三七生长发育和皂苷积累对低温的响应及其调控机制研究

Panax notoginseng (Burk)F. H. Chen, which belongs to the genus Panax in the Aralisceae, is one of the most well-known and valued Chinese medical herbs. It has been also designated as the first-place pillar industry for the development of the Yunnan pharmaceutical industry in Yunnan Province. Wenshan is the main genuine production area of Sanqi. In recent years, people began to expand Sanqi cultivation in other areas of Yunnan, as fewer lands in Wenshan are suitable to cultivate it due to the obstacle of continuous cropping. Owing to its special pharmacological effect, Sanqi production has increased dramatically in recent years. As its cultivation moved to the higher latitude and altitude, low-temperature has become one of the important factors influencing the growth and saponins accumulation. In this research, various methods such as molecular technique and ecological tools for traditional Chinese medicine resources will be applied. The impacts of the low-temperature on the biological characteristics photosynthetic physiology, lipid peroxidation and protecting enzymatic activity will be evaluated under natural and controlled conditions of low temperature. The relationships between the low-temperature and the chemical constituents from sanqi will be assessed using the two analytic methods: High Performance Liquid Chromatography (HPLC) and near infrared (NIR) spectroscopic methods, with the hope of evaluating how the low temperature affects the saponin accumulation and its quality. The cDNA Library of the differentially expressed genes from Sanqi under low-temperature stress will be developed using the SSH technology, and the regulatory mechanism and the genes relating to the accumulation of the notoginsenosides will be analyzed. The research outcome will help understand the adaptability of Sanqi to low temperature and provide the baseline information for a healthy development of the sanqi industry.

三七［Panax notoginseng (Burk)F.H.Chen］是五加科人参属多年生药用植物，是我国特有、传统名贵中药材，是云药产业发展的第一大支柱产业。由于三七皂苷独特的药理、药效，致使需求量剧增，同时受连作障碍影响，道地主产区文山州土地资源紧缺，现逐渐向高纬度、高海拔地区异地引种，低温已成为影响三七生长发育及其皂苷积累的一个主要因素。本研究采用中药资源生态学和分子生物学方法，通过自然和人工控温处理，揭示三七的生物学、光合生理、脂质过氧化及保护酶活性低温响应状况；利用高效液相色谱(HPLC)与近红外光谱双指标序列分析三七化学特征组/成分的变化差异，阐明低温对三七皂苷积累及其质量的影响。利用抑制消减杂交(suppression subtractive hybridization,SSH)技术构建三七低温胁迫差异表达基因cDNA文库，解析低温诱导三七皂苷积累相关基因及其调控机制。

本项目针对文山州砚山县等5个不同海拔、不同产地自然生长的三年生三七采收期株高等8个生物学表型性状分析，发现株高等8个生物学性状的表型值在5个不同产地间均存在显著差异，具有随着海拔增高、温度降低，三七生物学性状表型值增加的趋势。不同产地三七主根和剪口中4种单体皂苷及总皂苷含量均呈显著性差异，均表现为道地产区文山州砚山县的含量为最高。 .通过控温处理，发现低温有利于一年和二年生三七地下部分的生长发育，而三年生三七则随温度的增高有利于地下部分的生长发育。一年生三七地下部分三七总皂苷含量以10℃时最高，二年生三七地下部分三七总皂苷含量以20℃时最高，三年生三七主根中三七总皂苷含量以20℃为最高，而温度对三年生三七剪口中皂苷成分含量影响无显著差异。综上表明，三七道地产区、温度20℃左右较适宜三七的皂苷积累。.通过控温处理，发现低温显著抑制了三七的光合作用（Pn），自然生长的三七光合能力显著高于人工气候室培养的三七。低温处理对三七光合色素含量影响不显著。通过检测丙二醛（MDA），我们发现低温处理对不同生长年限三七脂质过氧化影响不显著。.通过不同温度（10 °C，15 °C，20 °C）诱导下三年生三七的转录组的变化，发现低温10°C、15°C诱导导致三七大量基因表达的变化。通过对野三七的转录组进行分析，对其中1条可能参与野三七三萜皂苷合成的鲨烯环氧酶基因PvfSE进行全长克隆，该基因开放阅读框为1887 bp，编码628个氨基酸，蛋白相对分子量为68.8 kDa，理论等电点为9.28，为不稳定蛋白。.项目研究申报的3项发明专利均已在三七生产上推广应用。