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Transgenic lines of melon (Cucumis melo L. var. makuwa cv. “Silver Light”) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens
|作者:||莫, 貝 伊
|關鍵字:||幾丁質酶真菌病原菌;Agrobacterium-tumefaciens;基因轉殖;農桿菌;東方甜瓜;Cucumis melo;Chitinase;Fungal pathogen;segregation analysis||出版社:||食品暨應用生物科技學系所||引用:||References Adams DJ (2004) Fungal cell wall chitinase and glucanases. Microbiology 150: 2029-2035 Akasaka-Kennedy Y, Tomita K, Ezura H (2004) Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in melon (Cucumis melo L.). Plant Sci 166:763–769 Anderson A, Moore L (1979) Host specificity in the genus Agrobacterium. Phytopathol 69: 320–323 Araki T, Funatsu J, Kuramoto M, Konno H, Torikata T (1992) The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase. J Biol Chem 267: 19944-19947 Ary MB, Richardson M, Shewry PR (1989) Purification and characterization of an insect α-amylase inhibitor/endochitinase from seeds of Jobís tears (Croixlachryma-jobi). 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東方甜瓜 (Cucumis melo L. var. makuwa cv. “Silver Light”）為一重要之水果作物，其主要分佈於熱帶及亞熱帶地區。然而，真菌引起的疾病會造成其產量下降。
本研究將抗真菌蛋白（AFP）與幾丁質酶（CHI）之融合基因轉殖入東方甜瓜中，探討該基因轉殖作物對於真菌枯絲核菌（Rhizoctonia solani）及尖孢鐮刀菌（Fusarium oxysporum）所引起疾病之抗性。轉殖方式乃利用農桿菌LBA4404（Agrobacterium tumefaciens strain LBA4404）行之，該農桿菌含有抗真菌蛋白（AFP）與幾丁質酶（CHI）基因，其啟動子為椰菜嵌紋病毒35S啟動子（Cauliflower mosaic virus, CaMV），篩選標記為新黴素磷酸轉移酶（neomycin phosphotransferase, nptII）基因。
利用農桿菌將pBI121- AFP- CHI基因轉殖入懸浮培養中之東方甜瓜子葉體後，再建立植物培養系統。以聚合酶鏈反應（polymerase chain reaction, PCR）確認基因是否轉殖入。南方墨點法（Southern blot）之結果確認PCR陽性轉殖株之AFP-CHI融合基因確實被轉殖入。RT-PCR分析結果則確認於個別基因轉殖株之AFP-CHI融合基因確實有表現。 西方墨點法（Western blot）分析結果顯示CHI蛋白累積於葉片上。分離分析（segregation analysis）顯示該基因可被遺傳至第一子代（T1）。
The oriental melon (Cucumis melo L. var. makuwa cv. “Silver Light”) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases.
In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. “Silver Light”) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR–positive lines. RT–PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T1 generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.
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