Browsing by Subject "VIGS"

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  • Liu, Yanbo (Helsingfors universitet, 2011)
    Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.
  • Zhao, Yafei (Helsingfors universitet, 2013)
    The transition from vegetative growth to flower formation is especially crucial for the reproduction of flowering plants. This transition is controlled through the regulatory activities of a group of genes named as floral meristem identity genes, of which LEAFY (LFY) is thought as the most important one. As a plant-specific transcription factor, LFY controls flower formation and floral patterning, which has been most intensively studied in the model annual plant Arabidopsis. In contrast to the plant architecture and flower morphology in Arabidopsis, Gerbera (Gerbera hybrida), belonging to the large sunflower family (Asteraceae), processes head-like inflorescences with different types of flowers distinct in floral morphs, sex and sometimes coloration. Within the last decades, a number of MADS-box and TCP transcription factor genes have been functionally characterized using stable transgenic plants. Recently, another functional assessment method using virus-induced gene silencing (VIGS) has been developed in Gerbera hybrida. In this study, the expression pattern of GhLFY was analyzed in wild-type Gerbera and TRV-based GhLFY silencing was conducted in two Gerbera cultivars – Terra Regina and Grizzly. It could be concluded that the activity of GhLFY is involved in regulating flower development. In VIGS:GhLFY lines, leaf-like organs emerged in disc flowers and the identity of stamen and carpel was interrupted. However, further VIGS trials are needed verify the observed phenotypes. At the same time, two potential lfy mutants – Pingpong and Marimbo were analyzed in both phenotype and genotype. These cultivars show phenotypic alteration in inflorescence development and floral organ structures that were distinct from WT Gerbera. Although the expression level of GhLFY did not change among these cultivars, but the GhLFY sequences contained amino acids mutation sites and four missing proline amino acids in Marimbo were detected. The role of these mutation sites need to be further analyzed in later experimental steps.
  • Deng, Xianbao (Helsingfors universitet, 2007)
    Virusinduced gene silencing (VIGS) vectors based on tobacco rattle virus (TRV) are now widely used for characterizing the function of plant genes. However, previous TRV vectors using RNA2 to carry the targeted gene sequence had difficulties to induce gene silencing on some plant species (Gerbera hybrida etc.) due to the obstacle of RNA2 movement. To achieve efficient gene silencing in those species, it is necessary to develop new TRV vectors, in which the targeted gene will be included in TRV RNA1 and the 16K gene will be replaced. Based on TRV RNA1, two new VIGS vectors M1 and M2 were developed through deletion part of 16K gene. Another mutant 16Kstop was also constructed to carry an early terminator in the 4th codon of 16K gene. The infectivity and gene silencing efficiency of the new constructs were assessed through a series of infection experiments. It was found that the infectivity of M1 and M2 was lower than wild TRV RNA1. M1 and M2 could induce PDS gene silencing on Nicotiana benthamiana, but their gene silencing efficiency was limited as compared with previous TRV VIGS vectors in which the PDS gene fragment was contained in RNA2. We also found that the 16K gene sequence, rather than the 16K protein, was required for efficient virus movement and accumulation.
  • Nyberg, Paulina (Helsingin yliopisto, 2020)
    Kasvit tuottavat ensisijaisen aineenvaihdunnan eli primaarimetabolian avulla tarvitsemansa amino-, nukleiini- ja rasvahapot. Näitä molekyylejä kasvit käyttävät kasvuun ja kehitykseen sekä elintoimintojensa turvaamiseen. Suuri osa primaarimetabolian geeneistä on hyvin konservoituneita eli ne ovat säilyneet lähes muuttumattomana kasvilajista toiseen miljoonien vuosien ajan. Sekundaarimetaboliitteja kasvit tuottavat vuorovaikuttaakseen ympäristönsä kanssa. Sekundaarimetaboliittien biosynteesin voi laukaista esimerkiksi kasvin kokema stressi, joka aiheutuu kasvinsyöjistä, taudinaiheuttajista, toisista kasveista tai jos kasvi altistuu liikaa ultraviolettisäteilylle. Ympäristötekijöillä, kuten pH:lla, veden saatavuudella, lämpötilalla ja maaperän suolapitoisuudella on myös suuri vaikutus sekundaarimetaboliittien biosynteesiin sekä vaihteluun saman kasvilajin sisällä. Suurin osa kasvien sekundaarimetaboliiteista syntetisoidaan sikimaatti-, isoprenoidi- ja polyketidireittiä pitkin. Polyketidit toimivat muun muassa pigmentteinä ja osana kasvin puolustusjärjestelmää. Gerberassa esiintyy harvinaista polyketidijohdannaista, 4-hydroksi-5-metyylikumariinia (HMC), jonka biosynteesistä 2-pyronisyntaasi G2PS2:n on päätelty vastaavan. Tässä tutkielmassa selvitettiin g2ps2:n osallisuutta HMC:n biosynteesiin käyttämällä Virus induced gene silencing (VIGS)- menetelmää sekä proteiinin ylituottoa gerberan kallussolukossa. VIGS- menetelmä ei toiminut tässä kokeessa g2ps2- geenin aikaisen ekspression takia. Kokeet gerberan Regina- lajikkeen kallussolukossa sen sijaan vaikuttivat lupaavilta ja kallus osoittautui hyödylliseksi menetelmäksi gerberan polyketidisyntaasien tutkimiseen.
