Orientadores: Gonçalo Amarante Guimarães Pereira, Juan Lucas Argueso / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T03:40:46Z (GMT). No. of bitstreams: 1 CarvalhoNetto_OsmarVazde_D.pdf: 3871803 bytes, checksum: 86689af3801aaa7c1f590f5061a729ba (MD5) Previous issue date: 2012 / Resumo: A fermentação alcoólica realizada por leveduras da espécie Saccharomyces cerevisiae é atualmente o principal processo em escala econômica para a produção de etanol a partir de fontes renováveis (bioetanol). Entretanto, as linhagens isoladas há quase duas décadas vêm perdendo gradualmente seu desempenho fermentativo ao longo dos anos, reduzindo o rendimento e a estabilidade do processo. Um dos fatores que contribuem para tais perdas está associado às significativas alterações operacionais do processo nestas duas décadas. Aliado a isto, pouco se conhece sobre as bases genéticas das principais linhagens comerciais utilizadas no Brasil, inviabilizando o sucesso de programas de melhoramento genético. Neste ponto, este trabalho propõe de forma inédita um estudo completo de caracterização, análise de expressão e manipulação gênica de PE-2, uma das principais linhagens usadas no processo Brasileiro. Para isto, dados da estrutura, composição e regulação do genoma foram gerados. A partir dessas informações foi desenvolvida, através de manipulações genéticas, uma linhagem auxotrófica para uracila (plataforma biológica) e outras com características de fermentação superiores ao isolado selvagem. O conceito de que o sucesso de manipulações pontuais depende de um conhecimento prévio detalhado da linhagem de interesse, compreendendo tanto suas bases genéticas quanto fisiológicas, foi validado neste estudo. Os dados aqui apresentados abrem novas perspectivas para o desenvolvimento de uma segunda geração de linhagens de S. cerevisiae ainda mais robustas, voltadas não apenas para a produção de bioetanol, como também para utilização em outros processos biotecnológicos / Abstract: The alcoholic fermentation performed by yeasts of the specie Saccharomyces cerevisiae is the main current process to produce ethanol from renewable resources (bioethanol) under economic scale. However, strains isolated two decades ago have been gradually losing their original fermentative fitness during the years, decreasing the process yield and stability. One of the factors that contribute for these losses is associated with the meaningful process operational changes along these years. In addition to that, there is a knowledge gap regarding the genetic bases of the commercial strains used on Brazil, not leading to success of the genetic improvements programs. Hereupon, this work propose in a first time way a complete characterization, expression analysis and genetic manipulation study of PE-2, one of the most important strain used on Brazilian process. To this, structure, composition and regulation data of the genome were produced. From such information, it was developed by genetic manipulations assays an auxotrophic yeast for uracile (biological plataform) and others strains with superior fermentative characteristics in comparison with the wild isolated. The concept that the success of precise manipulations depends on the previous detailed knowledge of the interest strain, regarding the genetic and physiology bases, was confirmed herein. The data presented here also establish new perspectives for the construction of a second generation strains even more robusts, designed not only for the bioethanol production, but also to be applied on different biotechnological processes / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Abstract The efforts to improve forest trees could be accelerated by means of genetic engineering. Thus, the performance and effects of genetically modified (GM) trees have been investigated in numerous studies, which have generally concluded that GM trees have similar effects on environment and/or other organisms as do conventionally bred trees. In the present study, GM silver birch (Betula pendula Roth) and hybrid aspen (Populus tremula L. × tremuloides