A study of pyrroloquinoline quinone on Pei-Chiao (Musa spp.) growth and biocontrol improvement

香蕉是世界上重要的糧食作物之一，但深受香蕉黃葉病害之影響導致產量受限，因此解決此問題為當前首要之任務。前人實驗結果證實有接種Burkholderia sp. 869T2的北蕉平均株高增加32.6公分，發病率降低21.1%。而在本研究中於溫室實驗中同樣驗證了869T2接種於北蕉之促進生長及抗病之效果，有效降低黃葉病的罹病程度。於轉錄基因體分析中發現869T2體內合成Pyrroloquinoline quinone (PQQ)之基因組有高表現量。藉由使用PQQ對北蕉進行生長測試，探討PQQ是否為促進北蕉生長之重要因子及抗香蕉黃葉病之作用。於添加PQQ作為促進生長因子之實驗結果中得知，添加100nM PQQ株高增加了2.2公分、健葉數增加了2.9葉及鮮重增加了1.1克；而在溫室感染實驗中PQQ組平均罹病度低於控制組。且於轉錄基因體中研判添加PQQ後北蕉促進生長及生物防治之反應，發現添加PQQ後可提高碳水化合物代謝、胺基酸代謝、光合作用、氧化作用及吉貝素合成酵素的高表現等可能為促進北蕉生長之因素，以及誘導植物抗病因子PR1之表現。Banana is one of the most important food crops in the world, but it is deeply affected by the fusarial wilt, which leads to limited production. Therefore, solving this problem is the current priority. The results of previous experiments confirmed that the average plant height of Pei-Chiao with Burkholderia sp. 869T2 inoculated increased by 32.6 cm, and the incidence decreased by 21.1%. In this study, the effect of 869T2 inoculation on Pei-Chiao to promote growth and disease resistance was also verified in the greenhouse experiment, which effectively reduced the degree of fusarial wilt. In the transcriptome analysis, it was found that the genome of Pyroloquinoline quinone (PQQ) synthesized by 869T2 in vivo has high expression. The growth test of the Pei-Chiao was to explore whether PQQ is an important factor for promoting the growth of the Pei-Chiao and the role of resistan fusarial wilt. In promoting growth analysis, adding 100nM PQQ was proved that the plant height increased by 2.2 cm, the number of healthy leaves increased by 2.9 leaves, and the fresh weight increased by 1.1 g. In the greenhouse infection experiment, the average fusarial wilt in the PQQ was lower than that in the control. After adding PQQ to the transcript gene, the Pei-Chiao promoted the growth and biological control reaction. It was found that the addition of PQQ can improve carbohydrate metabolism, amino acid metabolism, photosynthesis, oxidation and high performance of gemcitabine synthetase. It may be a factor that promotes the growth of the northern banana and induces the performance of the plant resistance factor PR1.摘要 i Abstract ii 表目錄 vi 圖目錄 vii 附圖 viii 附表 ix 第一章 前言 1 1.1 香蕉 1 1.1.1 香蕉分類命名 1 1.1.2 北蕉 2 1.2 香蕉黃葉病 2 1.2.1 分布 2 1.2.2 香蕉黃葉病病原菌 3 1.2.3 感染途徑 4 1.2.4 病徵 4 1.2.5 病害生態 5 1.2.6 抗病現況 5 1.3 植物內共生菌 5 1.3.1 型態 6 1.3.2 Burkholderia sp. 869T2 7 1.4 Pyrroloquinoline quinone (PQQ) 8 1.4.1 PQQ生合成途徑 9 1.5 氨基酸在植物中的重要性 11 1.6 研究動機 13 第二章 材料方法 14 2.1 培養基配置 14 2.2 組織培養香蕉種植 15 2.3 病土製作 15 2.4 病土感染 16 2.5 螢光顯微鏡觀察 16 2.6 Burkholderia sp. 869T2接種測試 16 2.6.1 內共生菌接種液製備 16 2.6.2 內共生菌接種於香蕉組培苗 16 2.6.3 接種內共生菌之香蕉組培苗之菌相觀察 17 2.6.4 接種內共生菌抗北蕉黃葉病測試 17 2.7 Pyrroloquinoline quinone(PQQ)促進北蕉生長及香蕉黃葉病生物防治測試 18 2.7.1 培養基添加PQQ促進北蕉苗生長測試 18 2.7.2 培養基添加PQQ北蕉苗抗黃葉病測試 19 2.7.2.1 組織培養感染種植測試 19 2.7.2.2 溫室感染種植測試 19 2.8 Pyrroloquinoline quinone(PQQ)改善北蕉生長次世代定序(Next Generation Sequencing) 19 2.9 統計分析 20 第三章 結果 21 3.1 利用含綠螢光蛋白香蕉黃葉病病原菌株Fusarium oxysporum f. sp. cubense tropical race 4觀察入侵植株情形 21 3.2 Burkholderia sp. 869T2接種測試 23 3.3 Pyrroloquinoline quinone(PQQ)添加促進北蕉生長測試 24 3.3.1培養基添加0nM、5nM、50nM、100nM及1000nM PQQ促進北蕉生長測試 25 3.3.2 提高培養基添加 PQQ濃度促進北蕉生長測試 27 3.4 組織培養添加PQQ抗北蕉黃葉病測試 29 3.4.1 組織培養測試 29 3.4.2溫室測試 30 3.5 利用NGS探討培養基添加Pyrroloquinoline quinone(PQQ) 後北蕉組培苗之轉錄基因體表達差異 34 3.5.1基因本體論(Gene Ontology ) 35 3.5.2 調控路徑(KEGG pathway) 36 第四章 討論 39 4.1 PQQ改善北蕉生長之表現 39 4.2 PQQ參與北蕉苗生長之基因轉錄體分析 40 第五章 結論 43 第六章 參考文獻 4