轉殖果膠分解酶基因於蝴蝶蘭抗細菌性軟腐病之研究

Studies on Resistance Against Bacterial Soft Rot by Transgenic Manipulation of Pectate Lyase Genes in Phalaenopsis

林映均(Ying-Chun Lin)

指導教授 ： 黃鵬林

共同指導教授 ： 杜宜殷(Yi-Yin Do)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10073

摘要

蝴蝶蘭 (Phalaenopsis spp.) 為近年來全球重要花卉，亦為臺灣重要之出口花卉。細菌性軟腐病 (bacterial soft rot) 使感病植株於葉片、花瓣或花梗皆出現水浸狀及軟腐等病癥，甚至造成植株腐爛、死亡，為影響蝴蝶蘭生產及貯運之重要病害。該病害主要由 Pectobacterium屬之病原菌所引起，於高溫高濕環境易發病、蔓延且難以防治。本研究利用基因轉殖方式，將 Pectobacterium chrysanthemi之果膠分解酶基因 (pectate lyase, pel) 轉入菸草及蝴蝶蘭，使該酵素作用產生之細胞壁分解物質如寡半乳糖醛酸 (oligogalacturonic acid, OGA)、半乳糖醛酸 (D-galacturonic acid) 等，先行誘導植物產生抗病物質、啟動抗病反應而達到抗病效果。本研究為取得抗病轉殖株並減低其程序性細胞自殺反應 (programmed cell death, PCD) 現象，分別使用持續表達之 CaMV 35S 啟動子及受水楊酸誘導之 PR1a 啟動子 (pathogenesis-related protein 1a promoter )，將 35Spro::pelZ 及 PR1apro::pelE 之構築質體 pGKZ3-8 及 pGK-PelE-GUS，以農桿菌媒介法轉殖入菸草葉片及蝴蝶蘭癒傷組織細胞中，以抗生素篩選及GUS活性組織化學染色分析，得到轉殖細胞並再生成植株。擬轉殖株經聚合酶連鎖反應 (polymerase chain reaction, PCR) 及南方氏雜交分析 (Southern blot analysis)，確認轉殖基因之完整性。進一步以不同轉殖系之轉殖株進行果膠分解酶活性測定、果膠分解產物半乳糖醛酸分析及接種抗病分析，蝴蝶蘭 35Spro::pelZ 轉殖株之果膠分解酶以薄層色層法 (Thin layer chromatography analysis) 分析顯示，其具有將果膠分解成單醣、雙醣及三醣之能力，相較於非轉殖株無法產生單醣，轉殖株之分解酶活性較佳。蝴蝶蘭 35Spro::pelZ、菸草 PR1apro::pelE 及蝴蝶蘭 PR1apro::pelE 經 Sulfamate / m-hydroxy diphenyl 法檢測轉殖株葉片，半乳糖醛酸含量皆較非轉殖株高，顯示轉殖之果膠分解酶 PelZ 及 PelE 皆可於植株中作用，並與非轉殖株具顯著差異，亦可間接證實 PR1a 啟動子受水楊酸誘導作用。以流式細胞儀分析轉殖株基因組小片段基因，蝴蝶蘭 35Spro::pelZ 轉殖株非整倍體基因片段較非轉殖株提高 3.4%，與 35Spro::pelE 轉殖株相比，非整倍體基因片段比例下降，程序性細胞自殺反應程度較低，且 35Spro::pelZ 轉殖株較 35Spro::pelE 轉殖株易出瓶存活。菸草 PR1apro::pelE 轉殖株經流式細胞儀分析，轉殖株非整倍體基因片段比例較非轉殖株高，水楊酸誘導後 PR1a 啟動子，轉殖株非整倍體基因片段比例下降。蝴蝶蘭及菸草轉殖株分別以 P. chrysanthemi 進行接種，相較於非轉殖株感病範圍皆較小；以 trypan blue 進行接種葉片染色，可見葉片感病處及其外圍皆形成死細胞，顯示轉殖株啟動抗病反應並具有抗病能力。本研究成功取得 35Spro::pelZ 及 PR1apro::pelE 蝴蝶蘭轉殖株，並經檢測證實具有較佳之抗病能力，未來可進一步育成新的抗病品種，並可作為育種之雜交母本，或研發生產生物製劑，期可解決軟腐病於蝴蝶蘭生產外銷之問題。

關鍵字

蝴蝶蘭 ； 細菌性軟腐病 ； 果膠分解酶基因 ； 半乳醣醛酸 ； PR1a 啟動子

並列摘要

The Phalaenopsis orchids are one of major flowers globally, and also the important export flowers in Taiwan. Bacterial soft rot causes the tissue maceration and soft rot in the leaves, petals, and flower stalks of infected plants, even resulting in plants decay or death. Therefore, bacterial soft rot considerably affects the production and storage of Phalaenopsis. The disease primarily caused by Pectobacterium spp. It’s hard to prevent the disease infection at high temperature and high humidity. In this study, we aim to produce transgenic plants by transfering pectate lyase (pel) genes of the Pectobacterium chrysanthemi to tobacco and Phalaenopsis. Thereby, the oligogalacturonic acid (OGA) and D-galacturonic acid, which produced by pectate lyase acting with the plant cell walls, induce the resistance mechanism in the plants. For resistant plants we chose the constitutive CaMV 35S promoter (35Spro) and salicylic acid-inducible pathogenesis-related protein 1a promoter (PRlapro) to drive the aimed genes. 35Spro::pelZ (pGKZ3-8), and PR1apro::pelE (pGK-PelE-GUS) transgenic tobacco and Phalaenopsis lines were generated by agrobacterium-mediated transformation, screened by antibiotics selection and GUS activity staining. Gene integration was confirmed by polymerase chain reaction and Southern analysis. Pectate lyase activity and the amount of the galacturonic acid were also determined. The pectate lyase activity of 35Spro::pelZ transgenic plant was measured by thin layer chromatography analysis. Enzymes of the transgenic plants have higher activities which produced the D-galacturonic acid and its di- and tri-galacturonic acid, while the untransformed plants produced di- and tri-galacturonic acid. The galacturonic acid contents in the leaves of the transgenic plants were determined by Sulfamate / m-hydroxy diphenyl method, and their levels are higher than the untransformed plants. Flow cytometric analysis was applied to monitor the programmed cell death of the transgenic plant cells. Increase in the subgenomic DNA masses in transgenic 35Spro::pelZ Phalaenopsis was found when compared with the untransformed plants. A lower proportion of subgenomic DNA masses in the transgenic 35Spro::pelZ Phalaenopsis was also found when compared with the transgenic 35Spro::pelE Phalaenopsis. The resistance of the transgenic plants were demonstrated by the inoculation with P. chrysanthemi and trypan blue staining. The infection areas were smaller and the programmed cell death occurred arounding the injection area in the transgenic plants. Transgenic 35Spro::pelZ Phalaenopsis and PR1apro::pelE Phalaenopsis were proved to be resistant against the bacterial soft rot. These studies showed allow the establishment of strategies aimed at the resistance of bacterial soft rot in Phalaenopsis.

並列關鍵字

Phalaenopsis ； bacterial soft rot ； pectate lyase gene ； D-galacturonic acid ； PR1a promoter

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