Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 15, 2010). Includes bibliographical references.

Universiẗat, Diss., 2004--Düsseldorf. / Includes bibliographical references (p. 225-233).

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a serious insect pest of wheat and barley. It affects the quality and yield of grain by sucking plant sap from the newest growth whilst toxic substances are injected that destroy plant tissue. The Russian wheat aphid also acts as a vector of plant viruses. The cultivation of aphid resistant cultivars is the preferred control strategy and nine resistance genes, designated Dn1 to Dn9, have been identified. Another undesignated gene, Dnx, was found in the wheat accession PI220127. Mapping of the resistance genes relative to known markers will improve their use in breeding programs. The dominant RWA resistance gene, Dn5, was identified in the accession PI294994 and mapped to chromosome arm 7DL. However, recent reports have placed Dn5 on ...

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Sugarcane is a crop that is farmed commercially due to the high amounts of sucrose that is stored within the mature internodes of the stem. Numerous studies have been done to understand sugar metabolism in this crop as well as to enhance sucrose yields. Until now sugarcane improvement strategies have been implemented through either breeding programs or transgenic manipulation. Public mistrust and regulatory hurdles, however, have made the commercialisation of transgenic crops difficult, expensive and timeconsuming. In this thesis two projects will address issues relating to the above. The first will address an effort to increase sucrose accumulation within the sugarcane culm. This was attempted via the expression of an Arabidopsis thaliana vacuolar pyrophosphatase (AtV-PPase) gene, linked to the maize ubiquitin promoter, in sugarcane callus. It was anticipated that increased activity of the tonoplast-bound AtV-PPase will result in increased sucrose accumulation in the vacuole. Transgenic sugarcane callus lines were tested for soluble sugar content which suggested no significant increase in sucrose content. However, this may change upon further assessment of sugarcane suspension cultures and glasshouse plants. The second project was concerned with the development of a novel sugarcane transformation technology that utilises only sugarcane sequences. This ‘cisgenic’ approach to sugarcane transformation will require a native sugarcane promoter, terminator, vector backbone and selection marker. It was attempted to first isolate a functional promoter as well as developing a selection system based on an endogenous selection marker. A promoter was amplified from sugarcane, using primers designed on a sorghum template, and its expression assessed using a GFP reporter gene. Unfortunately expression could not be confirmed in transgenic sugarcane callus. Currently, an alternative approach is followed by using short fragments of constitutively expressed genes to screen sugarcane Bacterial Artificial Chromosome (BAC) libraries to isolate their corresponding promoters. Lastly, it was attempted to develop a selection system for transgenic sugarcane based on resistance to the herbicide chlorosulfuron. A mutant acetolactate synthase (alsb) gene from tobacco, which has shown to confer resistance to the tobacco, was transformed into sugarcane callus. It was anticipated that this gene will confer chlorosulfuron resistance to transgenic sugarcane. If resistance is achieved, the corresponding sugarcane gene will be mutated via site-directed mutagenesis and checked if it also confers resistance to sugarcane. Results showed that although transgenic lines were generated, resistance development is still inconclusive. / AFRIKAANSE OPSOMMING: Suikerriet is ‘n kommersiële gewas wat verbou word as gevolg van die hoë hoeveelhede sukrose wat gestoor word in die volwasse tussenknope van die stam. Verskeie studies is al gedoen om suiker metabolisme in die gewas te ondersoek, sowel as om die sukrose opbrengs te verhoog. Huidige strategieë vir suikerriet verbetering word beywer deur middel van teel-programme of transgeniese manipulasie. Die kommersialiseëring van transgeniese gewasse word egter bemoeilik deur publieke wanpersepsies, sowel as regulatoriese uitdagings. Hierdie tesis beoog om