绞股蓝茎端入土无性繁殖的生理学机制及其环境适应性研究

Jiao-Gu-Lan (Gynostemma pentaphyllum) is an important medicinal plant in the Cucurbitaceae family. This medicinal plant is called “Southern China Ginseng” because it produces a high content of ginsenosides. Its southern nation-wide farming is expected to substitute Ginseng in the future. Although it is a perennial climbing herb, its above-ground organs only grow one season because they completely senesce prior to winter. There is a specific development feature during senescense, during which stems grow gravitropically and each shoot apex develops into a big adventitious bud. All adventitious buds gravitropically grow into soil and develop into multiple auxillary buds, from which new plants will start to grow in the new growth season. Here, this phenomenon is defined as “Gravitropism of Shoot Apex Propagation (GSAP)” Unfortunately, this GSAP has never been studied. Our hypothesis is that this growth phenomenon is induced by environmental factors. In addition, we hypothesize that there are two physiological reversions associated with this GSAP: 1) a change from negative gravitropism to garvitropism and 2) development from adult phase to juvenile adventurous buds. In this project, we propose to complete the following 6 experiments, 1) investigation and recording of GSAP in the field, 2) plant growth in growth chamber to testing induction factors of GSAP, 3) microscopical observation and anatomic characterization, 4) observation of amyloplast distribution and IAA immunolocalization in cells, 5) analysis of plant hormones during GSAP, 6) analysis of gene expression involved in mRNA and miRNA. The outcome will elucidate the physiological mechanism and ecological significance of GSAP, and will be important to develop new biotechnology to enhance propagation for Jiao-Gu-Lan agriculture.

绞股蓝为葫芦科绞股蓝属的重要药用植物，因能合成和积累人参皂苷类物质而被誉为“南方人参”。由于其种植区广，有望成为人参的代用品。作为多年生草质藤本，绞股蓝地上部分的生活期不足一年，其藤蔓在入冬枯死前，从空中垂下，顶芽发育为节间粗短的变态芽。变态芽长入土中，并分化出多个侧芽，越冬后萌发成新苗。我们称这一现象为茎端入土无性繁殖。这一罕见的无性繁殖现象一直被绞股蓝研究者所忽视。我们推测变态芽是由特定的环境因子所诱导，且体内可能发生两类生理学逆转：生长的向性由负的向地性转向正的向地性，发育阶段由成熟阶段恢复到幼态阶段。为此，本项目拟根据对自然生境中绞股蓝这种无性繁殖现象的观察和人工气候室摸拟实验，发现诱导变态芽形成的主要环境因子，建立绞股蓝顶端入土无性繁殖的实验体系，并采用实验形态学、生理学和分子生物学等方法，揭示绞股蓝这一无性繁殖的生理学机制和生态学意义，并为发展绞股蓝高效栽培技术提供理论依据。

绞股蓝秋末时其空中茎下垂生长，钻入土中转化为根状根以越冬，并在来春萌多个新芽。本研究表明，光是影响绞股蓝这种变化的主要环境因子。弱光或暗诱导下，正常茎端通过黄化膨大期、变态芽期发育为根状茎。各茎期的基本结构相似。膨大黄化期的生长素、赤霉素和茉莉酸含量均升至最高，应是茎端转化的必须期和关键期。激素从芽尖段、弯曲段、膨大段至稳定段的含量表现为先升后降趋势。赤霉素可能是茎端转化的主要激素，而茎转化中可溶性糖和淀粉含量呈连续下降，蔗糖和果糖平缓上升，还原性糖上升较快。.对绞股蓝地上茎向根状茎转变的3个发育阶段转录组测序，鉴定到5428个差异表达基因，这些与向地性相关的基因协调控制变态茎的重力响应。此外与苯丙烷生物合成、光周期、激素信号转导、碳水化合物代谢和其它与根状茎发育相关的差异基因也参与调控了绞股蓝茎的转变。.对9个小RNAs文库的测序获得GpmiR156a、GpmiR159、GpmiR166b-5p、GpmiR166e-3p共4个已知和Co.47071、Co.25160、Co.59333共3个预测的新miRNA。在茎转变中GpmiR156a、GpmiR159和Co.47071显著上调，而其他则显著下调。GpmiR166b-5p、GpmiR166e-3p、GpmiR156a和Co.47071在促进空中茎向根状茎的转变中起着重要作用。.同源克隆了GpMIR156a 和GpMIR166b并进行生物信息学分析，通过构建植物过表达载体转化拟南芥，发现GpmiR156a能促进营养器官发育和种子萌发，但抑制童期向成熟期的转变，而GpMIR166b在一定程度上影响营养器官的发育和延缓种子萌发，但能促进童期向成熟期发育的转变。.光质可以显著影响绞股蓝总皂苷的积累，红光显著提高总皂苷含量，其次是蓝光和白光，黑暗下含量最低。绞股蓝皂苷生物合成关键酶鲨烯合成酶SS和鲨烯环氧酶SE也受光质影响，红光下两者酶基因高水平表达，蓝光下次之，黑暗中表达最低。总皂苷含量与两种基因的表达水平呈显著正相关。.乙烯是重要的植物激素，外施乙烯利能不同程度地上调GpFPS、GpSS和GpSE基因的表达水平，且其表达模式在根、茎和叶间各异，而在同一器官中相似。在乙烯利处理后各器官中的总皂苷含量均有所上升，在各单体中以Rb3含量较高。.