多花黄精种子发芽过程转录组分析及ABA合成关键基因功能验证

Polygonati Rhizoma is an important and popular herbal medicine in China and could be used as food and drug, Guizhou province is one of the genuine producing areas of Polygonati Rhizoma. However, because of its low seed germinate rate and emergence rate, long-term tube propagation in practice results in low medicinal quality and yield and restricts its large-scale development. Polygonatum cyrtonema Hua seed germination, emergence time, upper and lower hypocotyl dormancy time and ABA content are different due to different origin. So it’s critical to understand the synthesis mechanism of ABA during Polygonatum cyrtonema Hua seed germination so as to regulate the ABA content of Polygonatum cyrtonema Hua seed during its germination and promote seed germination. Nevertheless, the researches for morphological development, variation of ABA content, transcriptome analysis and related genes of ABA synthesis during Polygonatum cyrtonema Hua seed germination have not been reported. Morphology and microstructure variation and ABA content variation law will be studied in our test, then Polygonatum cyrtonema Hua seeds treated by low temperature and primary roots will be used as test materials to establish ABA transcriptome and digital gene expression profile library of Polygonatum cyrtonema Hua seed germination with high-throughput transcriptome sequencing technology, so as to reveal ABA transcript information of Polygonatum cyrtonema Hua seed germination, analyze and compare the quantity, function and expression pattern of differential expression genes, systematically select key genes related ABA metabolic pathway and verify their function with RNAi technology, and reveal the relationship between ABA metabolism key gene expression and ABA content during upper and lower hypocotyl dormancy as well as mechanism of action of ABA metabolism key gene expression during Polygonatum cyrtonema Hua seed germination.

黄精是重要的常用大宗药材，可药食两用，贵州是其地道产区，因种子发芽和出苗困难，长期块茎繁殖致药材品质、产量下降和大规模发展受限。不同来源多花黄精种子发芽、出苗时间、上下胚轴休眠和脱落酸含量不同，为调控种子发芽时ABA含量、促进种子萌发，需要了解多花黄精种子发芽过程中ABA的合成机制，但多花黄精种子发芽的形态发育、ABA含量变化、转录组分析及ABA合成相关基因的研究还未见报道。项目研究其发芽过程的外观和显微结构变化及ABA含量变化规律，以低温处理前后的多花黄精种子和初生根茎为材料，应用高通量转录组测序技术构建多花黄精种子发芽ABA转录组和数字基因的表达谱文库，拟揭示多花黄精种子发芽ABA转录本信息，分析比较差异表达基因的数量、功能和表达模式，筛选参与ABA代谢关键基因，并对关键基因进行功能验证，揭示上下胚轴破除休眠时ABA代谢关键基因表达与ABA含量的关系及其在多花黄精种子发芽过程中的作用。

多花黄精是我国传统大宗药材，同时也是一种药食兼用药材，该药材在生产中存在的主要问题是种子具有休眠习性，出苗周期长达两年。从现有资料可知，植物种子休眠与其内源激素脱落酸含量密切相关，为探讨多花黄精种子休眠机理及脱落酸合成关键基因，本实验开展了多花黄精种子萌发过程中的形态发育、脱落酸含量变化规律、转录组测序与分析、脱落酸合成代谢途径相关基因筛选与验证等研究。.结果表明，多花黄精种子出苗周期长是由于其种子存在二次休眠习性，即上、下胚轴均具有休眠习性，刚采收的种子处于下胚轴休眠状态，经过近6个月的4℃沙藏，下胚轴首先解除休眠，接着长出初生根茎，然后进入二次休眠阶段，再次对带有初生根茎的种子进行长达7个月的4℃沙藏，上胚轴破除休眠，接着进入成苗阶段。脱落酸含量在此生理进程中呈现高→低→高→低的规律。对多花黄精种子此四个阶段的样品开展转录组测序，检测到88554条转录本，转录本表达率达到90.66%，通过开展分组间差异转录本GO和KEGG功能富集分析，获得了被归类到“代谢过程”、“细胞”、“催化活性”等基因本体以及“Protein processing in endoplasmic reticulum”、“Linoleic acid metabolism”、“Biosynthesis of secondary metabolites”等代谢途径的表达显著差异转录本。通过植物激素代谢途径分析，找到了脱落酸合成的代谢途径，在种子的这个生理进程中影响脱落酸合成的关键酶有3个，即“9-顺式-环氧类胡萝卜素双加氧酶”、“脱落醛氧化酶”和“脱落酸-8'-羟化酶”，三者呈线性单项排列，且编码此三个酶的基因均为一个，即K09840、K09842和K09843，注释到此3条基因的转录本数为13条。.多花黄精种子二次休眠习性的明确及脱落酸代谢途径的明晰，可为人工调控其萌发提供思路。