A lisina é um dos aminoácidos essenciais e um dos fatores limitantes ao uso de cereais como o milho na alimentação pois, sem suplementação, não permite a obtenção de uma dieta balanceada. A fim de melhorar a qualidade nutricional dos cereais, várias tentativas têm sido feitas utilizando o melhoramento genético. Com o advento das técnicas de engenharia genética é possível, atualmente, utilizar a biotecnologia para aprofundar estes estudos, a fim de desvendar os complexos mecanismos que controlam o fluxo de aminoácidos nos grãos. O presente trabalho tem como objetivo principal testar uma nova estratégia que se baseia na expressão de uma proteína quimérica, a zeolina, sob controle de um promotor endosperma específico. A zeolina é uma combinação de 421 aminoácidos da faseolina do feijão com 89 aminoácidos da y- zeína, inserida no genoma do milho sob controle de um promotor isolado da -kafirina de sorgo. Para que o objetivo do projeto fosse alcançado, o trabalho foi dividido nas seguintes etapas: i) Síntese da construção e clonagem nos vetores; ii) Transformação de embriões e calos de milho utilizando biobalística e Agrobacterium tumefaciens; iii) Cultura de tecidos para seleção e regeneração das plantas transformadas; iv) Verificação dos eventos de transformação através da análise do DNA, RNA e da proteína do transgene; v) Análises preliminares das plantas transformadas para o padrão das proteínas de reserva e perfil de aminoácidos dos grãos. A construção da zeolina foi amplificada por PCR com primers específicos e clonada nos vetores pCambia3301 e pTF102 sob controle do promotor da -kafirina. Os embriões e calos do milho híbrido HiII foram utilizados na transformação empregando-se a biobalística e Agrobacterium tumefaciens. No final do processo foram produzidas e multiplicadas oito plantas de milho transformadas com a zeolina, confirmadas por testes biológicos, PCR, sequenciamento e testes imunocromatográficos, representando seis eventos de transformação. A expressão gênica, verificada pela detecção do mRNA por PCR, foi constatada em seis eventos de transformação. Para detecção da proteína expressa pelo transgene foi utilizado anticorpo específico para faseolina, através do Western Blot. A banda relativa ao transgene foi detectada em três eventos de transformação. Nas análises preliminares não houve alteração no padrão das frações zeína, globulina, glutelina ou albumina nas plantas transformadas quando comparados com os controles. Também não houve diferenças significantes no teor de aminoácidos solúveis totais entre as plantas transformadas e os controles. Na determinação do perfil de aminoácidos no HPLC, o evento ZEO-3(3) apresentou as maiores concentrações para todos os aminoácidos analisados. Neste evento de transformação o teor de lisina foi de 25,76 mg/g de proteína, enquanto os controles apresentaram valores de 9,77 e 15,89mg/g de proteína. O evento ZEO-3(3) apresenta-se, portanto, como um candidato para estudos posteriores a fim de revelar os complexos mecanismos que controlam o fluxo de aminoácidos e o acúmulo das proteínas de reserva nos grãos de milho. / Lysine is an essential amino acid and a major factor limiting the use of cereals such as maize for food and feed as, without supplementation, does not allow obtaining a balanced diet. In order to improve the nutritional quality of cereals, several attempts have been made using the conventional breeding. With the advent of genetic engineering techniques it is now possible to use biotechnology to develop further studies and to unravel the complex mechanisms that control the flow of amino acids in grains. The present work aimed at testing a new strategy based on the expression of a chimeric protein, the zeolin, under the control of an endosperm specific promoter. The zeolin is a combination of 421 amino acids of bean phaseolin with 89 amino acids of the maize - zein inserted into the maize genome under the control of a promoter isolated from the protein -kafirin from sorghum. For the goal of the project to be reached, the work was divided into the following steps: i) Synthesis of the construction and the cloning vectors; ii) Transformation of embryos and callus of maize using the biolistic and Agrobacterium tumefaciens methods; iii) Tissue culture for selection and regeneration of transformed plants iv) Verification of transformation events through the analysis of the DNA, RNA and protein from the transgene; v) Preliminary analysis of transformed plants for the storage proteins pattern and amino acid profile of the grains. The zeolin construction was amplified by PCR with specific primers and cloned into the vectors pCambia3301 and pTF102 under the control of the promoter -kafirin. Embryos and callus of HiII hybrid were used in the transformation using the biolistic and Agrobacterium tumefaciens. At the end of the process of transformatio, eight maize plants transformed with zeolin were produced and multiplied, confirmed by biological tests, PCR, sequencing and immunochromatographic tests, representing six transformation events. The gene expression, verified by detection of mRNA by PCR, was found in six events of transformation. The protein translation, verified by Western Blot using an antibody specific for phaseolin, was found in three transformation events. In the preliminary analysis, the pattern of zein, globulin, glutelin or albumin fractions did not change comparing transformed plants with the controls. There were also no significant differences in the content of soluble amino acids among the Western positive transformed plants and the controls. In the HPLC amino acid profile, the event ZEO-3(3) showed the higher concentrations for all amino acids analyzed. In this transformation event the content of lysine was 25,76mg/g protein, while the controls exhibited values of 9,77 and 15,89mg/g protein. Therefore, ZEO-3(3) event presents itself as a candidate for further studies to reveal the complex mechanisms that control the flow of amino acids and accumulation of storage proteins in maize kernels.

