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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王俊能(Chun-Neng Wang) | |
dc.contributor.author | Ya-Ting Ke | en |
dc.contributor.author | 柯雅婷 | zh_TW |
dc.date.accessioned | 2021-06-17T06:11:04Z | - |
dc.date.available | 2020-11-16 | |
dc.date.copyright | 2018-11-16 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-11-05 | |
dc.identifier.citation | Albert, V.A., Soltis, D.E., Carlson, J.E., Farmerie, W.G., Wall, P.K., Ilut, D.C., Solow, T.M., Mueller, L.A., Landherr, L.L., Hu, Y., Buzgo, M., Kim, S., Yoo, M.J., Frohlich, M.W., Perl-Treves, R., Schlarbaum,S.E., Bliss, B.J., Zhang, X., Tanksley, S.D., Oppenheimer, D.G., Soltis, P.S., Ma, H., Pamphilis, C.W., and Leebens-Mack, J.H. (2005). Floral gene resources from basal angiosperms for comparative genomics research. BMC Plant Biol 5, 5.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71823 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:11:04Z (GMT). No. of bitstreams: 1 ntu-107-R05b21010-1.pdf: 3826363 bytes, checksum: ce78b6cd88573d9aec4144ee2f30f302 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員會審定書 II
致謝 III 摘要 IV Abstract VI Content VIII List of Figures and Tables X Abbreviations XI Chapter 1 Introduction 1 1.1 Virus-induced gene silencing (VIGS), a functional genetic approach 1 1.2 Cucumber mosaic virus (CMV) 4 1.3 Gesneriaceae 5 1.4 Titanotrichum oldhamii, a bulbiliferous plant with floral reversion 5 1.5 T. oldhamii, a good plant for applying VIGS system to study gene function 8 1.6 A marker gene, PHYTOENE DESATURASE (PDS), for rapid and visual evaluation of VIGS efficiency 9 1.7 Agroinfiltration, an approach for VIGS vector delivery 10 1.8 Aim of this study 10 Chapter 2 Materials and Methods 12 2.1. Plant materials and growth conditions 12 2.2. Molecular study 12 2.2.1. Total RNA extraction 12 2.2.2. First-strand cDNA synthesis 13 2.2.3. Standard polymerase chain reaction (PCR) 14 2.2.4. Agarose gel electrophoresis 16 2.2.5. Quantitative real-time PCR (qPCR) 17 2.3. Construction of pCMV-ToPDS infectious vector 18 2.4 Transformation of Agrobacterium with pCMV-ToPDS infectious vector 18 2.5 Agrobacterium-mediated inoculation 20 2.5.1 Incubation of Agrobacterium containing CMV infectious vector 20 2.5.2 Plant inoculation 21 2.6 VIGS efficacy evaluations 23 Chapter 3 Results 24 3.1 CMV could systemically infect in T. oldhamii 24 3.2 Construction of pCMV-ToPDS161 28 3.3 CMV-ToPDS161 could induce ToPDS silencing in leaves of T. oldhamii 30 3.4 The photobleaching phenotype persisted 21 days in upper non-inoculated leaves 34 3.5 CMV-ToPDS161 could induce gene ToPDS silencing in petals of T. oldhamii 37 3.6 The growth stage of T. oldhamii affected the VIGS frequency 41 3.7 Repeated inoculations did not extend the duration of photobleaching phenotype 45 3.8 The silencing activity and insert stability was not obviously different between vector with short (CMV-ToPDS87) and long (CMV-ToPDS161) ToPDS insert 47 Chapter 4 Discussion 52 4.1 The success of VIGS in T. oldhamii 52 4.2 No photobleaching observed in petals 53 4.3 Young developmental stages receive higher infection rate 55 4.4 The duration of VIGS in T. oldhamii was 3 weeks 56 4.5 The insert size has no difference in inducing silencing activity and insert stability 60 Chapter 5 Future aspects 62 Chapter 6 References 64 Chapter 7 Appendices 73 | |
dc.language.iso | en | |
dc.title | 俄氏草病毒誘導基因靜默系統之建立 | zh_TW |
dc.title | Establishment of virus-induced gene silencing (VIGS) system in Titanotrichum oldhamii | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 葉信宏 | |
dc.contributor.oralexamcommittee | 蔡文杰,鄭秋萍,陳仁治 | |
dc.subject.keyword | 俄氏草,病毒誘導基因靜默,Cucumber mosaic virus (CMV),phytoene desaturase (PDS),白化現象, | zh_TW |
dc.subject.keyword | Titanotrichum oldhamii,virus-induced gene silencing,Cucumber mosaic virus,PHYTOENE DESATURASE,photobleaching phenotype, | en |
dc.relation.page | 78 | |
dc.identifier.doi | 10.6342/NTU201800142 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-11-06 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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