植物第二型富含甘胺酸蛋白LsGRP1之特性與防禦相關功能研究

Characterization and functional studies of a plant class II glycine-rich protein LsGRP1 in plant defense

林家華(Chia-Hua Lin)

指導教授 ： 陳昭瑩

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00547

摘要

水楊酸與百合灰黴病菌誘導性百合防禦相關基因LsGRP1推定解碼一植物第二型富含甘胺酸蛋白。在本研究中，西方墨點檢測與串聯式質譜分析的結果顯示百合應具有14、16與23 kDa之葉部專一性表現的LsGRP1變體，其中14與16 kDa LsGRP1於不同生長時期之表現量均十分相近，但23 kDa LsGRP1的含量卻於老化時期急速衰減。透過免疫組織化學法、強化型綠色螢光蛋白成像與蛋白質萃取分析發現LsGRP1多累積於葉組織之表皮細胞與韌皮部，且14 kDa LsGRP1位於細胞膜而16與23 kDa LsGRP1弱鍵結於細胞壁。此外，水楊酸與百合灰黴病菌所誘導14 kDa LsGRP1與可被泛素抗體所辨識的23 kDa LsGRP1之累積，暗示23 kDa LsGRP1源自14 kDa LsGRP1之單泛素化並與百合誘導性抗病的發生有關，此特性未曾在其他植物第二型富含甘胺酸蛋白被報導。另一方面，大腸桿菌系統無法表現LsGRP1暗示其組成區段可能具有抗菌活性。使用化學合成的LsGRP1蛋白衍生性胜肽探討其抗菌特性，驗證對應富含半胱胺酸之C端片段的LsGRP1C為一兼具有效抗菌活性與廣泛抑菌圖譜之抗菌胜肽。進一步以SYTOX Green、H2DCFDA與DAPI染色探討LsGRP1C之作用機制，發現LsGRP1C經由影響細胞膜完整性與誘發真菌之類細胞程序性死亡以抑制真菌生長。甚者，LsGRP1C的免疫螢光定位聚集於真菌細胞表面，暗示位於植物細胞表面之LsGRP1能夠透過其C端區段之抗菌活性阻礙病原菌的入侵。綜合上述推斷，防禦相關蛋白LsGRP1在百合抵抗灰黴病菌的過程扮演重要角色，且其衍生性LsGRP1C為一具有實際應用潛力之抗菌胜肽。

關鍵字

防禦相關蛋白LsGRP1 ； 雙重次細胞定位 ； 泛素化 ； 植物第二型富含甘胺酸蛋白 ； 富含半胱胺酸抗菌胜肽 ； 膜通透性 ； 類細胞程序性死亡現象

並列摘要

Salicylic acid- and Botrytis elliptica-inducible defense-related LsGRP1 gene in lily presumably encodes a plant class II glycine-rich protein (GRP). In this study, the results of western blot detection and tandem mass spectrometer analysis revealed that three LsGRP1 variants of 14, 16 and 23 kDa expressed in the leaves of lily specifically, and the expression of 14 and 16 kDa LsGRP1 remained a similar level at different growth stages while the amount of 23 kDa LsGRP1 decreased at the senescence stage. As investigated by immunohistochemistry, LsGRP1 was found to accumulate in the epidermal and the phloem tissues of leaves. The subcellular localization assayed by EGFP imaging and protein extraction analysis revealed that 14 kDa LsGRP1 was located in the plasma membrane whereas 16 and 23 kDa LsGRP1 were weakly bound to the cell wall. Additionally, the accumulation of 14 kDa LsGRP1 and ubiquitin antibody-recognizable 23 kDa LsGRP1 was triggered by salicylic acid and B. elliptica, suggesting that 23 kDa LsGRP1 comes from mono-ubiquitinated 14 kDa LsGRP1 and is related to the occurrence of induced resistance in lily. This is a novel trait never reported for other plant class II GRPs. On the other hand, the failure in LsGRP1 expression using Escherichia coli system suggested the presence of antimicrobial activity in certain region of LsGRP1, and LsGRP1C corresponding to the cysteine-rich C-terminal region was considered an antimicrobial peptide according to its broad-spectrum and effective antimicrobial activity as assayed using chemically synthesized LsGRP1-derived peptides. Furthermore, the inhibition effect of LsGRP1C on fungal growth is possibly via alteration of the integrity of cell membrane and induction of programmed cell death-like phenomenon as revealed by SYTOX Green, H2DCFDA and DAPI staining assays. Moreover, immunofluorescence of LsGRP1C present at the outer layer of fungal cells was indicated and implied that plant cell surface-localized LsGRP1 might retard pathogen via the antimicrobial activity conferred by its C-terminal region. Thus, defense-related LsGRP1 playing an important role in the induced resistance of lily against B. elliptica was assumed; in addition, LsGRP1C derived from LsGRP1 is an antimicrobial peptide with a potential for practical use.

並列關鍵字

Defense-related LsGRP1 ； dual subcellular localization ； ubiquitination ； plant class II glycine-rich protein ； cysteine-rich antimicrobial peptide ； membrane permeability ； programmed cell death-like phenomenon

參考文獻

壹、 Characterization of the dual subcellular localization of Lilium LsGRP1, a plant class II glycine-rich protein 86

貳、 A potent antimicrobial peptide derived from the protein LsGRP1 of Lilium 104

口試委員審定書 I

誌謝 II

中文摘要 III