意大利蜜蜂工蜂中肠piRNA的鉴定与分析

【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|log₂ 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1 195, 1 018, 4 040和1 063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差异表达趋势的真实性。【结论】本研究从意大利蜜蜂工蜂中肠中鉴定到596个piRN，长度介于24~33 nt。不同长度分布范围的piRNA的首位碱基偏向性具有明显差异；piR-ame-11093, piR-ame-1111451,piR-ame-190949和piR-ame-932156具有靶向调控基因表达参与工蜂中肠发育过程的潜力。

Identification and analysis of piRNAs in the midgut ofApismelliferaligusticaworkers

【Aim】 PIWI-interacting RNAs (piRNAs) play a key regulatory role in crucialbiological processes such as development and immunity in insects. The objective of thisstudy is to enrich the information of Apis mellifera piRNAs and to provide a basis forfurther 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) omicsdata 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 smallnon-coding RNAs (ncRNAs) such as rRNAs and tRNAs, and the identification of piRNAsaccording to their length characteristics followed. The expression levels of piRNAs werecalculated and normalized using tags per million (TPM) arithmetic. DEpiRNAs in the Am7 vsAm10 comparison group were screened out according to the standard of |log₂ fold change|≥1and P≤0.05. Target mRNAs of DEpiRNAs were predicated with related software, and GO andKEGG annotation of DEpiRNAs was performed. The DEpiRNA-mRNA regulatory networks werevisualized 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 sixrandomly selected DEpiRNAs were validated by RT-qPCR. 【Results】 In total, we identified596 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 thefirst base of piRNAs in different lengths within Am7 and Am10 groups. Additionally, weidentified 41 DEpiRNAs in the Am7 vs Am10 comparison group, in which piR-ame-11093, piR-ame-111451,piR-ame-190949 and piR-ame-932156 could target 1 195, 1 018, 4 040 and 1063 mRNAs, respectively. These target mRNAs could be respectively annotated to 45functional terms and 45 pathways. Stem-loop RT-PCR results verified the true expressionof six piRNAs (piR-ame-1084826, piR-ame-11093, piR-ame-14476, piR-ame-24995,piR-ame-39500 and piR-ame-774987). RT-qPCR results exhibited that the expressiontrends of six DEpiRNAs (piR-ame-1084826, piR-ame-11093, piR-ame-14476,piR-ame-24995, piR-ame-39500 and piR-ame-774987) were in accordance with those in thesequencing data, confirming the reliability of sRNA-seq data and the authenticity ofdifferential expression trends of piRNAs in this study. 【Conclusion】 In this study, atotal of 596 piRNAs have been identified from the midgut of A. m. ligustica workers, with alength distribution of 24-33 nt. The bias of the first base of piRNAs with various lengthdistribution ranges has apparent differences, and piR-ame-11093, piR-ame-1111451, piR-ame-190949 and piR-ame-932156 are potentially involved in the developmental process ofthe midgut of A. m. ligustica workers via regulation of target gene expression.