胡瓜嵌紋病毒外鞘蛋白基因默化載體之構築及分析

Construction and Analysis of Silencing Plasmids Specific for Cucumber mosaic virus Coat Protein Gene

王以寧(En-Ning Wang)

指導教授 ： 黃鵬林

共同指導教授 ： 杜宜殷(Yi-Yin Do)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10289

摘要

核醣核酸干擾技術（RNA interference, RNAi）是默化基因的策略之一，由雙股RNA（dsRNA）誘導同源目標基因產物mRNA的降解作用。因此，應用核醣核酸干擾技術可作為植物抗病毒的轉殖策略。本試驗以胡瓜嵌紋病毒(Cucumber mosaic virus, CMV) 外鞘蛋白 ( coat protein, CP)基因作為RNAi抗病策略之目標默化基因。經比對CMV CP DNA序列，分別將保守性序列25 bp共5區和基因全長657 bp進行RNAi質體之構築。其構形為2x 35S CaMV啟動子、反義序列、香蕉ACC氧化酶基因 (1-aminocyclopropane-1-carboxylate oxidase, MAO1) 第一個隱子、順義序列和35S poly A 終結子。經聚乙二醇 (polyethylene glycol, PEG) 轉殖法將CMV CP報導質體分別與各默化質體共轉殖至蝴蝶蘭花瓣原生質體，以Real-time polymerase chain reaction進行CMV CP基因默化分析。其結果顯示，短片段默化質體piCR4和全長默化質體piCAS確實干擾並降低原生質體內CMV CP RNA含量，默化效率為30﹪。另一方面，藉由農桿菌媒介共培養轉殖法，將各默化質體穩定性轉殖至香蕉北蕉 (Musa AAA cv. Pei Chiao) 和邊沁菸草 (Nicotiana benthamiana) 。植株再生後，經GUS活性組織化學染色分析，各轉殖試驗事件獲得為數不等之擬轉殖株。邊沁菸草擬轉殖株DNA經專一性引子對進行PCR檢測，以及南方氏雜交分析，確定為轉殖株，此等植株可供日後接種CMV進行抗病試驗，以進一步瞭解轉殖株對CMV之抗病性。

關鍵字

胡瓜嵌紋病毒 ； 核糖核酸干擾技術 ； 基因轉殖 ； 基因默化

並列摘要

RNA interference (RNAi) in eukaryotic organisms is an effective strategy for gene silencing. It is involved in sequence-specific degradation of targeted RNA by RNA inducing silencing complex. RNAi makes a new strategy possible to create virus-resistance transgenic plants. In this study, we have selected five conserved regions, 25 bp in length, from Cucumber mosaic virus coat protein (CMV CP) nucleotide sequences and the full length coding region (657 bp) for construction of silencing vectors as effector plasmids. The constructs contuined including 2x 35S CaMV promoter, sense sequence, banana 1-aminocyclopropane-1-carboxylate oxidase gene (MAO1) intron 1, antisense sequence and 35S poly A terminator. Reporter plasmid encoding CMV CP was co-transformed with various effector plasmids via polyethylene glycol into protoplasts isolated from Phalaenopsis petals. The total RNA from transformed protoplasts was extracted for real-time polymerase chain reaction assay to identify the silencing efficiency. The CP RNA was reduced by 30% for the fourth homologous region (piCR4) and the full length silencing plasmid (piCAS). On the other hand, the effector plasmids were stably transformed into banana (Musa AAA cv. Pei Chiao) and Nicotiana benthamiana by Agrobacteria-mediated transformation system. Genomic DNA from regenerated transgenic Nicotiana benthamiana were extracted individually, and polymerase chain reaction and Southern analysis were used to confirm the transgenic plantlets.

並列關鍵字

Cucumber mosaic virus ； RNA interference (RNAi) ； gene transformation ； gene silencing

參考文獻

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