Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22969
標題: 木瓜幾丁質酶-抗真菌蛋白轉基因甜瓜之研究
Studies on Transgenic Melon Co-transformed with Papaya Chitinase and Antifungal Protein Genes
作者: 馮臺貞
Feng, Tai-Chen
關鍵字: Chitinase
幾丁質酶抗真菌蛋白
Antifungal Protein
melon
甜瓜
出版社: 生命科學系所
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摘要: 由木瓜中篩選而來的兩種抗真菌蛋白質:其一為chitinase (CpCHI),它能夠分解真菌細胞壁的幾丁質;另一為CpAFP,為植物防禦素(plant defensins)類似的胜肽,會與真菌細胞膜上的受體相結合,造成鉀離子流失及鈣離子大量流入,而達到抑制病原真菌或殺死病原真菌的效果,兩者均具有抗真菌活性但其作用機制不同,已有許多的論文指出將抗真菌蛋白質利用基因轉殖的方式轉殖進入植物中,可以提高植物對於病原真菌之防禦。 本實驗建立了Chitinase-CpAFP共轉殖基因(CHI-AFP)之pBI121轉殖載體開始,藉由農桿菌進行甜瓜之基因轉殖,由含康黴素之共同培養基初步篩選出具有CHI-AFP基因之轉基因株系,經由genomic DNA PCR 、Southern blot、 RT-PCR及Western blot 等分子基因方式的檢測分析顯示共有五個轉基因株系確實有轉入CHI-AFP基因至植株之的基因組中,可以正常轉錄出mRNA,並可正常的表現出CHI-AFP蛋白質。 經由植物組織培養、發根、馴化將此五個轉基因株系培養於溫室中,進行真菌(Rhizoctonia solani)感染之挑戰測試。結果顯示,對照組經接種R. solani四天後,開始有莖倒伏、莖基部乾枯的病徵現象產生,八天後整棵植株幾近完全的枯萎 ,轉基因株系的實驗組中,植株大部分可延緩R. solani病徵的出現,並具有較長的存活期至二十天左右,甚至有四株植株存活超過30天,明顯的延遲病徵發生,對於R. solani之感染具有防禦之能力。
In our previous studies, we cloned cDNAs encoding class IV chitinase (CpCHI) and antifungal protein (CpAFP) from papaya fruit by PCR-based subtractive hybridization. The recombinant CpCHI produced by Escherichia coli can hydrolyte glycol chitin, against the growth of Fusarium oxysporum f. sp. Pisi, and exhibit strong antifungal activity. The recombinant CpAFP produced by Piehia was showed with the anti-fungal activity toward Rhizoctonia solani. Chitinase and antifungal protein inhibit the growth of fungal via different mechanisms: chtininases degrade the chitin in the cell wall of fungal; antifungal proteins, small peptids of plant defensins, bind to the cell memnerane causing Ca2+ ion influx and cell damage. The cDNA encoding the bifunctional antifungal protein, chitinase-antifungal protein co-transformation protein (CHI-AFP), was expressed in transgenic Cucumis melo L. In 5 melon lines, stable integration of the CHI-AFP gene has been demonstrated by genomic DNA PCR and Southern blot. Expression of the CHI-AFP gene in the 5 transgenic melon lines was detected by RT-PCR and Western blot analyses. The performance of the transgenic melons expressing chitinase-antifungal protein co-transformation protein (CHI-AFP) in a R. solani infection assay revealed significantly enhanced protection against fungal attack , compared with the non-transfomation C. melo L.
URI: http://hdl.handle.net/11455/22969
其他識別: U0005-2307200914373600
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