Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

<p>Abstract</p> <p>Background</p> <p>Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as <it>Arabidopsis thaliana </it>do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied.</p> <p>Results</p> <p>Several molecular biology tools were used to isolate flower-specific gene promoters from <it>Oncidium </it>'Gower Ramsey' (<it>Onc</it>. GR). A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in <it>Onc</it>. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (<it>TI</it>) genes (<it>OnTI1</it>, <it>OnTI2 </it>and <it>OnTI3</it>), which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable <it>A. thaliana </it>transformation analyses.</p> <p>Conclusions</p> <p>By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.</p

Generation of Flowers with Novel Phenotypes and Flowering Time in Eustoma Grandiflorum by Using Methods of Biotechnology

本年度之主要工作目標是繼續完成分析已轉殖百合LMADS1，LMADS2及LMADS3及洋桔梗EgMADS1及EgMADS2基因各式構築體之轉殖基因洋桔梗株，觀察其整體性狀及基因表現情形，以期得到具花型及開花時間變異之植株，進而更加確立這三個基因之功能。在轉殖洋桔梗的植株中若出現重要的花型及開花時間之變異，則可立刻進行田間測試，並評估推廣到市場的可能性。並將繼續完成轉殖其他已由洋桔梗選殖之基因EgMADS3及EgSEP3 cDNA全長之sense及antisense之構築體入阿拉伯芥中作功能測試，得到轉基因植株後分析其性狀，接著再將構築體轉殖入洋桔梗中，並初步分析其性狀。另將繼續由洋桔梗及百合中選殖更多的與花形及開花時間相關的MADS box或其他基因，並完成其基因表現之分析，及完成各式轉殖構築體之構築，以利後續轉殖洋桔梗之應用。In this project, transgenic Eustoma grandiflorum plants ectopically expressed various forms of constructs for lily MADS box genes LMADS1, LMADS2 and LMADS3 will be analyzed. In addition, transgenic E. grandiflorum plants ectopically expressed EgMADS1 and EgMADS2 will also be analyzed. The phenotypes observed in transgenic plants will be examined to see if any floral organ conversion or flowering time alteration were generated. Any interested novel flower phenotype observed in transgenic E. grandiflorum will be evaluated for the potential use in the flower market directly. Constructs contained various forms of EgMADS3 and EgSEP3 cDNA will be transformed into Arabidopsis for functional analysis. The phenotypes observed in transgenic plants will be examined to see if any floral organ conversion or flowering time alteration were generated. These various constructs for EgMADS3 and EgSEP3 will also be transformed into E. grandiflorum for further functional analysis and application. In this project, we also plan to clone more MADS box genes associated with flower formation and flower transition from E. grandiflorum and lily for further analyses

Generation of Flowers with Novel Phenotypes in Lily by Using Methods of Biotechnology

In this project, constructs contained various forms of LAG cDNA will be generated and transformed into tobacco and Eustoma grandiflorum for further functional analysis.The phenotypes observed in transgenic plants will be examined to see if any floral organ conversion such as sepal to carpel or petal to stamen were generated.Any dominant negative mutations produced by transforming truncated LAG will also be analyzed.The results will help to confirm the function for LAG.Furthermore, any interested novel flower phenotype observed in transgenic E.grandiflorum will be evaluated for the potential use in the flower market directly.In addition, LAG proteins will be purified from bacterial expression system and used to generate antibody for further Western blot analysis.In this project, we also plan to clone the promoter region for LAG gene using IPCR strategy.The cloned promoter will be fused to report gene such as GUS or GFP and transformed into plants for expression analysis.The constructs described above will be transformed into lily using genegun approach to initiate the functional analysis for LAG in lily.本年度之工作目標將構築完成含LAG基因cDNA之各式構築體, 透過膿桿菌轉殖入菸草及洋桔梗中.並分析轉殖菸草及洋桔梗中LAG基因表現之情形, 及其整體性狀, 觀察是否花器中花萼及花瓣有轉形至雌蕊及雄蕊之形態.另外也將觀察轉含不同缺失之構築體是否會有dominant negative mutantion的出現, 以便說明在阿拉伯芥中得到的結果是否正確及具代表性.所得到的轉基因洋桔梗植株, 可用在時實際市場推廣到用.在LAG基因之表現探討上, 將進一步在細菌中表現及純化LAG之蛋白質, 並製備LAG之抗體, 透過Western blot之方法分析LAG之蛋白質表現與其mRNA之表現之相關性.本年度並將透過IPCR ( inverse PCR )的方式選殖出LAG基因之調控區( promoter ), 選殖出之LAG promoter將來可用來在花器中專一表達任何外來之基因產物, 在植物生產上有極高的應用價值以供將來分析應用.另外我們亦將進行LAG在鐵砲百合中轉殖之工作.我們計畫在本年度中將含LAG的各式構築體透過基因槍的方法先進行一些轉殖測試工作

Manipulation of Flowering Time to Solve the Problem for Production of Rosette in Eustoma Grandiflorum

