應用基因默化載體進行香蕉與蝴蝶蘭抗病毒基因轉殖之研究

Abstract

香蕉於感染胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)後，會產生發育不良之現象並且會降低產量。喜姆比蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)與齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)為兩種最普遍的蘭花病毒，罹病蘭花的商業價值會因而大幅降低。核酸干擾技術(RNA interference, RNAi)是植物降解病毒RNA之有效的策略。在先前的試驗中，分別選取數區具有高度同源性的CMV、ORSV及CymMV之外鞘蛋白胺基酸序列，構築可以產生25 bp小干擾片段的表現質體，並經由農桿菌轉殖系統以穩定性基因轉殖導入邊沁菸草。經由GUS化學活性染色分析及聚合酶連鎖反應確認後，得到數個轉殖系。以病毒接種轉殖株並依其外表型及反轉錄聚合酶連鎖反應結果分析抗病毒之效率，目前觀察到CMV外鞘蛋白基因默化構築轉殖系NbCR1與NbCR4有顯著的抗病性。ORSV及CymMV外鞘蛋白基因默化構築轉殖株接種ORSV及CymMV後，病徵皆呈現復原狀態，由於野生型邊沁菸草之病徵也呈現復原，接種之病毒濃度需進一步調整。

Once infected with Cucumber mosaic virus, banana became stunted and fruit production was decreased. Cymbidium mosaic virus and Odontoglossum ringspot virus are two most prevalent viruses of orchids .The commercial value of an orchid can be reduced considerably when it becomes diseased. RNA interference (RNAi) is a potent strategy for engineering plants with ability to degrade virus RNA. In previous study, several highly homologus regions of amino acid sequence of CMV, CymMV and ORSV coat protein were used to construct double-stranded 25 bp small interference expression plasmid. Expression plasmids were stably transformed into Nicotiana benthamiana by Agrobacteria-mediated transformation system. Putative transgenic lines were confirmed by GUS histochemical staining and polymerase chain reaction analysis. Transgenic lines were inoculated with viruses and analyzed by phenotype and reverse transcription polymerase chain reaction. Transgenic lines NbCR1 and NbCR4 were observed significantly resistant to CMV. Symptoms of transgenic line expressing ORSV or CymMV coat protein RNA interference construct of Nicotiana benthamiana after inoculation with ORSV or CymMV displayed recovery phenotype. Recovery phenotype also appeared on wild type of Nicotiana benthamiana. Virus concentration for plant inoculation remains to be determined.