boenoemde kwessies aan te spreek, deur middel van twee projekte. Die eerste projek poog om sukrose akkumulasie in sukerriet te verhoog. Dit was onderneem om die Arabidopsis thaliana vakuolere pirofosfatase (AtV-PPase) geen, wat verbind is met die mielie ubiquitien promoter, uit te druk in suikerriet kallus. Daar was verwag dat die verhoogde aktiwiteit van die tonoplast-gebonde AtV-PPase sal veroorsaak dat meer sukrose in die vakuool akkumuleer. Oplosbare suiker inhoud was getoets in transgeniese suikerriet kallus lyne, maar geen merkbare verhoging in sukrose inhoud was waargeneem nie. Hierdie mag egter verander met verdere ondersoeke in suikerriet suspensie-kulture en glashuis-plante. Die tweede projek het beywer om ‘n nuwe suikerriet transformasie tegnologie te ontwikkel, wat slegs van suikerriet genetiese materiaal gebruik maak. Hierdie ‘cisgeniese’ benadering tot suikerriet transformasie sal ‘n inheemse suikerriet promoter, terminator, vektor ruggraat en seleksie-merker, benodig. Dit was eers beoog om ‘n funksionele promoter te isoleer, sowel as om ‘n seleksie sisteem, gebasseer op ‘n inheemse seleksie merker, te ontwikkel. Deur gebruik te maak van primers wat op ‘n sorghum templaat gebasseer is, was ‘n promotor geisoleer vanuit suikerriet; die uitdrukking hiervan is bepaal deur gebruik te maak van ‘n GFP verklikker geen. Ongelukkig kon uitdrukking nie bevestig word in transgeniese suikerriet kallus nie. Tans word suikerriet Kunsmatige Bakterieële Chromosoom (KBC) biblioteke geskandeer, deur gebruik te maak van geen-fragmente van globaal-uitgedrukte gene, om ooreenstemmende suikerriet promoters te isoleer. Die tweede deel van die cisgeniese projek het beoog om ‘n seleksie sisteem vir transgeniese suikerriet te ontwikkel, wat gebasseer is op weerstand teen die plantdoder chlorosulfuron. Suikerriet kallus was getranformeer met ‘n mutante tabak geen – asektolaktaat sintase (alsb) – wat chlorosulfuron weerstand in tabak meebring. Daar was verwag dat die geen chlorosulfuron weerstand aan transgeniese suikerriet sou oordra. Indien weerstand ontwikkel, sal die ooreenstemende suikerriet geen deur gerigte mutagenese gemuteer word; dan sal dit kan bepaal word of weerstand ook oorgedra word aan suikerriet. Daar is bevind dat alhoewel transgeniese lyne gegenereer is, daar steeds nie ‘n konklusiewe bevestiging van weerstand ontwikkeling is nie.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Gene transfer from wild gras species to wheat is complicated by the simultaneous integration of large amounts of alien chromatin. The alien chromatin containing the target gene is inherited as a linkage block and the phenomenon is known as linkage drag. The degree of linkage drag depends on whether, and how readily, recombination occurs between the foreign and wheat chromatin. The S13 translocation line was developed by the department of Genetics, US. A cross was made between Chinese Spring and a leaf rust resistant Aegilops speltoides accession. Resistant backcross F1 was backcrossed to Chinese Spring and W84-17. S13 was selected from the backcross progeny and found to carry three rust resistance genes temporarily named LrS13, SrS13 and YrS13. Unfortunately, the resistance genes were completely linked to gametocidal (Gc) genes that were co-transferred from the wild parent. In wheat Gc genes cause reduced fertility, poor plant phenotype and hybrid necrosis. In order to use employ the rust resistance genes commercially they need to be separated from the Gc genes. At the onset of this study four putative shortened forms of the S13 translocation were provided. The four lines were identified in a homoeologous paring induction experiment (involving the test cross 04M127). This study aimed to achieve the following: (i) characterize the four recombinants with the use of molecular markers, (ii) use the knowledge gained to identify further recombinants in the 04M127 cross, (iii) identify the shortest (most useful) recombinant, and (iv) attempt to shorten the shortest recombinant form still further and thereby remove as many of the Gc genes as possible. In total, seven recombinants of the S13 translocation (04M127-1, -2, -3, -4, -7, -11 and -12; referred to as recombinant group A) were identified and characterised with microsatellite and SCAR markers. These recombinants have exchanged different amounts of foreign