红、蓝光对铁皮石斛栽培品质的影响及其分子机制初探

Light is one of the important ecological factors that affect the growth of Dendrobium officinale under facilities cultivation condition. It has shown that red and blue light quality can significantly affect the growth and development, and the accumulation of polysaccharides and alkaloid of Dendrobium officinale. In this study annual seedlings and triennial plants of Dendrobium officinale were exposed on red and blue LED light source to study the effect of different illumination and processing time on the content and component of polysaccharides and alkaloid of stem and leaf. And then, the samples of stem and leaf in the specific period of light treatment with the significant change of the contents of alkaloid and polysaccharides were chosen for transcriptome sequencing and analysis of differentially expressed genes. Finally screening the metabolism related genes as transcription factors, functional gene and the miRNA which influenced by red and blue light treatment were selected. Light response related genes and pathways were speculated with the expression spectrum analysis, the expression level of candidate gene were validated by RT-PCR. Using the existing database of miRNA, The target genes of miRNA which was related to candidate genes of transcription factors and miRNA which could regulate gene expression were selected through bioinformatics analysis.The interactions of them were validated by the yeast one-hybrid and degradome sequencing of miRNA. The molecular regulatory network of the influence of light quality on the metabolism of polysaccharides and alkaloid of Dendrobium officinale were constructed preliminarily. The research results will provide important basis for quality control and innovative breeding of Dendrobium officinale under artificial cultivation conditions.

光照是影响设施栽培铁皮石斛生长的重要生态因子之一。红、蓝光质可以显著影响铁皮石斛的生长发育及多糖和石斛碱的累积。本研究应用红色或蓝色LED对铁皮石斛一年生幼苗和三年生植株进行处理，研究光质及处理时间对茎、叶中多糖和石斛碱的组成、含量的影响规律，进而选择多糖和石斛碱含量显著变化的时期进行茎和叶的转录组测序和基因差异表达分析，以筛选出红光或蓝光处理下影响多糖、石斛碱合成代谢相关的转录因子、功能基因、miRNA。通过表达谱分析，推测与光质相关的基因和通路，并通过RT-PCR对候选基因进行表达水平的验证。通过生物信息学分析，利用已有的miRNA数据库，筛选与候选基因中的转录因子相关的靶基因及调控其表达相关miRNA，分别通过酵母单杂试验和miRNA降解组测序，验证其互作关系，初步构建光质影响铁皮石斛多糖和石斛碱代谢的分子调控网络。研究结果将为人工栽培条件下铁皮石斛的品质控制和创新育种提供重要基础。

红光和蓝光作为绿色植物叶片吸收的主要光谱，成为近年来研究光质对植物生长影响的最常用光源，越来越多研究表明红、蓝光源显著影响药用植物次生物质代谢和药效组分。铁皮石斛药材的人工设施栽培面积和需求量大增不断扩大，而中药材最重要的品质提升及其机理需要探究，基于此，本研究应用红色或蓝色LED对铁皮石斛植株进行处理，通过利用UPLC-TOF-MS/MS技术和DNA二代测序技术等方法，本项目针对红光、蓝光对铁皮石斛生长和活性物质代谢、累积的影响，借助代谢组学、转录组学、小RAN组学、降解组学，以及mRNA-miRNA和mRNA-代谢组等联合分析技术，初步获得了红蓝光对铁皮石斛活性物质代谢的数字表达谱、影响活性物质含量的基因的数字表达谱，影响活性物质含量的miRNA数字表达谱，获得miRNA和靶基因的作用和剪切位点。初步构建了铁皮石斛miRNA-基因的调控网络。综上所述，该项目在栽培学、生物化学、多组学、生物信息学等技术的综合运用，研究成果具有较强的科学意义，初步筛选了部分关键基因，将用于后续种质创新，在相关领域具有实际应用价值。