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Öğe Apomikt Boechera holboellii (hornem.) A. Löve ve D.Löve türünde DYAD gen ortoloğunun karekterizasyonu(Çanakkale Onsekiz Mart Üniversitesi, 2010) Sezer, Fatih; Taşkın, Kemal MelihBu çalışmada, apomikt ve seksüel Boechera türlerinde DYAD gen ortoloğunun yapısı ve ifadesi ortaya çıkarılmıştır. Boechera çiçek tomurcuklarından DYAD cDNA'sı RT-PCR aracılığı ile elde edilmiştir. SWITCH1/DYAD geni kardeş kromatid kohezyonu, mayotik kromozom organizasyonundan sorumlu bir gen olarak tanımlanmıştır. Ayrıca diploid gametlerin oluşumuna yol açması sebebi ile apomiksi sürecinde etkili bir gen olduğu düşünülmektedir. Bitkilerde eşeysiz tohum oluşumu anlamına gelen apomiksi, genetik olarak birbirinin aynı, anasal kopyalardan oluşan nesillerin oluşumuna yol açar. Apomiksinin zirai bitkilere aktarılması bitki yetiştiriciliğinde heterozigotluğun sabitlenmesi, hibrit özelliklerinin korunması ve hibritlerin nesiller boyunca çoğaltılabilmesine olanak tanıyacak olması nedeni ile gelecek vaat eden bir konudur. Apomiksinin altında yatan moleküler etmenler tam olarak bilinmiyor olsa da DYAD geni üzerinde yapılan çalışmalar bu gendeki mutasyonların apomiksinin önemli bir aşaması olan apomayoza yol açabildiğini göstermiştir. DYAD geni seksüel bir tür olan Arabidopsis thaliana bitkisinde tanımlanmış ve bu gene ait mutant Arabidopsis hatlarında yapısı ve fonksiyonu ortaya konmuştur. Bu çalışma ile doğal apomikt ve seksüel Boechera türlerine ait protein dizileri Arabidopsis SWITCH1/DYAD geni ile yüksek oranda benzerlik göstermektedir. Filogenetik analizler Boechera SWITCH1/DYAD geni ortoloğuna ait muhtemel protein dizisinin 6 farklı türe ait proteine benzerlik gösterdiğini ortaya koymuştur. Boechera muhtemel proteinleri bir fosfolipaz C domaini ve nüklear lokalizasyon sinyali içermektedir.Öğe Determination of the expression levels of DNA methyltransferase genes during a highly efficient regeneration system via shoot organogenesis in the diploid apomict Boechera divaricarpa(Springer, 2015) Taşkın, Kemal Melih; Özbilen, Aslıhan; Sezer, Fatih; Corduk, Nurşen; Erden, DamlaIn this study, we established the first efficient method to regenerate diploid apomict Boechera divaricarpa via shoot organogenesis. Hypocotyl explants were cultured on MS medium supplemented with plant growth regulators. The morphogenic potential of B. divaricarpa hypocotyl tissue was investigated to establish an efficient adventitious shoot regeneration system. We tested hypocotyls from 7-day-old in vitro seedlings. The effect of various concentrations of cytokinin and auxin on in vitro regeneration of these explants was also investigated. We found that callus induction and shoot regeneration were significantly affected by the concentrations and types of plant growth regulators. MS medium supplemented with 17.75 mu M benzylaminopurine and 0.53 mu M naphthaleneacetic acid gave the highest number of shoots per explant after 4 weeks in culture. We also determined the expression levels of three DNA methyltransferase genes under tissue culture conditions to understand their activities during callus induction and shoot regeneration. BdMET1 was expressed at low levels during both callus induction and shoot regeneration, while expression of BdDRM2 was high during callus induction and low during shoot regeneration. BdCMT3 was highly expressed in both hypocotyl explants and seedlings and may be a good candidate to induce epigenetic variations in diploid apomict B. divaricarpa shoots derived from tissue culture. Our study provides an efficient in vitro regeneration method and determines the expression levels during tissue culture of methyltransferase genes; it can be used as a new tool to understand the molecular biology of apomixis in B. divaricarpa.Öğe Genome-wide identification and expression analysis of SWI1 genes in Boechera species(Elsevier Sci Ltd, 2016) Sezer, Fatih; Yüzbaşıoğlu, Gözde; Özbilen, Aslıhan; Taşkın, Kemal MelihAs a mode of reproduction in plants, apomixis leads to the generation of clones via seeds. Apomictic plants form viable diploid female gametes without meiosis (apomeiosis) and produce embryos without fertilization (parthenogenesis). Apomeiosis, as a major component of apomixis, has recently been reported in some Arabidopsis thaliana mutants; dyad mutants of SWI1 showed developmental processes common to apomeiosis, such as producing functional diploid gametes. However, the orthologs of SWI1 genes in natural apomicts has not been previously reported. To identify the