chromatin for wheat chromatin, but were still associated with Gc genes, showing hybrid necrosis and seed shrivelling. Some of the recombinants have lost the undesirable „brittle rachis‟ phenotype which occurs in Ae. speltoides and the S13 translocation line. In plants VII having this trait, the rachis spontaneously disarticulates after the third spikelet upon ripening of the ear. Recombinant 3 appeared to be least affected by Gc genes and was therefore used in further attempts to shorten the translocation. Recombinant 3 was crossed with wheat (W84-17) and resistant F1 (heterozygous for the translocation) were test crossed with Chinese Spring nullisomic 3A tetrasomic 3B/D plants. Thirty five resistant testcross F1 plants were identified (named recombinant group B). The resistant group B recombinants as well as nine susceptible test cross F1 (which also appeared to be recombinant) were characterised making use of microsatellites and a SCAR marker. From the results it appeared that each of the 35 resistant plants exchanged substantial amounts of Ae. speltoides chromatin for wheat chromatin. The species chromatin that remained (and which contains LrS13) is probably located either close to the 3AS telomere or within the proximal regions of 3AS and 3AL. A SCAR marker that has been developed specifically for the S13 translocation provided useful confirmation of the presence of Ae. speltoides chromatin in the 35 recombinants. If the SCAR marker proves to be tightly linked to LrS13 it may eventually be used for marker assisted selection of the resistance or it may be employed in continued attempts to reduce the amount of foreign chromatin. Seedling rust resistance tests showed that the recombinants have lost SrS13 and YrS1 during recombination. An attempt was also made to develop additional markers that specifically detect the translocation in order to further characterise the group B recombinants. Published information on Ae. speltoides specific repeated and transposon sequences were obtained and used for primer design. Unfortunately, no suitable markers could be found and the primers that were designed tended to amplify the same fragments in both the wheat and species genomes. DArT markers were also employed in an attempt to characterise the 35 group B recombinants and controls. The DArT results provided an independent verification of the results obtained with the microsatellite markers. The DArT results confirmed that the group B recombinants exchanged large amounts of species chromatin for wheat chromatin. Even though the 35 resistant group B recombinants have undergone extensive recombination they still show signs of residual Gc effects. It is believed these effects can be removed by continued backcrossing to wheat accompanied by selection against Gc symptoms. While the effects of Gc genes per se were not studied, their properties were reminiscent of those of transposable elements. Indications were that complex interactions involving the Gc genes themselves as well as genetic factors in the wheat genome may have a drastic effect on the selective survival of recombinant gametes.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Biotechnology is an important tool that is used to isolate and characterise genes. It is also used to produce clones that are genetically and phenotypically similar. Many Arabidopsis thaliana transcription factors have been isolated and characterised, but many have yet to be fully described. MYB proteins are members of a super-family of multifunctional transcription factors that can also interact with other transcription factors in the control of pathways. To date, more than 126 AtMYBs have been identified, but most have not been fully characterised, particularly in terms of the molecular role(s) they play in plants. