【作者】 羊海军；

【导师】 崔大方； 刘仲健；

【作者基本信息】 华南农业大学 ， 植物学， 2019， 博士

【摘要】 传粉生物学是进化生物学研究的热点内容之一,对理解植物与生物或非生物传粉媒介、植物与环境之间的交互作用及协同进化关系具有重要意义。兰科是物种多样性最丰富的类群之一,拥有极其多样的遗传和形态变异,自达尔文时代起,兰科中就发现了许许多多的传粉系统和各式各样的传粉机制,这些生物学信息为探讨和理解兰花的起源与进化、繁殖生态和种群扩张等提供有效线索和证据。小沼兰(Oberioides microtatantha)是兰科中一个小型岩生物种,主要分布在中国华东至华南的低山地带,因其独特的生境适应,进化出了水流传粉机制,并通过环境诱导雌性不育调整自身雌雄性适合度,实现极端环境下的生存与繁衍,向人们展示了兰科植物对陆生环境的多样化适应策略。本文以小沼兰为研究对象,对分布于广东始兴和浙江宁波两地的野生居群进行了为期5年的调查研究,揭示了其传粉生物学机制,并初步探讨了其育性分子基础,主要结果如下:(1)通过多年多地的野外观察,发现小沼兰在野生条件下每年都需要通过长时间的夏眠来躲避炎热和干旱,期间仅以假鳞茎的形式生存,并借助苔藓、地衣等进行遮蔽防护。花序在冬季完成分化,当早春寒潮来临时,进入盛花期。由于此时,所处生境多为低温阴雨,严重制约动物活性,使小沼兰陷入缺乏生物传粉媒介的险境,通过长期进化,该种兰花的花部形态和开花模式已极度适应利用地表水流作为传粉媒介的传粉机制。本研究通过野外观察和模拟实验,证实了小沼兰水流传粉机制的成功有效,以水流为媒介进行混合交配,实现自交和异交,达到生殖保障和杂种优势双重利益。这是一种不同于其它水媒传粉和雨媒传粉的新的传粉机制,在兰科植物中尚属首次发现。(2)人工授粉实验显示小沼兰异花结实率(11.38%±2.10%)高于自花结实率(4.87%±0.85%)。鉴于花粉块完全失活率仅2.64%,意味着存在显著的雌性不育现象。本研究认为小沼兰的雌性器官结构完整,其不育性是功能型的,由此,在居群水平上,形成功能型雄-雄全同株的性系统,是对特殊生境和水流传粉机制的适应表现,具有独特性。(3)不同育性水平的小沼兰植株在光合特性、可溶性糖含量、淀粉积累情况和生活史型方面都表现出差异。高光可育组的表观量子效率(AQY)和最大净光合速率(Pmax)大于低光不育组而小于高光不育组,但近光饱和点和光补偿点均较后两者高。高光可育组的可溶性糖含量和淀粉积累量均最高。生活史定量划分结果显示,高光可育组、高光不育组和低光不育组分别以V0.2427C0.3577S0.399、V0.2202C0.3562S0.4236和V0.2760C0.3431S0.3809为分布中心。本研究表明,小沼兰对光强的响应具有多态性,对生活史型具有微调效应,光合同化产物的富足是小沼兰进行有性生殖的能源保障。同时,光照也刺激小沼兰形态渐变,导致花柄、子房、柱头等跟育性密切相关的器官组织在数量性状上发生微调,进而影响授粉。(4)利用转录组分析方法比较小沼兰可育组和不可育组的差异基因,共筛选出1184个差异unigene,富集得到22个GO term和10个KEGG代谢通路。结果表明小沼兰的育性相关基因涉及光合作用、碳水化合物代谢、信号传导、组蛋白修饰等多个生理过程,初步筛选了一些潜在的关键基因,包括Lhcb、Sn RK1、FBP、ADPG、SAI、HMT和HDAC等编码基因及产物,它们在小沼兰的育性调节方面发挥重要作用。(5)小沼兰不育花中与生长素相关的生长素响应因子ARF3下调表达、生长素抑制蛋白ARP上调表达,可能降低了花粉块与柱头不亲和性,阻滞了生长素从花粉块向子房传输的进程,干扰了生长素介导的授粉信号,从而无法诱导启动其下游基因的表达,导致授粉后子房不能及时膨大,进而阻滞胚珠发育,表明生长素也与雌性不育有关。更多还原

