香蕉條紋病調查與細胞病理學觀察及感染系統之建立

Abstract

香蕉條紋病毒(Banana streak virus, BSV)在香蕉產業上逐漸成為嚴重威脅，其特色在於其基因體可嵌入寄主基因體中，並且序列在活化後，可造成感染。本論文由香蕉研究所內具重要經濟性品種香蕉中檢驗出27個未被BSV感染之品種，在未來可用於無毒種苗的生產。並在3株含有B genome之香蕉並未檢測出含有BSV序列，與之前所報導BSV會分佈於B genome之香蕉並不一致。另外，以田間種植之北蕉、呂宋蕉、假呂宋蕉等為對象，調查香蕉條紋病之分佈。BSV在台灣分佈調查顯示Cavendish (cv. Pei Chiao, AAA)品種感染此病毒的比例極低，在130棵植株當中只有五棵有偵測到BSV，但皆無典型病徵表現。然而在呂宋蕉(Silk, Latundan, AAB)或假呂宋蕉(Silk, Mysore, AAB)中，BSV發生率極高，根據樣本病徵型態觀察中，只有一株呈現典型病徵，此與先前報導BSV序列在B genome香蕉中可嵌入寄主基因體但並未活化感染是一致的。然而目前仍然缺乏大規模快速且準確的方法可用於田間調查，來區別嵌入型與活化型之BSV，因此，本實驗根據非活化型病毒其RNA可能無法被寄主轉錄製造，發展利用RT PCR方法來進行快速檢測。為了觀察BSV所造成的細胞生理學上的變化，亦進行超薄切片，並以穿透式電子顯微鏡觀察，結果發現不論BSV活化或非活化，PCR測得病毒訊號之葉片，其葉脈附近的伴細胞中細胞核有膨大現象，且葉肉細胞中葉綠體所含之澱粉粒體積所佔比例亦較正常葉綠體中來的大。將實驗室所得之分離株進行基因體全長定序，與NCBI所發表之各BSV分離株進行比對，其序列與Banana streak mysore Virus相似(99% identity)。欲了解BSV之基因的功能，嘗試建立包含1.26倍BSV基因體全長之感染系統，接種後兩個月尚未出現BSV典型條紋病徵，以PCR檢測，只在接種葉上有測得BSV之訊號，因此，此感染系統尚有須改進及檢討之處。

Banana streak virus (BSV) is becoming a serious threat to banana industry. One character of BSV is that its sequence can integrate into the host genome and the episomal infection may occur after the activation of the BSV integrant. In this study, 27 out of 50 economically important banana collected in Taiwan Banana Research Institution were screened to be free of BSV by PCR detection and could be used for breeding programs. Three B genome containing bananas were BSV free which is inconsistent with previous studies that all B genome bananas contain BSV genome. Field survey in Taiwan showed that the incidence of BSV in cv. Cavendish was relatively low. Only five out of 130 cv. Cavendish contained BSV sequence and no visible symptoms were observed. In B genome containing cultivars, a high incidence of BSV was detected but only one sample showed typical streak symptoms. The result is in accordance with the prediction that the BSV integration infection occurred in B genome bananas. However, lacking of a rapid detection method that can be used in high throughput screening to differentiate integrated form and episomal form of BSV, we are not sure that how many BSV positive bananas contain episomal BSV infection. To overcome the problem, based on the report that the integrated DNA is highly methylated thus the transcription of mRNA may be inhibited, a RT-PCR method to examine the existence of RNA of integrated BSV was developed. In addition, cytopathological observation was conducted to understand ultrastrucural changes in banana leaves caused by BSV. Interestingly, in both BSV episomal infection and integrated in genome leaves, the nuclei of the companion cells enlarged. Enlargement of starch grains also appeared in the chloroplast of these plants. To reveal the phylogenetic relationships of BSV isolated in Taiwan, the complete genome of Taiwanese isolate was sequenced. Sequence analysis revealed that it shares highly sequence identity (99%) to Banana streak mysore virus. For further study of the gene function of BSV, an infectious clone containing 1.26 copies of BSV complete genome were constructed. However, the clone could not infect cv. Cavendish successfully.