野生苦瓜RIPs基因序列分析與表現

Analysis and Expression of the RIPs Gene in Wild Bitter Gourd ( Momordica charantia L. var. abbreviate Ser. )

黃明雅(Ming-Ya, Huang)

指導教授 ： 楊雯如

共同指導教授 ： 王自存(Tsu-Tsuen Wang)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.01777

摘要

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。

關鍵字

野生苦瓜 ； 核醣體失活蛋白基因 ； 序列相似度 ； 發育種子 ； 種子蛋白

並列摘要

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.

並列關鍵字

wild bitter melon ； RIPs gene ； sequence similarity ； developing seeds ； seed protein

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