Ferritin is an iron storage protein consisting of H and L chains to form a 24-subunit heropolymer. Ceruloplamin oxidizes Fe(II) and then loads the iron into ferritin. This research was conducted to determine which ferritin subunit is involved and whether a proposed iron-loading channel is required for iron loading by ceruloplasmin. Recombinant rat liver H and L chain ferritin homopolymers, designated as rH-Ft and rL-Ft, respectively, were produced using insect cell-baculovirus and Escherichia coli expression systems. The expressed rH-Ft strongly suppressed the growth of the host. The rH-Ft expressed in the E. coli contained approximately 150 iron atoms/ferritin and was observed to have protein damage, which was found to affect iron-loading by ceruloplasmin. The ferritin expressed in the E. coli system apparently was not proper for this iron loading study. Alternatively, the ferritins expressed in the insect cell-baculovirus system were utilized for this purpose . Ceruloplasmin was able to load iron into the rH-Ft, but not the rL-Ft. The initial rate of loading iron into the rH-Ft by ceruloplasmin was similar to that of native rat liver ferritin heteropolymer. Both the rH-Ft and the native rat liver ferritin could be maximally loaded with iron by ceruloplasmin up to 2,500 iron atoms/ferritin. When the rH-Ft or the native ferritin was present, the ferroxidase activity of ceruloplasmin was enhanced. No such enhancement was observed in the presence of the rL-Ft. This suggests that ceruloplasmin only associates with the ferritin H, but not L, chain during iron loading. The role of an a-helix bundle channel in iron loading by ceruloplasmin was investigated by using sitedirected mutagenesis. The channel in the rH-Ft was closed by mutation E62K and H65G to form a K62 to El07 salt bridge, which is thought to exist in the L chain. Conversely, the salt bridge in the channel of the L chain was removed by mutation K58E and G61H to form a channel similar to that in the four-a-helix bundle of the H chain. The initial rate of loading iron into the rL-FT mutant by ceruloplasmin was 50% of that for loading iron into the rH-Ft. When 500 atoms of iron per ferritin were used for loading, 98% loaded into the rH-Ft by ceruloplasmin in 5 minutes, but only 30% loaded into the rL-Ft mutant in the same time. The ferroxidase activity of ceruloplasmin was enhanced in the presence of the rHFt and its mutant, but not in the presence of the rL-Ft or its mutant. These results indicate that the association of ceruloplasmin and ferritin is required and the a-helix bundle channel is a channel for iron loading.

