建立魚針草毛狀根培養生產洋丁香酚?之研究

Establishment of Hairy Root Culture in Anisomeles indica O. Kuntze for the Production of Acteoside

郭宗儒(Tsung-Ju Kuo)

指導教授 ： 詹効松

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2011年)

摘要

魚針草(Anisomeles indica O. Kuntze)，為唇形科(Labiatae)之一年生草本植物，其全草含有洋丁香酚?(acteoside)，具有提高性器官臟器係數，改善睪丸型態結構、抑制人類血癌細胞(HL-60)增生、抑制由黃菊毒素B1(AFB1)所誘導肝癌細胞增生，並有抗氧化與抗肝毒等藥理活性。本研究利用農桿根群菌(Agrobacterium rhizogenes)菌株ATCC 15834及ATCC 43057轉殖魚針草所誘導的毛狀根(hairy root)，於液態培養基中建立二代謝產物洋丁香酚?之生產系統。經感染獲得毛狀根經PCR鑑定證實為轉殖根系，根系經固態培養基培養28天後，挑選高生質量根系HR10-11，其生質量為1.25 g dw/L，並以HPLC分析其洋丁香酚?含量為10.94 mg/g，其值均高於魚針草植株不同組織部位(莖：1.23 mg/g；不定根:3.09 mg/g)；比較毛狀根在不同基礎鹽類配方(1/2 MS, 1/2 WPM, B5, WPM)之液態培養基中生長情況，發現WPM培養基最適合魚針草毛狀根生長；魚針草毛狀根於WPM液態培養基之時序培養試驗，於第5週為對數生長期中，有最高洋丁香酚?含量為11.6 mg/g，而於第7週進入生長停滯期，有最佳生質量為7.5 g/L，洋丁香酚?產量為83.6 mg/L；以WPM鹽類配方為基礎培養基，進一步針對培養基內蔗糖、硝酸銨、硝酸鈣、硫酸銅、磷酸鉀濃度分別進行調整，添加不同濃度誘引劑(離層酸、茉莉酸甲酯、水楊酸、酵母誘引劑)，尋求對生質量、洋丁香酚?含量與產量之影響，結果毛狀根顯示培養於蔗糖濃度為50 g/L有最高魚針草毛狀根生質量(12.02 mg/L)，為控制組的1.66倍，毛狀根培養於茉莉酸甲酯濃度為5 mg/L時有最高的洋丁香酚?含量(18.72 mg/g)，為控制組的1.66倍，並且毛狀根培養於硝酸銨濃度為800 mg/L有最高洋丁香酚?產量(129.61 mg/L)，為控制組的1.66倍。

關鍵字

毛狀根 ； 魚針草 ； 洋丁香酚?

並列摘要

Anisomeles indica O. Kuntze belongs Labiatae, the major active ingredients mainly exist in the whole plant, including ovatodiolide, acteoside and apigenin. In the previous studies, the acteoside shown increase the coefficient of sexual organs significantly and improved pathology changes of testes. Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation and significantly inhibited the cytotoxicity induced by aflatoxin B1 (AFB1) and used anti-oxidant, anti-inflammatory. In this study, the hairy-root cultures were established by infecting sterile leave segments of Anisomeles indica with Agrobacterium rhizogenes strains ATCC15834 and ATCC43057. The integration of foreign gene into the genome was confirmed by PCR using rolB and rolC gene specific primers in different transgenic lines. The transformed hairy roots are characterized by high growth rate and genetic stability. After 28 days cultivation on soild-agar plate, the hairy root line (HR10-11) which exhibited high biomass (1.25g dw/L) and acteoside content (10.94 mg/g) was obtained. The acteoside content of hairy root was higher than that in different parts of plant (stem: 1.23 mg/g；adventitious roots:3.09 mg/g ). The hairy roots were cultured on 3 different media such as 1/2MS, B5 and WPM to obtain the suitable culture medium. The result shown the WPM was the most suitable media for in vitro hairy root culture. A time-course experiment was carried out in Erlenmeyer flask with WPM medium, in the exponential phase (fifth week) of growth, the highest acteoside content (11.6 mg/g) was obtained, furthermore, in the stationary phase (seventh week) of growth, the maxium biomass (7.5 g dw/L) and acteoside production (83.6 mg/L) were obtained. For increasing the biomass, acteoside accumulation and production of hairy root, the nutritional composition of WPM, such as sucrose, ammonium nitrate, calcium nitrate, copper sulfate and potassium phosphate and elicitors (aba meja sa and yeast elicitor) were conducted to evaluated. The results shown the maximum biomass of 12.02 g dw/L was obtained with 50 g/L sucrose, it was 2 fold compared to the control. Moreover, a 1.66 fold increases in acteoside content (18.72 mg/g) was observed with 5 mg/L MeJA in the WPM medium. Meanwhile, the maximum acteoside production was obtained with supplementing 800 mg/L ammonium nitrate.

並列關鍵字

Hairy root ； Anisomeles indica O. Kuntze ； Acteoside

參考文獻

林李昌、李鎮宇、林昆彥、葉茂生、蔡新生。2006。何首烏毛狀根誘導與Emodin及Physcion之成份分析。作物、環境與生物資訊 3, 265-274

陳威臣、詹?松、李秋儀、曹進義、劉禎祺、李艷琪、夏奇鈮。2008。丹參毛狀根培養建立與其丹參酮產量之研究。台灣農業研究。57, 305-316。

羅子庭。2009。丹參毛狀根液態培養生產丹參酮之探討。朝陽科技大學生

劉選頡。2010。建立粉防己毛狀根培養生產粉防己鹼之研究。朝陽科技大

Amdoun, R., Khelifi L., Khelifi-Slaoui M., Amroune S., Benyoussef E.-H., Thi Dao Vu., Assaf-Ducrocq C. and Gontier E. 2009. Influence of minerals and elicitation on Datura stramonium L. tropane alkaloid production: Modelization of the in vitro biochemical response. Plant Science. 177, 81-87.