水楊酸誘導葵百合表現 LsGRP1 及累積過氧化氫之分析

Abstract

植物對病原之入侵具有多重防禦機制，包括先天及誘導性抗病。依抗病特性不同，可將植物誘導抗性分為 systemic acquired resistance (SAR)、induced systemic resistance (ISR) 及過敏性反應。本實驗室研究葵百合誘導抗病性時，施以水楊酸可有效地降低灰黴病的發病程度，並選殖葵百合受水楊酸誘導表現的基因，獲得一個全長度 cDNA，編碼一含138個胺基酸的蛋白質(LsGRP1)，富含甘胺酸，N端具有訊息序列，C 端富含半胱胺酸。本研究純化 LsGRP1 重組蛋白，並製備 anti-LsGRP1 抗體，利用此抗體偵測 LsGRP1 及其類似 GRPs 於葵百合上受到水楊酸誘導表現之情形，結果顯示 LsGRP1 相關蛋白質主要分布於表皮及維管束周鞘。LsGRP1 除了富含甘胺酸外，也含有許多酪胺酸，酪胺酸為極性分子，易與其他極性分子產生氫鍵結合；生體外試驗即指出 LsGRP1 可能具有蛋白質間交互鍵結的能力。分析水楊酸誘導葵百合累積過氧化氫情形，發現過氧化氫大量表現的部位與 LsGRP1 相關蛋白質類似；且在水楊酸誘導24 小時，過氧化氫及 LsGRP1 均大量表現。此等結果暗示 LsGRP1及其類似 GRPs於葵百合中可能具有蛋白質間交互鍵結的能力，扮演強化植物細胞壁的角色。

Plant defense mechanism induced by pathogen attack includes systemic acquired resistance (SAR), induced systemic resistance (ISR) and hypersensitive response (HR). In the study of SA-induced disease resistance, a cDNA named LsGRP1 (Lilium ‘Star Gazer’ glycine-rich protein 1) was cloned from Lilium hybrid ‘Star Gazer’. In this study, His-LsGRP1 protein was purified from a Escherichia coli strain harboring recombinant plasmid and used to prepare anti-LsGRP1 antiserum which was used to examine the location of SA-induced LsGRP1 and its homologues in ‘Star Gazer’ leaves. The LsGRP1 and its homologues were mainly localized in the leaf epidermis and vascular bundle sheath cells. The glycine-rich domain of LsGRP1 contains several tyrosine-residues which might cross-link to other Tyr-residues in vivo. In vitro experiments indicated that LsGRP1 might have oxidative cross-linking capability. As shown, SA-induced hydrogen peroxide (H2O2) was accumulated in a similar spatial pattern as LsGRP1 and/or its homologues. Moreover, accumulation of H2O2 and LsGRP1-related mRNA transcript simultaneously increased 24 hr after SA treatment. These results indicated that LsGRP1 and/or its homologues might exhibit cross-linking capability and facilitate the fortification of plant cell walls.