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The study is aimed to determine the role of avocado DGAT1 and PDAT1 in seed oil synthesis. Triacylglycerol (TAG) has a nutritional and industrial value and is essential for plant growth. DGAT1 and PDAT1 catalyze the final step of TAG Assembly. We hypothesized that both PaPDAT1 and PaDGAT1, although predominantly expressed in non-seed tissues, could contribute to oil accumulation in seeds. Agrobacterium transformants with PaPDAT1 and PaDGAT1 cloned in pCAMBIA were generated to test this. Subsequently, the Agrobacterium-mediated transformation of Arabidopsis mutant lines and camelina was carried out by floral dipping. The T1 camelina seeds expressing the genes of interest were selected using fluorescence screening. Homozygous T3 lines were generated. The transgenic camelina seeds were evaluated for TAG content and fatty acid composition relative to wild-type seeds. Line D1 3-3-2 expressing PaDGAT1 and line P1 7-8 expressing PaPDAT1 showed a significant increase in C18:1 compared to the wild type.

Plants synthesize and store oil, mostly as triacylglycerols (TAG), in seeds that is transcriptionally controlled by WRINKLED1 (WRI1), an APETALA2 (AP2) transcription factor. Among the four Arabidopsis WRI paralogs, WRI2 is nonfunctional, while the others are expressed in a tissue-specific manner. Additionally, two rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid diacylglycerol acyltransferase (PDAT) catalyze the terminal step in TAG assembly and contribute to oil accumulation. Avocado (Persea americana) mesocarp, a non-seed tissue, accumulates significant amounts of TAG (~70% by dry weight) enriched with heart-healthy oleic acid. The oil accumulation stages in mesocarp coincide with the high expression of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1. The strong preference for oleic acid demonstrated by the avocado mesocarp TAG biosynthetic machinery represents lucrative biotechnological opportunities, yet functional implication of these genes is not explored. Using structural analyses, we showed that PaWRI2 is a relatively stable protein, has a single intact AP2 DNA-binding domain, and has different C-terminal properties compared to its ortholog in Arabidopsis. Through transient expression, we demonstrated that PaWRI2 is functional and drives TAG accumulation in Nicotiana benthamiana leaves, unlike Arabidopsis WRI2. Additionally, co-infiltration of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1 further increased the lipid content and oleic acid levels in ‘benth’ leaves. Quantitative real-time PCR (qPCR) analyses of >46 fatty acid biosynthetic pathway genes revealed that several were distinctly up- or down-regulated by the expression of PaWRI2 and PaWRI1. Further yeast-one-hybrid assay showed a unique characteristic of PaWRI2 being autoregulated and by PaWRI1. Also, both the proteins could bind to AW-box promoter elements in specific avocado genes. Deletion of the C-terminally-located ordered region in both the proteins further improved the lipid content with an altered composition in the leaf tissue. In conclusion, avocado WRI2 is capable of transactivation of fatty acid biosynthesis genes and TAG accumulation, synergistically with DGAT1 and PDAT1, in non-seed tissues. This study provides a functional role for WRI2 in a basal angiosperm species that is likely lost in modern angiosperms and thus provides basis for mechanistic differences in the transcriptional regulation of lipid biosynthesis among different plant species and between seed and non-seed tissues.

