NAC特异基因调控铁皮石斛原球茎原分生组织形成的分子机理研究

Protocorm, derived from the dormant globular embryo in the matured seeds, is a specific structure during the process of seed germination in orchids. It usually can be devided into four stages including embryo activation, protocorm formation, protocorm maturation and protocorm degeneration. The promeristem formation in the early stage of protocorm maturation is the key process during the protocorm development and is an essential event for the SAM (shoot apical meristem) formation. And the process of SAM formation of a protocorm is different from that of embryo development in other monocotyledonous and dicotyledonous plants. However, the mechanism underlying it remains unknown. It will be a model to investigate the molecular mechanism of the specific establishment of SAM in orchids. Some NAC genes that are specifically expressed in the protocorms of Dendrobium candidum Wall ex Lindl were identified with expression pattern-based screening in our previous studies, in which a NAC gene, DcNACA3, is specifically expressed in the promeristem of protocorm. In the present work, the potential role of NAC genes in protocorm development were to be investigated through the transcriptome analysis of the protocorms among the stages of protocorm formation, promeristem formation and SAM formation, especially in the process of promeristem formation, and molecular mechanism underlying it will be elucidated.

兰科植物原球茎是成熟种子中未进行辐射对称向两侧对称转换、并具部分成熟胚特质的球形休眠原胚重启细胞分裂后形成的，历经种胚活化及原球茎的形成、成熟与退化等时期，而发生在成熟期早期的原分生组织(PM)形成是原球茎发育进程的关键及顶端分生组织(SAM)形成的必要事件，这与水稻、拟南芥等植物的SAM建立不同，是一个难得的研究模型来揭示兰科植物SAM建立的独特调控机制,而迄今关于其分子调控机制仍属未知。前期在铁皮石斛原球茎中发现一些差异表达的NAC转录因子家族成员，其中DcNAC3在PM形成期特异表达。本项目拟通过对原球茎形成期、PM形成期及SAM形成期的铁皮石斛原球茎转录谱进行分析,并在此基础上选择在PM特异表达的DcNAC3等特异基因进行功能与调控网络研究，探讨兰科植物PM形成以建立SAM的独特分子调控机制,这对认识兰科植物个体发生的分子机理有重要意义，对其种质繁育与保护也有重要作用。

兰科植物胚胎在发育早期模式建成后未进行辐射对称向两侧对称转换，种子成熟时胚胎仍处于未分化的球形期，萌发时按原球茎(protocorm)发育程序建立基本形体构图(body plan)，与水稻、拟南芥等植物胚胎发育过程中的形态发生有显著区别，因而对原球茎发育进程的研究对理解植物形态发生的分子机理有重要意义。铁皮石斛种子原球茎的发育进程经历了丰富的形态变化，可分为“突破种皮（倒三角形或锥形）、球形、顶端发生、椭球形、哑铃形、生根“等6个阶段，而在球形期发生的原分生组织的形成是整个原球茎形态发生过程中的关键事件；然后，原球茎底端逐渐退化、至最后完全退化，并成为具幼叶及一条根的幼苗。经转录组测序分析发现，在铁皮石斛原球茎发育过程中获得34,085条非冗余全长转录本及28,159个可变剪接转录本。基于原球茎时期转录组数据和文献报道，筛选了26个表达最稳定的基因和5个常用的管家基因作为测试基因，选择出ASS+APH1L组合作为在原球茎各时期基因表达谱分析的内参基因；并以此对34个NAC基因在原球茎各时期的表达进行定量分析，发现DcNAC4特异优势表达于原球茎退化时期和PLB，DcNAC5特异优势表达于种胚活化时期，DcNAC6特异优势表达于原球茎退化时期和根，DcNAC14特异优势表达于PLB， DcNAC17特异表达于叶原基和维管系统形成时期，DcNAC23特异优势表达于原球茎形成时期，DcNAC28特异优势表达于花，DcNAC29、DcNAC40特异优势表达于原球茎形成时期花和PLB。经亚细胞定位及转录活性分析，发现DcNAC1、DcNAC13和DcNAC44三条基因参与铁皮石斛种子从萌发到顶端分生组织SAM形成期过程中的发育进程。以上研究结果对阐明铁皮石斛原球茎发育进程的分子机理有重要意义。