Zona pellucida glycoproteins (ZP) are important proteins for maturation of the oocytes in eukaryotes, these proteins are encoded by cluster of zp genes. zp2.3 and zp3.5 genes are expressed during the developing and maturation of the oocytes in zebrafish ovaries. Both of the gens have a CCAAT box in their promoter regions, playing a big role in the expression of the both genes in zebrafish oocytes. The transcription of the genes in the eukaryotes requires transcription factors to initiate and promote the transcription, the transcription factors can bind to the promoter region and initiate the transcription process. The nuclear factor y (NFY) regulates the genes by binding to the CCAAT boxes in their promoter regions, it consist from many subunit such as NF-YA and NF-YB. Here in this study we characterize the expression pattern of NF-YA and NF-YB by screening these genes expression in several organs and tissues, also to determine its roles in the expression of the zp2.3 and zp3.5 genes in the adultzebrafish ovary.

Yes / As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved fromsponges to humans, and play vital roles inmany physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including sixα and five β subunitswere cloned and characterized fromsilkworm. Our results showed that integrins fromsilkwormown more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins performdiverse functions in hemocytes of silkworm. Overall, our results provide a newinsight into the functional and evolutionary features of integrins. / National Basic Research Programof China (No. 2012cb114603), the Research Fund for the Doctoral Program of Higher Education of China (20130182110003), the Natural Science Foundation of Chongqing (cstc2013jcyjys0007), and the Fundamental Research Funds for the Central Universities (SWU111014).

Les éléments transposables (ETs) sont des séquences d'ADN mobiles retrouvées dans les génomes de toutes les espèces où ils ont été recherchés. Moteurs de l'évolution, ces éléments mobiles, présents en de nombreuses copies dans les génomes, ont joué un rôle majeur dans la dynamique des génomes en engendrant des mutations et des réarrangements chromosomiques.Néanmoins, étant des constituants majeurs des génomes, ils doivent être finement régulés dans le but de préserver l'intégrité génomique, et ainsi de conserver l'équilibre entre variabilité et stabilité des génomes. Afin de protéger l'information génétique de l'hôte transmise à la descendance, la régulation des ETs au niveau des gonades est effectuée par la voie des piRNAs, voie d'ARN interférent conservée chez les animaux. Bien qu'elle soit relativement bien décrite chez la drosophile et la souris, certaines étapes de cette voie restent encore incomprises. Durant ma thèse, j’ai exploré différents aspects de la biologie des clusters de piRNAs, en prenant comme modèle d’étude le locus flamenco. Le cluster de piRNAs flamenco est le producteur majeur de piRNAs dans les cellules folliculaires des ovaires de Drosophila melanogaster. Tout d'abord, j'ai analysé les fenêtres spatio-temporelles de l’expression du cluster de piRNAs flamenco tout au long du développement de la drosophile,de l'embryon à l'âge adulte. Ensuite, j'ai recherché, in vivo, la séquence des transcrits de flamenco qui serait suffisante pour induire l'adressage d'un transcrit chimérique à la voie de maturation des piRNAs. J'ai également exploré l'impact de certains facteurs sur la prise en charge de transcrits artificiels par la voie des piRNAs. Enfin, je me suis intéressée à la régulation génique que pourraient effectuer les piRNAs provenant de flamenco dans les ovaires de drosophile en recherchant, par des approches bioinformatiques et de biologie moléculaire, les gènes potentiellement reconnus, et par conséquent, régulés par les piRNAs de flamenco. L'ensemble de ces axes de recherche in vivo permettront d'avancer dans la compréhension de la biologie des clusters de piRNAs ainsi que sur les mécanismes moléculaires mis en jeu lors de la biogenèse des piRNAs chez la drosophile. / Transposable elements (TEs) are defined such as mobile DNA sequences found in genomes ofall species where they were searched. As evolutionary drivers, these mobile elements, presentin many copies in genomes, have played a major role in the genome dynamics by generatingmutations and chromosomal rearrangements. Nevertheless, being major genome constituents,they must be finely regulated in order to preserve the genomic integrity, and thus, to maintainthe balance between variability and stability of genomes. In order to protect the geneticinformation of the host transmitted to the offspring, the gonadal TE regulation is carried outby the piRNAs pathway, an interfering RNA pathway conserved in animals. Although this isrelatively well described in Drosophila and in mouse, some steps of piRNA pathway are stillmisunderstood. During my thesis, I explored various aspects of piRNA cluster biology, usingthe flamenco locus as a model. This piRNA cluster is the main piRNA producer in thefollicular cells of Drosophila melanogaster ovaries. First, I analyzed the spatio-temporalwindows of flamenco piRNA cluster expression throughout the Drosophila development,from embryo to adulthood. Then, I searched, in vivo, the flamenco transcript sequence thatwould be sufficient to induce the addressing of a chimeric transcript to the piRNA processingpathway. I also explored the impact of some factors on the management of artificialtranscripts by piRNAs. Finally, I was interested in the gene regulation that flamenco-derivedpiRNAs could make in Drosophila ovaries by searching, through bioinformatics andmolecular biology approaches, the potentially recognized genes, and therefore, regulated byflamenco piRNAs. All of these in vivo research axes will advance in the understanding of thebiology of piRNA clusters as well as the molecular mechanisms involved in the piRNAbiogenesis in Drosophila.

