Molecular Cloning and Expression of a Sweet Potato Cysteine Protease SPCP1 from Senescent Leaves

本研究報告從甘藷老化葉片分離出一條全長的cDNA稱之SPCP1，SPCP1其ORF含有1020個核苷酸（339氨基酸）且與不同物種包括赤楊、阿拉伯芥、紫雲英、油菜、胡蘿蔔、棉花、大麥、鳶尾、苜蓿、菸草、水稻、蓖麻、及白花三葉草等類似木瓜酶的半胱胺酸蛋白分解酶胺基酸序列具有高相似性（約58%至74%）。半定量RT-PCR及西方墨點雜交結果顯示SPCP1在自然老化及黑暗、ethephon、及ABA誘導的老化葉片中會被活化及增加表現量，然而於成熟的綠色葉片、莖、及根中SPCP1並無顯著增加。葉片老化過程中葉綠素開始降解的時間比SPCP1基因表現早。1 mM ethephon處理對於甘藷懸浮培養細胞亦會誘導SPCP1的表現，Evan blue染色結果顯示ethephon處理濃度高至2 mM時對懸浮培養細胞亦無顯著影響，然而10 mM濃度時大部份的細胞會死亡。依據這些實驗結果我們結論甘藷SPCP1為有功能的老化相關基因，其mRNA及蛋白質的表現於天然的或誘導的老化葉片及懸浮培養細胞皆顯著增加，SPCP1的功能與角色可能與老化過程中葉綠素開始降解及細胞死亡無關。

關鍵字

半胱胺酸蛋白分解酶； Ethephon ；葉片老化； SPCP1 ；甘藷

並列摘要

In this report a full-length cDNA, SPCP1, was isolated from senescent leaves of sweet potato (Ipomoea batatas (L.) Lam). SPCP1 contained 1020 nucleotides (339 amino acids) in the open reading frame, and exhibited high amino acid sequence homologies (ca. 58% to 74%) with papain-like cysteine proteases of Alnus glutinosa, Arabidopsis thaliana, Astragalus sinicus, Brassica napus, Daucus carota, Gossypium hirsutum, Hordeum vulgare, Iris hollandica, Medicago truncatula, Nicotiana tabacum, Oryza sativa, Ricinus communis, Trifolium repens. Semi-quantitative RT-PCR and Western blot hybridization showed that SPCP1 gene expression was enhanced significantly in natural senescent leaves and in dark-, ethephon-, and ABA-induced senescent leaves, whereas, was almost not detected in mature green leaves, stems, and roots. Initiation of chlorophyll degradation is earlier than the SPCP1 gene expression during leaf senescence. SPCP1 expression was also induced in sweet potato suspension cells treated with 1 mM ethephon. Evan blue staining showed that suspension cells were not significantly affected by ethephon treatment up to 2 mM, however, most of the cells died when treated with 10 mM ethephon. In conclusion, sweet potato SPCP1 is likely a functional, senescence-associated gene and its expression levels were significantly enhanced at mRNA and protein levels in natural and induced senescent leaves and suspension cells. The physiological role and function of SPCP1 were likely not in association with initiation of chlorophyll degradation and cell death during senescence.

並列關鍵字

Cysteine protease ； Ethephon ； Leaf senescence ； SPCP1 ； Sweet potato

國際替代計量

Molecular Cloning and Expression of a Sweet Potato Cysteine Protease SPCP1 from Senescent Leaves

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