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 and MYB32 have been reported to be negative regulators of general phenylpropanoid metabolism. It has been reported that the five transcription factors mentioned above are likely to negatively regulate flavonoid biosynthesis, even though they may have different target genes. Studies on AtMYB13, AtMYB14 and AtMYB15 reported that they are likely regulators of general phenylpropanoid metabolism. The mentioned roles of the eight AtMYB transcription factors means that they can be manipulated in order to see what effect they have on primary and secondary metabolites in plants. The transcription factors ligated into the pUBI510-GRFCA vector were then used to transform sugarcane callus (Chapter 3). Sugarcane produces sucrose which makes up 70% of the sugar produced in the world, making sugarcane a commercially important and profitable plant. The sugarcane callus was transformed via particle bombardment. The transcription factors AtMYB3, AtMYB6, AtMYB7, AtMYB13 and AtMYB32 were successfully incorporated into the genomic DNA of the sugarcane callus. The data obtained for callus over-expressing AtMYB3, AtMYB13 and AtMYB32 on solid media and the callus in liquid media were contradictory (i.e callus on solid media producing more sucrose than the wildtype whereas the same transgenic line will poduce less sucrose that the wildtype in liquid media or vice versa). However, AtMYB13 transgenic lines produced more sucrose than the wildtype. Transgenic lines of AtMYB7 all produced less sucrose as compared to the wildtype both on solid and in liquid media. The transcription factors which resulted in increased production of starch when over-expressed were AtMYB7 and AtMYB13. The data obtained for AtMYB6 transgenic lines was highly inconsistent in lines grown on same media and across the two media. The effects of these transcription factors in the overall metabolism of the sugarcane callus, either on MSC3 solid or liquid media, could not be fully determined from the GC-MS analysis as there was no consistent phenotypic effect between different transgenic lines for any of the MYB transcription factors used. In Chapter 4, a micropropagation strategy was developed and phytochemicals and their biological activities were determined for the medicinal plant Salvia repens. Salvia plants have been found to be medicinally important due to the secondary metabolites, particularly the essential oils that they produce. The plant extracts have been found to have many biological activities such as antibacterial, anti-inflammatory, antioxidant and anticancer activities. Salvia repens was successfully germinated in vitro,with 60% germination being achieved in MS media containing 1x10-5 times diluted smoke water following scarification for 12 min in 75% (v/v) H2SO4. Success rates of 100% were achieved in the hardening off process when the seedlings were moved into the greenhouse. Germination of S. repens ex vitro was 100% in an autoclaved soil mixture of 1:1 (v/v) sand and vermiculite. Importantly the medicinal value of S. repens produced in vitro or ex vitro was not lost as the GC-MS metabolite analysis showed that the plants produced the chemicals that are medicinally important. Metabolite extracts of S. repens were for the first time reported to be active against fungi with MIC values lower than 1 mg/ml over 4-5 d period against four Fusarium spp. tested. Lastly (Chapter 5), transcription factors AtMYB6 and AtMYB13 were used to trasnform Salvia stenophylla via Agrobacterium-mediated transformation, in order to determine whether the over-expression of these transcription factors could up-regulate the production of medicinally and commercially important secondary metabolites in S. stenophylla. Whilst both A. tumefaciens and A. rhizogenes strains were utilised for the transformation procedure, transformation was only achieved using A. rhizogenes and no transformants could be generated from the A. tumefaciens-treated material. Transgenic hairy roots did not produce any of the medicinally important metabolites. The GC-MS analysis of the transgenic root material identified mainly sugars and other primary metabolites. / AFRIKAANSE OPSOMMING: Biotegnologie is 'n belangrike instrument wat gebruik kan word om gene te isoleer en te karakteriseer. Dit word ook gebruik om klone wat geneties en fenotipies identies is te produseer. Baie Arabidopsis thaliana transkripsiefaktore is al geïsoleer en gekarakteriseer, maar baie moet nog volledig beskryf word. MYB proteïene is lede van 'n super-familie van multifunksionele transkripsiefaktore wat ook interaksie het met ander transkripsiefaktore tydens die beheer van metaboliese weë. Tot op hede is meer as 126 AtMYBs geïdentifiseer, maar die meeste is nie volledig gekarakteriseer nie, veral nie ten opsigtigte van die molekulêre rol(le) wat hulle in plante speel nie. