Generation of drought-resistant transgenic cereals using transcription factors isolated from wheat grain

Tese de doutoramento em Ciências da Saúde, ramo de Medicina, na especialidade de Medicina Interna (Cardiologia), apresentada à Faculdade de Medicina da Universidade de Coimbr

Tissue specific promoters from rice and wheat for modifying grain characteristics

Trabalho final do 6º Ano Médico com vista à atribuição do grau de mestre no âmbito do ciclo de estudos de Mestrado Integrado em Medicina, apresentada à Faculdade de Medicina da Universidade de CoimbraA tiroidite, inflamação da glândula tiroideia, é, juntamente com o bócio, a afecção endócrina mais frequente, sendo comummente encontrada em medicina ambulatória. Associada a uma função tiroideia normal, aumentada ou diminuída (frequentemente com evolução de uma condição para outra), as circunstâncias da sua descoberta podem ser variadas e a distinção dos seus vários tipos baseia-se, essencialmente, no panorama clínico, rapidez de instalação sintomática, história familiar e presença ou ausência de sintomas prodrómicos e dor no pescoço. Vários critérios podem ser utilizados para a sua classificação, nomeadamente histológicos e clínicos (acompanhada ou não de dor tiroideia), sendo, contudo, a evolução da doença o critério classicamente utilizado. Assim, de acordo com este último, os diferentes subtipos de tiroidite podem ser agrupados em: tiroidite aguda, tiroidite subaguda ou tiroidite crónica. A primeira é uma forma dolorosa de tiroidite extremamente rara causada por uma infecção bacteriana, fúngica ou parasitária da tiróide e surgindo, sobretudo, na criança/adolescente e adulto jovem. Nas tiroidites subagudas, podemos encontrar a tiroidite subaguda granulomatosa ou de De Quervain – causa mais frequente de dor tiroideia, eventualmente, de origem viral – e as tiroidites subagudas linfocíticas – tiroidites esporádica indolor, do pós-parto, iatrogénica (interferão, interleucina-2, lítio), tóxica (amiodarona), por irradiação (iodo 131, irradiação externa) ou traumática (cirurgia, punção, traumatismo externo). Finalmente, nas tiroidites crónicas, caracterizadas, portanto, por um maior tempo de evolução, podemos destacar a tiroidite de Riedel, de natureza fibrótica e fisiopatologia desconhecida, e a tiroidite de Hashimoto, a tiroidite mais frequente e causa mais comum de hipotiroidismo nas regiões com aporte suficiente de iodo e da qual as tiroidites esporádica indolor e do pós-parto se aproximam pelo seu carácter autoimune. O diagnóstico destas afecções é feito pelo contexto e achados clínicos, incluindo a presença ou ausência de dor, textura e autoanticorpos. Adicionalmente, o grau de absorção de iodo radioactivo pela iii glândula é reduzido na maioria dos pacientes com inflamação viral, induzida por radiação, traumática, autoimune ou induzida por drogas. O tratamento é, primeiramente, dirigido ao alívio sintomático da dor tiroideia e restabelecimento do eutiroidismo.Thyroiditis, inflammation of the thyroid gland, is, along with goiter, the most frequent endocrine pathology, being commonly found in ambulatory medicine. Associated with normal, elevated or depressed thyroid function (often with evolution from one condition to another), the circumstances of its discovery are varied and the distinction of its several types is based primarily on the clinical setting, rapidity of symptom onset, family history and presence or absence of prodromal symptoms and neck pain. Different criteria can be used to its classification, namely histological and clinical (with thyroid pain or not). Nevertheless, the evolution of the disease is the classically used criterion. Thus, accordingly with this one, the several subtypes of thyroiditis can be divided into: acute thyroiditis, subacute thyroiditis and chronic thyroiditis. The first one is an extremely rare painful form of thyroiditis cause by a bacterial, fungic or parasitic infection of the thyroid and it in children/adolescents and young adults. In subacute thyroiditis, we can have the subacute granulomatous or de De Quervain thyroiditis – most common cause of thyroid pain, eventually of viral original – and the subacute lymphocytic thyroiditis – sporadic painless, postpartum, iatrogenic (interferon, interleukin-2, lithium), toxic (amiodarone), by irradiation (iodine 131 and external irradiation) or traumatic (surgery, puncture or external traumatism) thyroiditis. Finally, in chronic thyroiditis, characterized, therefore, by a longer time of evolution, we can mention Riedel’s thyroiditis, of fibrotic nature and unknown physiopathology, and Hashimoto’s thyroiditis, the most frequent thyroiditis and the most common cause of hypothyroidism in the regions with an adequate iodine supply, from which the sporadic painful and postpartum thyroiditis are close by their autoimune character. Diagnosis is by clinical context and findings, including the presence or absence of pain, tenderness and autoantibodies. In addition, the degree of radioactive iodine uptake by the gland is reduced in most patients with viral, radiation-induced, traumatic, autoimmune, or drug-induced inflammation of the thyroid. Treatment primarily is directed at symptomatic relief of the thyroid pain and restoration of euthyroidism

A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions

Published: 26 June 2018Background: The relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating. Results: A simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results. Conclusions: The DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.Ainur Ismagul, Nannan Yang, Elina Maltseva, Gulnur Iskakova, Inna Mazonka, Yuri Skiba, Huihui Bi, Serik Eliby, Satyvaldy Jatayev, Yuri Shavrukov, Nikolai Borisjuk and Peter Langridg

Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield

First published online: July 27, 2015Heterotrimeric nuclear factors Y (NF-Ys) are involved in regulation of various vital functions in all eukaryotic organisms. Although a number of NF-Y subunits have been characterized in model plants, only a few have been functionally evaluated in crops. In this work, a number of genes encoding NF-YB and NF-YC subunits were isolated from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius was investigated. TaNF-YB4 was isolated as a result of two consecutive yeast two-hybrid (Y2H) screens, where ZmNF-YB2a was used as a starting bait. A new NF-YC subunit, designated TaNF-YC15, was isolated in the first Y2H screen and used as bait in a second screen, which identified two wheat NF-YB subunits, TaNF-YB2 and TaNF-YB4. Three-dimensional modelling of a TaNF-YB2/TaNF-YC15 dimer revealed structural determinants that may underlie interaction selectivity. The TaNF-YB4 gene was placed under the control of the strong constitutive polyubiquitin promoter from maize and introduced into wheat by biolistic bombardment. The growth and yield components of several independent transgenic lines with up-regulated levels of TaNF-YB4 were evaluated under well-watered conditions (T1-T3 generations) and under mild drought (T2 generation). Analysis of T2 plants was performed in large deep containers in conditions close to field trials. Under optimal watering conditions, transgenic wheat plants produced significantly more spikes but other yield components did not change. This resulted in a 20-30% increased grain yield compared with untransformed control plants. Under water-limited conditions transgenic lines maintained parity in yield performance.Dinesh Yadav, Yuri Shavrukov, Natalia Bazanova, Larissa Chirkova, Nikolai Borisjuk, Nataliya Kovalchuk, Ainur Ismagul, Boris Parent, Peter Langridge, Maria Hrmova and Sergiy Lopat

Characterization of the wheat gene encoding a grain-specific lipid transfer protein TdPR61, and promoter activity in wheat, barley and rice

The TaPR61 gene from bread wheat encodes a lipid transfer protein (LTP) with a hydrophobic signal peptide, predicted to direct the TaPR61 protein to the apoplast. Modelling of TaPR61 revealed the presence of an internal cavity which can accommodate at least two lipid molecules. The full-length gene, including the promoter sequence of a TaPR61 orthologue, was cloned from a BAC library of Triticum durum. Quantitative RT-PCR analysis revealed the presence of TaPR61 and TdPR61 mainly in grain. A transcriptional TdPR61 promoter-GUS fusion was stably transformed into wheat, barley, and rice. The strongest GUS expression in all three plants was found in the endosperm transfer cells, the embryo surrounding region (ESR), and in the embryo. The promoter is strong and has similar but not identical spatial patterns of activity in wheat, barley, and rice. These results suggest that the TdPR61 promoter will be a useful tool for improving grain quality by manipulating the quality and quantity of nutrient/lipid uptake to the endosperm and embryo. Mapping of regions important for the promoter function using transient expression assays in developing embryos resulted in the identification of two segments important for promoter activation in embryos. The putative cis-elements from the distal segment were used as bait in a yeast 1-hybrid (Y1H) screen of a cDNA library prepared from the liquid part of the wheat multinucleate syncytium. A transcription factor isolated in the screen is similar to BES1/BLZ1 from Arabidopsis, which is known to be a key transcriptional regulator of the brassinosteroid signalling pathway.Nataliya Kovalchuk, Jessica Smith, Natalia Bazanova, Tatiana Pyvovarenko, Rohan Singh, Neil Shirley, Ainur Ismagul, Alexander Johnson, Andrew S. Milligan, Maria Hrmova, Peter Langridge and Sergiy Lopat

Biolistic DNA Delivery in Turfgrass Embryonic Callus Initiated from Mature Seeds

We describe a protocol for the establishment and preparation of creeping bentgrass (Agrostis stolonifera L.) cultivar “Penn A-4” embryonic calli, biolistic transformation, selection, and regeneration of transgenic plants. The embryonic callus is initiated from mature seeds, maintained by visual selection under the dissecting microscope and subjected to bombardment with plasmid DNA containing a bialaphos-resistance (bar) gene. PCR, Southern, and Northern blot analyses are used to confirm the transgene integration and expression

Characterization of the wheat endosperm transfer cell-specific protein TaPR60

The TaPR60 gene from bread wheat encodes a small cysteine-rich protein with a hydrophobic signal peptide, predicted to direct the TaPR60 protein to a secretory pathway. It was demonstrated by heterologous expression of recombinant TaPR60 protein that the signal peptide is recognized and cleaved in yeast cells. The full-length gene including promoter sequence of a TaPR60 orthologue was cloned from a BAC library of Triticum durum. A transcriptional promoter-GUS fusion was stably transformed into wheat, barley and rice. The strongest GUS expression in wheat and barley was found in the endosperm transfer cells, while in rice the promoter was active inside the starchy endosperm during the early stages of grain filling. The TaPR60 gene was also used as bait in a yeast two-hybrid screen. Five proteins were identified in the screen, and for some of these prey proteins, the interaction was confirmed by co-immunoprecipitation. The signal peptide binding proteins, TaUbiL1 and TaUbiL2, are homologues of animal proteins, which belong to proteolytic complexes, and therefore may be responsible for TaPR60 processing or degradation of the signal peptide. Other proteins that interact with TaPR60 may have a function in TaPR60 secretion or regulation of this process. Examination of a three dimensional model of TaPR60 suggested that this protein could be involved in binding of lipidic molecules.Nataliya Kovalchuk, Jessica Smith, Margaret Pallotta, Rohan Singh, Ainur Ismagul, Serik Eliby, Natalia Bazanova, Andrew S. Milligan, Maria Hrmova and Peter Langridge, et al