Michx.) lines were utilized to study the influence of transgenes to the transcription of related endogenous genes and to the production of soluble phenolic compounds in relation to ectomycorrhizal symbiosis or herbivory. The GM silver birch lines had altered lignin composition, whereas the hybrid aspen lines produced the hemoglobin of Vitreoscilla sp. (VHb). The Pt4CL1a lines were generated using biolistic transformation and monitored under greenhouse conditions for three growing seasons. The Pt4CL1a and PtCOMT silver birch lines, with altered lignin syringyl/guaiacyl ratio, had also reduced transcript levels of endogenous genes, Bp4CL1 and BpCOMT, respectively. This indicates that these members of the 4CL and COMT multigene families are likely to contribute to the monolignol biosynthesis pathway of silver birch. No unintended effects were detected in the PtCOMT or Pt4CL1a lines in relation to ECM symbiosis or performance of insect larvae. Moreover, in soluble phenolic compounds, alterations were found mainly in cinnamic acid derivatives, a group of compounds involved in the biosynthesis of monolignols. In addition, the responses of the studied hybrid aspen lines that were exposed to herbivory for 24 hours were found to be comparable. Furthermore, the proportional weight gain of lepidopteran larvae was alike when fed with leaves of the VHb and non-transgenic hybrid aspen lines. Taken together, no unintended changes were found in the GM silver birch lines with altered lignin composition or in the VHb hybrid aspen lines. However, it is acknowledged that these short-term studies that were conducted under controlled conditions have certain limitations. / Tiivistelmä Puiden ominaisuuksia on mahdollista muuttaa geenitekniikkaa käyttämällä huomattavasti perinteistä jalostusta nopeammin. Geneettisen muuntamisen vaikutuksia puiden ominaisuuksiin ja vuorovaikutussuhteisiin on selvitetty useissa tutkimuksissa geenitekniikkaan liitettyjen riskien arvioimiseksi. Muunnettuja kohdeominaisuuksiaan lukuun ottamatta geneettisesti muunnettujen (GM) puiden ei ole yleisesti ottaen tutkimuksissa havaittu eroavan ympäristövaikutuksiltaan perinteisellä jalostuksella tuotetuista puista. Tässä työssä tutkittiin siirrettyjen geenien vaikutuksia GM-rauduskoivun (Betula pendula Roth) sekä hybridihaavan (Populus tremula L. × tremuloides Michx.) endogeenisten geenituotteiden ja liukoisten fenoliyhdisteiden määriin. Lisäksi työssä tarkasteltiin ligniinirakenteeltaan muunnettujen rauduskoivulinjojen ektomykorritsasymbioosia sekä ligniinimuunnettujen ja Vitreoscilla sp. -bakteerin hemoglobiinia (VHb) tuottavien hybridihaapalinjojen lehtien laatua perhostoukkien ravintona. Biolistisella geeninsiirrolla tuotetuista Amerikan haavan 4-kumaraattikoentsyymi A-ligaasi -geeniä (Pt4CL1) ilmentävistä rauduskoivulinjoista yhdessä havaittiin ligniinin syringyyli- ja guaiasyyliyksikköjen suhteessa muutos. Havaittu muutos aiheutui todennäköisesti koivun Bp4CL1-geenituotteiden määrän vähenemisestä. Myös kaffeaatti/5-hydroksylaatti O-metyylitransferaasi -geeniä (PtCOMT) ilmentävissä, ligniinirakenteeltaan muunnetuissa rauduskoivulinjoissa havaittiin endogeenisen BpCOMT-geenin tuotteiden määrän väheneminen. Tulokset viittaavat siihen, että Bp4CL1- ja BpCOMT-geenien tuottamat entsyymit toimivat rauduskoivun monolignolien biosynteesissä. Ligniiniominaisuuksiltaan muunnettujen rauduskoivujen liukoisista fenoliyhdisteistä todettiin muutoksia ensisijaisesti kanelihappojohdannaisissa, jotka liittyvät läheisesti monolignolien biosynteesireittiin. Ektomykorritsasymbioosissa tai perhostoukkien kasvunopeudessa ei havaittu kasvien geneettisestä muuntamisesta johtuvia eroja. Merkitseviä eroja ei todettu myöskään hybridihaapalinjojen herbivoria-vasteissa. On kuitenkin otettava huomioon, että kaikki tutkimuksen kokeet suoritettiin kasvihuoneissa käyttäen vasta juveniilivaiheessa olevia kasveja. Jotta abioottisten ja bioottisten ympäristötekijöiden sekä GM-puiden vuorovaikutusta olisi mahdollista arvioida kokonaisvaltaisesti, puita pitäisi tutkia pitkäaikaisissa kenttäkokeissa.