La régulation de la transcription est l‟un des processus cellulaires des plus fondamentaux et constitue la première étape menant à l‟expression protéique. Son altération a des effets sur l‟homéostasie cellulaire et est associée au développement de maladies telles que le cancer. Il est donc crucial de comprendre les règles fondamentales de la fonction cellulaire afin de mieux cibler les traitements pour les maladies. La transcription d‟un gène peut se produire selon l‟un des deux modes fondamentaux de transcription : en continu ou en burst. Le premier est décrit comme un processus aléatoire et stochastique qui suit une distribution de Poisson. À chaque initiation de la transcription, indépendante de la précédente, un seul transcrit est produit. L‟expression en burst se produit lorsque le promoteur est activé pour une courte période de temps pendant laquelle plusieurs transcrits naissants sont produits. Apportant la plus grande variabilité au sein d‟une population isogénique, il est représenté par une distribution bimodale, où une sous-population n‟exprime pas le gène en question, alors que le reste de la population l‟exprime fortement. Les gènes des eucaryotes inférieurs sont pour la plupart exprimés de manière continuelle, alors que les gènes des eucaryotes supérieurs le sont plutôt en burst. Le but de ce projet est d‟étudier comment l‟expression des gènes a évolué et si la transcription aléatoire, ou de Poisson, est une propriété des eucaryotes inférieurs et si ces patrons ont changé avec la complexité des organismes et des génomes. Par la technique de smFISH, nous avons étudié de manière systématique quatre gènes évolutivement conservés (mdn1+, PRP8/spp42+, pol1+ et cdc13+) qui sont continuellement transcrits dans la levure S. cerevisiae. Nous avons observé que le mode d‟expression est gène-et-organisme spécifique puisque prp8 est exprimé de manière continuelle dans la levure S. pombe, alors que les autres gènes seraient plutôt exprimés en légers burst. / Regulating transcription is one of the most fundamental cellular processes and the first step of a long cascade of processes leading to protein expression. Altering transcriptional output often has major effects on cellular homeostasis and is associated with many disease phenotypes, such as cancer. Understanding the fundamental rules governing transcription regulation is therefore instrumental in understanding cellular function as well as in finding disease treatments. Transcription of a gene can occur through two fundamental different modes: “continuous” or “bursting”. Continuous transcription is defined as stochastic process where a promoter is always in its “on” state and each initiation event is independent of the previous. Bursting transcription occurs when a promoter is activated for a short time and multiple mRNAs are produced during the “on” state, followed by long periods of transcription inactivity. It leads to greater variability in an isogenic population, as expression is often bimodal as a sub-population does not express a given gene. Bursting expression is frequently observed in higher eukaryotes, while a continuous pattern seems to be common in lower eukaryotes. The goal of this project is to study how gene expression patterns evolved. We investigate whether Poisson-like transcription is a property of lower eukaryotes and whether transcription patterns have changed when organisms and genomes evolved into more complex systems. Using smFISH, we have systematically determined expression patterns of four evolutionarily conserved genes; mdn1+, PRP8/spp42+, pol1+ and cdc13+, previously shown to be continuously expressed in the yeast S. cerevisiae. Expression was studied in the yeast S. pombe, as an example for another lower eukaryote as well as in human cell-lines. We observe that expression patterns are organism-and-gene specific suggesting that expression patterns have evolved to fulfill gene specific functions.