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 en MYB32 is gevind om negatiewe reguleerders van algemene fenielpropanoied-metabolisme te wees. Daar is ook berig dat dié vyf transkripsiefaktore moontlik flavenoied-biosintese negatief kan reguleer, selfs al kan hulle verskillende teikengene hê. Studies op AtMYB13, AtMYB14 en AtMYB15 het berig dat hulle waarskynlik reguleerders van algemene fenielpropanoied-metabolisme is. Die genoemde rolle van die agt AtMYB transkripsiefaktore beteken dat hulle gemanipuleer kan word om te bepaal watter effek hulle op primêre en sekondêre metaboliete in plante het. Die transkripsiefaktore, wat in die pUBI510-GRFCA vektor geligeer was, is toe gebruik om suikerriet-kallus te transformeer (Hoofstuk 3). Suikerriet vervaardig sukrose wat tot 70% van die suiker wat in die wêreld geproduseer word opmaak. Dít maak suikerriet 'n kommersieel belangrike en winsgewende plant. Die suikerriet-kallus is getransformeer deur middel van partikel-bombardering. Die transkripsiefaktore AtMYB3, AtMYB6, AtMYB7, AtMYB13 en AtMYB32 was suksesvol in die DNA van die suikerriet-kallus opgeneem. Data wat verkry was vir kallus wat AtMYB3, AtMYB13 en AtMYB32 ooruitgedruk het op soliede media en kallus in vloeibare medium was teenstrydig (m.a.w. kallus op soilede media wat meer sukrose as die wildetipe op soliede media geproduseer het, terwyl dieselfde transgeniese lyn minder sukrose as die wildetipe geproduseer het in vloeibare medium, en anders om). Nietemin, het AtMYB13 transgeniese lyne meer sukrose geproduseer as die wildetipe. Transgeniese lyne van AtMYB7 het almal minder sukrose geproduseer as die wildetipem op beide soliede en vloiebare media. Die transkriopsiefaktore wat gelei het tot 'n styging in stysel produksie wanneer hulle ooruitgedruk was was AtMYB7 en AtMYB13. Data wat verkry is van die AtMYB6 transgeniese lyne was hoogs veranderlik in lyne wat op dieselfde medium gegroie was en oor die twee media. Die effek van hierdie transkripsiefaktore op die algehele metabolisme van die suikerriet-kallus, hetsy op MSC3 soliede of vloeibaremedia, kon egter nie van die GC-MS analise ten volle bepaal word aangesien daar geen konsekwente fenotipiese effek tussen die verskillende transgeniese lyne vir enige van die gebruikte MYB transkripsiefaktore was nie. In Hoofstuk 4 was ‘n mikropropagerings strategie ontwikkel. Fitochemikalieë en hul biologiese aktiwiteite was ook bepaal vir die medisinale plant Salvia repens. Salvia plante is gevind om medisinaal belangrik te wees as gevolg van die sekondêre metaboliete, veral die essensiële olies, wat hulle produseer. Dit is ook bevind dat die plant-ekstrakte baie biologiese aktiwiteite soos anti-bakteriese, anti-inflammatoriese, anti-oksidant en anti-kanker aktiwiteite het. Salvia repens is suksesvol ontkiem in vitro, met 60% ontkieming wat bereik is in MS media met 1x10-5 maal verdunde rook-water na insnyding vir 12 min in 75% (v/v) H2SO4. Suksessyfers van 100% was behaal in die afhardingsproses wanneer die saailinge na die glashuis verskuif was. Ontkieming van S. repens ex vitro was 100% in 'n geoutoklaveerde grondmengsel van 1:1 (v/v) sand en vermikuliet. Gewigtig het die medisinale waarde van S. repens wat in vitro of ex vitro geproduseer was nie verlore gegaan nie. Die GC-MS data metaboliete analise het aangetoon dat die plante die medisinaal belangrike chemikalieë geproduseer het. Metaboliet-ekstrakte van S. repens was vir die eerste keer na berig aktief teen swamme, met MIK waardes laer as 1mg/ml oor ‘n tydperk van 4-5 d, teen vier Fusarium spp wat getoets was. Laastens (Hoofstuk 5), transkripsiefaktore AtMYB6 en AtMYB13 was gebruik om Salvia stenophylla te transformeer deur Agrobacterium-bemiddelde transformasie, om sodoende te bepaal of die ooruitdrukking van hierdie transkripsiefaktore die produksie van medisinale en kommersieël-belangrike sekondêre metaboliete in S. stenophylla kan verhoog. Alhoewel beide A. tumefaciens en A. rhizogenes stamme gebruik was vir die transformasie proses, kon transformasie slegs deur die gebruik van A. rhizogenes bereik word. Geen transformante kon gegenereer word vanuit die A. tumefaciens behandelde materiaal nie. Transgeniese harigewortels het geen van die medisinaal belangrike metaboliete vervaardig nie. Die GC-MS analise van die transgeniese wortel materiaal het hoofsaaklik suikers en ander primêre metaboliete geïdentifiseer.