【Abstract】 Pollination biology is one of the hotspots in evolutionary biology and is important to understand the interaction and co-evolution between plants and biological or abiotic pollinators,and also between plants and environment.Orchidaceae is one of the most species diversity families with extremely diverse genetic and morphological variations.Up to date,many pollination systems and various pollination mechanisms have been found in Orchidaceae.These biological information on pollination provide the effective clues and the evidence for exploring and understanding the origin and evolution,the reproductive ecology and the population expansion of orchids.Oberonioides microtatantha is a small lithophytic species in Orchidaceae.It is mainly distributed in the low mountain area from East China to South China.Because of the adaptation to the habitat with low temperature and rainy in the period of flowering,the orchid has evolved the mechanism of water flow pollination and can adjust its female suitability by inducing functional female sterility to achieve its survival and reproduction in the extreme environment.In the study,the wild populations of O.microtatantha in Shixing,Guangdong Province and Ningbo,Zhejiang Province were investigated for five years and theri pollination mechanism and the molecular basis of fertility were discussed.The main results were as follows:(1)Under the wild conditions,O.microtatantha need a long-term aestivation to avoid the heat and drought stress.During this period,they only survive in the form of pseudobulbs,and rely on the moss and the lichens to shield themselves.The inflorescences usually finish their tissue differentiation in winter and are in full bloom when the cold spell comes in early spring.At this time,the habitat is mostly low temperature and rainy,which seriously restricts the animal activity,so that the orchid is lacking of the biologicalpollinators,while the flower feature and flowering pattern of the orchid has been adapted to the extreme environment and takes the water flow as its abiotic pollinator.With the field observation and the simulation experiments of water-flow polliation,the new pollination mechanism is confirmed in success.In the orchid,mixed mating is carried out by water flow to realize self-and cross-polination,and to achieve the double benefits from reproductive security and heterosis.The water flow pollination mechanism is different from the others hydrophily pollination and is the first report in Orchidaceae.(2)According to the results of manual pollination,it was showed that the fruit-set ratio of cross-pollinated flowers(11.38% ± 2.10%)was higher than that of self-pollinated flowers(4.87% ± 0.85%).In consideration of the complete inactivated pollinia is only 2.64% in total,it means that there is a significant phenomenon of female sterility in O.microtatantha.This study considered the female organ structurally-complete and its sterility was functional.Therefore,the sexual system of O.microtatantha is a functional andro-andromonoecy at the level of population,which is an adaptation to the unique habitats and the water-flow pollination.(3)There are differences in photosynthetic characteristics,soluble sugar content,starch accumulation and life history types among the three different groups.The apparent quantum efficiency(AQY)and the maximum net photosynthetic rate(Pmax)of the high-light fertile group(HF)were higher than those of the low-light sterile group(LNF)and the high-light sterile group(HNF),but the light saturation estimate(LK)and the light compensation point(LCP)were higher than those of the latter two groups.The soluble sugar content and the starch accumulation in the high-light fertile group were the highest.The results of quantitative classification based on PCA of life cycle forms in the three groups were V0.2427C0.3577S0.399,V0.2202C0.3562S0.4236 and V0.2760C0.3431S0.3809 respectively.This study showed that the response to light intensity was polymorphic in O.microtatantha and it would led to a fine-tuning for the life cycle form.A sufficient supply of photosynthetic assimilation products was help for improving the female fertility of the orchid.The light also would induce the morphological gradient resulting in the change of the quantitative traits of organs,such as petioles,ovaries and stigmas,which were closely related to fertility,and thus affect pollination.(4)Basing on RNAseq analysis,total 1184 differentially expressed unigenes were screened and 22 GO terms and 10 KEGG metabolic pathways were enriched.The results showed that the fertility-related genes involved in several physiological processes including photosynthesis,carbohydrate metabolism,signal transduction,histone modification,etc.Some potential key genes,including Lhcb,Sn RK1,FBP,ADPG,SAI,HMT and HDAC coding genes and products,were preliminarily screened.They played an important role in regulating the fertility of O.microtatantha.(5)The down-regulated expression of auxin response factor ARF3 and the up-regulated expression of auxin-repressed proteins in the sterile flowers suggested that auxin was closely related to the fertility.This study proposed a hypothesis that these auxin-related genes may regulate the transport of auxin from pollinia to ovary,interfere with auxin-mediated pollination signal to stimulate or inhibit the enlargement of ovary and the development of ovule.更多还原