半夏珠芽发育的激素调节和关键调控基因克隆研究

Banxia (Pinellia ternata), which is distributed widely in East Asia, is an important medicinal plant. Bulbil, which is formed in the lower part of the petiole, is the key vegetative reproduction organ in wild. It is also a sink organ that can compete with tuber in soil. So, the development of bulbil could affect the economic coefficient in culture. The development of bulbil has common characteristics comparing with other reproductive organs, such as zygotic embryo, apomictic embryo and somatic embryo. However, researching work about the development of bulbil is very little to date. Therefore, the work to analyze the role of phytohormones during bulbil development and to isolate key genes that control the development of bulbil is very valuable for the culture of Banxia or for revealing the difference among various reproductive organs.. P. ternata, P. cordata and P. integrifolia (1, 2, and 0 bulbil formed in the petiole respectively) were selected as materials in this proposal to accomplish works as follows: ①morphologic and anatomic observations in wild or in vitro induction for the bulbil development; ②analyzing the variation of phytohormones and other physiological parameters during the development of bulbil; ③isolation and cloning of key genes that involved in the control of bulbil development based on the SSH technology or transcriptome sequencing; function analysis for genes that had been isolated through genetic transformation and quantifying of gene expression.

半夏是东亚广布的一种重要中药植物，其叶柄下部形成的珠芽是自然无性繁殖器官。半夏的珠芽发育既是生产用种的重要生产途径，也对“收获量/用种量比”或经济系数具有重要影响。珠芽发育同无融合生殖胚、体细胞胚等发育现象具有内在的联系，但相比较而言目前对珠芽发育的激素调控和分子机理研究还非常少。因此，探讨半夏珠芽发育的激素调控机制并分离其关键调控基因具有重要的实用价值和理论价值。本研究选取每叶发育珠芽一个的半夏为主要实验材料，参照每叶发育二个珠芽或不发育珠芽的滴水珠或石蜘蛛，拟通过形态解剖学研究与珠芽体外发生试验来揭示半夏的珠芽发育过程；通过内源植物激素测定和观察施用外源激素的影响来探讨珠芽发育的激素调控机制；通过SSH技术或转录组（表达谱）测序来进行珠芽发育关键基因片段的分离和筛选；通过转基因试验和表达量测定来分析验证基因功能。

生殖发育是植物发育生物学的重要研究内容，植物的无性生殖发育相对于有性生殖发育而言研究相对滞后，特别是植物的珠芽无性繁殖发育。本项目拟选取自然条件下每叶生一个珠芽的半夏(Pinellia ternata)、生两个珠芽的滴水珠(P. cordata)和不生珠芽的石蜘蛛(P. integrifolia)等三种具有不同数量珠芽发育的半夏属近缘植物进行珠芽发育的形态、解剖学观察，分析珠芽发育过程中不同时期不同组织的激素含量变化，筛选珠芽发育调控关键基因并进行分离克隆、表达特征分析与功能验证，为进一步探讨植物珠芽发育的调控机制奠定基础。研究发现，被子植物的珠芽发生通常起始于不同位置的薄壁细胞，发育过程包括启动期、原基形成期、器官分化/膨大期和成熟脱落期(半夏属植物无此时期)。珠芽发生过程中各种植物激素含量发生变化，IAA和CTK在半夏珠芽发育中起促进作用。通过转录组和表达谱测序分析，在半夏、滴水珠和淡黄花百合的珠芽发育组织中PEBP基因家族成员及生长素极性运输蛋白编码基因AUX1 LAX同源基因都具有差异表达特性，显示这两类基因在珠芽发育过程中可能发挥调节作用。通过在这些植物中的基因CDS克隆、序列分析及表达分析，结果进一步显示它们在珠芽发育调节中的重要作用。对于克隆PteMFT基因(来源于单珠芽发生植物半夏)的功能验证通过构建超表达载体转化无珠芽发生植物石蜘蛛以及在半夏中对该基因进行CRISPR/Cas9基因编辑的方式进行。已获得遗传转化石蜘蛛植株，但表型观察还在进行中；而半夏的CRISPR/Cas9基因编辑载体遗传转化试验也还在进行中。.