This thesis investigates the effect of Russian wheat aphid (RWA; Diuraphis noxia) infestation on the defence responses of the bread wheat line, PI 137739, on a molecular level. PI 137739 is known to contain the RWA resistance gene, Dn1. The study was conducted by utilising and combining a vast array of molecular biological techniques. Chapter 1 introduces the reader to a summary of the resistance responses observed within infested plants. A detailed description of the Russian wheat aphid follows and the genes responsible for RWA resistance in wheat is discussed. A brief report of research performed on the bread wheat genome is given and the biochemical defence responses of plants against insect infestation are discussed. This is followed by a concise description of resistance (R) genes and resistance gene categories in plants. The last discussion concerns microarray technology, a molecular tool utilised during this study. Chapter 2 aims at identifying genes involved in resistance against RWA infestation; specifically, genes containing the conserved nucleotide binding site¬leucine rich repeat (NBS-LRR) motif. Genomic, as well as complementary DNA (cDNA), was utilised in order to compare functional gene expression in wheat infested with the RWA. This was executed by employing PCR-based methods, single-pass sequencing and basic local alignment search tool (BLAST) analyses. Chapter 3 introduces suppression subtractive hybridisation (SSH) as a tool to further identify NBS-LRR or other resistance-related sequences in RWA infested wheat plants. SSH allows the comparative analysis of differential gene expression in RWA infested and uninfested wheat in order to identify resistance-¬related genes expressed in the infested, resistant wheat plants. The effect of RWA infestation on wheat resistance responses was examined further in chapter 4 through microarray analysis. The aim was the introduction and establishment of the microarray technique and to test the feasibility of using microarrays for differential gene expression and regulation studies. Microarray slides were assembled in order to monitor the up- and down¬regulation of genes at different time intervals - day 2, day 5 and day 8 - of RWA infestation. Clones isolated throughout this study were assembled on microarray slides and probed with control and RWA infested RNA. Differential gene regulation was assessed and further confirmed through Northern blot analyses, as well as quantitative real-time PCR. The thesis concludes with a general summary of the results obtained in chapter 5 and future prospects are outlined. / Thesis (PhD(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

No description available.

With the introduction of antibiotics in animal feed becoming less popular, the agricultural industry has begun a shift towards the use of probiotics in animal feed. Since there is no current method to evaluate the risks of using genetically modified probiotics in animal feed. The goal of this project was to create a genetically modified model organism for risk assessment. The genetic marker for that was chosen was GFP that was to be expressed on the surface of the cell. The fluorescent properties allow for visualisation of the genetically modified bacteria and the surface expression would allow for the easy capture and recovery of the bacteria for culturing and cell counts. Genome wide screens were performed using the CW PRED algorithm to locate proteins with LPXTG motif for cell wall anchoring. 16 hypothetical proteins were detected and 6 were selected as candidates for possible surface display of GFP. Of these candidates, the novel L. casei protein LSEI_2320 was found to be expressed at the mRNA during early growth by RT PCR and at then protein level during stationary phase with western blot. This LPXTG protein was found at the surface of L. casei ATCC334 during stationary phase and late stationary phase with immunofluorescence microscopy. A genetically modified L. casei ATCC334 was constructed using the surface protein LSEI_2320 locus as a region for recombination with the pRV300 suicide plasmid. Genetic modification of the locus by the insertion of a GFP reporter region just before the predicted signal peptide site resulted in the abrogation of the expression of LSEI_2320 from the cell surface at the late stationary phase. It appears that this particular gene is not necessary to cell survival even though it is abundantly expressed on the cell surface and can be used as a location for genetic modification in L. casei ATCC334.

No description available.

