| Summary: | 碩士 === 國立臺灣大學 === 生命科學系 === 107 === Virus-induced gene silencing (VIGS) is an efficient tool for gene function analysis in non-model plants lacking transformation platform. Titanotrichum oldhamii (Gesneriaceae) is a potential model plant for the study of floral reversion. Floral reversion is a process that allows the flower meristem to revert to vegetative bulbils in late flowering stage enabling asexual reproduction post flowering. Previous studies found the expression of some floral identity genes were associated with the transition of flowers to bulbil formation in T. oldhamii. However, whether these genes really involved in floral reversion remained elusive. In this study, we developed a CMV-based VIGS system for gene functional analysis in T. oldhamii. CMV viral vectors containing T. oldhamii phytoene desaturase (ToPDS) gene fragment were agroinfiltrated with Agrobacterium strain LBA4404 into leaves of T. oldhamii. The newly emerged leaves showed photobleaching, and down-regulation of ToPDS was confirmed through qRT-PCR analysis. The photobleaching persisted for 21-42 days post inoculation in T. oldhamii, and the repeated inoculations did not extend the photobleaching period. The inoculation of CMV vector containing ToPDS fragment in the leaves or the base of inflorescence also caused down-regulation of ToPDS in petals but color fading was not observed. After testing the VIGS frequency (the percentage of CMV-ToPDS infiltrated plants showing ToPDS down-regulation) in four growing stages of T. oldhamii, it was found the VIGS frequency was higher in vegetative stage (50-56%) than in flowering stage (12.5-37.5%). The different insert sizes of CMV viral vectors carrying 87 bp or 161 bp ToPDS insert were also compared. The results showed the 87 bp and 161 bp insert delivered similar silencing extent. In conclusion, CMV-based VIGS system worked in T. oldhamii. This VIGS protocol not only can be used to study the function of interesting gene in T. oldhamii in the future, but also serves the first successful VIGS case within somewhat 3200 Gesneriaceae species.
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