地黄中梓醇积累及生物合成关键基因的筛选与验证

Catalpol is the main active ingredient of Rehmannia glutinosa and has been demonstrated having hypoglycemic and neuroprotective effects. Preclinical studies on the effect of catalpol on hypoglycemic activity have been performed. Prestudies reveal that catalpol content are variable among different tissues at different stages. A better understanding the catalpol biosynthesis and accumulation mechanism in R. glutinosa and finding the key genes that regulate catalpol production will help us to enhance catalpol production using biotechnological approaches. In order to achieve these aims, firstly, we aim to investigate the temporal and spatial gene expression patterns using RNA-seq and analyze the catalpol contents in different tissues at different stages; secondly, we intend to get a set of candidate genes that potentially regulate catalpol biosynthesis by establishing the correlation between the gene expression level and the catalpol contents; finally we will further analyze these genes by detailed expression analysis to identify key genes that play significant regulation roles in catalpol biosynthesis and we will select a few key genes to investigate their in vivo functions by transplant method. The studies proposed in this project will definitely establish a foundation for understanding the catalpol biosynthesis, and it also provide a model for studying the key genes that are involved in plant's second metabolites biosynthesis.

梓醇为地黄的主要有效成分，具有降血糖及神经保护作用。以梓醇为降糖新药的研发已完成临床前研究。地黄的不同组织器官梓醇含量存在差异。因此，了解梓醇在地黄生长发育期的积累，研究与筛选梓醇合成关键基因，对合理利用地黄资源、提高梓醇含量具有现实意义。本课题以地黄不同生长发育期的根、茎、叶、花等组织器官为材料，利用高通量测序技术进行数字表达谱分析（RNA-Seq），对每个基因的表达量与同样品的梓醇含量进行相关性分析，具有显著性相关的基因为梓醇合成的关键基因，并利用实时荧光定量表达分析方法及转基因技术对筛选的关键基因进行验证，为地黄梓醇生物合成的调控打下了良好的工作基础。本课题同时对上万条基因的表达与梓醇合成的关系进行研究，为植物次生代谢产物合成关键基因的发掘提供了新思路和新方法。

1、随着叶龄的增加地黄叶片、叶柄中总环烯醚萜苷和梓醇含量逐渐降低，以幼嫩叶片、幼嫩叶柄中含量最高；块根中总环烯醚萜苷和梓醇含量与块根直径密切相关；9月份茎中总环烯醚萜苷和梓醇含量较8月份提高2-3倍。.2、对地黄叶片、叶柄、茎和块根共16个组织萜类生物合成41个酶基因进行表达量分析表明： DXR3表达量与梓醇、总环烯醚萜苷含量显著相关；地黄叶片中GPPS.LSU2和GPPS.SSU的相对表达量与梓醇和总环烯醚萜苷含量显著相关。.3、不同叶龄叶片基因数字表达谱GO分类分析结果：在负相关的基因中与梓醇合成密切相关的基因303个，包括形成碳氧键连接酶基因46个，碳碳裂解酶活性基因46个等；在正相关的基因中与梓醇合成密切相关的基因130个，包括作用于CH-OH基团供体氧化还原酶活性基因66个，作用于CH-CH基团供体氧化还原酶活性基因37个等。.4、利用建立的遗传转化体系，构建了萜类合成关键酶基因GPPS.SSU的遗传转化体系，得到转基因的苗33株。实验组和野生组相比，生长至50天后地黄北京1号的L1~L4叶片中梓醇的含量有所增加，L3变化最显著，L5~L8叶片中梓醇的含量有所减少，L7变化最显著；生长至70天后实验组和野生组相比，北京1号的L6~L8叶片中梓醇的含量有所增加，L7变化最显著，其余叶片中梓醇的含量变化不明显。