Storage Protein Genes Research Articles

Introduction of chromosome segment carrying the seed storage protein genes from chromosome 1V of Dasypyrum villosum showed positive effect on bread-making quality of common wheat

Development of wheat- D. villosum 1V#4 translocation lines; physically mapping the Glu - V1 and Gli - V1 / Glu - V3 loci; and assess the effects of the introduced Glu - V1 and Gli - V1 / Glu - V3 on wheat bread-making quality. Glu-V1 and Gli-V1/Glu-V3 loci, located in the chromosome 1V of Dasypyrum villosum, were proved to have positive effects on grain quality. However, there are very few reports about the transfer of the D. villosum-derived seed storage protein genes into wheat background by chromosome manipulation. In the present study, a total of six CS-1V#4 introgression lines with different alien-fragment sizes were developed through ionizing radiation of the mature female gametes of CS--D. villosum 1V#4 disomic addition line and confirmed by cytogenetic analysis. Genomic in situ hybridization (GISH), chromosome C-banding, twelve 1V#4-specific EST-STS markers and seed storage protein analysis enabled the cytological physical mapping of Glu-V1 and Gli-V1/Glu-V3 loci to the region of FL 0.50-1.00 of 1V#4S of D. villosum. The Glu-V1 allele of D. villosum was Glu-V1a and its coded protein was V71 subunit. Quality analysis indicated that Glu-V1a together with Gli-V1/Glu-V3 loci showed a positive effect on protein content, Zeleny sedimentation value and the rheological characteristics of wheat flour dough. In addition, the positive effect could be maintained when specific Glu-V1 and Gli-V1/Glu-V3 loci were transferred to the wheat genetic background as in the case of T1V#4S-6BS · 6BL, T1V#4S · 1BL and T1V#4S · 1DS translocation lines. These results showed that the chromosome segment carrying the Glu-V1 and Gli-V1/Glu-V3 loci in 1V#4S of D. villosum had positive effect on bread-making quality, and the T1V#4S-6BS · 6BL and T1V#4S · 1BL translocation lines could be useful germplasms for bread wheat improvement. The developed 1V#4S-specific molecular markers could be used to rapidly identify and trace the alien chromatin of 1V#4S in wheat background.

Over-expressing GsCBRLK/SCMRP Enhances Alkaline Tolerance and Methionine Content in Transgenic Medicago sativa

Drought and saline-alkaline stresses, significantly affect growth and productivity of plants. The reaction of plant to environmental stresses is controlled by numerous genes via transcriptional regulation and protein phosphorylation. A stress-responsive kinase gene, GsCBRLK, has been cloned from a Glycine soja cDNA library under salinity, drought, and cold stresses. Over-expression of GsCBRLK in transgenic Arabidopsis resulted in enhanced tolerance to high salinity and ABA. SCMRP is a kind of storage protein gene coding high-sulfur-containing amino acid. In this study, we constructed an expression vector BEOCBRLK-SCMRP and transformed both GsCBRLK and SCMRP genes into alfalfa. The transgenic alfalfa grew well after NaHCO3 treatment(100 and 150 mmol L–1) for 14 days; whereas, the wild type plants exhibited discoloration and stunted growth, even death. The MDA content and relative membrane permeability caused by alkaline stress in transgenic plants varied significantly compared to those in the wild type(P 0.05). Moreover, the superoxide dismutase(SOD) activity in transgenic plants under alkali stress increased than that of the wild type. Amino acid content assay showed that the transformants had higher methionine content than the non-transformed plants. These results indicated that the transgenic alfalfa carrying both GsCBRLK and SCMRP possesses enhanced alkaline tolerance and rich methionine simultaneously.

