俄氏草病毒誘導基因靜默系統之建立

Establishment of virus-induced gene silencing (VIGS) system in Titanotrichum oldhamii

柯雅婷(Ya-Ting Ke)

指導教授 ： 王俊能

共同指導教授 ： 葉信宏

國立臺灣大學/生命科學院/生命科學系/碩士(2018年)

https://doi.org/10.6342/NTU201800142

摘要

病毒誘導基因靜默(VIGS)是可用於研究基因功能之技術。因Cucumber mosaic virus (CMV)有廣泛的寄主， CMV被應用在很多非模式物種上建立VIGS系統。苦苣苔科的俄氏草(Titanotrichum oldhamii)是一種具有可以研究成花反轉潛能的物種。晚期，俄氏草的花分生組織會反轉為營養性的珠芽並利用珠芽行無性繁殖。先前的研究中已發現許多可能跟成花反轉相關的調控基因，但尚未清楚他們確切的功能為何，因此希望能夠在俄氏草建立VIGS系統來研究基因功能。本研究選取一旦靜默就能清楚且快速看到葉片白化的性狀表現的phytoene desaturase (PDS)基因來測試CMV在俄氏草的VIGS效果。在將攜帶ToPDS基因片段的CMV載體(CMV-ToPDS)利用農桿菌LBA4404接種在營養時期四個月大的植株的葉片21天後，發現新長的葉片有明顯白化的現象產生，藉由qPCR在白化的葉片也偵測到ToPDS的表現量明顯下降，證實了基因靜默效果。葉片白化的效果大約可維持接種後的21到42天。而重複接種CMV-ToPDS並無法延長白之效。將攜帶ToPDS的CMV載體接種在開花時期的葉片和花序基部21天後，花色並沒有任何的改變，但是藉由qPCR仍然發現ToPDS的表現量在花瓣有明顯下降，也可偵測到CMV的存在，因此證實了基因靜默效果在花也有作用。在檢測俄氏草的各個時期有多少比例的植株成功誘發VIGS後，顯示營養時期的50-56%比開花時期的12.5-37.5%有更高的比例能夠成功誘發VIGS，顯示植株越年輕VIGS的效率越好。此外，我們也比較病毒載體攜帶基因片段的長短是否會影響VIGS的效率，結果顯示攜帶較短(87 bp)或較長(161 bp)的ToPDS片段的病毒載體在基因靜默程度並無明顯差異。本研究中，我們成功的在俄氏草建立了VIGS系統，將來可利用此系統進行相關的基因功能研究，而且這也是在苦苣苔科的物種裡第一個成功建立VIGS系統的案例。

關鍵字

俄氏草 ； 病毒誘導基因靜默 ； Cucumber mosaic virus (CMV) ； phytoene desaturase (PDS) ； 白化現象

並列摘要

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.

並列關鍵字

Titanotrichum oldhamii ； virus-induced gene silencing ； Cucumber mosaic virus ； PHYTOENE DESATURASE ； photobleaching phenotype

參考文獻

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