原局资源昆虫培育与利用重点实验室,云南昆明650224

年份：2019

卷号：32

期号：6

起止页码：14-20

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD2019_2020】；

基金：国家自然科学基金面上项目(31772542);国家林业与草原局948项目(2014-4-66)

语种：中文

中文关键词：中华紫胶虫;RNA干扰;FAD基因;L4440

外文关键词：Kerria chinensis;RNA interference;FAD gene;L4440

分类号：S759.7

摘要：[目的]引入细菌表达dsRNA干扰系统对中华紫胶虫FAD基因进行RNA干扰,通过检测干扰后FAD基因表达量与个体泌胶量动态变化,对FAD基因功能进行初步分析,为验证紫胶合成相关基因功能提供科学基础。[方法]中华紫胶虫FAD基因和L4440载体经双酶切之后,利用T4连接酶连接,构建FAD-L4440重组质粒,并转入HT115感受态细胞中,IPTG诱导表达dsRNA后,通过虫体涂抹法将dsRNA转染到紫胶虫体内。用RT-qPCR检测干扰后FAD基因的表达量并测定干扰后个体泌胶量的变化。[结果]RNA干扰后FAD基因表达量明显降低,其中,以低浓度菌液处理后12 h和中浓度菌液处理后24 h相对于对照组有显著差异,分别降低了90.90%和86.52%,中浓度在72 h时干扰效率高于其他两个组,个体泌胶量与对照组比较有显著下降。[结论]本研究构建了中华紫胶虫FAD基因的RNA干扰载体,在基因功能验证中引入了细菌表达dsRNA的RNAi系统,使用虫体涂抹法将其导入虫体内引起FAD基因表达量与紫胶虫个体泌胶量显著下降,为RNAi转染过程中不适用注射法、喂食等转染方法的昆虫提供技术参考,为后续解析紫胶合成的分子机理提供技术基础与科学依据。
[Objective]In order to verify the function of FAD gene of Kerria chinensis,the RNAi system by bacterial expression of dsRNA was used and the function of FAD gene was preliminarily analyzed to provide references for verifying the function of genes related to lac synthesis.[Method]The FAD gene of K.chinensis and L4440 vector were digested by double enzymes primarily,then the FAD-L4440 recombinant plasmid was constructed by T4 ligase ligation and transferred into HT115 competent cells as well.After the expression of dsRNA induced by IPTG,the dsRNA was transfected into lac insect by spreading it to the trunks.Detected the FAD gene expression after RNAi by RT-qPCR and the amount of individual lac secreted was also measured timely.[Result]The expression of FAD gene significantly decreased.There was a significant difference between the control group and the low concentration bacterial solution treated for 12 hours and the medium concentration bacterial solution treated for 12 hours,which decreased by 90.79%and 85.46%respectively.The interference efficiency of medium concentration at 72 hours was higher than that of the other two groups and there was a significant difference in the amount of individual lac secretion in comparison with the control group.[Conclusion]In this study,the RNAi vectors of FAD gene of K.chinensis were successfully constructed.In the verification of gene function,the RNAi system was used for bacterial expression of dsRNA.The expression of FAD gene and the amount of individual lac secretion decreased significantly after imported into the insect body.It provides a new technical reference for insects which not suitable for injection and feeding for RNAi transfection and it also provides a technical and scientific basis for the verification of molecular mechanism related to lac biosynthesis.