牡丹内源酚类物质与IAA反应产物结构与生理功能解析

The problems as poor rhizogenesis and rooting quality, low survival rate of transplanted plantlets in vitro are the main factors affecting the establishment of Paeonia suffruticosa propagation system. Chinese and foreign scholars have explored a variety of training methods and culture conditions for getting a breakthrough,however, the results were not satisfying. Phenolic metabolic activities of Paeonia suffruticosa, which belong to plant species for the high phenols content, would surely have greater impacts on growth and differentiation of plantlets in vitro. Preliminary studies, the qualitative and quantitative analysis on the polyphenols of Paeonia plantlets in vitro and the dynamic change law of polyphenols during the process of rooting cultivation were determined, the research group identified three kinds of phenolic compounds ,chlorogenic acid caffeic acid and catechol existed in the plantlets in vitro of Paeonia, could react with indole-3-acetic acid (IAA) to form new products under the condition of polyphenol oxidase (PPO) catalytic. Preliminary identification of chlorogenic acid reaction products, we found the structure of a new product from reaction products was similar to IAA's, and presumably it could be the IAA-phenolic complex proposed by the previous studies. Based on the previous research, the reaction system would be further improved and the reaction products was separated and its structure was also identifed in this study. Meanwhile, the aspects of stability and physical activity of reaction products and its effect on the rooting of Paeonia plantlets in vitro were studied to reveal the relationship of phenolic metabolism with rooting in the root initiation process of Paeonia suffruticosa plantlets in vitro,and the regulation measures affect on the rooting of Paeonia suffruticosa plantlets in vitro was raised to provide theoretical and technical support for establishing efficient plant regeneration system of Paeonia suffruticosa.

牡丹试管苗培养中存在的根系发生难、质量差、移栽成活率低等问题一直是制约其快繁体系建立的主要因素。中外学者探讨了各种培养方法和条件，但均未获得突破。牡丹属酚类物质含量较高的植物种类，酚类物质的各种代谢活动对牡丹试管苗生长和分化将产生较大的影响。课题组前期研究中，定性定量分析了牡丹试管苗酚类物质及其在生根过程中的变化规律，明确了绿原酸、咖啡酸和儿茶酚3种牡丹内源酚类物质在PPO催化下可与IAA反应形成新产物。经对绿原酸的反应产物初步鉴定，新产物中含有与IAA结构相类似的物质，推测可能是前人研究提出的"IAA-酚酸复合物"。本研究拟在前期工作基础上，进一步完善反应体系、分离和鉴定产物结构。同时，通过对反应产物的稳定性和生理活性及其对牡丹试管苗生根影响等方面的研究，初步揭示牡丹试管苗生根过程中酚类物质代谢与其根系发生的关系，并在此基础上提出调控措施，为建立牡丹高效植株再生体系提供理论和技术支撑。

牡丹试管苗培养中存在的根系发生难、质量差、移栽成活率低等问题一直是制约其快繁体系建立的主要因素。牡丹属酚类物质含量较高的植物种类，酚类物质的各种代谢活动对牡丹试管苗生长和分化将产生较大的影响。课题组前期研究中，定性定量分析了牡丹试管苗酚类物质及其在生根过程中的变化规律，明确了绿原酸、咖啡酸和儿茶酚3种牡丹内源酚类物质在PPO催化下可与IAA反应形成新产物。经对绿原酸的反应产物初步鉴定，新产物中含有与IAA结构相类似的物质，推测可能是前人研究提出的“IAA-酚酸复合物”。本研究拟在前期工作基础上，进一步完善反应体系、分离和鉴定产物结构。同时，通过对反应产物的稳定性和生理活性及其对牡丹试管苗生根影响等方面的研究，初步揭示牡丹试管苗生根过程中酚类物质代谢与其根系发生的关系，并在此基础上提出调控措施，为建立牡丹高效植株再生体系提供理论和技术支撑。主要研究结果如下：3种酚类物质均与IAA反应均可产生3种相同的物质，且三种酚类物质均在PH=5.0时与IAA反应的产物率最高，在反应6 h后趋近稳定状态；分离提纯后发现，产物1和产物2互为同分异构体，其结构为吲哚乙酸和甲基结合物，产物3为吲哚-3-甲醛；产物1和产物3均具有生长素活性，均能促进小麦芽鞘的伸长和绿豆下胚轴不定根的发生，但活性均小于IAA，产物3对绿豆下胚轴不定根形成有显著作用；产物1稳定性大于IAA；通过免疫荧光组织化学定位发现，牡丹试管苗在生根期间内源IAA主要分布于维管束中；外源添加产物发现，产物1对牡丹试管苗生根作用不显著，产物3对其生根有促进作用，且牡丹试管苗POD和PPO酶活性在根原基诱导期均处于升高趋势，而IAAO处于下降趋势；通过免疫荧光定位法发现，IAA在茎基部主要分布在维管束中，且一直作用于根原基形成的部位，且信号的强弱与其含量相一致。