- 學位論文
探討青花菜冷休克蛋白CSPs與RNA結合蛋白GR-RBP2基因及其表現
Exploration of Cold Shock Domain-Containing Proteins and Glycine-Rich RNA Binding Protein 2 Genes in Brassica oleracea var. italica and Investigation of Their Expressions
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摘要
青花菜 (Brassica oleracea var. italica) 採收後於20℃儲放2至3日即老化並失去商品價值。首先分析青花菜之蛋白質體，發現RNA結合蛋白GR-RBP2 (glycine-rich RNA binding-protein 2) 可能與青花菜採後老化相關，此外，除了GR-RBP2外，冷休克結構域蛋白 (cold shock domain containing-protein，CSP) 也同為富含甘胺酸之蛋白質 (glycine-rich protein)，兩者於阿拉伯芥被證實具有RNA伴護蛋白 (RNA chaperone) 活性且其表現皆受低溫所誘導，因此該兩種蛋白質可能皆參與青花菜老化過程之生理反應。另一方面在採後處理技術中，冷休克處理 (cold shock treatment，CST) 被發現能夠延緩部分蔬果老化，故假設CST造成之低溫能誘導此類蛋白質產生，以穩定mRNA結構及轉譯進而延緩採後老化。本研究欲分析青花菜CSP及GR-RBP2與老化之關聯性，並分析其表現是否受CST所誘導，以說明CST誘導CSP及GR-RBP2表現而延緩採後老化之可能性。利用次世代定序建立青花菜轉錄體資料庫，其中45,051個特定基因 (unigenes) 分別有6個註解到CSP基因 (BoCSPs)，2個註解到GR-RBP2 (BoGR-RBP2s)。將青花菜小花貯藏於20℃下3天、5℃及0℃下15天，以青花菜花苞葉綠素損失率作為老化指標，分析其與上述8個基因表現量之相關性。結果顯示BoCSP1_1、BoCSP3、BoCSP4_1、BoGR-RBP2_1及BoGR-RBP2_2與老化呈現負相關。將青花菜小花浸泡0℃冰水浴30分鐘進行CST後放置於5℃貯藏，並於第0、16、24、40及48小時收樣，分析BoCSPs與BoGR-RBP2s之表現量是否受CST所誘導，結果顯示BoCSP2、BoCSP3、BoCSP4_1及BoCSP4_2表現量可受CST所提升。前述兩項實驗結果顯示，BoCSP3與BoCSP4_1與老化呈負相關，且其表現量受CST所提升，說明冷休克處理可能經由誘導CSP之產生，得以延緩青花菜老化。未來可深入探討其受CST調控之訊息傳遞路徑及其於採後生理之作用機制。
並列摘要
Storage life for broccoli (Brassica oleracea var. italica) is only 2 to 3 days at 20℃. Results in proteomic analysis of senescing broccoli suggesting its involvement in postharvest broccoli senescence. In Arabidopsis thaliana, GR-RBP2 and cold-shock domain-containing protein (CSP), another cluster of glycine-rich protein superfamily, were found to have RNA chaperone activity and to be cold-inducible. On the other hand, cold shock treatment (CST) was reported to delay the postharvest senescence of various horticultural crops, indicating that expression of cold-inducible GR-RBP2 and CSPs might be induced by CST. The induction of CST thus resulted in the stabilization of mRNA and translation and further delayed the postharvest senescence. In this study, correlation among broccoli senescence and expressions of GR-RBP2 and CSPs was analyzed, and the expressions of these genes after CST were also investigated to reveal the possibility that CST induced the expression of GR-RBP2 and CSPs which could further delay postharvest senescence. There were 6 broccoli CSPs (BoCSPs) and 2 broccoli GR-RBP2s (BoGR-RBP2s) found within the established broccoli transcriptome data base containing 45,051 unigenes. Expression of BoCSPs and BoGR-RBP2s in broccoli florets stored at 20℃ for 3 days and at 5℃ and 0℃ for 15 days were determined. Furthermore, correlation between the gene expressions and total chlorophyll loss, the senescence indicator, was analyzed to explore genes that might be related to floret senescence. BoCSP1_1, BoCSP3, BoCSP4_1, BoGR-RBP2_1, and BoGR-RBP2_2 were found to be negatively correlated to senescence. To explore CST-inducible BoCSPs and BoGR-RBP2s, floret was treated with CST and stored at 5℃ and sampled at 0, 16, 24, 40, and 48 h. The results showed that CST could lead to enhancement in the expression BoCSP2, BoCSP3, BoCSP4_1, and BoCSP4_2. Among these genes, BoCSP3 and BoCSP4_1 were found to be negatively correlated with senescence and CST-inducible, which revealed the possibility that CST induced the expression of CSPs which could further delay postharvest senescence. Further researches could be conducted to reveal the signal transduction pathway of CSP in CST and the role of CSP in postharvest physiology.
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