以人工改造之microRNA防治菸草嵌紋病毒屬病毒

Abstract

在植物體內，以基因靜默(gene silencing)的策略來抵抗病原入侵是一種天然又有效的方式。目前學者們在植物體內發現兩種與基因靜默機制相關的小RNA分子，分別為干擾性小RNA分子(small interfering RNA, siRNA)及微小RNA分子(microRNA, miRNA)。以上兩種小RNA分子所引導的基因靜默途徑，經過近幾年的研究已被發展成具有序列專一性的抗病毒策略。本研究中，我們嘗試利用miRNA的基因靜默途徑，使茄科植物具有抵抗四種重要菸草嵌紋病毒屬病毒(tobamoviruses)的能力，此四種病毒包含菸草嵌紋病毒(Tobacco mosaic virus, TMV)、番茄嵌紋病毒(Tomato mosaic virus, ToMV)、菸草微綠嵌紋病毒(Tobacco mild green mosaic virus, TMGMV)以及番椒微斑駁病毒(Pepper mild mottle virus, PMMoV)。首先我們從阿拉伯芥的基因體DNA將miR159a的前驅物片段(precursor miR159a)擴增出來，接著將miR159a的前驅物片段中的miRNA和miRNA*(反意股)序列以tobamovirus病毒RNA複製酶(RNA dependent RNA polymerase, RdRp)、移動蛋白(movement protein, MP)或鞘蛋白(coat protein, CP)的高保守性序列進行置換，以構築帶有一個或兩個人工改造之微小RNA分子(artificial miRNA, amiRNA)的DNA片段，再進一步將amiRNA序列選殖至雙偶載體(binary vector)中。為了能快速了解不同amiRNA序列對抗病毒的能力，我們將這些構築在雙偶載體內的amiRNA以agroinfiltration的方式短暫表現於菸草(Nicotiana benthamiana)上，在處理7到9天後，接種TMV及ToMV並觀察分析。我們發現不同序列的amiRNA能展現出不同程度的抗病能力，分別表現出延遲發病或是病徵減輕的情形。將amiRNA表現載體轉入菸草，並接種TMV進行抗病測試，結果發現在某些轉基因菸草上能觀察到病毒感染的進程較為延緩，使病毒在植物上位葉所造成的病徵較輕微，且能延長植物的存活時間。我們相信此種抗病毒策略能廣泛的被使用在茄科植物上，並且在將來可能成為新穎且受歡迎的植物病毒防治方法。

Gene silencing is a natural and powerful defense strategy in plants. So far, there are two major types of small RNAs have been found in plant: small interfering RNA (siRNA) and microRNA (miRNA). Both of them have been used to develop virus-resistant plants by means of inducing sequence-specific gene silencing and thus suppressing virus invasion. In our study, we tried to employ artificial microRNA (amiRNA) as a control strategy against four tobamoviruses including Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), and Pepper mild mottle virus (PMMoV). We first cloned the precursor miR159a sequence from Arabidopsis thaliana, and then replaced the 21 nucleotides of mature miRNA and the miRNA*(antisense) sequence by viral consensus sequences within RNA dependent RNA polymerase (RdRp), movement protein (MP), and capsid protein (CP) genes. Several different amiRNA constructs containing one or two kinds of viral sequences were prepared and further cloned the amiRNA sequence into a binary vector. To rapidly evaluate the antiviral ability, amiRNA was transiently expressed in Nicotiana benthamiana by agroinfiltration and challenged with TMV and ToMV after 7-9 days. The results showed that the amiRNA with different sequences revealed different degrees of resistance to the viruses. The transgenic plants that expressed these amiRNAs were prepared and then challenged with TMV. The systemic symptoms displayed on the inoculated transgenic plants were delayed, and the plants survived longer than the control inoculated plants. We believe that this antiviral strategy can be used widely in other solanaceous plants, and it may become a new and popular control method for plant viruses in the future.