The seed storage globulins found among virtually all spermatophytes comprise a multi-gene family of proteins with ancient evolutionary origins. The two main groups of storage globulins include the legumins (11S) and vicilins (7S), both of which play a main role in protein deposition and storage in the seed endosperm. Composed of two cupin domains (bicupin), these proteins have been recently noted not only for their close structural relationships among the two subfamilies (7S and 11S) but also for their similarity to other proteins such as germin-like proteins (GLP's), bacterial oxalate decarboxylases, and other cupin containing proteins. Previous studies have investigated the evolutionary relationships among the legumin and vicilin groups, as well as their presumed evolutionary link to other cupin containing proteins; however these have each come short of any comprehensive resolved evolutionary history of the globulin family. This study focuses first on resolving the relationships among the cupin super-family in relation to the storage globulins, as well as the GLP's, which have been postulated to be the single domain ancestors of the bicupin storage globulins. Nucleotide coding sequences for both N-terminus and C-terminus cupin domains of the storage globulins, including conserved non-cupin domain helical repeats and inter-domain spacers were aligned to a comparably sized set of single cupin coding sequences (CDS). The phylogenetic relationships among the two globulin domains as well as the single cupin genes were elucidated using Bayesian inference of tree likelihoods. Further phylogenetic analysis was performed on the complete CDS's for all storage globulin sequences in the study, using an appropriate out-group of similar overall domain architecture determined by the overall topology of the cupin super-family. This globulin muti-gene tree was used, along with an alignment corresponding to structurally resolved portions of the mature globulin peptides, to perform an analysis of patterns of selection among the various lineages of cupin-containing globulins. The results of these analyses provide evidence for a common origin of all cupin containing genes. The GLP and storage globulin domains do not appear to be immediate ancestors of one another, but are grouped with the fungal spherulins as well, suggesting that the single cupin genes which gave rise to these groups had already diverged prior to the rise of land plants. The storage globulin gene tree provides evidence supporting the notion that true legumins and vicilins were recruited as seed storage proteins independent of one another, after their divergence. This is evidenced by the fact that they comprise two separate groups each with basal non-storage 11S/7S-like proteins. Additional insight into the differentiating selection pressures provides a clearer picture of how similar suites of physicochemical properties came under selection after the recruitment of the 11S and 7S families as seed specific proteins. Regions under strong destabilizing selection correspond to regions known to be of importance in the overall structure of storage globulins. Strong destabilizing selection at the pore of the globulin subunit suggests that this region may have undergone more functional diversification than previously thought to have occurred among the legumins and vicilins.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24655号 / 農博第2538号 / 新制||農||1097(附属図書館) / 学位論文||R5||N5436(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 白岩 立彦, 教授 中﨑 鉄也, 教授 丸山 伸之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

<p>The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The <i>napA</i> gene encodes for a <i>Brassica napus</i> 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The <i>napA</i> promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to <i>napA</i> through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster.</p><p>We also show that the RY/G cluster is responsible for repression of <i>napA</i> expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the <i>napA</i> RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the <i>napA</i> RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.</p>

The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The napA gene encodes for a Brassica napus 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The napA promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to napA through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster. We also show that the RY/G cluster is responsible for repression of napA expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the napA RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the napA RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.

No description available.

The effect of gamete storage on sperm quality has received considerable attention in recent years. Previous studies have shown that spermatozoa stored in vivo or in vitro for a long time can lost their motility and fertilization capacity. Moreover, it have been concluded that male fertilization potential is highly dependent, not only on spermatozoa motility parameters, but also on organic components including protein composition of seminal plasma. On the other hand, spermatozoa motility and protein profiles of seminal plasma are highly dependent on conditions of storage. Therefore, additional data about effects of in vivo and in vitro storage on quality sperm parameters and protein composition of seminal plasma are essential for development of fish artificial reproduction methods. In the current work the influence of in vitro and in vivo storage on parameters of sperm motility, DNA integrity, antioxidant defensive system and seminal plasma protein composition were studied. As a conclusion, the results of this study provide new data on sperm quality and quantity parameters of chondrostean and teleost fish species with respect to in vivo and in vitro storage capacities, which should be beneficial for the development of aquaculture of these species. The data confirmed that protein patterns in seminal plasma varied during in vivo storage, depending on time of sperm collection. Furthermore, the altered proteins are probably involved in enzymatic pathways that regulate spermatozoa movement. In practice, the results presented in this thesis should help to improve management and optimize the development of protocols for artificial reproduction.