La glande mammaire d'une vache laitière haute productrice, produit quotidiennement une quantité de lait équivalente à 10% du poids de l'animal. L'alimentation, la génétique et le processus de différenciation terminale du tissu mammaire peuvent impacter significativement la productivité de l'animal et la composition du lait. L'activité intense de biosynthèse et de sécrétion de la glande mammaire fait intervenir un large répertoire de gènes dont l'expression peut aujourd'hui être analysée de façon globale et simultanée grâce à des outils tels que les puces à ADN. C'est ce type d'approche que nous avons mis en œuvre, dans ce travail de thèse, afin d'établir les réseaux de gènes qui participent au processus de différenciation terminale du tissu mammaire et qui permettent d'expliquer les variations de composition du lait observées avec la mutation K232A au locus DGAT1 bovin. Pour ce faire, nous avons utilisé chez la chèvre un dispositif en boucle qui a permis d'analyser, au moyen d'un répertoire de 22 000 sondes oligonucléotidiques bovines (22K), l'évolution du profil d'expression génique du tissu mammaire au cours de la gestation. Nous avons ainsi pu montrer que ce profil subit au cours du développement de l'organe et de sa différenciation terminale des changements profonds qui définissent 3 étapes majeures : i) le déclenchement de la différenciation fonctionnelle qui intervient avant le premier tiers de la gestation ; ii) un remodelage du tissu au cours duquel les structures qui produiront le lait (acini) se mettent en place et prolifèrent pour envahir progressivement l'organe - cette phase est marquée par l'expression de gènes de la réponse immunitaire ; iii) l'acquisition du phénotype sécrétoire, matérialisée par le déclenchement de la synthèse lipidique à la parturition. Afin de déterminer les mécanismes cellulaire sous-jacents aux variations de composition du lait observées avec la mutation K232A au locus DGAT1, nous avons entrepris la comparaison du transcriptome de tissu mammaire bovin à partir d'un dispositif expérimental génétiquement bien défini et contrôlé. A chaque animal de génotype GC/GC (A232), correspondait soit sa pleine sœur (n=4), soit sa demi-sœur (n=2) de génotype AA/AA (K232), au même numéro de lactation et au même nombre de jours de lactation. Nos résultats ont confirmé une baisse significative du TB (41.6 vs. 51.6 g/kg) et des AG à chaîne moyenne et insaturés (C14:1 et C16:1) et une augmentation des AG à chaîne moyenne et saturée (C14:0) et longue et insaturée (C18:1 cis11, C18:1 cis12, C18:2 n-6, C18:3 n-3) avec le variant GC/GC (A232) en comparaison au variant AA/AA (K232). Les globules gras étaient également plus petits avec ce variant. L'analyse du transcriptome à partir d'ARN extraits des biopsies de tissu mammaire a montré que les gènes spécifiant les acteurs des voies de synthèse du lactose, des lipides et des protéines étaient sur-exprimés chez les individus de génotype GC/GC (A232). La liste des gènes de la biosynthèse des lipides sur-exprimés avec ce génotype expliquent, pour partie, les différences de composition fine en acides gras observées en suggérant des voies alternatives du métabolisme du diacylglycérol.

In the present study samples from four native Turkish Cattle Breeds / South Anatolian Red (n= 48), East Anatolian Red (n= 34), Anatolian Black (n= 42) and Turkish Grey (n=46) and elite bulls of Holstein (n=21) were genotyped with respect to two milk production enhancer genes, Prolactin (PRL) and Diacylglycerol acyltransferase (DGAT1), and one disease (Complex Vertebral Malformation) causing gene (SLC35A3). A allele frequency for PRL gene, believed to be positively associated with the milk yield in cattle, ranged between 0.5645 (Anatolian Black) - 0.7558 (South Anatolian Red). K allele frequency which is thought to be related with the milk fat content in cattle varied between 0.7794 (East Anatolian Red) - 0.9250 (Anatolian Black). Complex Vertebral Malformation gene was not observed in any of the examined individuals (n= 164), hence, SLC35A3 locus was monomorphic. Pairwise Fst values based on the two polymorphic loci revealed that breeds are not significantly different from each other with respect to these two genes. Correlations, but weak, between the PRL A allele frequency and milk yield and similarly DGAT1 K allele and milk fat content was observed, Principle Component Analysis generated two compound axis based on the two polymorphic loci. Positions of the breeds on the first axis were correlated with the milk fat content of the breeds, perfectly. Again, positions of the breeds on the second axis were correlated with the milk yield of the breeds. Furthermore, PCA revealed that both A of PRL and K of DGAT1 genes seemed to have contributions in milk yield Results are believed to be useful for the management efforts of Turkish native cattle breeds.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Many Euonymus species express an acetyltransferase enzyme in their seeds which catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol (DAG) producing unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAG). The presence of the sn-3 acetate group gives acetyl-TAG with unique physical properties over regular triacylglycerol (TAG) found in vegetable oils. The useful characteristics of acetyl-TAG oil offer advantages for its use as emulsifiers, lubricants, and 'drop-in' biofuels. One enzyme, Euonymus alatus diacylglycerol acetyltransferase (EaDAcT), responsible for acetyl-TAG synthesis in nature was previously isolated from the seeds of Euonymus alatus (burning bush) and expressed in the oilseed crop Camelina sativa. Expression of EaDAcT successfully led to production of high levels of acetyl-TAG in camelina seeds. To further increase acetyl-TAG accumulation in transgenic camelina seeds, multiple strategies were examined in this study. Expression of a new acetyltransferase enzyme (EfDAcT) isolated from the seeds of Euonymus fortunei, which was previously shown to possess higher in vitro activity and in vivo acetyl-TAG levels compared to EaDAcT, increased acetyl-TAG accumulation by 20 mol%. Suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation to 90 mol% in selected transgenic line. Studying the regulation of EfDAcT transcript, protein, and acetyl-TAG levels during seed development further provided new insights on the factors limiting acetyl-TAG accumulation.