  • Nguyen, Cuong Xuan (Helsingfors universitet, 2012)
    Phytoene desaturase (PDS) plays a key role in the carotenoid biosynthesis in plants. Knocked-down the expression of PDS gene by virus induced gene silencing (VIGS) shows the photobleached phenotype in infected plants so that it has been used as a marker or a long time in VIGS systems with range of plant species. Tobacco rattle virus (TRV) based VIGS system, which uses PDS as the visual marker has been successfully applied and showed the white phenotype in Gerbera hybrida. However, 2-pyrone synthase (PS) gene, which encodes the first enzyme in gerebrin/parasorboside biosynthesis, is significantly reduced in these infected VIGS albino sectors. The transcription level of 2PS gene was also strongly suppressed in leaves treated with photobleaching herbicide, Norflurazon (NF), which inhibits the activity of PDS. Thus, down-regulation of 2PS gene in photobleaching sectors is caused by silencing PDS gene rather than by reacting of gerbera to TRV in VIGS treatment. Interestingly, expression of 2PS in transgenic tobacco (Nicotina tabacum SR1) causes photooxidative bleaching of the leaves. The reduction of ??-carotene in white leaves which analyzed by thin layer chromatograph (TLC) is the main reason; however, the interference between gerberin/parasorboside and carotenoid biosynthesis in these transgenic plants is still unclear. To overcome the effect of overexpression 2PS gene, exogenous mevalonic acid lactone (MAL) could be applied to partially rescue this transgenic phenotype at the seedling stages.
  • Zhang, Yao (Helsingfors universitet, 2012)
    The evolution of floral novelties involves activities of several transcription factors. The large Asteraceae plant family has inflorescences consisting of different types of flowers. TCP transcription factors have recently been reported to regulate this complex structure. Gerbera hybrida, a common ornamental crop, has been used as a model plant for flower developmental studies in Asteraceae. It can be genetically transformed; however, transformation is laborious and time-consuming. Calendula officinalis also belongs to the Asteraceae plant family. It has working micropropagation methods, and it grows and flowers faster than Gerbera hybrida. In addition, Calendula officinalis genes possess high sequence similarity to Gerbera hybrida genes and thus provide an opportunity for functional testing of large numbers of genes. For functional assessment, virus induced gene silencing (VIGS) is a rapid approach, as it circumvents the need to establish stable transgenic lines that requires efficient tissue culture and time-consuming whole-plant regeneration steps. In this study, twelve Calendula officinalis cultivars were tested for VIGS. Calendula showed potential as a new model for the Asteraceae plant family as all twelve different cultivars could be infected by tobacco rattle virus (TRV) via observing the fluorescence caused by pTRV2: GFP; and they all showed photobleaching silencing phenotype caused by PHYTOENE DESATURASE (GhPDS) gene from gerbera. Especially the cultivars „Touch of Red/Orange? and „Princess Orange? turned out to be the most efficient in silencing. Virus dynamics was successfully traced by fluorescence encoded by pTRV2: GFP construct. However, using the construct pTRV2: GFP: PDS, it was observed that the GFP gene could not be applied as a marker gene to indicate the silencing area, because fluorescence could not be observed in photobleaching area. The calendula PDS was also tested. More similar sequence corresponding to the plant?s endogenous gene could cause more visible silencing symptom, but quantitative polymerase chain reaction results showed no statistically significant difference on gene silencing efficiency between GhPDS and CoPDS fragments. VIGS was also tested for silencing of TCP domain transcription factor genes using gerbera specific gene fragments. It was observed that the VIGS approach was not suitable for functional testing of CYCLOIDEA-clade genes in Calendula officinalis as identification of the putative phenotypes was highly disturbed by variable inflorescence phenotype of the tested cultivar.
  • Deng, Xianbao; Elomaa, Paula; Nguyen, Cuong X.; Hytonen, Timo; Valkonen, Jari P. T.; Teeri, Teemu H. (2012)