Anti-Neutrophile Cytoplasmatische Antikörper verursachen nekrotisierende Vaskulitiden kleiner Blutgefäße. Die Serinprotease PR3 ist ein ANCA-Zielantigen, welches von zirkulierenden ANCA auf der Zellmembran erkannt wird. ANCA aktivieren neutrophile Granulozyten, die dann die nekrotisierende Vaskulitis verursachen. Das Membran-PR3 Expressionsmuster ist bimodal wobei mPR3-niedrig- und mPR3-hoch-exprimierende Zellen existieren. Wir testeten die Hypothese, dass ein Membranrezeptor eine hohe mPR3-Expression vermittelt. Wir verwendeten humane neutrophile Granulozyten, neutrophil-differenzierte Stammzellen und transfizierte HEK293 Zellen. Wir identifizierten das Glykoprotein CD177 als einen mPR3-präsentierenden Rezeptor. CD177 zeigte eine spezifische Bindung von reifem PR3-Protein, nicht aber von einem unprozessierten PR3. Wir separierten die mPR3-Zellpopulationen und führten Durchflusszytometrie, Giemsa-Färbung, Western Blot-Experimente und RT-PCR für die PR3 und CD177 mRNA-Expression durch. Wir fanden, dass die mPR3hoch neutrophilen Granulozyten PR3- und CD177-Protein enthielten, während in den mPR3niedrig neutrophilen Granulozyten nur PR3, aber kein CD177 detektierbar war. Die CD177-Regulation vollzog sich auf transkriptioneller Ebene, da die Zellen, die negativ für das CD177-Protein waren auch keine mRNA transkribierten. Um die Grundlage der fehlenden CD177-Transkription zu analysieren, identifizierten wir den Transkriptionsstart von CD177 für eine anschließende Mutations- und SNP-Analyse. Die CD177-Sequenzen der proteinkodierenden Regionen und der Intron-Exon-Übergänge der beiden Zellpopulationen waren identisch. Jedoch fanden wir, dass das CD177-Gen einer monoallelischen Expression unterliegt. Es wurde dabei maternale als auch paternale monoallelische Expression detektiert. In weiterführenden Untersuchungen soll der Regulationsmechanismus der monoallelischen CD177-Expression charakterisiert werden. / Anti-Neutrophil Cytoplasmic Antibodies cause necrotizing small-vessel vasculitis. The serine protease PR3 provides a main ANCA target antigen and is recognized by circulating ANCA on the neutrophil cell surface. ANCA activate neutrophils and activated neutrophils cause vasculitis. The membrane-PR3 expression pattern is bimodal in that low and high mPR3 expressing cells can be distinguished. We tested the hypothesis that a membrane receptor mediates mPR3high expression. We studied human neutrophils, neutrophilic differentiated CD34-positive hematopoietic stem cells and transfected HEK293 cells. We identified the glycoprotein CD177 as an mPR3 presenting receptor. CD177 demonstrated specific binding of mature, but not of unprocessed pro-PR3. We separated the two mPR3 populations and performed cytometry analysis, Giemsa staining, western blot analysis and RT-PCR for PR3 and CD177 expression. We detected PR3 and CD177 protein in mPR3high expressing neutrophils, whereas only PR3, but no CD177 was found in mPR3low expressing cells. Regulation took place on a transcriptional level because cells that were negative for CD177 protein were also negative for mRNA. To further study this finding, we identified the CD177 transcription start for a subsequent mutation and SNP analysis. CD177 sequences of the protein-coding regions and the intron-exon regions did not differ in both populations. However, we found a monoallelic CD177 expression and were able to detect maternal as well as paternal allele expression. Future experiments will elucidate the mechanisms that control monoallelic CD177 gene expression.

No description available.