relationship between the SWI1 gene and the apomeiosis process, we isolated and sequenced SWI1 orthologs from Boechera species, including apomictic and sexual species. Boechera species are close relatives of A. thaliana and thus are advantageous model species for apomixis research. The SWI1 cDNAs were obtained by RT-PCR from apomictic and sexual Boechera young flower buds. We sequenced partial SWI1 transcripts that were 650 bp for B. holboellii and 684 by for B. stricta. These SWI1-like sequences showed 86% similarity for B. holboellii and 92% for B. stricta to the A. thaliana SWI1 transcript. We also used available genome data and amplified genomic sequences for SWI1 orthologs in B. holboellii and B. stricta. The predicted proteins contain a phospholipase C domain and a nuclear localization signal. Sequence analysis did not show significant mutations related to apomixis, and phylogenetic analysis showed that SWI1-like sequences were common across plant families, regardless of the presence of a sexual or apomictic reproduction system. We also investigated the expression levels of SWI1 mRNA in the B. holboellii and B. stricta young unopened flower buds and found that relatively high levels of expression occurred in apomicts. (C) 2016 Elsevier Ltd. All rights reserved.Öğe Identification and expression of strigolactone biosynthesis and signaling genes and the in vitro effects of strigolactones in olive (Olea europaea L.)(John Wiley & Sons Ltd, 2024) Özbilen, Aslıhan; Sezer, Fatih; Taşkın, Kemal MelihStrigolactones (SLs), synthesized in plant roots, play a dual role in modulating plant growth and development, and in inducing the germination of parasitic plant seeds and arbuscular mycorrhizal fungi in the rhizosphere. As phytohormones, SLs are crucial in regulating branching and shaping plant architecture. Despite the significant impact of branching strategies on the yield performance of fruit crops, limited research has been conducted on SLs in these crops. In our study, we identified the transcript sequences of SL biosynthesis and signaling genes in olive (Olea europaea L.) using rapid amplification of cDNA ends. We predicted the corresponding protein sequences, analyzed their characteristics, and conducted molecular docking with bioinformatics tools. Furthermore, we quantified the expression levels of these genes in various tissues using quantitative real-time PCR. Our findings demonstrate the predominant expression of SL biosynthesis and signaling genes (OeD27, OeMAX3, OeMAX4, OeMAX1, OeD14, and OeMAX2) in roots and lateral buds, highlighting their importance in branching. Treatment with rac-GR24, an SL analog, enhanced the germination frequency of olive seeds in vitro compared with untreated embryos. Conversely, inhibition of SL biosynthesis with TIS108 increased lateral bud formation in a hard-to-root cultivar, underscoring the role of SLs as phytohormones in olives. These results suggest that modifying SL biosynthesis and signaling pathways could offer novel approaches for olive breeding, with potential applicability to other fruit crops.Öğe Identification of reference genes for real-time quantitative polymerase chain reaction based gene expression studies on various Olive (Olea europaea L.) tissues(Taylor & Francis Ltd, 2018) Hürkan, Kaan; Sezer, Fatih; Özbilen, Aslıhan; Taşkın, Kemal MelihReference genes are essential for the normalisation of the expression data of quantitative real-time PCR for the purposes of validation. Although several reference genes have been validated for the olive, comprehensive analyses including an excessive number of candidate reference genes still require study in various olive tissue samples. In this work, a total of 40 candidate reference genes were tested for their stability in 8 different olive tissues (root, apical bud, lateral bud, pedicel, young leaf, mature leaf, fruit mesocarp, and seed) with the utilisation of the most popular software programs including GeNorm, NormFinder, BestKeeper, and Delta C-t. The analyses of expression stability of candidate reference genes using quantitative real-time PCR demonstrated ubiquitin-conjugating enzyme (UBC1) as the most stable reference gene for the studied tissues of the olive. The GeNorm software also calculated the optimum reference gene combinations as two which consist of UBC1 and the Clathrin adaptor complex medium subunit (CLATHRIN) genes. This study provides the most stable reference gene combination for normalisation of target genes for quantitative real-time PCR gene expression studies on the olive.