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標題: 缺失性竹嵌紋病毒衛星核酸重組機制之研究
The common possible mechanisms for recombination in defective satellite BaMV RNAs
作者: 賴威延
Lai, Wei-Yen
關鍵字: satBaMV RNA;竹嵌紋病毒衛星核酸;defective satBaMV;recombination;缺失性竹嵌紋病毒;重組作用
出版社: 生物科技學研究所
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竹嵌紋病毒衛星核酸(Bamboo mosaic virus satellite RNA, satBaMV)全長為836個核苷酸之單股正極性核酸分子,依賴其輔助病毒竹嵌紋病毒 (Bamboo mosaic virus, BaMV)進行複製、包被及系統性移動,並可轉譯出P20蛋白。在前人的研究(顏思敏,2008)中,從不同來源的感病竹子中分離到不同的缺失性竹嵌紋病毒衛星核酸(satBaMV defective RNA, D-RNA),在本研究接種實驗中得知缺失性衛星核酸影響BaMV的RNA複製之能力低於野生型衛星核酸F4。而且共同接種野生型衛星核酸F4和缺失性核酸的實驗中得知,這些缺失性核酸在接種初期會影響野生型衛星核酸F4的複製,但在系統葉上這些缺失性衛星核酸並不具干擾輔助病毒或野生型衛星核酸F4複製之特性。這些缺失性核酸中以分離株F4-4-3出現頻率最高,利用Mfold軟體預測其缺失的104個核苷酸序列,可能為一個stem loop的二級結構,同時利用genedoc軟體針對不同來源的竹嵌紋病毒衛星核酸進行序列比對,發現僅在台北植物園麻竹群體之衛星核酸不具有重複性序列GAGATCG且不產生缺失性衛星核酸,因此推測缺失性衛星核酸產生的原因可能為RNA depended RNA polymerase 合成負股核酸時,受此二級結構以及重複序列影響而發生模板重組作用。為了證明以上的推測,於野生型衛星核酸重複性序列內設計不同的核苷酸突變,進行接種實驗,結果得知衛星核酸突變株透過重組機制產生缺失性衛星核酸的效率比起野生型有明顯下降的趨勢。即代表此重複性序列對於竹嵌紋衛星核酸重組扮演關鍵性的角色。本實驗首度在Potexvirus中證實具有缺失性衛星核酸,並預測其可能的重組機制為Similarity-Assisted Recombination,這些結果將有助於了解竹嵌紋病毒衛星核酸的演化過程。

Bamboo mosaic virus satellite RNA (satBaMV) is a linear RNA molecule of 836 nucleotides (nt) which contains an ORF for a 20 kDa protein. It completely dependents on BaMV for replication, encapsidation and systemic movement. In previous study, we had isolated different defective satBaMV RNAs from different sources of natural infected bamboo. The efficacies of defective satBaMV RNAs in affecting the level of BaMV accumulation were less than that of wt satBaMV RNA. SatBaMV D-RNAs had little effect on wt satBaMV RNA accumulation in coinoculated leaves, however, wt satBaMV became the dominant sequence accumulated in systemic leaves. Among the defective satBaMV RNAs, isolate F4-4-3 was cloned most frequently. An 104-nucleotide fragment deleted in F4-4-3 defective satBaMV RNA can fold a stem loop structure as predicted by Mfold software. Similar defective satBaMV RNAs are absent in BaMV isolated from Dendrocalamus latiflorus Munro. SatBaMV therefrom are lack of sequence repeats (GAGATCG). We hypothesize that possible origin of defective satBaMV RNA F4-4-3 is due to the existent stem loop structure and sequence repeats (GAGATCG) resulting in the “ template recombination ” during the synthesis of minus template. In order to demonstrate this hypothesis, we designed different deletion mutants of wt satBaMV RNA to elucidate the required sequences for the recombination of satBaMV RNAs in inoculated and systemic leaves. The results showed that the duplicate sequence GAGATCG plays a crucial role for recombination of satBaMV RNA, which was revealed by the absence of defective satBaMV RNAs in mutated strains. Our studies confirmed the existence of defective satellite RNAs in Potexviruses and established the possible recombination mechanism.
其他識別: U0005-0108200923151000
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