A soja constitui uma das culturas mais importantes mundialmente, tanto social quanto economicamente. Consequentemente, informações moleculares sobre processos de desenvolvimento, bem como conhecimento detalhado das interações entre condições estressoras e a resposta da planta a fatores ambientais são necessários. A identificação e caracterização de genes que respondem a condições ambientais específicas constituem um passo inicial no entendimento dos processos adaptativos. Proteínas ricas em glicina (GRPs) são polipeptídeos contendo um grande número do aminoácido glicina em sua estrutura primária. Os genes codificantes de GRPs são regulados ao longo do desenvolvimento e regulados por auxina, ABA, frio, ferimentos, luz, ritmo circadiano, salinidade, seca, patógenos e encharcamento. Entretanto, há pouca informação sobre GRPs de plantas e seus papéis no desenvolvimento e resposta a estresses. As GRPs podem ser divididas em quatro classes (I, II, III, IV) de acordo com sua estrutura primária e presença de domínios característicos. A classe IV é composta por proteínas ligantes de RNA. Domínios adicionais permitem dividir a classe IV de GRPs em quatro subclasses (IVa, IVb, IVc, IVd). A subclasse IVc é representada por proteínas contendo um cold-schock domain (CSD) e dedos de zinco CCHC tipo retrovirais. O objetivo do presente estudo foi: (i) identificar e caracterizar os genes codificantes de classe IV de GRPs, (ii) verificar a padrão de expressão dos genes codificantes da subclasse IVc de GRPs e (iii) produzir plantas de soja transgênicas expressando o gene AtGRP2, o qual foi mostrado estar envolvido na floração e desenvolvimento da semente em Arabidopsis, e também poderia desempenhar um papel na aclimatação ao frio. Um total de 47 genes codificantes da classe IV de GRPs foi identificado no genoma da soja: 19 da subclasse IVa, sete da IVb, seis da IVc e 15 da IVd. Análises in silico indicaram uma expressão preferencial de todos os genes codificantes da subclasse IVc em tecidos em desenvolvimento. Análises de RT-qPCR revelaram que plantas jovens e maduras exibem uma expressão mais alta em folhas do que em outros órgãos, com exceção dos genes GRP2L_4/5 que tiveram expressão mais alta em sementes. GRP2L_4/5 e GRP2L_2 foram induzidos em resposta a baixas temperaturas. Sob estresse com ABA a expressão de todos os genes foi reprimida em folhas e/ou raízes, com exceção do gene GRP2L_2 que foi induzido em raízes. Em resposta a infecção com Phakopsora pachyrhizi, a expressão de GRP2L_2 e GRP2L_3 foi mais alta e precoce no genótipo suscetível quando comparada com o resistente, enquanto que a resposta de GRP2L_4/5 e GRP2L_6 foi mais tardia no genótipo resistente. Ainda, embriões somáticos secundários das cultivares Bragg, IAS-5 e BRSMG 68 Vencedora de soja foram usados para introduzir o gene AtGRP2 no genoma da soja por bombardeamento e sistema bombardeamento/Agrobacterium. Seis eventos de transformação independentes foram confirmados por PCR. No presente momento as plantas estão em desenvolvimento em frascos de vidro. No presente estudo a classe IV de GRPs em soja foi identificada e caracterizada. Este é o primeiro passo para elucidar o papel destas proteínas em plantas. / Molecular information on plant developmental process, as well as detailed knowledge of the interaction between stress conditions and plant response to environmental factors are essential for understanding the adaptive response. Glycine-Rich Proteins (GRP) have the amino acid glycine well represented in their primary structure. The genes encoding GRPs are developmentally regulated and induced by auxin, ABA, cold, wound, light, circadian rhythm, salinity, drought, pathogens, and flooding. However, there is scarce information about plant GRPs and its role on development and stress response. The GRPs can be divided into four classes (I, II, II and IV) according to their primary structure and the presence of characteristic domains. Class IV is composed by RNA-binding proteins. Additional domains permit to split class IV GRPs into four subclasses (IVa, IVb, IVc and IVd). Subclass IVc is represented by proteins containing a Cold-Shock Domain (CSD) and retroviral-like CCHC zinc fingers. The goal of the present study was: (i) to identify and characterize the genes encoding class IV GRPs, (ii) to verify the relative expression of genes encoding subclass IVc GRPs and (iii) to produce transgenic soybean plants expressing the AtGRP2 gene, which was shown to be involved in Arabidopsis flower and seed development, and can also play a role in cold acclimation. A total of 47 genes encoding class IV GRPs were found in the soybean genome: 19 from IVa, seven from IVb, six from IVc and 15 from IVd subclasses. In silico analyses indicated a preferential expression of all genes encoding subclass IVc GRPs in tissues under development. RT-qPCR analyses revealed that both young and mature plants exhibit relative higher expression of subclass IVc GRPs in leaves than in other organs, with exception of GRP2L_4/5 genes that have higher expression in seeds. The GRP2L_4/5 and GRP2L_2 were up-regulated in response to low temperatures. Under ABA stress the expression of all genes was down-regulated in leaves and roots, with exception of GRP2L_2 gene that was up-regulated in roots. In response to Phakopsora pachyrhizi infection, GRP2L_2 and GRP2L_3 expression was higher and earlier in the susceptible genotype when compared with that of the resistant one, while GRP2L_4/5 and GRP2_6 respond later in the resistant genotype. Furthermore, secondary somatic embryos of Bragg, IAS-5 and BRSMG 68 Vencedora soybean cultivars were used to introduce the AtGRP2 gene into the soybean genome by particle bombardment and bombardment/Agrobacterium system. Six independent Bragg transformation events were confirmed by PCR. In the present moment the plants are under development in glass flasks. In the present study the soybean class IV GRPs were identified and characterized. This is the first step to elucidate the role of these proteins in plants.

