香蕉抗萎縮病及ACC氧化酶基因默化轉殖株分析

Analysis of Transgenic Banana Plants with Enhanced Resistance against Banana Bunchy Top Virus or with Reduced Biosynthesis of Ethylene through silencing of 1-Aminocyclopropane-1-Carboxylate Oxidase Genes

張紹強(Shao-Chiang Chang)

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(2011年)

https://doi.org/10.6342/NTU.2011.10041

摘要

香蕉萎縮病是一種在熱帶及亞熱帶地區香蕉普遍發生的疾病，患病植株矮化，葉片叢生於假莖頂端成掃帚狀，葉柄、葉脈及中肋有深綠色斷續之線條，罹病蕉株往往不能抽穗而影響果實生產。本研究室先前以農桿菌媒介法轉殖香蕉萎縮病病毒之W2 DNA內之Rep基因反義序列，經抗生素篩選後得到轉殖植株，這些轉殖香蕉並經病毒接種試驗顯示具有抗病性。
本研究以南方氏雜交分析抗萎縮病香蕉轉殖株，將轉殖株分為六群。為了瞭解轉殖株T-DNA插入染色體的位置，以IPCR (Inverse polymerase chain reaction)與APCR (Anchored polymerase chain reaction)分析轉殖株邊界序列，分別獲得四段左邊界序列與六段右邊界序列。經序列比對分析發現邊界序列額外帶有部分農桿菌之Ti Plasmid序列，並且在邊界保守序列有缺失之現象。所獲得之邊界序列可作為轉殖株之分子標誌。
為了探討抗萎縮病轉殖香蕉之抗病機制，經抽取轉殖香蕉Total RNA，並濃縮小分子量RNA，進行小片段RNA北方雜交分析。結果顯示轉殖株具有與轉殖之反義序列互補之siRNA序列。進一步接種香蕉萎縮病毒後，進行小片段RNA北方雜交分析，發現接種病毒後具有較高之siRNA累積。推斷抗萎縮病轉殖株之抗病機制，係經PTGS (post-transcriptional gene silencing)默化了病毒W2 DNA內的Rep基因，而達到抗病之效果。
此外本研究並分析了香蕉ACC氧化酶默化轉殖株之邊界序列，獲得一段於Mh-ACO2默化轉殖株之右邊界序列，將可作為轉殖株之分子標誌。

關鍵字

抗病毒 ； ACC氧化酶PTGS ； siRNA ； Flanking sequence

並列摘要

Banana bunchy top disease is a serious disease in tropics caused by Banana bunchy top virus (BBTV). The symptoms includes dwarfism in plants and clustering of leaves at the top giving a broom-like appearance on pseudostem. The veins and petioles turn dark green. Infected banana plants often do not bear fruits. In our research, we transformed the antisense sequence of BBTV additional Rep gene to banana to produce transgenic banana. The transgenic banana plants obtained exhibited resistance against BBTV.
To exploit the flanking sequences as molecular markers, IPCR (Inverse polymerase chain reaction) and APCR (Anchored polymerase chain reaction) analyse were carried to determine the border sequences of transgenic lines. Six right border sequences and four left border sequences were obtained. The sequence alignment analysis showed that some additional Agrobacterium Ti plasmid sequences were present and some conserved sequences were missing in the border sequences. The flanking sequences of the transgenic plants can be used as molecular markers.
To elucidate the mechanism of BBTV resistant transgenic banana lines, we used northern blot to determine the accumulation of the small RNA population. The results showed siRNA sequence complementary to transgene accumulated in the transgenic lines. Additionally, infection by BBTV was found to induce the higher siRNA accumulation when compared to un-infected plants. It could be inferred that the resistance mechanism is through post-transcriptional gene silencing of the Rep gene within the BBTV.
Additionally, in this study the flanking sequences of the transgenic banana lines silenced for ACC oxidase gene were determined. In Mh-ACO2 silencing banana line we got a right border sequence that could be employed as molecular markers.

並列關鍵字

Virus resistance ； ACC oxidase ； PTGS ； siRNA ； Flanking sequence

參考文獻

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