Arabidopsis SAG protein containing the MDN1 domain participates in seed germination and seedling development by negatively regulating ABI3 and ABI5

Three proteins containing a midasin homologue 1 (MDN1) domain from the yeast Solanum chacoense and Arabidopsis thaliana have important functions in yeast survival, seed development, and female gametogenesis. In this study, a novel protein containing the MDN1 domain from Arabidopsis negatively regulated abscisic acid (ABA) signalling during seed germination. Seeds of a T-DNA insertion line of this gene exhibited increased sensitivity to ABA during seed germination and seedling development (named sag). By contrast, seeds with overexpressed AtSAG (OX2) were less sensitive to ABA. The seeds of the sag mutant showed similar sensitivity to high concentrations of mannitol and NaCl during these stages. AtSAG was also highly expressed in germinating seeds. However, ABA-induced AtSAG expression remained almost unchanged. ABA-responsive marker genes, including ABI3, ABI5, Em1, Em6, RD29A, and RAB18, were upregulated in sag mutants but were downregulated in OX2. Genetic analyses indicated that the function of AtSAG in ABA signalling depended on ABI3 and ABI5. The expression of some target genes of ABI3 and ABI5, such as seed storage protein and oleosin genes, was induced higher by ABA in sag mutants than in wild-type germinated seeds, even higher than in abi5 mutants. This finding indicated that other regulators similar to ABI3 or ABI5 played a role during these stages. Taken together, these results indicate that AtSAG is an important negative regulator of ABA signalling during seed germination and seedling development.

Nucleotide diversity of 11S seed storage protein gene and its implications for ecological adaptation of Oryza nivara

AbstractThe mechanism by which a new species arises and adapts to its environment is a fundamental question in evolutionary biology. Seed characteristics such as seed size and nutrient composition are important fitness‐related traits and have been shown to vary greatly among populations and species. However, the significance of variation in seed traits in plant adaptation and speciation remains unclear. We carried out a population genetic study on nucleotide variation of one 11S seed storage protein gene (Pss) of Oryza rufipogon Griff. and O. nivara Sharma & Shastry, two closely related wild rice species. By comparatively examining the genetic variation pattern of the regulatory and coding regions of Pss and fragments of six reference loci across different chromosomes, we found significantly lower polymorphisms at coding regions of the gene (PssI) in O. nivara relative to O. rufipogon. Neutrality tests indicated that the reduction of polymorphisms at PssI in O. nivara was caused by positive selection rather than population demography, implying a role of selection on the 11S seed protein gene. Further phylogenetic and principal component analyses also support the hypotheses that the origin of O. nivara was associated with the adaptive divergence on the coding region of Pss. It is most likely that higher reproductive effort would be favored when O. nivara arose from O. rufipogon populations and adapted to the environment change.

Molecular markers and plant breeding

The achievements of modern biotechnology allow to modernize significantly the traditional plant breeding. The use of molecular codominant markers reduces considerably the quantity of breeding material and promotes the selection of genotypes, which posses desirable genes in the homozygous state. Molecular marking systems of agronomically important simple and quantitative traits have been developed using mono- and multiloci systems. Markers of the plant type and development rate, alleles of the storage protein genes, Wx-genes, short-stem genes, etc., have been created and tested at the South Biotecnology Center, National Academy of Agricultural Sciences. The technology of the application of DNA-typing for the identification and registration of varieties that has been developed in the South Biotecnology Center is of great importance for the systematization of germplasm sources and the protection of the rights of breeders.

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.

Transcriptome analysis of embryo maturation in maize

BackgroundMaize is one of the most important crops in the world. With the exponentially increasing population and the need for ever increased food and feed production, an increased yield of maize grain (as well as rice, wheat and other grains) will be critical. Maize grain development is understood from the perspective of morphology, hormone responses, and storage reserve accumulation. This includes various studies on gene expression during embryo development and maturation but a global study of gene expression of the embryo has not been possible until recently. Transcriptome analysis is a powerful new tool that can be used to understand the genetic basis of embryo maturation.ResultsWe undertook a transcriptomic analysis of normal maturing embryos at 15, 21 and 27 days after pollination (DAP), of one elite maize germplasm line that was utilized in crosses to transgenic plants. More than 19,000 genes were analyzed by this method and the challenge was to select subsets of genes that are vitally important to embryo development and maturation for the initial analysis. We describe the changes in expression for genes relating to primary metabolic pathways, DNA synthesis, late embryogenesis proteins and embryo storage proteins, shown through transcriptome analysis and confirmed levels of transcription for some genes in the transcriptome using qRT-PCR.ConclusionsNumerous genes involved in embryo maturation have been identified, many of which show changes in expression level during the progression from 15 to 27 DAP. An expected array of genes involved in primary metabolism was identified. Moreover, more than 30% of transcripts represented un-annotated genes, leaving many functions to be discovered. Of particular interest are the storage protein genes, globulin-1, globulin-2 and an unidentified cupin family gene. When expressing foreign proteins in maize, the globulin-1 promoter is most often used, but this cupin family gene has much higher expression and may be a better candidate for foreign gene expression in maize embryos. Results such as these allow identification of candidate genes and promoters that may not otherwise be available for use. mRNA seq data archived in NCBI SRA; Accession number: ACC=SRA060791 subid=108584.