A soja constitui uma das culturas mais importantes mundialmente, tanto social quanto economicamente. Consequentemente, informações moleculares sobre processos de desenvolvimento, bem como conhecimento detalhado das interações entre condições estressoras e a resposta da planta a fatores ambientais são necessários. A identificação e caracterização de genes que respondem a condições ambientais específicas constituem um passo inicial no entendimento dos processos adaptativos. Proteínas ricas em glicina (GRPs) são polipeptídeos contendo um grande número do aminoácido glicina em sua estrutura primária. Os genes codificantes de GRPs são regulados ao longo do desenvolvimento e regulados por auxina, ABA, frio, ferimentos, luz, ritmo circadiano, salinidade, seca, patógenos e encharcamento. Entretanto, há pouca informação sobre GRPs de plantas e seus papéis no desenvolvimento e resposta a estresses. As GRPs podem ser divididas em quatro classes (I, II, III, IV) de acordo com sua estrutura primária e presença de domínios característicos. A classe IV é composta por proteínas ligantes de RNA. Domínios adicionais permitem dividir a classe IV de GRPs em quatro subclasses (IVa, IVb, IVc, IVd). A subclasse IVc é representada por proteínas contendo um cold-schock domain (CSD) e dedos de zinco CCHC tipo retrovirais. O objetivo do presente estudo foi: (i) identificar e caracterizar os genes codificantes de classe IV de GRPs, (ii) verificar a padrão de expressão dos genes codificantes da subclasse IVc de GRPs e (iii) produzir plantas de soja transgênicas expressando o gene AtGRP2, o qual foi mostrado estar envolvido na floração e desenvolvimento da semente em Arabidopsis, e também poderia desempenhar um papel na aclimatação ao frio. Um total de 47 genes codificantes da classe IV de GRPs foi identificado no genoma da soja: 19 da subclasse IVa, sete da IVb, seis da IVc e 15 da IVd. Análises in silico indicaram uma expressão preferencial de todos os genes codificantes da subclasse IVc em tecidos em desenvolvimento. Análises de RT-qPCR revelaram que plantas jovens e maduras exibem uma expressão mais alta em folhas do que em outros órgãos, com exceção dos genes GRP2L_4/5 que tiveram expressão mais alta em sementes. GRP2L_4/5 e GRP2L_2 foram induzidos em resposta a baixas temperaturas. Sob estresse com ABA a expressão de todos os genes foi reprimida em folhas e/ou raízes, com exceção do gene GRP2L_2 que foi induzido em raízes. Em resposta a infecção com Phakopsora pachyrhizi, a expressão de GRP2L_2 e GRP2L_3 foi mais alta e precoce no genótipo suscetível quando comparada com o resistente, enquanto que a resposta de GRP2L_4/5 e GRP2L_6 foi mais tardia no genótipo resistente. Ainda, embriões somáticos secundários das cultivares Bragg, IAS-5 e BRSMG 68 Vencedora de soja foram usados para introduzir o gene AtGRP2 no genoma da soja por bombardeamento e sistema bombardeamento/Agrobacterium. Seis eventos de transformação independentes foram confirmados por PCR. No presente momento as plantas estão em desenvolvimento em frascos de vidro. No presente estudo a classe IV de GRPs em soja foi identificada e caracterizada. Este é o primeiro passo para elucidar o papel destas proteínas em plantas. / Molecular information on plant developmental process, as well as detailed knowledge of the interaction between stress conditions and plant response to environmental factors are essential for understanding the adaptive response. Glycine-Rich Proteins (GRP) have the amino acid glycine well represented in their primary structure. The genes encoding GRPs are developmentally regulated and induced by auxin, ABA, cold, wound, light, circadian rhythm, salinity, drought, pathogens, and flooding. However, there is scarce information about plant GRPs and its role on development and stress response. The GRPs can be divided into four classes (I, II, II and IV) according to their primary structure and the presence of characteristic domains. Class IV is composed by RNA-binding proteins. Additional domains permit to split class IV GRPs into four