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標題: 應用胡瓜嵌紋病毒2b基因建構轉基因抗性及2b基因產物製備專一性抗血清
Applying Cucumber mosaic virus 2b gene to construct transgenic resistance and producing specific antiserum against 2b protein
作者: 陳德旭
Chen, De-Shiu
關鍵字: CMV
transgenic resistance
出版社: 植物病理學系所
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摘要: 胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV) 是一種對經濟作物危害甚大的植物病毒,並在許多國家中造成了重大的經濟損失。針對胡瓜嵌紋病毒的危害有許多的防治策略,包含了抗病育種及建構轉基因抗性。與傳統抗病育種相較,建構轉基因抗性所需時間較少,因而成為現今在植物上建構病毒抗性的主流策略。胡瓜嵌紋病毒含有三條主要的核糖核酸,其上大部分的基因都有被直接應用於獲取轉基因抗性的實例,做為後轉錄基因默化抑制子的 2b 基因雖有應用部分序列以人造 miRNA 在轉基因植物中表現的方式構築抗性,卻未有直接應用全長度基因建構抗性的研究。蕃茄的胡瓜嵌紋病毒分離株 (CMV-peets) 藉由簡併式引子對選殖 2b 基因,經與已知的CMV 2b基因的核酸序列比對,可知 CMV-peets 的 2b 基因序列與屬於 IB 亞群的 CMV 之 2b 基因序列有極高的相同度。將 CMV-peets 2b 基因的轉譯架構導入蛋白質表現載體,透過大腸桿菌 (Escherichia coli) 表現出 2b 重組蛋白。以純化的 CMV 2b 重組蛋白製備抗血清,所產生的抗血清可應用於西方轉漬法以偵測同源抗原,但未能於生體內偵測到 CMV-peets 2b 蛋白。另一方面, 構築 5 種包含 2b 基因在內的轉基因構築,包括正向與反向的 CMV 2b ORF (pCMV2b-S 和 pCMV2b-AS)、正向與反向的 CMV 2b ORF 及兩側翼序列 (pCMV2b-EXT-S 和 pCMV2b-EXT-AS)、和 CMV 2b ORF 的反向重複序列 (pCMV2b-IR)。藉由農桿菌 (Agrobacterium tumefaciens) 導入模式植物菸草 (Nicotiana benthamiana) 中以測試應用 CMV 2b 基因建構病毒抗性之可行性。實驗結果顯示,五種構築中 pCMV2b-IR 在 R0 轉基因植株提供最佳的抗病性,其餘四種單一基因構築的載體亦可提供抗性於轉基因植株。
Cucumber mosaic virus (CMV) causes severe damage and economical loss to many commercial crops. Many approaches, including conventional resistance breeding and transgenic resistance, have been developed to confer resistance in plants against Cucumber mosaic virus. Comparing to the time-consuming conventional measures, transgenic approaches are currently the main stream of building resistance against viruses in plants. CMV contains three major RNAs, most genes on these RNAs have been applied to generate transgenic resistance in the literature. The 2b gene has applied to generate transgenic resistance with expressing artificial RNA in plants, however, never applied in the form of full length. In this study, the 2b gene of a tomato isolate of CMV (CMV-peets) was amplified with a degenerated primer pair and the product was cloned. Sequence analysis showed that the nucleotide of the 2b gene of CMV-peets is highly homologous to that of CMV isolates belong to subgroup IB. The 2b open reading frame was introduced into protein expression vector to express 2b recombinant protein in E.coli. The expressed CMV 2b protein was purified and applied to produce a polyclonal antiserum. The produced antiserum was applicable to detect homologous antigen in vitro, but failed to detect CMV 2b protein for immunoblot tests. In the mean time, five constructs consist of 2b ORF in sense or anti-sense orientations (pCMV2b-S and pCMV2b-AS, respectively), 2b ORF with flanking sequences in sense or anti-sense orientations (pCMV2b-EXT-S and pCMV2b-EXT-AS, respectively), and 2b ORF in inverted repeat form (pCMV2b-IR), were transferred into tobacco (Nicotiana benthamiana) mediated by Agrobacterium tumefaciens. Our results indicated all five constructs confer transgenic resistance to plants and the inverted repeat construct has the best efficiency among the five constructs.
其他識別: U0005-0608200822262300
Appears in Collections:植物病理學系



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