臺灣博碩士論文加值系統

核醣體失活蛋白（RIPs, Ribosome inactive proteins），可抑制目標細胞蛋白質生合成，一般認為與植物防禦有關，但相關研究不多，主要著重在抗癌、抗腫瘤及抗愛滋病毒的藥物研究。目前已發表的苦瓜RIPs多由栽培種的種子分離出來，野生苦瓜（Momordica charantia L. var. abbreviata Ser.）為苦瓜的變種，則沒有關於RIPs的相關研究。本試驗根據葫蘆科的RIPs序列保守區設計引子，以野生苦瓜基因組DNA為模版，擴增的片段（mca600）有609個核苷酸，可演繹出203個胺基酸，包含RIPs活性區域及RNA結合區域，與苦瓜屬RIPs相似度為47.7-58 %，mca600極有可能是由尚未發表的RIPs基因擴增而來。mca600與具有細胞毒性的括樓（Trichosanthin kirilowii）RIPs, trichosanthin相似度高（61.5%），根據記載野生苦瓜成熟種子有毒，可能與RIPs在成熟果實及種子累積有關。將野生苦瓜發育種子分成5級，以mca600為探針進行北方雜合分析，結果顯示發育第至3級種子的表現量最多，然而在種子發芽期間及葉片處理ABA、SA及MJ後皆無表現；mca600探針也無法偵測到栽培種發育第3級種子的基因表現訊息，顯示野生種與栽培種在種子生成的RIPs可能是由不同的基因所控制。
以SDS-PAGE分析發育種子的初抽蛋白，觀察到33kD蛋白開始出現的時間和RIPs基因表現的時間一致，以分子量推測RIPs基因的初級產物約為33kD，因此33kD蛋白可能含有RIPs基因的初級產物，在種子中後期累積，成為儲藏蛋白。發育種子及成熟乾燥種子，沒有觀察到明顯的30kD蛋白累積，但當種子發芽後第0-8天可以觀察到，30kD蛋白可能是由RIPs的33kD蛋白裂解而來。依據本試驗結果及前人研究假設RIPs基因先以未成熟蛋白的形式儲藏在成熟種子，RIPs活性較低，待種子萌發再裂解為30kD的成熟蛋白，活性提升，可能與野生苦瓜苗期早期的抗病力有關。

Ribosome inactive proteins (RIPs) are a group of proteins that inhibit protein synthesis of the target cell. In Cucurbitaceae, RIPs have been identified from the extracts of roots and seeds of different species. Research was mainly focused on their anti-tumor and anti-HIV activity; however, their role in plant physiology was not yet clear. Due to the wild bitter gourd (Momordica charantia L. var.abbreviata Ser.) is much more tolerant to environmental stresses; it is a good material to study for the relationship between RIPs and plant defense. Using a set of degenerate primers, designed according to conserved domain of RIPs in Cucurbitaceae, a 609 bp PCR product was amplified (mca600) from the genomic DNA of the wild bitter gourd. The deduced amino acid sequence contained two RIPs active regions and two RNA binding regions. The similarity of amino acid sequence between mca600 and RIPs in Momordica was 47.7~58 % revealed the possibility that mca600 might amplified from a different RIPs gene as compared with the published RIPs gene in Momordica. Besides, the expression of RIPs gene from any other cultivated bitter gourd could not be detected by the mca600 probe. Therefore, the mca600 might be relevant to a RIP gene different from the published RIPs gene in Momordica. Surprisingly, mca600 shared 61.5% similarity with trichosanthin, a RIPs in Trichosanthes kirilowii. Since trichosanthin is not only anti-tumor and anti-HIV but also toxic to intact animals, the biological character of the RIPs in wild bitter gourd is worth to be investigated. The result of northern blot analysis indicated that the RIP gene we identified was seed specific and expressed in the developing seeds from 14 to 22 DAP. Besides, a 33KD protein appeared on the SDS-PAGE according to the same time course. The predicted size of RIPs is 30 kD, but it did not appeared on the SDS-PAGE until seed germination. According to this study and others’ investigation, we hypothesize that 33kD protein contained the unmature protein of RIPs in wild bitter gourd and it accumulated during middle-post developmented seed. The 33 kD protein would be proteolyted to smaller one, a 30 kD protein, after germination, which could offer nutrient and defence ability to seedling.