subclasses (IVa, IVb, IVc and IVd). Subclass IVc is represented by proteins containing a Cold-Shock Domain (CSD) and retroviral-like CCHC zinc fingers. The goal of the present study was: (i) to identify and characterize the genes encoding class IV GRPs, (ii) to verify the relative expression of genes encoding subclass IVc GRPs and (iii) to produce transgenic soybean plants expressing the AtGRP2 gene, which was shown to be involved in Arabidopsis flower and seed development, and can also play a role in cold acclimation. A total of 47 genes encoding class IV GRPs were found in the soybean genome: 19 from IVa, seven from IVb, six from IVc and 15 from IVd subclasses. In silico analyses indicated a preferential expression of all genes encoding subclass IVc GRPs in tissues under development. RT-qPCR analyses revealed that both young and mature plants exhibit relative higher expression of subclass IVc GRPs in leaves than in other organs, with exception of GRP2L_4/5 genes that have higher expression in seeds. The GRP2L_4/5 and GRP2L_2 were up-regulated in response to low temperatures. Under ABA stress the expression of all genes was down-regulated in leaves and roots, with exception of GRP2L_2 gene that was up-regulated in roots. In response to Phakopsora pachyrhizi infection, GRP2L_2 and GRP2L_3 expression was higher and earlier in the susceptible genotype when compared with that of the resistant one, while GRP2L_4/5 and GRP2_6 respond later in the resistant genotype. Furthermore, secondary somatic embryos of Bragg, IAS-5 and BRSMG 68 Vencedora soybean cultivars were used to introduce the AtGRP2 gene into the soybean genome by particle bombardment and bombardment/Agrobacterium system. Six independent Bragg transformation events were confirmed by PCR. In the present moment the plants are under development in glass flasks. In the present study the soybean class IV GRPs were identified and characterized. This is the first step to elucidate the role of these proteins in plants.

A soja constitui uma das culturas mais importantes mundialmente, tanto social quanto economicamente. Consequentemente, informações moleculares sobre processos de desenvolvimento, bem como conhecimento detalhado das interações entre condições estressoras e a resposta da planta a fatores ambientais são necessários. A identificação e caracterização de genes que respondem a condições ambientais específicas constituem um passo inicial no entendimento dos processos adaptativos. Proteínas ricas em glicina (GRPs) são polipeptídeos contendo um grande número do aminoácido glicina em sua estrutura primária. Os genes codificantes de GRPs são regulados ao longo do desenvolvimento e regulados por auxina, ABA, frio, ferimentos, luz, ritmo circadiano, salinidade, seca, patógenos e encharcamento. Entretanto, há pouca informação sobre GRPs de plantas e seus papéis no desenvolvimento e resposta a estresses. As GRPs podem ser divididas em quatro classes (I, II, III, IV) de acordo com sua estrutura primária e presença de domínios característicos. A classe IV é composta por proteínas ligantes de RNA. Domínios adicionais permitem dividir a classe IV de GRPs em quatro subclasses (IVa, IVb, IVc, IVd). A subclasse IVc é representada por proteínas contendo um cold-schock domain (CSD) e dedos de zinco CCHC tipo retrovirais. O objetivo do presente estudo foi: (i) identificar e caracterizar os genes codificantes de classe IV de GRPs, (ii) verificar a padrão de expressão dos genes codificantes da subclasse IVc de GRPs e (iii) produzir plantas de soja transgênicas expressando o gene AtGRP2, o qual foi mostrado estar envolvido na floração e desenvolvimento da semente em Arabidopsis, e também poderia desempenhar um papel na aclimatação ao frio. Um total de 47 genes codificantes da classe IV de GRPs foi identificado no genoma da soja: 19 da subclasse IVa, sete da IVb, seis da IVc e 15 da IVd. Análises in silico indicaram uma expressão preferencial de todos os genes codificantes da subclasse IVc em tecidos em desenvolvimento. Análises de RT-qPCR revelaram