Introgression of a novel Thinopyrum intermedium St-chromosome-specific HMW-GS gene into wheat

Thinopyrum intermedium has been hybridized extensively with wheat (Triticum aestivum L.) and several genes for disease resistance have been introgressed to cultivated wheat. However, there are very few reports about the Th. intermedium-derived seed storage protein genes which have been transferred into a wheat background by chromosome manipulation. Our aim is to identify several wheat–Th. intermedium ssp. trichophorum derivatives, and document these lines by genomic in situ hybridization (GISH), molecular markers and seed storage protein analysis. We found that a novel Th. intermedium 1St#2 chromosome-specific high-molecular-weight glutenin subunit (HMW-GS) was transferred to the wheat–Thinopyrum derivative lines. The genomic sequence of the Thinopyrum-derived HMW-GS was characterized and designated Glu-1St#2x, since it resembled x-type glutenins in both the N-terminal domain and C-terminal domain. It is much shorter than that of reported HMW-GS genes. The Glu-1St#2x sequence was successfully expressed in Escherichia coli and resulted in the identical weight to the native protein. The GISH and newly developed chromosome Thinopyrum-specific DNA markers enabled physically location of Glu-1St#2x to the region FL0.60–1.00 on Th. intermedium 1St#2L chromosome arm. Phylogenetic analysis revealed that the Glu-1St#2x evolved earlier than other x-type HMW-GS homoeologues in modern wheat genomes. The effect of Glu-1St#2x on protein content, sodium dodecyl sulphate sedimentation value and improvement of solvent retention capacity in wheat background suggested that Th. intermedium chromosome 1St#2 may have potential for improvement of wheat end-product quality.

Iron Biofortification of Myanmar Rice

Iron (Fe) deficiency elevates human mortality rates, especially in developing countries. In Myanmar, the prevalence of Fe-deficient anemia in children and pregnant women are 75 and 71%, respectively. Myanmar people have one of the highest per capita rice consumption rates globally. Consequently, production of Fe-biofortified rice would likely contribute to solving the Fe-deficiency problem in this human population. To produce Fe-biofortified Myanmar rice by transgenic methods, we first analyzed callus induction and regeneration efficiencies in 15 varieties that are presently popular because of their high-yields or high-qualities. Callus formation and regeneration efficiency in each variety was strongly influenced by types of culture media containing a range of 2,4-dichlorophenoxyacetic acid concentrations. The Paw San Yin variety, which has a high-Fe content in polished seeds, performed well in callus induction and regeneration trials. Thus, we transformed this variety using a gene expression cassette that enhanced Fe transport within rice plants through overexpression of the nicotianamine synthase gene HvNAS1, Fe flow to the endosperm through the Fe(II)-nicotianamine transporter gene OsYSL2, and Fe accumulation in endosperm by the Fe storage protein gene SoyferH2. A line with a transgene insertion was successfully obtained. Enhanced expressions of the introduced genes OsYSL2, HvNAS1, and SoyferH2 occurred in immature T2 seeds. The transformants accumulated 3.4-fold higher Fe concentrations, and also 1.3-fold higher zinc concentrations in T2 polished seeds compared to levels in non-transgenic rice. This Fe-biofortified rice has the potential to reduce Fe-deficiency anemia in millions of Myanmar people without changing food habits and without introducing additional costs.

