摘要
【目的】Piwi蛋白互作RNA(PIWI-interacting RNA,piRNA)在昆虫的发育和免疫等重要生物学过程发挥重要的调控作用。本研究旨在丰富西方蜜蜂Apis mellifera的piRNA信息,并为进一步探究差异表达piRNA(differentially expressed piRNA,DEpiRNA)调控意大利蜜蜂Apis mellifera ligustica工蜂中肠发育的分子机理提供基础。【方法】基于已获得的意大利蜜蜂7日龄(Am7)和10日龄(Am10)工蜂中肠的small RNA(sRNA)组学数据,对piRNA进行预测和分析。将质控后的sRNA数据比对西方蜜蜂参考基因组,再将比对上的序列标签(tags)进一步比对数据库以滤除rRNA和tRNA等小非编码RNA(non-coding RNA,ncRNA),进而根据piRNA的长度特征鉴定piRNA。采用TPM(tags per million)算法对piRNA表达量进行计算和归一化处理。根据|log_(2) fold change|≥1且P≤0.05的标准筛选Am7 vs Am10比较组的DEpiRNA。通过相关软件预测DEpiRNA的靶mRNA并进行GO和KEGG数据库注释。利用Cytoscape软件对DEpiRNA-mRNA调控网络进行可视化。通过Stem-loop RT-PCR对随机选取的Am7与Am10两组共有的6个piRNA的表达进行验证。利用RT-qPCR对随机挑选的6个DEpiRNA的表达趋势进行验证。【结果】从意大利蜜蜂工蜂中肠中共鉴定到596个piRNA,长度介于24~33 nt,且Am7和Am10组中不同长度分布范围的piRNA的首位碱基偏向性具有明显差异。在Am7 vs Am10比较组共筛选出41个DEpiRNA,其中piR-ame-11093,piR-ame-1111451,piR-ame-190949和piR-ame-932156可分别靶向1195,1018,4040和1063条mRNA。上述靶mRNA可分别注释到45个功能条目和45条通路。Stem-loop RT-PCR验证结果显示6个piRNA(piR-ame-1084826,piR-ame-11093,piR-ame-14476,piR-ame-24995,piR-ame-39500和piR-ame-774987)均真实表达。RT-qPCR结果显示共有6个DEpiRNA(piR-ame-1084826,piR-ame-11093,piR-ame-14476,piR-ame-24995,piR-ame-39500和piR-ame-774987)的表达趋势与测序数据中的表达趋势一致,证实了本研究中sRNA-seq数据的可靠性和piRNA差异表达趋
【Aim】PIWI-interacting RNAs(piRNAs)play a key regulatory role in crucial biological processes such as development and immunity in insects.The objective of this study is to enrich the information of Apis mellifera piRNAs and to provide a basis for further exploration of the molecular mechanism underlying differentially expressed piRNA(DEpiRNA)regulation of the development of the midgut of A.m.ligustica workers.【Methods】piRNAs were predicated and analyzed based on previously gained small RNA(sRNA)omics data from the midguts of the 7-day-old(Am7)and 10-day-old(Am10)workers of A.m.ligustica.The sRNA data after quality control was mapped to the reference genome of A.mellifera,the mapped tags were then further mapped to database to filter out small non-coding RNAs(ncRNAs)such as rRNAs and tRNAs,and the identification of piRNAs according to their length characteristics followed.The expression levels of piRNAs were calculated and normalized using tags per million(TPM)arithmetic.DEpiRNAs in the Am7 vs Am10 comparison group were screened out according to the standard of|log_(2) fold change|≥1 and P≤0.05.Target mRNAs of DEpiRNAs were predicated with related software,and GO and KEGG annotation of DEpiRNAs was performed.The DEpiRNA-mRNA regulatory networks were visualized with Cytoscape software.The expression of six randomly selected piRNAs shared in Am7 and Am10 was verified by Stem-loop RT-PCR,and the expression trends of six randomly selected DEpiRNAs were validated by RT-qPCR.【Results】In total,we identified 596 piRNAs from the midgut of A.m.ligustica workers.The length distribution of the above-mentioned piRNAs was among 24-33 nt,and there was obvious difference in the bias of the first base of piRNAs in different lengths within Am7 and Am10 groups.Additionally,we identified 41 DEpiRNAs in the Am7 vs Am10 comparison group,in which piR-ame-11093,piR-ame-1111451,piR-ame-190949 and piR-ame-932156 could target 1195,1018,4040 and 1063 mRNAs,respectively.These target mRNAs could be respectively annotated to 45 f
作者
范小雪
隆琦
孙明会
郭意龙
赵浩东
宋岳梅
康育欣
顾小雨
陈大福
郭睿
FAN Xiao-Xue;LONG Qi;SUN Ming-Hui;GUO Yi-Long;ZHAO Hao-Dong;SONG Yue-Mei;KANG Yu-Xin;GU Xiao-Yu;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)
出处
《昆虫学报》
CAS
CSCD
北大核心
2022年第6期684-694,共11页
Acta Entomologica Sinica
基金
国家自然科学基金项目(32172792,31702190)
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)
福建省病原真菌与真菌毒素重点实验室开放课题(郭睿)
福建省大学生创新创业训练计划项目(202010389016,202010389162)
福建农林大学优秀研究生学位论文资助基金(范小雪)。
