荔枝早期体细胞胚发生相关microRNA的鉴定与分析

Identification and Analysis of microRNA Associated with Early Somatic Embryo Formation in Litchi

荔枝(Litchi chinensis Sonn.)种胚发育状态直接影响种子和果实大小及其品质,但迄今为止对其发育分子机理尚缺乏系统研究与了解。本研究利用‘御金球’荔枝幼叶诱导的胚性愈伤组织(EC)和早期体胚(SE)进行小RNA高通量测序。利用生物信息学方法鉴定体胚发生相关microRNA(miRNA),预测其靶基因,筛选潜在的miRNA-靶基因互作对;并利用实时荧光定量PCR(RT-qPCR)方法对差异miRNA与miRNA-靶基因的关系进行验证。在EC和SE小RNA文库中共鉴定得到属于564个家族的1127个已知miRNAs。与EC相比,有88个miRNAs在SE中差异表达,包括51个上调和37个下调miRNAs。其中80个miRNAs预测得到581个靶基因,多为转录因子和参与初级、次生代谢途径的酶基因。根据基因表达模式共筛选得到167个具有负调控关系的miRNA-靶基因对,涉及胚胎发育、激素信号、氧化还原和细胞壁建成等生物学过程。RT-qPCR结果表明12个miRNAs的表达在胚性愈伤和体胚发生阶段变化显著,且4个miRNA-靶基因对具有负调控表达模式,这些结果与小RNA测序和前期转录组结果吻合。荔枝早期体胚发生相关miRNA的鉴定和分析以及miRNA-靶基因调控网络图的绘制为后续深入研究体胚发育分子机制提供理论依据。

The developmental condition of lychee seed embryos has a direct impact on the size and quality of seeds and fruits,yet there is still a dearth of comprehensive research and understanding of their molecular development mechanisms.In this study,small RNA high-throughput sequen cing was pe rformed on embryogenic calli(EC)and early somatic embryos(SE)produced by young leaves of'Yujinqiu'litchi.Using bioinformatics methods,the researchers were able to identify microRNAs(miRNAs)associated with somatic embryogenesis,predict their target genes,and screen for potential miRNA target gene interactions;and the relationship between differential miRNA and miRNA target genes was validated using real-time fluorescence quantitative PCR.In the EC and SE small RNA libraries,1127 known miRNAs from 564 families were discovered.In SE,88 miRNAs were differentially expressed as compared to EC,with 51 upregulated and 37 downregulated miRNAs.Eighty miRNAs were predicted to produce 581 target genes,the majority of which are transcription factors and enzyme genes involved in primary and secondary metabolic pathways.Based on gene expression patterns,167 miRNA target gene pairings with negative regulatory interactions were identified,involving biological processes such as embryonic development,hormone signaling,redox,and cell wall construction.The expression of 12 miRNAs altered considerably during embryogenic callus and somatic embryogenesis,as validated by RT-qPCR,and four miRNA target gene pairs displayed negative regulatory expression patterns.These findings agreed with those of small RNA sequencing and early transcriptome analysis.The discovery and study of miRNAs associated with early somatic embryogenesis in litchi,as well as the mapping of miRNA target gene regulatory networks,provide a theoretical foundation for future research into the molecular mechanisms of somatic embryogenesis.