Öğe Improving the adventitious rooting ability of hard-to-root olive (Olea europaea L.) cultivar cuttings through inhibiting strigolactone biosynthesis(2022) Len, Aslıhan Özbi; Sezer, Fatih; Taşkın, Kemal MelihStrigolactones (SLs) are synthesized in roots and control plant development. As phytohormones, SLs regulate plant architecture, including roots. Recently, the inhibiting effects of SLs on adventitious rooting have been identified. Olive (Olea europaea L.) is consumed for oil and table in Mediterranean countries and is an economically important crop. Turkey is one of the countries with the highest olive production. Olive has mostly propagated asexually via cuttings, however, the rooting capacities of some agriculturally important olive cultivars are very low. Indole Butyric Acid (IBA) is commonly used to promote the rooting of olive cuttings, however, it can be inadequate. Ayvalık is an easy-to-root cultivar and one of the most common cultivars grown for oil production and Domat is a hard-to-root cultivar in which IBA is insufficient for inducing rooting. In our study, the effects of synthetic SLs rac-GR24 and SLs biosynthesis inhibitor TIS108 on the rooting ability of olive cuttings were investigated. As a result, the adventitious rooting ability was increased when a hard-to-root cultivar was treated with TIS108, indicating a promising future for olive-cutting rooting. Therefore, our study will provide potentially new tools for propagation strategies using SLs in fruit trees.Öğe Molecular characterization of tocopherol biosynthesis genes from Olea europaea (L.) cv. Ayvalik(Tubitak Scientific & Technological Research Council Turkey, 2017) Sezer, Fatih; Taşkın, Kemal MelihVitamin E is a group of compounds that includes metabolites known as tocopherols, which have high antioxidant activities. Tocopherols are synthesized in plants, and their beneficial effects on human health have been reported for diseases such as coronary heart disease and cancer. Here we report the full-length transcripts encoding vitamin E biosynthetic enzymes from fruit mesocarp tissues using the rapid amplification of cDNA ends (RACE) method for the first time in olives. We characterized the structure of the genes 4-hydroxyphenylpyruvate dioxygenase (PDS1), homogentisate phytyltransferase 1 (HPT1), vitamin E defective 3 (VTE3), tocopherol cyclase (VTE1), and gamma tocopherol methyltransferase (GTMT), which are responsible for tocopherol biosynthesis in the olive cultivar Ayvalik. Although PDS1 is widespread in all organisms, HPT1, VTE3, VTE1, and GTMT are only present in photosynthetic organisms. We isolated total RNA from the dissected mesocarp tissues of fruit collected at 15-day intervals between October and December 2014. We compared the expression levels of genes using quantitative RT-PCR and determined the tocopherol content using the high-performance liquid chromatography-fluorescence detection (HPLC-FLD) technique during fruit maturation in the Ayvalik cultivar. High OeHPT1, OeVTE3, OeVTE1, and OeGTMT expression was noted in young fruit. However, the OePDS1 mRNA did not exhibit significant expression changes during maturation. The alpha-tocopherol content varied between 26.78 and 21.05 mg/100 g and was highest at the early stages of fruit development. In addition, expression studies and tocopherol content revealed that tocopherol biosynthesis in olive is more active at the early stages of fruit maturation.Öğe Phylogenetic and Expression Analysis of CENH3 and APOLLO Genes in Sexual and Apomictic Boechera Species(Mdpi, 2022) Bakin, Evgeny; Sezer, Fatih; Özbilen, Aslıhan; Kılıç, İrem; Üner, Buket; Rayko, Mike; Taşkın, Kemal MelihApomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO gene encoding aspartate glutamate aspartate aspartate histidine exonuclease is one of the very few described genes associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Furthermore, we characterize the structure of CENH3 by bioinformatic tools, study expression levels of APOLLO and CENH3 transcripts by Real-Time Polymerase Chain Reaction RT-PCR in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of meiosis and before and after fertilization. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until the 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes. The results obtained may indicate to a role of the CENH3 and APOLLO genes in the development of apomixis in species of the genus Boechera.