The structure of 11S cruciferin has been solved; however, how the individual subunits contribute to its physico-chemical and functional properties are not well known. The cruciferin isoforms in Arabidopsis thaliana, CRUA, CRUB, and CRUC, were investigated with respect to their molecular structures and the relationship of structural features to the physico-chemical and functional properties of cruciferin using homology modeling and various analytical techniques. Comparison of these models revealed that hydrophobicity and electrostatic potential distribution on the surface of the CRUC homotrimer had more favorable interfacial, solubility, and thermal properties than those of CRUA or CRUB. Flavor binding and pepsin digestion were associated with hypervariable regions (HVRs) and center core regions, respectively, moreso for CRUA and CRUB homotrimers than for CRUC. Chemical imaging of a single cell area in wild type (WT) and double-knockout seeds (CRUAbc, CRUaBc, and CRUabC) using synchrotron FT-IR microscopy (amide I band, 1650 cm-1, νC=O) showed that seed storage proteins were concentrated in the cell center and protein storage vacuoles, whereas lipids were closer to the cell wall. Secondary structure components of proteins of double-knockout lines did not show major differences. Changes in protein secondary structure components of pepsin-treated CRUabC (CRUC) mutant were minimal, indicating low enzyme accessibility. A three-step chromatographic procedure allowed isolation of the hexameric form of cruciferin with high purity (>95%). Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic analysis of the secondary structure of these proteins revealed cruciferins were folded into higher order secondary structures; 44−50% β-sheets and 7−9% α-helices. The relative subunit ratio was approximately 1:3:6 (CRUA:CRUB:CRUC) in the WT cruciferin. The Tm values of purified cruciferin at pH 7.4 (μ = 0.0) were in the order of WT = CRUA = CRUB < CRUC. The order of surface hydrophobicity as determined by ANS (1-anilinonaphthalene-8-sulfonate) probe binding was CRUA > CRUB = WT >> CRUC. Intrinsic fluorescence studies revealed a compact molecular structure for the CRUC homohexamer compared to the CRUA and CRUB homohexamers. The order of emulsion forming abilities was CRUA = CRUB > WT > CRUC (no emulsion formation) and the order of heat-induced network structure strength was WT > CRUA = CRUB > CRUC (no gel formation). The inability of CRUC to form gels or emulsions may be attributed to its low surface hydrophobicity and molecular compactness. At pH 2.0, CRUC hexamers dissociated into trimers which allowed the formation of an O/W emulsion and heat-induced network structures. Solubility of cruciferin as a function of pH at low ionic strength gave two minima around pH 4 and 7.4 yielding a “W” shape solubility profile deviating from the typical “U” or “V” shape solubility profile of other 11S globulins. The high ionic strength (μ = 0.5) was not favorable for emulsification, heat-induced gel formation, or solubilization for all cruciferins. Furthermore, the CRUA and CRUB homohexamers exhibited rapid pepsinolysis, while the CRUC homohexamer and WT heterohexamer were digested more slowly. Although fairly well conserved regions were found in the primary structure of these three cruciferin subunits, differences were found in the hypervariable regions and extended loop regions resulting in slight differences in 3D structures and interactions that occur during association to form superstructures, such as hexamers. These differences were reflected in the physico-chemical and techno-functional properties of hexamers and trimers composed of each subunit. In silico predictions for certain functionalities were highly correlated with empirical data from laboratory experiments.