縮寫字表 1
中文摘要 2

第一章 前言 3

第二章 前人研究 6
一、核醣體失活蛋白：Ribosome inactive proteins 的活性 6
二、RIPs的三種類型 6
三、RIPs對病原菌的攻擊 7
四、核醣體失活蛋白的分佈情形 8
1. RIPs於生物界的分佈 8
2. RIPs於植物的分佈 8
3. 植物RIPs於各器官的分佈 9
4. 植物RIPs於細胞層次的分佈 9
五、種子發育及發芽後RIPs的表現 10
1. 種子發育期間RIPs基因與活性之表現 10
2. 種子發芽後RIPs基因與活性表現 10
六、RIPs受到環境誘導 11


七、植物RIPs的生合成與攻擊 12
1. RIPs在自身細胞的生合成 12
2. RIPs攻擊目標細胞 13
八、栽培種苦瓜的種子發育及RIPs相關研究： 14

第三章 材料與方法 16
一、 植物材料的準備 16
二、 野生苦瓜RIPs基因探針之製備 17
1. 引子之選擇 17
2. 野生種及栽培種苦瓜基因組DNA之抽取 17
3. 核酸探針取得與序列分析 18
4. 探針的DIG標定 18
三、 RNA之抽取 19
四、 北方氏雜合分析 19
五、 SDS-PAGE(聚丙烯醯胺膠體電泳)分析 20
1. 吸光值線性回歸公式建立與蛋白質濃度的測定 20
2. SDS-PAGE電泳分析及CBR染色 21





第四章 結果 22
一、 mca600序列特徵 22
二、 mca600與苦瓜屬RIPs的胺基酸相似度 23
三、 mca600 與28種植物RIPs的相似度 23
四、 苦瓜蛋白質組成 25
1. 野生苦瓜各部位蛋白質的組成 25
2. 26個苦瓜品系種子蛋白質組成 25
五、 苦瓜種子發育期間RIPs基因表現與蛋白質變化情形 26
1. 野生苦瓜種子的發育 26
2. 野生苦瓜發育種子RIPs基因的表現 27
3. 野生種與栽培種RIPs基因的表現 27
4. 野生苦瓜種子發育時期蛋白質的變化 28
六、 苦瓜種子發芽期間RIPs基因表現與蛋白質變化情形 28
1. 野生苦瓜種子發芽後RIPs基因的表現 28
2. 野生苦瓜種子發芽後蛋白質組成的變化情形 28
3. 栽培種‘瓊一號苦瓜’（品系71號）蛋白質組成的變化情形 29
七、植物生長調節劑對野生苦瓜RIPs基因表現的影響 29




第五章 討論 31
一、 mca600序列分析 31
二、 mca600與苦瓜屬RIPs的胺基酸相似度 31
三、 mca600 與28種植物RIPs的相似度 32
四、 野生苦瓜RIPs基因表現 33
五、 野生苦瓜RIPs蛋白的功能 34

Abstract 36

參考文獻 37

圖表目錄
圖 1. 三種類型RIPs蛋白的初級結構示意圖 42
圖 2. 三種類型RIPs成熟蛋白的結構示意圖 42
圖 3. mca600片段電泳情形與序列。 43
圖 4. mca 600演繹胺基酸序列與α-momorcharin, β-momorcharin、ricin A鏈及b-32胺基酸序列對比結果。 44
圖 5. 植物界3種類型的RIPs的胺基酸序列之間的演化相關性。 47
圖 6. 12.5% SDS-PAGE分析野生苦瓜各部位蛋白質的組成。 48
圖 7. 12.5% SDS-PAGE分析26個苦瓜品系種子蛋白質的組成。 49
圖 8. 野生苦瓜第3、4、5級種子發育的情形以及乾燥種子外型。 50
圖 9. 野生苦瓜RIPs基因於不同成熟度種子的基因表現情形。 51
圖 10. 野生苦瓜果實發育至黃熟期果實生長的變化情形。 51
圖 11. 苦瓜野生種（品系59）及栽培品系17、26、37、108及121的果實外觀。 52
圖 12. 苦瓜野生種（品系59）及栽培品系17、26、37、108及121之3級種子RIPs基因的表現。 53
圖 13. 苦瓜品系17、26、37、59、108及121擴增RIPs基因。 53
圖 14. 野生苦瓜不同成熟度種子蛋白12.5% SDS-PAGE分析情形。 54
圖 15. 野生苦瓜不同時期幼苗生長情形。 55
圖 16. 野生苦瓜不同時期幼苗RIPs基因的表現情形。 55
圖 17. 野生苦瓜不同時期幼苗於12.5% SDS-PAGE分析情形。 56
圖 18. 瓊一號苦瓜（品系71）不同時期幼苗於12.5% SDS-PAGE分析情形。 56
圖 19. 野生苦瓜RIP基因於不同濃度的ABA、SA、MJ處理離體葉片後之基因表現分析。 57

表 1. 苦瓜屬RIPs核酸（胺基酸）序列相似度比對。............................................45
表 2. 參與演化樹狀圖比對的RIPs序列相關資料。 46
表 3.種子的蛋白質組成分析參試的26個苦瓜品系 49
表 4. 苦瓜野生種（品系59）及栽培品系17、26、37、108及121的果實特徵。 52

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