Os impactos ambientais causados pela queima dos combustíveis fósseis e pela sua manipulação, aliados ao crescente preço dos combustíveis, têm fomentado a procura de novos recursos renováveis e o desenvolvimento de novas tecnologias que suportem as necessidades desse mercado. Os biocombustíveis são recursos biodegradáveis e renováveis, que vêm se revelando uma alternativa economicamente viável. No entanto, a atual geração de biocombustíveis possui alguns pontos negativos, tais como: utilização de solos férteis e competição com a indústria de alimentos, uma vez que utiliza culturas como soja, milho e cana-de-açúcar, produtos de extrema importância econômica para seus países produtores. Por estes motivos, há um crescente interesse em explorar outras matérias-primas possíveis, em especial as voltadas exclusivamente para a geração de energia. Neste contexto, as microalgas vêm se mostrando uma opção bastante interessante. Estes organismos apresentam um alto potencial para tal, pois possuem alta taxa de crescimento e capacidade de produzir grande quantidade de óleo. Além disso, a produção do biocombustível por estes organismos pode ser otimizada tanto pela modificação das condições de cultivo (engenharia bioquímica), como através da manipulação genética das linhagens (engenharia genética). Neste trabalho, ambas as estratégias foram utilizadas com o intuito de se aumentar a quantidade de lipídeo produzido pela linhagem CC424 da microalga modelo Chlamydomonas reinhardtii. A via metabólica escolhida para a manipulação genética foi o ciclo do glioxilato, sendo as duas enzimas-chave desse ciclo, isocitrato liase (icl) e malato sintase (ms), os alvos. O plasmídeo pSL18 foi utilizado como vetor da transformação nas microalgas. Seis tipos de linhagens transformantes foram obtidas: duas delas subexpressando os genes icl e ms separadamente, duas subexpressando esses genes e duas contendo duplas transformações, ou seja, uma delas subexpressando ambos os genes ao mesmo tempo e a outra superexpressando os mesmos. Quando se subexpressou ambas as enzimas ao mesmo tempo, houve um aumento significativo na quantidade de lipídeos neutros da célula. Além disso, essa linhagem transgênica foi submetida à escassez de nitrogênio, o que acentuou ainda mais esse resultado. Enquanto em meio normal a diferença entre a quantidade de lipídeos foi de 1,5 vezes, em escassez de nitrogênio essa diferença foi de aproximadamente 3 vezes, corroborada pela diferença nos níveis de expressão gênica, que também foi em torno de 3 vezes. Além disso, a linhagem transgênica também mostrou um aumento em cada um dos ácidos graxos analisados individualmente, revelando uma grande quantidade de todos os tipos de C16 e C18, ácidos graxos importantes para que o biodiesel se adeque ao regulamento da Agência Nacional de Petróleo, Gás Natural e Biocombustíveis. Apesar de maior quantidade de lipídeos em relação à linhagem selvagem, a nova linhagem transgênica Dupla-ICL-MS-anti não mostrou nenhum efeito deletério crítico. Tanto a produção de biomassa, quanto a quantidade de clorofila a, proteínas totais e carboidratos totais se mantiveram estáveis após a introdução da mutação. Esses resultados sugerem que as enzimas do ciclo do glioxilato, sabidamente ligadas ao catabolismo de ácidos graxos, podem ser utilizadas como alvos promissores para a otimização de linhagens já utilizadas comercialmente na produção de biodiesel. / The environmental impacts caused by gases emitted from burning fossil fuels and their manipulation, combined with rising fuel prices, has stimulated demand for new renewable resources and developing new green technologies that support the industry and market needs. Biofuels are biodegradable and renewable resources, which come out to be an economically viable alternative. However, the current generation of biofuels has some disadvantages, such as: use of fertile soils and competition with the food industry, once it uses crops such as soybeans, corn and sugar cane, products of extreme economic importance to the producing countries. For these reasons, there is a growing interest in exploring other possible raw materials, especially those that are geared exclusively for power generation. In this context, microalgae have shown to be a very interesting option. These organisms have a high potential because they have fast growth rate and the ability to produce large amounts of oil. In addition, biofuel production by these organisms can be optimized for both the modification of culture conditions (biochemical engineering), and through the genetic manipulation of microalgae strains (genetic engineering). In this work, the two strategies have been used in order to increase the amount of lipid produced by the strain CC424 from the model organism Chlamydomonas reinhardtii. The metabolic route chosen for genetic manipulation is the glyoxylate cycle, and the two key enzymes of this cycle, isocitrate lyase (icl) and malate synthase (ms), the targets. The plasmid pSL18 was used as a vector of transformation in the microalgae. Six types of transformant strains were obtained, two of them overexpressing the ms and icl genes separately, two underexpressing these genes and two double transformations, one of them overexpressing both genes at the same time the other one underexpressing them. The strain underexpressing both enzymes at the same time, showed a significant increase in the amount of neutral lipids. In this mutant, the shortage of nitrogen led to an even greater increase in these lipids. While in normal media the difference between the amount of lipids was 1.5 times, under nitrogen starvation the difference was approximately 3 times, corroborated by the difference in gene expression levels, which was also about 3 times. Moreover, the mutant strain also showed an increase in each of the individual fatty acids analyzed, revealing a large amount in all kinds of C16 and C18 fatty acids, important for biodiesel that suits the regulation of Agência Nacional de Petróleo, Gás Natural e Biocombustíveis. Although the mutant Dupla-ICL-MS-anti produces higher amounts of lipids compared to the wild type, the strain showed no critical negative effects. Both the production of biomass and the amount of chlorophylla, total protein and total carbohydrates remained stable after the introduction of the mutation. These results suggest that the enzymes of the glyoxylate cycle, which are linked to the catabolism of fatty acids, can be used as promising targets for the optimization of strains already used commercially in the production of biodiesel.

