文心兰侧花瓣转录组分析与调控唇瓣化变异相关转录因子的挖掘

Transcriptome Analysis of Lateral Petals of Oncidium and Mining of Transcription Factors Related to The Regulation of Labialization Variation

文心兰因其独特的花型和艳丽的颜色是市场上重要的鲜切花。文心兰花瓣极易产生花型多样性,为了探究调控文心兰侧花瓣唇瓣化的分子机制,对柠檬绿文心兰(Oncidium flexuosum ‘Honey Angel’)及其侧花瓣唇瓣化变异种(突变体)的侧花瓣进行RNA提取和转录组分析。测序结果共获得45 525 415个高质量reads,鉴定出52 253个unigenes。利用NR、Swissprot、PFAM、GO、NT、KO、KOG七大数据库分别进行基因功能注释,分别注释到31 828、23 174、21 369、21 367、20 830、10 523、6465条unigenes。经表达量比较,在柠檬绿文心兰和突变体之间获得差异表达基因3120条。GO功能富集分析显示,共有1537条差异表达基因注释到生物过程、细胞组分和分子功能3个功能群组。KEGG功能富集分析显示,将690个差异表达基因富集到不同的代谢通路,其中代谢途径富集的基因最多。兰花唇瓣器官发育的研究更多地集中在MADS-box基因家族中,在该转录组数据中不仅筛选到5个显著差异表达MADS-box基因家族成员,还挖掘到其他可能与文心兰唇瓣发育相关的NAC、TCP、MYB及WRKY等转录因子。运用RT-qPCR对这些转录因子基因进行表达量验证,定量结果与RNA-seq数据一致,且这些转录因子大部分在文心兰唇瓣化侧花瓣中具有高表达,说明其可能正向调控着文心兰唇瓣器官的发育。综上所述,本研究挖掘到大量参与文心兰唇瓣化变异的关键转录因子,为兰花唇瓣发育分子机制及调控网络研究提供参考依据。

Oncidium is an important fresh-cut flower in the market because of its unique flower pattern and brilliant color.The petals of Oncidium are highly susceptible to flower diversity.RNA extraction and transcriptome analysis were performed on the lateral petals of O.flexuosum‘Honey Angel’and variants with two lateral petals converted into lips to explore the molecular mechanism regulating the labialization of Oncidium lateral petals.A total of 45525415 high-quality reads were obtained from the sequencing results and 52253 unigenes were identified.The seven databases of NR,Swissprot,PFAM,GO,NT,KO and KOG were used to annotate gene functions and annotated to 31828,23174,21369,21367,20830,10523,6465 unigenes.Upon expression comparison,3120 differential genes were obtained between O.flexuosum‘Honey Angel’and the mutant.A total of 1537 differentially expressed genes were annotated into three functional groups:biological processes,cellular components and molecular functions.KEGG functional enrich-ment analysis enriched 690 differentially expressed genes into different metabolic pathways,with the most genes en-riched by metabolic pathway.To date,the study of lip formation in orchids has focused more on the MADS-box gene family,not only five significantly differentially expressed MADS-box genes were screened in this transcriptome data,but also other transcription factor such as NAC,TCP,MYB and WRKY that may be related to the lip formation in On-cidium were also discovered.RT-qPCR was used to verify the expression of key genes,and the quantitative results were consistent with RNA-seq data,most of the genes were highly expressed in the lip-like lateral petals,indicating that the genes may positively regulate the development of the lip of Oncidium.In summary,this study excavated a large number of key transcription factors involved in the variation of labialization in Oncidium,which would provide a certain refer-ence for the study of the molecular mechanism and regulatory network of lip formation of orchid.