大車前草內生菌之分離、鑑定與其合成麥角甾苷能力探討

Isolation and identification of Plantago major endophytes and their potential to biosynthesize verbascoside

黃立中(Li-Zhong Huang)

指導教授 ： 楊禮亘

國立虎尾科技大學/文理學院/生物科技系碩士班/碩士(2017年)

摘要

車前草植物(Plantago family)常在世界各地的傳統醫學中被使用，而且有些車前草植物已經被廣泛研究，而其中在傳統及現代醫學最被廣泛使用之車前草物種則為大車前草(Plantago major)。如要藉由大量栽種藥用植物以獲取足夠之活性分子可能會與糧食作物競爭耕地面積或因不當農業操作而使土壤劣化。在植物組織內且不對植物造成任何明顯病症狀之微生物稱內生菌，被報導具有合成生物活性分子等各種功能，因此本實驗由大車前草分離內生菌株並初步鑑定與測試能否合成目標生物活性分子-麥角甾苷。本實驗先將取得之車前草成熟植株(約16 cm高)，依植物檢索表比對發現其列屬於大車前草種(Plantago major)，並將車前草植株依其根、莖葉、花穗部分分離內生菌。大車前草根部內生菌總數為7.92x103(CFU/g)、莖葉內生菌總數為2.56x103(CFU/g)、花穗內生菌總數則為1.87x104(CFU/g)，內生菌分離鑑定於根部共分離得到18株、莖葉組織為23株、花穗組織為12株，經16S rRNA之3F引子初步基因定序與比對後，發現根部含13種不同種之菌株並以Bacillus菌屬居多(54%)；莖葉含11種不同種之菌株並以Bacillus菌屬居多(64%)，同時經全長定序後發現一株疑似新種之菌株；花穗含10種不同種之菌株並以Sphingomonas、Kineococcus菌屬較多(各占20%)，結果可發現花穗部分之內生菌株含有最多不同的菌屬(66%)，另外從全部部位中挑選14株內生菌株進行後續研究。菌株功能性分析中，發現14株內生菌株皆具有固氮能力、僅有LS1402具有溶磷能力、LS1417與LS1424具有鐵載體合成功能。於麥角甾苷生物合成實驗，因麥角甾苷其中一個結構為咖啡酸，然而本實驗篩得之14株內生菌株部分菌株會被低濃度之咖啡酸抑制，例如於第三天培養時LS1434、LS1435、LS1438、LS1454、LS1460會被25 µg/ml咖啡酸濃度抑制，甚至LS1454會在5 µg/ml咖啡酸濃度被抑制，因此無法直接投入以麥角甾苷前驅物-咖啡酸進行生物合成研究。接種內生菌於50%NB+大車前草葉片水萃液中，上清液結果可以發現培養一天時，LS1412可增加Verbascoside約27.66%的訊號面積，但因僅為圖譜訊號面積相互比較而未進行濃度定量比較，因此尚無法確定其結果，然而培養一天情形下，LS1450與其它菌株處理組與控制組相比，第3分鐘HPLC沖提時間下的波峰明顯較低，且其它菌株處理組別及控制組於培養第三天時該波峰才會明顯下降，推論LS1450會比其它菌株處理組率先利用第3分鐘沖提時間下的化合物進行反應；另外以超音波破碎內生菌菌塊，發現每個菌株處理組皆有麥角甾苷之訊號反應，而LS1412、LS1417、LS1434、LS1450菌塊雖在培養一天時與控制組相比分別增加Verbascoside訊號面積2.39倍、1.88倍、1.77倍、1.14倍，但無法判定此結果為內生菌攝入、化合物附著於菌體表面或菌株本身具合成能力之緣故。

關鍵字

大車前草 ； 內生菌 ； 16S rRNA ； 麥角甾苷生物合成

並列摘要

Plantago family is often used in traditional medicine around the world, and some of which have been extensively studied, and the most used in traditional and modern medicine is Plantago major. If medicinal plants are grown in large scale for extracting enough active molecules, it may compete with the food crop area or lead to soil degradation because of inappropriate agricultural management. Endophytes are microbes which live in plant tissues and without any obvious disease symptoms, they have been reported to possess functions of nitrogen fixation, phosphorus-solubilizing or bioactive compound biosynthesis activities. This study isolated and identified endophytes of Plantago major, then detect whether selected endophytes could synthesize verbascoside in vitro. This study isolated endophytes from root, leave, and flower tissues of Plantago major. The endophyte colony forming units are 7.92x103 (CFU/g), 2.56x103 (CFU/g) or 1.87x104 (CFU/g) in root, leave and flower, respectively. We isolated 18, 23 and 12 endophyte isolates from the root, leaf and flower of Plantago major, respectively. After using 16S rRNA gene sequencing technique, we obtained 13 unique endophytes in root and the Bacillus genus was the majority (54%), 11 unique endophytes in leaf and Bacillus was the majority (64%), 10 unique endophytes isolate from flower and Sphingomonas and Kineococcus were the majority (20% respectively). In addition, we found the diversity in different genus (66%) was highest in the flower tissues. Then we chose 14 isolates for further analysis. In the functional analysis of 14 endophytes, all of which have found to fix nitrogen. LS1402 had phosphorus-solubilizing ability. LS1417 and LS1424 had siderophore producing function. In the verbascoside biosynthesis assay, because one of verbascoside precursors is caffeic acid, but some endophytes growth are inhibited at low caffeic acid concentration, such as in day 3 culture, the LS1405, LS1417, LS1456 were inhibited in 100 µg/ml, and LS1434, LS1435, LS1438, LS1454, S1460 were inhibited in 25 µg/ml, and even LS1454 was inhibited in 5 µg/ml. So the use of precursor for verbascoside biosynthesis using caffeic acid is not possible. When we inoculated endophytes in 50% NB+water extract of Plantago major leaf, the results showed that the supernatant of LS1412 increased 27.66% peack area of verbascoside as compared to control, however, the actual concentration was not determined. LS1450 will use the compound in 3 minute elution time. In addition, we use sonicator to destruct the microbe, we found pellets of LS1412, LS1417, LS1434 and LS1450 increased verbascoside peak area 2.39, 1.88, 1.77 and 1.44 times in day 1 culture respectively, which may due to adsorption, intake by endophytes, incomplete cleansing or may possess verbascoside biosynthesis ability.

並列關鍵字

Plantago major ； Endophyte ； 16S rRNA ； Verbascoside ； Biosynthesis

參考文獻

1. 丁俊榮。2013。連作對結球甘藍(Brassica oleracea var. capitata)根部可培養型內生細菌之影響及其功能性研究。國立虎尾科技大學生物科技系碩士論文。

4. 林志生、邱聖壹。2010。光生物反應器於微藻培養之研究與產業化的進展。農業生技產業季刊，22：44-51。

8. 張惠如、鍾文全。2012。植物的身分證-DNA條碼。種苗科技專訊，79：9-12。

10. 黃瑞彰、江汶錦。2011。微生物肥料在瓜果類作物應用研究。農業生技產業季刊，28：53-58。

13. 楊秋忠、沈佛亭。2008。溶磷微生物肥料之特性及效益。農業生技產業季刊，16: 57-59。