To face the effects potentially caused by attending WTO, the development and enhancement offlower industry is necessary for Taiwan’s agriculture. In plants, the transition from vegetative toreproductive stage is controlled by endogenous and environmental factors such as age, hormone,day length and temperature. These pathways regulate the expression of FT, SOC1, LFY and AP1to promote flowering. Exposed to low temperature, termed vernalization, activates VIN3 thatrepressed the expression of FLC and FVP, and induced the expression of AGL24 and AGL19 toaccelerate flowering. Eustoma grandiflorum is a popular and important plant in flower market dueto its abundant floral colors, shape and longevity. However, seedlings of E. grandiflorum grownunder high temperature produced rosette that limit its flower production and caused a significantdecrease of its market value. Therefore, to find a way by using biotechnology to solve thisproblem is urgent and definitely will benefit to the agriculture. The purpose of this project is toselect heat-tolerant, non rosette-forming E. grandiflorum through transgenic approach. Full lengthcDNA for EgFT、EgLFY and EgAP1 (EgMADS3/6) will be isolated and characterized from E.grandiflorum. Furthermore, the transgenic system for E. grandiflorum has been successfully setup in our laboratory. Ectopic expression of MADS box genes from orchid (OMADS1) in E.grandiflorum generated phenotype by flowering early. These results revealed the possibility forgenerating heat-tolerant E. grandiflorum through transgenic approach. In the project, EgFT,EgLFY, and EgAP1 (EgMADS3/6) will be transferred into Arabidopsis and E. grandiflorum forfunctional analysis. Transgenic E. grandiflorum will be selected to test the rosette forming at hightemperature. Moreover, EgSVP、EgFLC、EgVIN3 will also be cloned and characterized.Transgenic E. grandiflorum over-expression of EgVIN3 or down regulation of EgSVP、EgFLC byRNA interference will be generated and then examined the rosette forming at high temperature.All transgenic lines of E. grandiflorum shown the repression of the rosette formation in hightemperature will be further characterized, evaluated, and subjected into field test and finally intoflower market. Furthermore, EST library specific for high or low temperature will be constructedfor E. grandiflorum. Genes specifically expressed at high temperature will be sequenced andanalyzed to investigate the clues to rosette formation. Finally, the heat-tolerant, nonrosette-forming E. grandiflorum will be generated.台灣加入世界貿易組織後，花卉產業是台灣農業的發展重點。花卉產業由於政府資源的投入，期望將臺灣轉變成亞太農業生物科技的重鎮。植物由營養期轉變為生殖期的過程受到內在與外在因子的影響，包括植物年紀、荷爾蒙、光照與溫度。這些途徑影響FT、SOC1、LFY 與AP1 的表現，因此促進植物進入生殖時期。而低溫的春化作用造成阿拉伯芥早開花是因為低溫促進VIN3 的表現，VIN3 再經由3 個不同路徑-抑制FLC、FVP 及促進AGL24 或AG19 的表現，因此使阿拉伯芥提早進入生殖期。洋桔梗在日本屬於高級花卉，而台灣在2006 年栽培面積已高達65 公頃，出口價值為2,300 萬，其花型與花色種類多、切花吸水性佳且耐儲運、周年均可供貨，因此是深具市場潛力的新興花卉。洋桔梗在產業栽培上最主要的問題是高溫會造成苗株簇生化 (rosette)，因此延長栽培時間，增加生產成本。所以，培育高溫環境下不會簇生化的品種是洋桔梗育種上的首要目標。本實驗室對洋桔梗開花誘導的研究深感興趣，已選殖出EgFT、EgLFY、EgAP1 (EgMADS3/6)之基因全長。本實驗室亦成功建立洋桔梗的基因轉殖技術，將文心蘭OMADS1 或阿拉伯芥FYF 基因轉殖至洋桔梗中大量表現後，轉基因植物都會出現提早開花的性狀，這些結果對本計劃擬培育克服高溫簇生化的洋桔梗品種有很大的信心。本計劃先將選殖出全長的EgFT、EgLFY 與EgAP1(EgMADS3/6)基因轉殖入阿拉伯芥測試，再轉殖入洋桔梗大量表現後，以高溫環境測試轉基因植物抗簇生化的情形。本計劃同時要選殖洋桔梗春花作用的關鍵基因EgSVP、EgFLC、EgVIN3 等，經由以RNA 干擾技術來抑制EgSVP或EgFLC 基因的表現，或是大量表現EgVIN3 後，將這些轉基因洋桔梗以高溫環境來測試植物簇生化的情況。上述本計畫中所有洋桔梗轉基因植物若通過簇生化的測試實驗，亦即高溫生長下仍正常開花不會簇生化，則可立刻進行田間測試，並經評估後推廣到市場。本計劃同時要將高溫 (33/28℃) 與涼溫 (23/18℃) 培育之洋桔梗小苗進行EST library 之建立，經由序列分析在高溫育苗時特異表現之cDNA clones 後，選殖春花作用相關的基因或其他造成簇生化的基因，來深入研究洋桔梗簇生化的分子機制。最終亦將這些基因轉殖到洋桔梗中進行簇生化的測試實驗，培育出高溫不簇生的洋桔梗