Öğe Structure and expression of dna methyltransferase genes from apomictic and sexual Boechera species(Elsevier Sci Ltd, 2017) Taşkın, Kemal Melih; Özbilen, Aslıhan; Sezer, Fatih; Hürkan, Kaan; Güneş, ŞebnemIn this study, we determined the structure of DNA methyltransferase (DNMT) genes in apomict and sexual Boechera species and investigated the expression levels during seed development. Protein and DNA sequences of diploid sexual Boechera stricta DNMT genes obtained from Phytozome 10.3 were used to identify the homologues in apomicts, Boechera holboellii and Boechera divaricaipa. Geneious R8 software was used to map the short -paired reads library of B. holboellii whole genome or B. divaricarpa transcriptome reads to the reference gene sequences. We determined three DNMT genes; for Boechera spp. METHYLTRANSFERASEI (MET I), CHROMOMETHYLASE 3 (CMT3) and DOMAINS REARRANGED METHYLTRANSFERASE 1/2 (DRM2). We examined the structure of these genes with bioinformatic tools and compared with other DNMT genes in plants. We also examined the levels of expression in silique tissues after fertilization by semi -quantitative PCR. The structure of DNMT proteins in apomict and sexual Boechera species share common features. However, the expression levels of DNMTgenes were different in apomict and sexual Boechera species. We found that DRM2 was upregulated in apomicticBoechera species after fertilization, Phylogenetic trees showed that three genes are conserved among green algae, monocotyledons and dicotyledons. Our results indicated a deregulation of DNA methylation machinery during seed development in apomicts. (C) 2016 Elsevier Ltd. All rights reserved.Öğe Synthesis of SMZ derivatives and investigation of effects on germination, root, and plant growth of Arabidopsis thaliana L.(Tubitak Scientific & Technological Research Council Turkey, 2019) Güngör, Tuğba; Cömert Önder, Ferah; Sezer, Fatih; Özbilen, Aslıhan; Taşkın, Kemal Melih; Ay, MehmetA series of sulfonamide derivatives were synthesized by reactions with various functional groups containing benzenesulfonyl chlorides and aniline derivatives under different substitution reaction conditions. The structures of SMZ derivatives were confirmed with melting point, FT-IR, H-1 NMR, C-13 NMR, and LC-MS/MS techniques. In order to investigate the cytotoxic effects of these derivatives, we used a model plant species. The synthesized compounds (S1-S5) and sulfamethazine (SMZ) as a positive control were applied to Arabidopsis thaliana seeds. Our results indicated that S3 and S4 induced shorter roots and lower wet weight in plants. Plants treated with S2 and S5 showed no growth effects, similar to the untreated control group, while S1 slightly reduced root length and wet weight. These results suggest that S3 and the newly synthesized S4 derivatives have potential for use as herbicides since they possess cytotoxic effects on A. thaliana plants.Öğe Zeytin (Olea europaea L.) tokoferol biyosentezinde rol alan genlerin karakterizasyonu(Çanakkale Onsekiz Mart Üniversitesi, Lisansüstü Eğitim Enstitüsü, 2015) Sezer, Fatih; Taşkın, Kemal MelikZeytin (Olea europaea L.) ekonomik açıdan önemli, yaygın olarak yetiştiriciliği yapılan, yüksek E vitamini içeriğine sahip ve insan sağlığı açısından faydaları birçok çalışmaya konu olmuş bir Akdeniz bitkisidir. Sadece bitkilerde üretilen E vitamini tokoferoller olarak bilinen ve yüksek antioksidan özellikleri olan metabolitlerdir. Çoğunlukla yağ bitkileri tarafından üretilen E vitamininin kalp damar hastalıkları ve kanser gibi insan sağlığı açısından önemli hastalıklarda olumlu etkileri vardır. Tokoferollerin biyosentezinde bir grup metil transferaz ve siklaz enzimleri rol almaktadır. Zeytin türünde yakın zamanda transkriptomik çalışmalarla bazı süreçlerde rol alan genlere ait diziler ortaya çıkarılmış olmasına karşın henüz bu türde E vitamini biyosentezinden sorumlu genler tanımlanmamıştır. Bu tez çalışmasında zeytin tokoferol biyosentezinde rol alan genlerin tam uzunluktaki transkiptleri RACE (Rapid Amplification of cDNA Ends) yöntemi ile elde edilmiştir. İlgili genlerin anlatımları meyve gelişiminin değişik evrelerinde real time PZR ile belirlenmiş ve aynı süreçlerde HPLC metodu ile tokoferol miktarları ortaya çıkarılmıştır. Çalışmada HPT1, VTE3, TCYC ve GTMT genlerinin zeytin meyvesi erken gelişim dönemlerinde anlatımlarının daha yüksek olduğu ortaya çıkarılmıştır. Biyosentez yolunda görev alan PDS1 genlerinin anlatımları ise zeytin gelişiminden etkilenmemiştir. HPLC analizleri de benzer şekilde erken dönemdeki meyvelerden elde edilen zeytinyağında daha yüksek konsantrasyonlarda E vitamini miktarının olduğunu göstermiştir.
