利用RNA-Seq技术鉴定拟南芥不定芽再生相关的转录因子

Identifying transcription factors involved in Arabidopsis adventious shoot regeneration by RNA-Seq technology

摘要:

转录调控是不定芽再生过程的主要调控方式之一，但具体机制尚需进一步研究。为此，利用基于Illumina HiSeq? 2000测序平台的RNA-Seq技术分析了不定芽再生缺陷突变体be1-3和野生型WS在愈伤形成和不定芽再生过程以及野生型WS从脱分化向再分化转变过程差异表达的转录因子 (Transcription factor，TF) 编码基因。结果表明：与野生型WS相比，be1-3在脱分化过程差异表达的TF编码基因有155个，其中表达量上调的97个，表达量下调的58个；在再分化过程差异表达的TF编码基因有68个，其中表达量上调的40个，表达量下调的28个；而在野生型WS从脱分化向再分化转变的过程，总共检测到231个差异表达的TF编码基因，包括160个表达量上调的基因和71个表达量下调的基因。其中，MYB-related (v-myb禽成髓细胞瘤病毒癌基因) 家族的不定芽相关转录因子基因ART1在be1-3突变体脱分化阶段的表达量提高了3 217倍，是表达量上调最大的TF编码基因。进一步研究发现，该基因过量表达导致愈伤形成和不定芽再生缺陷，并抑制了幼苗特别是主根的生长发育，表明该基因是愈伤形成和不定芽再生过程的一个负调控因子。本研究不仅加深了人们对不定芽再生转录调控机制的认识，而且为今后不定芽再生相关转录因子的研究提供了大量候选基因信息。

Abstract:

Transcriptional regulation is one of the major regulations in plant adventious shoot regeneration, but the exact mechanism remains unclear. In our study, the RNA-seq technology based on the Illumina HiSeq? 2000 sequencing platform was used to identify differentially expressed transcription factor (TF) encoding genes during callus formation stage and adventious shoot regeneration stage between wild type and adventious shoot formation defective mutant be1-3 and during the transition from dedifferentiation to redifferentiation stage in wildtype WS. Results show that 155 TFs were differentially expressed between be1-3 mutant and wild type during callus formation, of which 97 genes were up-regulated, and 58 genes were down-regulated; and that 68 genes were differentially expressed during redifferentiation stage, with 40 genes up-regulated and 28 genes down-regulated; whereas at the transition stage from dedifferentiation to redifferention in WS wild type explants, a total of 231 differentially expressed TF genes were identified, including 160 up-regualted genes and 71 down-regulated genes. Among these TF genes, the adventious shoot related transcription factor 1 (ART1) gene encoding a MYB-related (v-myb avian myeloblastosis viral oncogene homolog) TF, was up-regulated 3 217 folds, and was the highest up-regulated gene during be1-3 callus formation. Over expression of the ART1 gene caused defects in callus formation and shoot regeneration and inhibited seedling growth, indicating that the ART1 gene is a negative regulator of callus formation and shoot regeneration. This work not only enriches our knowledge about the transcriptional regulation mechanism of adventious shoot regeneration, but also provides valuable information on candidate TF genes associated with adventious shoot regeneration for future research.