Philosophiae Doctor - PhD / Starch is a major carbohydrate reserve in many plants, providing energy during heterotrophic growth and it is contained in large amounts in staple foods such as potatoes, wheat, maize, rice, sorghum and cassava. Apart from being a major product for use in the food industry, starch is also attracting interest from the biofuels industry as a source of bioethanol. This study reports on the development of genetic tools aimed at increasing starch production in sorghum (Sorghum bicolor L Moench), a crop of key agronomic importance worldwide by exploiting a new discovery of a transcription factor gene that regulates starch accumulation in Arabidopsis thaliana namely LEAFY COTYLEDON I (LECl). Ectopic over expression of this gene in arabidopsis has previously been shown to induce a massive hyper accumulation of starch in vegetative tissues. Therefore, we set out to investigate the function of its orthologous gene counterpart in sorghum with the aim of manipulating starch yield directly. Deduced protein sequence analyses showed that the putative sorghum LEAFY COTYLEDON I gene (SbLEC1) cloned in this study shares an overall high amino acid sequence identity (70 %) with the arabidopsis LEC 1, while the functional central B domain shows an even higher percentage sequence identity (91 %) with the same region of arabidopsis LEC 1. The putative SbLEC1 protein shares 14 out of the 16, signature ammo acids characteristic of the Central B Domain with arabidopsis. Furthermore, the putative SbLEC1 protein was also shown to share a significantly high sequence identity (> 80 %) with other well-characterized LEC1 protein sequences from organisms such as maize, rice, rapeseed as well as other organisms documented in the NCB I database. Similarly, much of the sequence similarity lies within the functional central B domain compared to any other region. Gene expression profiling using semi-quantitative PCR showed that SbLEC1 transcripts accumulated in developing seeds as well as in embryogenic calli tissue and no SbLEC 1 transcripts were detectable in leaf, root or sheath tissue. In order to confirm that the identified transcription factor is a functional ortholog, the full cDNA encoding putative SbLEC 1 transcription factor was identified, isolated and cloned from the sweet sorghum MN 1812 genotype. Plant transformation gene constructs based on the pCAMBIA1305.2 binary vector harbouring the transcription factor gene under the control of different promoter sequences were then assembled and immobilized into Agrobacterium tumefaciens strain LBA4404 in preparation for sorghum and arabidopsis transformation. Transient GUS expression studies showed that the five SbLEC1 gene constructs developed in this study were successfully transformed into arabidopsis (Ws ecotype) and sorghum (variety MN1812) callus and cell suspension cultures. The transformed tissues thus represent essential tools that are useful to evaluate the effect of over expressing the putative SbLEC1 protein. Transient GUS expression assays also further revealed differences in efficiency among promoters in driving transgene expression. Transient GUS activity was highest for the maize ubiquitin promoter (MUbi1), followed by the sorghum LEC1 promoter (SLECP), the arabidopsis LEC1 promoter (ALECP) and lastly the maize alcohol dehydrogenase promoter (MAdh1). The ability of the putative SbLEC 1 gene to complement the arabidopsis lecI mutation was also investigated and our findings were not conclusive as they only revealed partial complementation. A detailed comparison of SbLECI full cDNA sequences isolated and cloned from twenty-eight different F2 population plants from different sorghum varieties revealed the existence of sequence variation within the SbLEC 1 gene, which appeared to be allelic. The allelic variation was further shown to affect the amino acid composition of the putative SbLEC 1 protein. Heterologous protein expression studies of the SbLECI gene using an E. coli system showed that the predicted 29.16 kDa putative SbLEC 1 protein could be expressed in vitro both as an development of an efficient tissue culture protocol is a prerequisite for plant genetic engineering, this study also reports on the evaluation of thirteen sorghum genotypes from different genetic backgrounds for their in vitro culture response. A tissue culture protocol for three previously unexplored sorghum genotypes namely Agricol white, AS4 and MNI812 was established. The effect of plant genotype, explant and medium composition on in vitro culture response was highly significant (95 % Cl) in this study. Taken together, the findings in our study demonstrate efforts to draw a baseline foundation for the development of molecular technologies that can be used to increase starch production in sweet sorghum as a water efficient and sustainable feedstock for biofuel production.

