基于RNA-Seq的拟南芥不定芽再生过程的基因表达谱分析

Analysis of RNA-Seq-based expression profiles during adventitious shoot regeneration in Arabidopsis thaliana

[目的]本研究旨在分析拟南芥愈伤组织形成和不定芽再生2个阶段基因的差异表达情况,探究不定芽再生背后的相关基因调控机制。[方法]采用拟南芥不定芽再生两步培养法培养根外植体,利用RNA-Seq高通量测序技术分别对愈伤组织诱导培养基(CIM)培养0 d根外植体(C0)、CIM培养4 d根外植体(C4)、芽诱导培养基(SIM)培养3 d根外植体(S3)进行基因测序,利用软件分析基因表达情况。[结果]将C4的基因表达量和C0进行比对,共检测到4 332个差异表达基因,其中1 399个基因表达上调,2 933个基因表达下调;在不定芽再生阶段(S3与C4的基因表达量进行比对)检测到2 910个差异表达的基因,其中2 231个基因表达上调,679个基因表达下调。对差异表达基因进行转录物功能注释分析发现:明显富集的功能分类多与激素合成、代谢等生物过程有关。在愈伤组织形成阶段一些与细胞分裂素分解代谢相关的CKX基因和与生长素活性相关的GH3家族基因表达量明显增加,而与细胞分裂素合成相关的多个基因表达量却明显降低。在不定芽再生阶段与细胞分裂素合成相关基因表达量明显增加,CKX家族部分成员表达量则明显降低。同时,还发现多个转录因子基因表达量在不同阶段发生显著变化,比如AP2/EREBP家族的部分成员。[结论]对拟南芥不定芽再生过程基因表达谱进行检测和对比分析,获得了大量基因表达谱分析数据,为揭示不定芽再生过程的基因调控机制提供了有效的数据和理论依据。

[Objectives]To study the underlying regulation mechanisms of adventitious shoot regeneration,we analyzed the dynamic transcriptome profiles of two step procedure during Arabidopsis thaliana adventitious shoot regeneration.[Methods]In this study,adventitious shoots were regenerated from root explants through the two-step process which means preincubation on an auxin-rich callus induction medium（CIM） before transferred to a cytokinin-rich shoot induction medium（SIM）.We obtained the transcriptome data from three developmental time-points（0,4 d on CIM and 3 d on SIM in vitro culture）.[Results]To identify the differentially expressed genes（DEGs） during callus formation,we compared the expression profiles of 4 d CIM to 0 d CIM and detected 4 332 DEGs in total,among which 1 399 genes were upregulated and 2 933 were downregulated.Moreover,we identified DEGs during adventitious shoot regeneration by comparing expression profiles of 3 d SIM to 4 d CIM.Finally,we identified a total of 2 910 DEGs including 2 231 up-regulated and 679 down-regulated genes.We performed the Gene Ontology（GO） enrichment analysis of the DEGs and our results suggested that those genes with highly enriched GO terms were associated with phytohormone biosynthesis and degradation.During callus formation,some genes involved in cytokinin oxidase CKX and indole-3-acetic acid amido synthetase GH3 gene family were highly expressed,while some members of cytokinin synthetase were downregulated.During adventitious shoot regeneration,some genes related to cytokinin synthetase were upregulated,and member genes of CKX family were downregulated.Additionally,we investigated the expression changes of some transcription factor during the two different stages,including AP2/EREBP family members.[Conclusions]Here,we compared and analyzed the transcriptome profiles of two stages during adventitious shoot regeneration of Arabidopsis by RNA sequencing technology.The transcriptome data will provide a valuable reference for future research on regulatory mechanisms underlying plant adventitious shoot regeneration.