The Native Structure and Composition of the Cruciferin Complex in Brassica napus

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300-390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

Efficiency for increasing seed oil content using WRINKLED1 and DGAT1 under the control of two seed-specific promoters, FAE1 and Napin

Seed storage oils are essential resources for not only human and animal diets but also industrial applications. The primary goal of this study was to increase seed oil content through comparative analysis of two seed-specific promoters, AtFAE1 from Arabidopsis Fatty Acid Elongase 1 gene and BnNapin from Brassica napus seed storage protein gene. AtWRI1 and AtDGAT1 genes encoding an AP2-type transcription factor and a Diacylglycerol Acyltransferase 1 enzyme, respectively, were expressed under the control of AtFAE1 and BnNapin promoters in Arabidopsis. The total seed oil content in all transgenic plants was increased by 8-11% compared with wild-type seeds. The increased level of oil content in AtWRI1 and AtDGAT1 transgenic lines under the control of both promoters was similar, although the activity of the BnNapin promoter is much stronger than that of AtFAE1 promoter in the mature stage of developing seeds where storage oil biosynthesis occurs at a maximum rate. This result demonstrates that the AtFAE1 promoter as well as the BnNapin promoter can be used to increase the seed oil content in transgenic plants.

Isolation and Characterization of an Endosperm-Specific Promoter from Wheat (Triticum aestivum L.)

Genes coding for avenin-like proteins (ALP) represent a new family of wheat storage protein genes. To find a wheat endosperm-specific promoter, a 1644-bp fragment upstream of the ALP type-B gene (GenBank accession number JN622144) was isolated. The important promoter elements of the ALP type-B gene were ascertained through sequence analysis which revealed that this fragment contains the TATA and CAAT boxes, which are important elements in gene expression. A prolamin box containing an endosperm motif and a GCN4-like motif (GLM) is present at about 300 bp upstream of the translation start site. The promoter sequence has two ESP-like elements and one of them is followed by an RY motif with the nucleotides CATG overlapping. The RY motif is considered the core functional sequence in a promoter. In an attempt to confirm the promoter activity, a series of 5'-deletions of the promoter were fused with the beta-glucuronidase (GUS) gene, and the constructs were stably introduced into tobacco plants. GUS staining confirmed that the AVL type-B promoter is an endosperm-specific promoter in tobacco seeds. Quantitative analysis of GUS expression in transgenic plants showed that even the shortest 5'-deletion, i.e. a 290-bp promoter sequence within the prolamin box, was sufficient to drive GUS expression in the endosperm. The highest expression level was found in transgenic plants containing the 5'-deletion vector construct pALP-8. This suggests that the ESP-like element overlapping with the RY motif may play a crucial role in the regulatory function of the promoter.

Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

BackgroundCereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.ResultsWe designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and γ-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.ConclusionsQuantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences.

Rapid Divergence of Prolamin Gene Promoters of Maize After Gene Amplification and Dispersal

Seeds have evolved to accommodate complicated processes like senescence, dormancy, and germination. Central to these is the storage of carbohydrates and proteins derived from sugars and amino acids synthesized during photosynthesis. In the grasses, the bulk of amino acids is stored in the prolamin superfamily that specifically accumulates in seed endosperm during senescence. Their promoters contain a conserved cis-element, called prolamin-box (P-box), recognized by the trans-activator P-box binding factor (PBF). Because of the lack of null mutants in all grass species, its physiological role in storage-protein gene expression has been elusive. In contrast, a null mutant of another endosperm-specific trans-activator Opaque2 (O2) has been shown to be required for the transcriptional activation of subsets of this superfamily by binding to the O2 box. Here, we used RNAi to knockdown Pbf expression and found that only 27-kDa γ- and 22-kDa α-zein gene expression were affected, whereas the level of other zeins remained unchanged. Still, transgenic seeds had an opaque seed phenotype. Combination of PbfRNAi and o2 resulted in further reduction of α-zein expression. We also tested the interaction of promoters and constitutively expressed PBF and O2. Whereas transgenic promoters could be activated, endogenous promoters appeared to be not accessible to transcriptional activation, presumably due to differential chromatin states. Although analysis of the methylation of binding sites of PBF and O2 correlated with the expression of endogenous 22-kDa α-zein promoters, a different mechanism seems to apply to the 27-kDa γ-zein promoter, which does not undergo methylation changes.