Made available in DSpace on 2014-06-11T19:26:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-09-24Bitstream added on 2014-06-13T18:54:05Z : No. of bitstreams: 1 vechetini_me_me_jabo.pdf: 579041 bytes, checksum: 90bdfc2c0590f6beb86275f97fa9a321 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Este estudo foi conduzido com 125 novilhas da raça Nelore de três rebanhos: Nelore Seleção (NeS), Nelore Tradicional (NeT), ambos selecionados para peso ao sobreano, e Nelore Controle (NeC), selecionado para a média deste peso, com o objetivo de avaliar polimorfismos do tipo PCR-RFLP's nos genes OGA T1 e LEPTlNA. Os rebanhos analisados pertencem ao Programa de Melhoramento Genético das Raças Zebuínas, da Estação Experimental de Zootecnia de Sertãozinho - SP - Brasil. O DNA genômico foi extraído de leucócitos pelo método salino. Para a amplificação dos fragmentos de gene foram utilizados oligonucleotídeos iniciadores previamente descritos na literatura. Os produtos de PCR foram digeridos com enzimas de restrição para a detecção dos polimorfismos OGAT1 - Eael e LEPTlNA - BsaAI, e submetidos a eletroforese em gel de agarose. O OGAT1K (Iisina) foi o alelo que apresentou maior freqüência n nos rebanhos. As maiores freqüências do genótipo OGA T1KK foram encontradas nos rebanhos selecionados NeS e NeT, respectivamente, seguido pelo heterozigoto OGAT1KA. Nenhum animal apresentou o genótipo OGAT1AA (alanina). As freqüências alélicas e genotípicas do OGA T1 não diferiram entre NeS e NeT, mas foram estatisticamente diferentes (p<0,05) entre esses e o rebanho controle NeC. Em relação ao hormônio Leptina, as freqüências dos alelos foram LEP A = LEP G = 0,50 em toda a população. O heterozigoto LEP AG foi o genótipo que apresentou maior freqüência para os três rebanhos A maior freqüência do genótipo LEP GG foi observada no rebanho controle (NeC), mas as freqüências alélicas e genotípicas entre os rebanhos não foram estatisticamente diferentes. / This study was conducted with 125 Nelore heifers from three different herds: selection (NeS), traditional (NeT), both selected for yearling weight, and contrai (NeC), selected for mean yearling weight, and had the objective of looking for PCR-RFLP's polymorphisms in DGA T1 and LEPTlN genes. The herds analysed belong to the Zebu Breeding Pragram, of the Animal B:-8eding Experiment Station of Sertãozinho - SP - Brazil. The DNA was extracted fram leukocytes by the saline protocol, and to amplify the gene fragments primers previously described were used. The PCR products were digested by restriction enzymes to detect the DGA T1 - Eael e LEPTlNA - BsaAI polymorphisms, and were submitted to electraphoresis. The DGAT1K allele (Iisine) presented the larger frequency in the population. The highest frequencies of the DGA T1KK were found in the NeS e NeT herds, respectively, followed by the DGA T1KA. Neither animal presented the DGA T1AA genotype (alanine). Selected herds (NeS and NeT) allelic and genotypic frequencies for this locus were not significanttly different. Howerver, significant differences (p<0.05) were detected between the selected (NeS and NeT) and the control herds (NeC). In respect of the Leptin hormone, the allele frequencies were LEP A = LEP G = 0,50 in all the population. The LEP AG was the most frequent genotype in all the three herds. The control group (NeC) presented the LEP GG largest frequency, however, no significant differences were observed among the three herds for this locus.

