摘要
【目的】miRNA在昆虫的生长、发育和代谢等诸多生物学进程中发挥关键调控作用。本研究对ace-miR-3759-y进行表达和序列验证,并通过过表达与敲降ace-miR-3759-y探究其对中华蜜蜂Apis cerana cerana幼虫靶基因表达及重量的影响,以期为ace-miR-3759-y调控幼虫发育的分子机理研究提供依据。【方法】通过Stem-loop RT-PCR和Sanger测序验证ace-miR-3759-y的表达和序列。通过饲喂模拟物(Mimic)和抑制物(Inhibitor)对中华蜜蜂幼虫血淋巴中的ace-miR-3759-y进行过表达和敲降。利用RT-qPCR检测ace-miR-3759-y的过表达和敲降效果及靶基因相对表达量。利用电子天平对幼虫进行称重。【结果】ace-miR-3759-y在中华蜜蜂幼虫肠道和血淋巴中真实存在和表达。相较于Mimic-NC组,ace-miR-3759-y在Mimic-ace-miR-3759-y组4日龄和5日龄幼虫血淋巴中显著上调(P<0.05),在6日龄幼虫血淋巴中上调表达但组间差异不显著(P>0.05)。相较于Inhibitor-NC组,Inhibitor-ace-miR-3759-y组4、5和6日龄幼虫血淋巴中均显著下调表达(P<0.05)。过表达ace-miR-3759-y后,CIC在4、5和6日龄幼虫血淋巴中的表达量均显著下调(P<0.05);CKI在4日龄和5日龄幼虫血淋巴中的表达量显著下调(P<0.05),在6日龄幼虫血淋巴中下调表达但组间差异不显著(P>0.05)。敲降ace-miR-3759-y后,CIC在5日龄幼虫血淋巴中显著上调表达(P<0.05),在4日龄和6日龄幼虫血淋巴中上调表达但组间差异不显著(P>0.05);CKI在4日龄幼虫血淋巴中显著上调表达(P<0.05),在5日龄和6日龄幼虫血淋巴中上调表达但组间差异不显著(P>0.05)。此外,与Mimic-NC组相比,Mimic-miR-3759-y组的6日龄幼虫的体重下降,但组间差异不显著(P>0.05);与Inhibitor-NC组相比,Inhibitor-miR-3759-y组的6日龄幼虫体重上升,但组间差异不显著(P>0.05)。【结论】通过饲喂Mimic和Inhibitor的方法在中华蜜蜂幼虫血淋巴中实现了ace-miR-3759-y的有效过表达及敲降,ace-miR-3759-y负调控CIC和CKI的表达,ace-miR-3759-y对幼虫体重无显著影响。
[Objectives]To determine the role of ace-miR-3759-y in target gene expression and body weight of Apis cerana cerana larvae in order to provide a foundation for further study of the ace-miR-3759-y-regulated molecular mechanism underlying larval development.[Methods]The expression and sequence of ace-miR-3759-y were confirmed by Stem-loop RT-PCR and Sanger sequencing.A.cerana cerana larvae were fed Mimic and Inhibitor to overexpress and knockdown ace-miR-3759-y in larval hemolymph.RT-qPCR was then used to determine the effect of ace-miR-3759-y overexpression and knockdown on the target genes CIC and CKI,and on larval weights which were measured using electronic scales.[Results]ace-miR-3759-y was both present and expressed in the hemolymph of A.cerana cerana larvae.Treatment with Mimic significantly up-regulated its expression in 4-and 5-day-old larvae relative to the control group.It was also upregulated in 6-day-old larvae,but this was not significant.Conversely,treatment with Inhibitor significantly down-regulated ace-miR-3759-y expression in 4-,5-and 6-day-old larvae relative to the control.After overexpression of ace-miR-3759-y,the expression of CIC in 4-,5-and 6-day-old larvae was significantly down-regulated,whereas that of CKI was significantly down-regulated in 4-and 5-day-old larvae.It was also down-regulated in 6-day-old larvae,but not significantly so.Knockdown of ace-miR-3759-y significantly up-regulated the expression of CIC in 5-day-old larvae.Expression was also up-regulated in 4-and 6-day-old larvae but this was not significant.Knockdown of ace-miR-3759-y also up-regulated CKI in 4-,5-and 6-day-old day-old larvae,although the degree of upregulation in the latter two age classes was not significant.Moreover,treatment with Mimic caused a non-significant decrease in the body weight of 6-day-old larvae relative to the Mimic control group and treatment with Inhibitor caused a non-significant increase in the body weight of 6-day-old larvae relative to the Inhibitor control group.[Conclusion]Effective overexpression and knockdown of ace-miR-3759-y in A.cerana cerana larvae can be achieved by feeding mimic and inhibitor.ace-miR-3759-y negatively regulates expression of CIC and CKI but has insignificant effects on body weight.
作者
吴鹰
叶亚萍
钱加珺
张佳欣
顾小雨
牛庆生
陈大福
郭睿
WU Ying;YE Ya-Ping;QIAN Jia-Jun;ZHANG Jia-Xin;GU Xiao-Yu;NIU Qing-Sheng;CHEN Da-Fu;GUO Rui(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Apitherapy Research Institute,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Jilin Province Institute of Apicultural Science,Jilin 132000,China)
出处
《应用昆虫学报》
CAS
CSCD
北大核心
2023年第1期87-95,共9页
Chinese Journal of Applied Entomology
基金
国家自然科学基金(202210389136,202210389121)
财政部和农业农村部:国家现代农业产业技术体系(CARS-44-KXJ7)
福建农林大学硕士生导师团队项目(郭睿)
福建农林大学动物科学学院(蜂学学院)科研扶持项目(郭睿)
福建省大学生创新创业训练计划(202210389136,202210389121)
作者简介
共同第一作者:吴鹰,E-mail:wy569703@163.com;共同第一作者:叶亚萍,15259907927@163.com;共同通讯作者:郭睿,E-mail:ruiguo@fafu.edu.cn;共同通讯作者:陈大福,dfchen826@fafu.edu.cn