RNA INTERFERENCE OF BROAD GENE EXPRESSION MIMICS ANTIMETAMORPHIC EFFECT OF PYRIPROXYFEN ON THE BEET ARMYWORM, Spodoptera exigua

A larva-to-pupa metamorphosis is induced by a low or undetectable level of juvenile hormone (JH) during last instar in holometabolous insects. An exogenous application of JH agonist, pyriproxyfen (PYR), inhibited pupal metamorphosis of the beet armyworm, Spodoptera exigua. Last instar larvae of S. exigua exhibited increase of body size at first 3 days along with active feeding behavior. Also, at this period, prothoracic gland increased in size, while corpora allata remained little change. Storage proteins were accumulated in hemolymph plasma from penultimate to last instars, during which two storage protein genes (SeHex and SeSP1) were actively expressed. A Broad-Complex 1 (BRC1) gene of S. exigua (SeBRC1) was partially cloned and showed a specific expression at the last instar in all tested tissues including hemocytes, fat body, epidermis, gut, nerve, and salivary gland. Knockdown of SeBRC1 expression by its specific double-strand RNA mimicked the antimetamorphic effect induced by PYR treatment. PYR treatment at early last instar inhibited expression of SeBRC1, but did not that of other nuclear receptor, βFTZ-F1. These results indicate that a transcriptional factor, SeBRC1, plays a crucial role in pupal metamorphosis of S. exigua.

The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein

Glutelin is a major seed storage protein, accounting for 60–80 % of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.

Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity.

From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula

• The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

Characterization of upstream sequences from the 8S globulin gene of Vigna radiata

Seeds rich in protein in nature, are ideal bioreactors for economic production and storage of valueadded recombinant proteins and enzymes for industries. The upstream region of the seed storage protein gene is able to provide an attractive promoter for seed-specific expression of heterologous genes. Our previous research showed that 8S globulin occupied the majority of total soluble protein stored in seeds of mung bean ( Vigna radiata ), a rich source of protein, indicating that the promoter of this gene could be a seed-specific promoter with high activity. To improve the expression of heterologous proteins in plants to act as a bioreactor, the putative seed-specific promoter of 8S globulin gene was characterized in this study. Hence, this potential promoter of beta subunit gene of 8S globulin (8SGb) was isolated by genome walking. Analysis with various promoter prediction softwares showed that the promoter sequence possessed many common motifs related to gene transcription in the seed (such as W-box, ABRE element, E-box, etc.). The putative promoter was subsequently cloned into the binary vector pBI121-GFP by replacing the CaMV 35S promoter. The resultant construct designated as pBI-8SGb-GFP was transformed to mung bean cotyledon mesophyll protoplasts. Reporter gene GFP was expressed high in cotyledon protoplasts, which was detected by confocal microscopy, demonstrating the specific activity of 8SGb promoter in driving gene expression. This study also proved that the 8SGb promoter is an efficient regulatory element for plant seeds to act as a bioreactor. Key words : Seed-specific, promoter, genome walking, Vigna radiata.

Evaluation of seed storage protein gene 3′-untranslated regions in enhancing gene expression in transgenic rice seed

3' untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. We evaluated the potential of the 3'-UTRs of 9 seed storage protein (SSP) genes as terminators in enhancing the expression of the β-glucuronidase (gus A) reporter gene driven by the glutelin GluB-3 promoter in stable transgenic rice lines. Six of the 3'-UTRs significantly enhanced the activity of the GluB-3 promoter without changing its tissue specificity but altered its expression pattern in endosperm. With the 3'-UTRs of GluB-5, GluA-2 and GluC, the expression of gus A was higher by 3.12-, 2.45- and 2.14-fold, respectively, than with the Nos terminator. These three 3'-UTRs, combined with GluC, Ubi-1 and CaMV35S promoters, also increased GUS levels in stable transgenic rice lines or in transient expression in protoplasts, which indicated that the enhancements were independent of the promoter sequence. The increase in protein production was accompanied by altered mRNA levels, which suggests that the enhancements were due to increased transcript level. The 3'-UTRs of GluB-5, GluA-2 and GluC, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds. The 9 SSP 3'-UTRs could function as faithful terminators in mono- or multi-gene transformation avoiding homology-based gene silencing.