摘要
【目的】对拟黑多刺蚁(Polyrhachis vicina Roger)成虫热激蛋白90(Heat shock protein 90,HSP90)基因进行RNA干扰(RNAi),为分析hsp90基因的功能及将RNAi技术用于害虫防治提供参考。【方法】采用Trizol法提取拟黑多刺蚁总RNA,反转录合成cDNA第一链用于hsp90基因的克隆。利用T7RiboMAXTM Express RNAi System将hsp90合成双链RNA(double-stranded RNA,dsRNA),将其配制成120,60,30,15和7.5ng/μL的溶液饲喂拟黑多刺蚁雌蚁、雄蚁和工蚁成虫进行RNAi,共饲喂14d,记录成虫死亡率,确定dsRNA最适质量浓度,并以此剂量对各品级成虫进行干扰,通过荧光实时定量PCR,检测干扰效果及干扰hsp90基因对EcR、USP mRNA表达的影响。【结果】成功获得拟黑多刺蚁hsp90基因片段,其长度523bp,并合成了dsRNA。饲喂dsRNA后发现,dsRNA质量浓度越大,拟黑多刺蚁死亡率越高,在dsRNA质量浓度为120ng/μL时,饲喂5d后成虫全部死亡;30ng/μL是dsRNA干扰的最适质量浓度。以30ng/μL dsRNA干扰后,荧光实时定量PCR结果表明,拟黑多刺蚁每个品级成虫hsp90基因mRNA均被沉默。hsp90基因被干扰后,EcR的表达没有显著变化,USP的表达显著降低。【结论】采用饲喂法成功干扰了拟黑多刺蚁hsp90基因mRNA的表达;hsp90基因与USP基因表达有一定的相关性。
【Objective】This paper involve the function of hsp90 gene in Polyrhachis vicina Roger by using RNA interference technology,and provide the reference for the prevention and control of pests.【Method】The total RNA was extracted by Trizol,reverse transcription synthetic cDNA first chain used for hsp90 gene cloning,double strand RNA(dsRNA)of hsp90 gene was synthesized by T7 RiboMAXTM Express RNAi System and the ants including female,male and wokers were fed by dsRNA at 120,60,30,15 and 7.5ng/μL in 14 days.The adult mortalities were recorded,the most suitable dsRNA concentration was confirmed,and all grade of adults was taken by this dose interferece.The interference effect and interference hsp90 gene effecting EcR as well as USP mRNA expression was detected by fluorescent real-time quantitative RT-PCR.【Result】P.vicina hsp90 dsRNA was gotten by the synthesis according to hsp90 gene of fragment length 523 bp dsRNA was fed to P.vicinaants,the mortality increased along with the in-creased dsRNA concentration,P.vicinaants were all died in 120ng/μL in 5days.30ng/μL maybe a appropriate choice.Under condition of 30ng/μL,total RNA was extracted,the analysis of Florescent realtime quantitative RT-PCR indicates hsp90 mRNA of P.vicinaants were suppressed in all grades.There are no significant change in the expression of EcR mRNA of P.vicinaand an obovious decrease of USP mRNA of P.vicinaafter interfering hsp90.【Conclusion】The interference of hsp90 has been achived successfully as firstly,and preliminary analyze indicates that a correlation may be exited between hsp90 and USP.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2015年第4期191-196,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金面上项目(31171195)
