菌根真菌诱导金钗石斛生物碱含量提高的分子作用与表达机制

Dendrobium nobile, a basic plant of traditional Chinese medicine of Dendrobium recorded by every version of Chinese Pharmacopoeia, belongs to two national level rare and endanger medicinal Orchidaceae plant. As it is rich in dendrobine, its economical value and market requirement is huge. For that its reproductive capacity low, its survival rate of the tissue culture seedlings after being transplanted low, its content of medical component low, and its artificial cultivation cost high, so it is not easy to plant but need to be symbiotic with mycorrhizal fungi in the plant life history under field condition. Therefore, it is always the first choice to search the mycorrhizal fungus promoting plant growth and enhancing plant quality in the field of mycorrhizal biology. A mycorrhizal fungus MF23 （Mycena sp.）isolated from Orchidaceae plant could promote the growth and alkaloid content of D. nobile, but the molecular effective mechanism about mycorrhiza inteaction is not clear also not reported. For that reason, it is not sure that the inducing-time quantity using the fungus as an inductive way and the plant harvested parts, which seriously limit the fungus to application in agriculture. In view to those reasons, this research on basis of our previous results plans to firstly detect the activity of three key enzymes in the course of alkaloid synthese in D. nobile induced by the mycorrhizal fungus, and then to study the expressive characteristic and function of the three enzymatic genes on the condition of fungal induction in the level of gene transcription and protein expression. This aim is to investigate the molecular expressive mechanism of alkaloid biosynthesis in D. nobile under induction of mycorrhizal fungi. The results will provide scientific proofs about taking advantage of endophytic fungus to promote the production of D. nobile and provide molecular information of mycorrhiza interaction research.

金钗石斛是历代《中国药典》收载的中药石斛基源植物和国家二级珍稀濒危兰科药用植物、富含石斛碱，经济价值和市场需求巨大。由于其繁殖力低、组培苗移栽成活率低、药用成分含量低、人工培养成本高，自然条件下需与真菌共生才能完成其生活史。因此，寻求促其生长和品质提高的菌根真菌是该领域研究和开发的首选。本课题组从兰科植物中分到促进金钗石斛生长和生物碱含量提高的真菌MF23(Mycena sp.)，究其分子作用机制不详并未见报道；导致使用该菌在诱导时间和采收石斛部位等方面把握不足，严重影响该菌的推广应用。鉴如此，本项目拟在前期研究基础上，研究该菌诱导金钗石斛生物碱合成途径中三种关键酶活性变化；并在基因转录水平和蛋白质表达水平上，研究这几个酶基因受真菌诱导的表达特征和表达功能；以期探究真菌诱导金钗石斛生物碱合成的分子作用与表达机制，为利用内生真菌促进金钗石斛生产提供科学依据，为菌根互作研究提供分子生物学资料。

早期研究显示，小菇属真菌Mycena sp.对促进金钗石斛的生长和有效成分含量的提高有较好的效果，但其作用机制不详。为进一步促进内生真菌的应用，提高金钗石斛品质，了解MF23对金钗石斛促生机理和作用机制，本研究从形态、细胞、化学和转录表达4个水平，分别在单纯人工琼脂基质和自然树皮基质的培养条件下，开展了不同培养时期内生真菌MF23（Mycena sp.）诱导金钗石斛的共生促生机制探究。研究结果如下： .在生长形态上，MF23对琼脂培养基上的金钗石斛组培苗茎的生长有显著的促进作用。在共生培养的第9周，相对于对照组，接菌组金钗石斛的茎粗、茎鲜重、茎干重和茎折干率分别增加了57.69%、21.34%、83.41%和50.90%。.在化学水平上，对共生培养1、3、6、9周金钗石斛接菌组和对照组的多糖和石斛碱含量进行检测。结果显示，在共生培养的第9 周，接菌组金钗石斛多糖、石斛碱含量急剧上升，分别为对照的4.04倍和2.99倍。.根部显微观察显示，金钗石斛皮层细胞中出现大量的菌丝的时间与形态促生和化学成分显著变化的时间基本一致，推测MF23菌在金钗石斛的生长和有效成分含量提高上发挥作用。.通过对共生培养1周和9周的金钗石斛茎构建了转录组文库，发现差异基因分别为1388个和2646个，并显著富集到碳水化合物代谢、应激防御等代谢通路。qRT-PCR分析结果显示，金钗石斛多糖含量的增加很可能与MF23激活其光合作用、淀粉和蔗糖代谢、果糖和甘露糖代谢中的基因上调表达相关，而且，细胞色素P450酶、氨基转移酶和甲基转移酶等基因可能参与石斛碱代谢后修饰作用过程。.为更加贴近自然条件，对MF23在树皮基质上培养的金钗石斛诱导与在琼脂基质上进行了比较，结果基本相似，只是在作用时间节点较为迟延，后劲促生作用更为明显，作用基因也基本雷同。.此外，本研究还对金钗石斛转录组SSR位点信息进行了归纳分析，并对3个关键酶基因HMGS、HMGR和MVD的全长cDNA及表达进行了分析检测，为金钗石斛SSR特异性引物的开发及MVA途径中关键酶基因功能的研究奠定了基础。