海芋potyvirus之基因體序列分析、快速檢測及應用RNAi防治其感染之研究

Abstract

中文摘要 海芋為天南星科，馬蹄蓮屬之多年生球根花卉。為廣受喜愛之觀賞花卉，因此也成為國際間重要之經濟花卉作物。海芋原產非洲，引進台灣已超過十年，除了組織栽培技術已開發成熟外，也培育出許多新品系。然而病毒病害防治仍為彩色海芋培育之重點。目前被報導過可感染海芋之植物病毒已有9屬18種。其中長絲狀病毒之Potyyvirus是最早被報導，且已知可感染之病毒種類較多的一屬。Potyvirus為單鏈正意股RNA病毒，其基因體大小約10 kb。而在台灣以Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV)以及Zantedeschia mild mosaic virus (ZaMMV) 和Zantedeschia mosaic virus (ZaMV)等四種potyvirus為感染海芋之重要病毒。由於Potyyvirus屬病毒為目前已知數量最多之植物病毒，為能便於檢測及了解其分子特性，因此首先發展能快速選殖及分析potyvirus全長度基因體序列之策略。此方法應用Potyvirus屬之多組廣效性引子對進行RT-PCR並配合terminal transferase修飾病毒5’端序列，以RT-PCR獲得病毒5’端序列，經解序及序列分析後可獲得病毒之正確全長度序列。廣效性引子對之設計則是利用目前已知病毒之胺基酸序列進行多序列排倂分析，分別選取選取NIb、CI及HC-Pro基因之高保守性區域設計出NIbF1、CIF2/CIR2及HCF4/HCR4等引子。以此廣效性引子對分別對七種不同之potyvirus進行RT-PCR測試，可獲得預期大小之PCR產物。利用此策略成功獲得兩種海芋重要病毒ZaMMV及ZaMV之5'端及全長序列。經序列分析後發現ZaMV與Konjac mosaic virus (KoMV)應為同種病毒。另一方面，為能快速且同時檢測DsMV、TuMV、ZaMMV以及ZaMV四種病毒，利用病毒專一性引子對研發多引子對RT-PCR檢測法，並加入植物之nad5 mRNA之專一性引子對，作為增幅植物樣品的內在對照。經由專一性及靈敏度測試後，發現此方法可在一次反應中成功的偵測出不同病毒，且其靈敏度高於利用抗體進行之酵素連結抗體免疫法(ELISA)二十五倍以上。除檢測方法開發外，本論文亦希望研究利用基因沉寂(RNA silencing)機制引發植物對入侵病毒的抗性作用。同樣以DsMV、TuMV、ZaMMV以及ZaMV四種病毒為對象。分析其胺基酸序列中高保守性區域，依照TuMV序列設計數種專一性引子對，藉由PCR將不同長度之HC-Pro, NIa及CP基因片段擴增並選殖至LITMUS 38i載體，用以產生病毒雙股RNA (dsRNA)。當以dsRNA與TuMV RNA共同接種於Nicotiana benthamiana植物，可得知較短之dsRNA片段對於病毒侵入之干擾效果較長片段為差。為進一步在植物上進行分析，將兩段分別位在HC-Pro及NIa基因，且感染效果較佳之病毒片段，利用農桿菌轉型方式(Agrobacterium-mediated infiltration)在植物上表現其雙股之hairpin RNA (hpRNA)。除了有效的干擾TuMV感染外，似乎對於另一種potyvirus，Bean yellow mosaic virus (BYMV)也稍具干擾性。因此將上述兩段含hpRNA之載體送入菸草，並獲得轉基因菸草。對於T0及T1植物初步分析可發現，80%以上的hpRNA轉基因菸草皆對TuMV感染具有抗性。

Abstract Calla lily (Zantedeschia spp.), belonging to the family Araceae, are perennial bulbous flowers. Because calla lily is a favorite ornamental flower, it becomes an important economic flower crop worldwide. Calla lily original from Africa, and have been introduced into Taiwan more than 10 years. Tissue culture technique has been developed for calla lily propagation and many calla hybrids have been bred in Taiwan. However, the viral disease control is important in calla lily cultivation. There are already 18 viruses have been reported which belonging to 9 genus. Potyirus is first reported and mainly calla lily-infecting virus in the field. Potyvirus is a positive sense stranded RNA virus with ~10 kb genome. Four viruseses are important in Taiwan, including Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Zantedeschia mosaic virus (ZaMV), and Zantedeschia mild mosaic virus (ZaMMV). For detection and molecular characterization of potyviruses, the largest plant virus, full-length cloning and sequencing strategy was developed. The RT-PCR-based methods for detection and identification of virus are based on the use of degenerate primers for RT-PCR amplification, combined with modified 5’RACE by using terminal transferase to modify the 5’ end sequence of potyviruses genome. The complete sequence would be identified by cloning and sequencing. The degenerate primers were designed from conserved sequences in the viral genome. According to the sequence alignment, theNIbF1, CIF2/CIR2, and HCF4/HCR4 were designed from potyviral NIb, CI and HC-Pro-coding regions. Expected PCR products were amplified by these primers from seven potyviruses. Complete genome sequences of ZaMMV and ZaMV, were successfully characterized. The sequence analysis reveals that ZaMV is the same with the Konjac mosaic virus (KoMV). On the other hand, in order to save time and simultaneous detection of DsMV, TuMV, ZaMV and ZaMMV in field, a multiplex RT-PCR assay was developed for these calla potyviruses. Specific primers for each virus were designed based on the sequences of 3’ terminal region of respective viruses. To prevent the false negative results, a primer pair specific to plant mitochondrial nad5 mRNA was used as an internal control of RT-PCR. After specific and sensitivity test, the multiplex RT-PCR can rapidly detect multiple targets in one single assay, and the detection sensitivity of multiplex RT-PCR was 25-625 times higher than that of I-ELISA depending on the virus. Furthermore, a control method that prevents the virus infection using the mechanism of RNA silencing was investigated. A dsRNA expression and screening system was used to obtain highly efficient interference fragments from the consensus regions of four calla lily-infecting potyviruses, DsMV, TuMV, ZaMMV and ZaMV. The viruses were chosen for multiple sequence alignment. Several TuMV specific primers were designed within the conserved regions of HC-Pro, NIa and CP genes. Different fragments were PCR amplified and cloned into LITMUS 38i vector to produce viral dsRNA. Mechanical inoculation of different dsRNA transcripts with target virus on tobacco plants induced different levels of interference with virus infection. Two of the most effective fragments (located in HC-Pro and NIa genes) were further analyzed by using Agrobacterium tumefaciens infiltration (agoinfiltration) as transient expression system for hairpinRNA (hpRNA) expression. The successful interferences of TuMV infection were observed in the infiltrated leaves. Besides, Bean yellow mosaic virus (BYMV), another calla lily-infecting virus, was interfered by TuMV hpRNAs transiently expressed in plants. Several lines of transgenic Nicotiana benthamiana plants transformed with hpHC and hpNIa fragments were obtained. Over 80% of T0 and T1 transgenic plants revealed resistance response to TuMV infection.