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A cana-de-açúcar vem assumindo um papel de destaque na atual conjuntura nacional, impulsionada principalmente pela produção de etanol, que vai de encontro com a crescente preocupação mundial na busca por fontes de energias renováveis e menos impactantes ao ambiente. Por essa razão, é preciso assegurar o contínuo desenvolvimento técnico-científico do setor sucroalcooleiro nacional, mantendo o Brasil na posição de vanguarda na produção de biocombustíveis. Ante a disponibilidade de inúmeras ferramentas biotecnológicas, tornou-se possível avançar com maior celeridade na compreensão dos campos da genética e fisiologia da cana-de-açúcar. Neste trabalho é demonstrado a transformação genética via biobalística da cultivar RB835486. No processo foram usadas duas construções para silenciamento gênico via RNA de interferência (RNAi), com genes quiméricos do tipo shRNA (short harpin RNA) para silenciamento dos genes SspTIP1;1 e SspPIP1;4, em co-tranformação com o gene marcador npt- II. Os dois genes alvo selecionados codificam aquaporinas, proteínas transmembrana responsáveis pelo transporte de água na planta. Estes genes foram identificados anteriormente por seu possível envolvimento no processo de acúmulo de sacarose. A co-integração dos cassetes de silenciamento gênico e do gene marcador ocorreu em 13 plantas, sendo obtidas três linhagens para o gene SspTIP1;1 e 10 linhagens para o gene SspPIP1;4. Dentre elas, duas linhagens SspTIP1;1 e cinco linhagens SspPIP1;4 foram analisadas via RT-PCR, quanto a possíveis modificações nos níveis de expressão dos genes alvos. Nas duas linhagens transgênicas avaliadas para silenciamento do SspTIP1;1, não houve redução em sua expressão em relação ao controle não transformado, possivelmente devido a efeitos de posição. Nas outras cinco linhagens transgênicas avaliadas para silenciamento do SspPIP1;4, houve redução significativa em seus níveis de expressão em três linhagens em relação ao controle não transformado. Nestas plantas serão realizadas as análises fisiológicas a fim de validá-las funcionalmente quanto ao transporte de água e acúmulo de sacarose. / Sugarcane has taken a leading role in the current national economy, mainly boosted by ethanol production, which meet the growing global concern on searching for renewable energy and with low impact on the environment. Therefore, it is necessary to ensure the continuous technical and scientific development of the national sugar and ethanol sector, maintaining the leading position of Brazil in biofuel production. By the availability of numerous biotechnology tools, it became possible to advance more rapidly in understanding the fields of genetics and physiology of sugarcane. This work demonstrated the genetic transformation of the cultivar RB835486 via biolistic assay. In the process it was used two constructs for gene silencing via RNA interference (RNAi) with chimeric genes of the type shRNA (short harpin RNA) for silencing of the genes SspTIP1;1 and SspPIP1;4, co-transformed with the marker gene npt- II. The two selected target genes encode aquaporins, transmembrane proteins which are responsible for water transport in plants. These genes were previously identified for their possible involvement in the process of sucrose accumulation. The co-integration of both, the cassette gene silencing and gene marker was observed in 13 plants, three strains were obtained for the gene SspTIP1;1 and 10 strains for gene SspPIP1;4. Among them, two strains of SspTIP1;1 and five strains of SspPIP1;4 were analyzed by RT-PCR, searching for possible changes in the levels of target gene expression. In the two transgenic lines evaluated for silencing SspTIP1;1, no reduction in expression compared to control non-transformed was obtained, possibly due to effects of position insertion of the gene in the genome. The other five transgenic lines evaluated for silencing of SspPIP1;4, a significant reduction in their expression levels was obtained in three strains when compared to the control untransformed plants. These silenced plants will be physiologically analyzed to validate their function on water transport and sucrose accumulation.