Resumo: Este estudo foi conduzido com 125 novilhas da raça Nelore de três rebanhos: Nelore Seleção (NeS), Nelore Tradicional (NeT), ambos selecionados para peso ao sobreano, e Nelore Controle (NeC), selecionado para a média deste peso, com o objetivo de avaliar polimorfismos do tipo PCR-RFLP's nos genes OGA T1 e LEPTlNA. Os rebanhos analisados pertencem ao Programa de Melhoramento Genético das Raças Zebuínas, da Estação Experimental de Zootecnia de Sertãozinho - SP - Brasil. O DNA genômico foi extraído de leucócitos pelo método salino. Para a amplificação dos fragmentos de gene foram utilizados oligonucleotídeos iniciadores previamente descritos na literatura. Os produtos de PCR foram digeridos com enzimas de restrição para a detecção dos polimorfismos OGAT1 - Eael e LEPTlNA - BsaAI, e submetidos a eletroforese em gel de agarose. O OGAT1K (Iisina) foi o alelo que apresentou maior freqüência n nos rebanhos. As maiores freqüências do genótipo OGA T1KK foram encontradas nos rebanhos selecionados NeS e NeT, respectivamente, seguido pelo heterozigoto OGAT1KA. Nenhum animal apresentou o genótipo OGAT1AA (alanina). As freqüências alélicas e genotípicas do OGA T1 não diferiram entre NeS e NeT, mas foram estatisticamente diferentes (p<0,05) entre esses e o rebanho controle NeC. Em relação ao hormônio Leptina, as freqüências dos alelos foram LEP A = LEP G = 0,50 em toda a população. O heterozigoto LEP AG foi o genótipo que apresentou maior freqüência para os três rebanhos A maior freqüência do genótipo LEP GG foi observada no rebanho controle (NeC), mas as freqüências alélicas e genotípicas entre os rebanhos não foram estatisticamente diferentes. / Abstract: This study was conducted with 125 Nelore heifers from three different herds: selection (NeS), traditional (NeT), both selected for yearling weight, and contrai (NeC), selected for mean yearling weight, and had the objective of looking for PCR-RFLP's polymorphisms in DGA T1 and LEPTlN genes. The herds analysed belong to the Zebu Breeding Pragram, of the Animal B:-8eding Experiment Station of Sertãozinho - SP - Brazil. The DNA was extracted fram leukocytes by the saline protocol, and to amplify the gene fragments primers previously described were used. The PCR products were digested by restriction enzymes to detect the DGA T1 - Eael e LEPTlNA - BsaAI polymorphisms, and were submitted to electraphoresis. The DGAT1K allele (Iisine) presented the larger frequency in the population. The highest frequencies of the DGA T1KK were found in the NeS e NeT herds, respectively, followed by the DGA T1KA. Neither animal presented the DGA T1AA genotype (alanine). Selected herds (NeS and NeT) allelic and genotypic frequencies for this locus were not significanttly different. Howerver, significant differences (p<0.05) were detected between the selected (NeS and NeT) and the control herds (NeC). In respect of the Leptin hormone, the allele frequencies were LEP A = LEP G = 0,50 in all the population. The LEP AG was the most frequent genotype in all the three herds. The control group (NeC) presented the LEP GG largest frequency, however, no significant differences were observed among the three herds for this locus. / Orientador: Lúcia Galvão de Albuquerque / Coorientador: Maria Eugênia Zerlotti Mercadante / Banca: Humberto Tonhati / Banca: Lenira El Faro Zadra / Mestre