que plantas jovens e maduras exibem uma expressão mais alta em folhas do que em outros órgãos, com exceção dos genes GRP2L_4/5 que tiveram expressão mais alta em sementes. GRP2L_4/5 e GRP2L_2 foram induzidos em resposta a baixas temperaturas. Sob estresse com ABA a expressão de todos os genes foi reprimida em folhas e/ou raízes, com exceção do gene GRP2L_2 que foi induzido em raízes. Em resposta a infecção com Phakopsora pachyrhizi, a expressão de GRP2L_2 e GRP2L_3 foi mais alta e precoce no genótipo suscetível quando comparada com o resistente, enquanto que a resposta de GRP2L_4/5 e GRP2L_6 foi mais tardia no genótipo resistente. Ainda, embriões somáticos secundários das cultivares Bragg, IAS-5 e BRSMG 68 Vencedora de soja foram usados para introduzir o gene AtGRP2 no genoma da soja por bombardeamento e sistema bombardeamento/Agrobacterium. Seis eventos de transformação independentes foram confirmados por PCR. No presente momento as plantas estão em desenvolvimento em frascos de vidro. No presente estudo a classe IV de GRPs em soja foi identificada e caracterizada. Este é o primeiro passo para elucidar o papel destas proteínas em plantas. / Molecular information on plant developmental process, as well as detailed knowledge of the interaction between stress conditions and plant response to environmental factors are essential for understanding the adaptive response. Glycine-Rich Proteins (GRP) have the amino acid glycine well represented in their primary structure. The genes encoding GRPs are developmentally regulated and induced by auxin, ABA, cold, wound, light, circadian rhythm, salinity, drought, pathogens, and flooding. However, there is scarce information about plant GRPs and its role on development and stress response. The GRPs can be divided into four classes (I, II, II and IV) according to their primary structure and the presence of characteristic domains. Class IV is composed by RNA-binding proteins. Additional domains permit to split class IV GRPs into four subclasses (IVa, IVb, IVc and IVd). Subclass IVc is represented by proteins containing a Cold-Shock Domain (CSD) and retroviral-like CCHC zinc fingers. The goal of the present study was: (i) to identify and characterize the genes encoding class IV GRPs, (ii) to verify the relative expression of genes encoding subclass IVc GRPs and (iii) to produce transgenic soybean plants expressing the AtGRP2 gene, which was shown to be involved in Arabidopsis flower and seed development, and can also play a role in cold acclimation. A total of 47 genes encoding class IV GRPs were found in the soybean genome: 19 from IVa, seven from IVb, six from IVc and 15 from IVd subclasses. In silico analyses indicated a preferential expression of all genes encoding subclass IVc GRPs in tissues under development. RT-qPCR analyses revealed that both young and mature plants exhibit relative higher expression of subclass IVc GRPs in leaves than in other organs, with exception of GRP2L_4/5 genes that have higher expression in seeds. The GRP2L_4/5 and GRP2L_2 were up-regulated in response to low temperatures. Under ABA stress the expression of all genes was down-regulated in leaves and roots, with exception of GRP2L_2 gene that was up-regulated in roots. In response to Phakopsora pachyrhizi infection, GRP2L_2 and GRP2L_3 expression was higher and earlier in the susceptible genotype when compared with that of the resistant one, while GRP2L_4/5 and GRP2_6 respond later in the resistant genotype. Furthermore, secondary somatic embryos of Bragg, IAS-5 and BRSMG 68 Vencedora soybean cultivars were used to introduce the AtGRP2 gene into the soybean genome by particle bombardment and bombardment/Agrobacterium system. Six independent Bragg transformation events were confirmed by PCR. In the present moment the plants are under development in glass flasks. In the present study the soybean class IV GRPs were identified and characterized. This is the first step to elucidate the role of these proteins in plants.

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