天山雪蓮之組織培養研究

Studies on tissue culture of Saussurea involucrate

邱雅婷(Ya-ting Chiu)

指導教授 ： 蔡新聲

朝陽科技大學/理工學院/生化科技研究所/碩士(2011年)

摘要

天山雪蓮 (Saussurea involucrata) 屬菊科鳳毛屬，可用於治療許多疾病，如關節炎、癌症、高血壓及婦科疾病，主要生長於新疆天山、昆崙山海拔4000公尺雪線以上的高原寒帶地區，由於生長環境的特異，加上長期以來對於此資源掠奪及開發，自然生長的天山雪蓮己不足以供應市場所需，因此本研究乃利用組織培養的方法大量繁殖，建立最佳的馴化系統以提供大量優良藥源，並利用生物技術的方法生產二次代謝產物，提高代謝物之品質和產量。本研究利用反應曲面法 (Response Surface Methodology, RSM) 進行大量繁殖的試驗設計，針對培養基內基本MS鹽類、BA和NAA組成進行測試，結果顯示三個因子最佳濃度分別為3/4 MS，1 mg/L BA及1.5 mg/L NAA，配合藥包紙之透氣處理50天後利用幼苗基部誘導所獲得芽體數為13個，約為不經透氣處理的3倍；將小苗移到1 mg/L IBA培養基中培養二星期後，再移至無添加賀爾蒙的培養基，培養50天可獲得最高平均鮮重690 mg，平均根數為4.77條，且移植的存活率達到100%；癒合組織誘導方面，以莖段為培殖體，0.5 mg/L 2,4-D及0.1 mg/L BA為誘導癒合組織之最佳條件；以HPLC進行分析結果發現，經馴化4個月的植株，rutin含量為78.626 mg/g，為野生植株的1.7倍；syringetin於健化兩個月之葉片含量最高，為藥材的1.4倍，其次則為野生藥材；quercetin則皆偵測不到其含量；利用添加水楊酸誘導癒合組織之藥用成分表現，rutin及syringetin分別高於未添加瓶苗9倍及2倍；於CHI基因選殖方面，利用RACE反應，搭配同源性比對後所設計的引子，成功選殖到GsCHI之基因，全長為666 bp，並構築於pET28a之蛋白表現載體，經IPTG誘導並利用NiNTA管柱，經SDS-PAGE確認為單一蛋白，做為往後基因工程相關研究使用。

關鍵字

反應曲面設計法 ； 天山雪蓮 ； 組織培養

並列摘要

Saussurea involucrate is one of the most important medicinal plants in China. This species is mainly distributed to snowy mountains at an altitude of 4000–5000 m. It has used in the Chinese system of traditional medicine for over 2000 years for the treatment of many diseases such as arthritis, cancer, lumbago, hypertension and gynecological disease. Over exploitation and ecological destruction of the natural habits in recent years leads to severe shortage of this species. While the market demands are ever increasing. The potential of micropropagation can be exploited for rapid multiplication of this plant species and it may be an important venue to meet the ever increasing demand. The isolation of active constituents from the wild plant is economically not viable. Thus, the use of plant tissue and cell culture methods can be an effective means to grow the plants at large scale and meet up the ever increasing demand. The present study describes the use of Response Surface Methodology (RSM) in order to achieve the suitable medium for micropropagation of S. involucrate. Shooting was observed from nodal segments after seven weeks of culture on MS medium supplemented with BA and NAA. An average 13 shoots were obtained on 3/4 MS supplemented with 1.0 mg/L BA and 1.5 mg/L NAA with ventilation treatment (culture containers were first sealed with two layers of AF and incubated for two weeks, then replaced by four layers of dispense paper {DP}and maintain for another five weeks). Rooting was induced in 100% plantlets, when containers sealed with two layers of non-permeable aluminum foil for two weeks in 1/2 MS supplemented with 1.0 mg/L IBA followed by transfer of plantlets on MS medium without PGR for next five weeks in vessels enclosed by 3 layers of DP paper as ventilation closure. Callus was induced from the stem and internodal segments on 1/2 MS supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA. Comparative HPLC analysis was done to detect syringetin, rutin and quencetin content from dry weight extracts of crude drug, in vitro grown plant, greenhouse grown plant and callus. Higher (1.7 fold) rutin content was obtained in four months old greenhouse grown plant (78.62 mg/mL) compared to crude drug (45.49 mg/mL). A 1.7 fold increase in syringetin content was recorded in two months old green house grown plants compared to wild plant. We could not detect quercetin in any of the analytes. The accumulation of metabolites further enhanced with the addition of 3.0 mg/L of salicylic acid, which leads to 9 and 2 fold increase in rutin and syringetin contents respectively. RACE was done to isolate 666 bp full lengths GsCHI gene (an important gene of the flavonoid pathway). The gene was cloned and their nucleotide sequences were analyzed using bioinformatics tools (BLAST, Clustal etc.). The GsCHI gene was over expressed in expression vector (pET-28a+) in order to get active protein. The recombinant protein was purified using NiNTA column and purity was confirmed by single band obtained on SDS PAGE. The robust micropropagation system in S. involucrate was established to ensure its viability in natural habitat and addition of salicylic acid can be used to enhance rutin and syringetin contents in in vitro plants. The isolated gene can be used to generate designer plants with enhance metabolite contents.

並列關鍵字

RSM ； Response Surface Methodology ； Saussurea involucrate

參考文獻

陳威臣、夏奇鈮、葉茂生、蔡新聲，「鹽類濃度、蔗糖、植物生長素及培養容器瓶口覆蓋物對高氏柴胡組培苗發根與馴化之影響」，中華農業研究，第五十三卷，第四期，第249-260頁 (2004)。

陳威臣、蕭雴柱、賴建洲、蔡新聲，「培養基組成及培養基容器覆蓋物對康乃馨組織培養苗玻璃質化與發根的影響」，中華農業研究，第四十七卷，第四期，第364-376頁 (1998)。

古新梅、蔡新聲，「培養基組成分對康乃馨試管苗玻璃質化的影響」，中華農業研究，第四十三卷，第一期，第51-62頁 (1994)。

黃婷葳，「樹豆癒合組織之抗氧化能力及異黃酮含量分析」，碩士論文，大同大學生物工程學系研究所，台北 (2007)。

羅子庭，「丹參毛狀根液態培養生產丹參酮之探討」，碩士論文，朝陽科技大學生化科技研究所，台中 (2009)。