<p>Synthetic biology reprograms organisms to perform non-native functions for beneficial reasons. An important practice in synthetic biology is the ability to edit DNA to change a base pair, disrupt a gene, or insert a new DNA sequence. DNA edits are commonly made with the help of homologous recombination, which inserts new DNA flanked by sequences homologous to the target region. To increase homologous recombination efficiency, a double stranded break is needed in the middle of the target sequence. Common methods to induce double stranded breaks use nucleases, enzymes that cleave ribonucleotides (DNA and RNA). The most common nucleases are restriction enzymes, which recognize a short, fixed, palindromic DNA sequence (4-8 base pairs). Because of the short and fixed nature of the recognition sites, restriction enzymes do not make good candidates to edit large chromosomal DNA. Alternatively, scientists have turned to programmable endonucleases which recognize user-defined DNA sequences, often times much larger than the recognition sites of restriction enzymes (15-25 base pairs). Programmable endonucleases such as CRISPR-based systems and prokaryotic Argonautes are found throughout the prokaryotic kingdom and may differ significantly in activity and specificity. To compare activity levels among endonuclease enzymes, activity assays are needed. These assays must clearly delineate dynamic activity levels of different endonucleases and work with a wide variety of enzymes. Ideally, the activity assay will also function as a positive selection screen, allowing modifications to the enzymes via directed evolution. Here, we develop an <i>in vivo</i> assay for programmable endonuclease activity that can also serve as a positive selection screen using two plasmids, a lethal plasmid to cause cell death and a rescue plasmid to rescue cell growth. The lethal plasmid houses the homing endonuclease, I-SceI, which causes a deadly double-stranded break at an 18 base pair sequence inserted into an engineered <i>E. coli</i> genome. The rescue plasmid encodes for a chosen endonuclease designed to target and cleave the lethal plasmid, thereby preventing cell death. With this, cell growth is directly linked to programmable endonuclease activity. Three endonucleases were tested, SpCas9, eSpCas9, and xCas9, displaying recovered growth of 49.3%, 26.1%, and 16.4% respectively. These values translate to kinetic enzymatic activity and are congruent with current literature findings as reported values find WT-SpCas9 to have the fastest kinetics cleaving around 95% of substrate within 15 seconds, followed closely by eSpCas9 cleaving 75% of substrate within 15 seconds and finally trailed by xCas9 cleaving 20% of substrate in about 30 seconds. The differences between each endonuclease’s activity is exacerbated in our <i>in vivo</i> system when compared to similar <i>in vitro</i> methods with much lower resolution. Therefore, slight differences in activity between endonucleases within the first few minutes in an <i>in vitro</i> assay may be a few percentages different whereas in our <i>in vivo</i> assay, these differences in activity result in a more amplified signal. With the ability to display the dynamic response of enzymes, this assay can be used to compare activity levels between endonucleases, give insight into their kinetics, and serve as a positive selection screen for use in directed evolution applications. </p>

No description available.

Soybean cyst nematode (SCN), a major pathogen of soybean worldwide, causes huge losses in soybean production. Various approaches including cloning of genes to combat this devastating disease help to better understand the cellular function and immune responses of plants. Membrane fusion genes are the important regulatory parts of vesicular transport system, which works through packaging of intracellular compounds and delivering them to apoplast or nematode feeding sites to induce an incompatible reaction. The incompatible nature of membrane fusion proteins such as SNAP25, Munc18, Syntaxin, Synaptobrevin, NSF, Synaptotagmin and alpha-SNAP are conserved in eukaryotes and regulate the intracellular function to combat abiotic and biotic stress in plants. Overexpression of these genes in G. max [Williams 82(PI518671)] which is a susceptible cultivar of soybean to nematodes resulted in a reduction of the SCN population providing further insights of molecular and genetic approaches to solve the SCN problems in agriculture.