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-06-08T20:29:50Z No. of bitstreams: 2 Tese - Alex Silva da Cruz - 2015.pdf: 4189617 bytes, checksum: f21e8815a51e44f144dd811005f32bdd (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-09T11:41:47Z (GMT) No. of bitstreams: 2 Tese - Alex Silva da Cruz - 2015.pdf: 4189617 bytes, checksum: f21e8815a51e44f144dd811005f32bdd (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Made available in DSpace on 2016-06-09T11:41:47Z (GMT). No. of bitstreams: 2 Tese - Alex Silva da Cruz - 2015.pdf: 4189617 bytes, checksum: f21e8815a51e44f144dd811005f32bdd (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2015-10-19 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Genomic selection in a dairy cattle breeding is a new strategy in national livestock. Genome wide association study (GWAS) is known as a strategy that involve the use of molecular markers panels distributed throughout the genome which are selected for the identification of chromosomal regions that are important for the interest traits. The aim of this study was apply the GWAS strategy for 305-day milk yield in Girolando cows. We did the genotype from 404 Girolando and after quality control analysis remained 337 individuals and 45.622 markers. The GWAS analysis resulted in 52 SNPs associated to 305-day milk yield. Of these, 23 SNPs were linked to Known genes and only 3 SNPs were linked to NUB1, SLC24A2 and DGAT1 genes that already were associated with cattle lactation. The other SNPs have no relationships described in the cattle lactation literature. In addition, the milk production QTL analysis resulted in 52 SNPs and 14 genes linked or close to 1Mb of the SNP marker. The ARS-BFGL-NGS-414 SNP on BTA19 at 47.9Mbp is located near to GH1 gene. This gene is commonly accepted as causal gene for Quantitative Character Locus of milk production mainly affecting the yield in liters and solid milk components. Thus, our data suggest that NUB1 and SLC24A2 genes could be considered as candidate genes to understand the milk production in Girolando breed. Like this DGAT1 and GH1 genes are valuable predictive markers to be added to genomic selection of dairy cattle in breeding program. / A seleção genômica, aplicada em bovino em associação à produção de leite é uma inovação estratégica na pecuária nacional, e que poderá se tornar uma ferramenta prática importante para a atividade. Estudos de Associação Ampla do Genoma (GWAs) caracteriza-se como uma estratégia que envolve o uso de painéis de marcadores moleculares distribuídos por todo o genoma, selecionados para a identificação de regiões dos cromossomos associadas com um fenótipo de interesse. O objetivo deste estudo foi aplicar a estratégia de GWAS para a característica de lactação total ajustada em 305 dias de vacas Girolando. Inicialmente, foram genotipados 404 vacas Girolando que após procedimento de controle de qualidade resultou em um total de 337 indivíduos e 45.622 marcador. O GWAS resultou em 52 SNPs associados a lactação ajustada em 305 dias. Destes, 23 SNPs apresentaram-se ligados a genes conhecidos e somente 3 SNPs estão ligados aos genes NUB1, SLC24A2 e DGAT1, descritos relacionados a lactação em bovinos. Os demais SNPs não apresentam relações descrita na literatura a lactação em bovinos. Para o QTL de produção de leite (MY), dos 52 SNPs, foram identificados 14 genes ligados ou próximos a 1Mb de distância do SNP marcador. Em particular, o SNP ARS-BFGL-NGS-414 associado ao QTL de lactação bovina, constituído de aproximadamente 47,9 Mbp localizado no BTA19 está localizado muito próximo do gene GH1 (Hormônio do Crescimento 1), comumente aceito como gene causal para o Lócus de Caráter Quantitativo (QTL) de produção de leite, afetando principalmente o rendimento em litros e componentes sólidos do leite. Dessa forma, nossos dados sugerem que os genes NUB1, e SLC24A2 poderiam ser considerados como genes candidatos para ajudar a explicar a produção de leite em animais da raça Girolando, assim como os genes DGAT1 e GH1, são considerados como valiosos marcadores preditivos a serem adicionados à seleção genômica do gado leiteiro em programas de melhoramento.