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1.
Proc Natl Acad Sci U S A ; 121(4): e2305745121, 2024 Jan 23.
Article in English | MEDLINE | ID: mdl-38236731

ABSTRACT

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 µg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 µg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.


Subject(s)
Oryza , Paramyxovirinae , Viral Vaccines , Animals , Chickens , Newcastle disease virus , Oryza/genetics , Universal Design , Epitopes , Antibodies, Viral
2.
Plant Biotechnol J ; 21(12): 2546-2559, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37572354

ABSTRACT

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.


Subject(s)
Classical Swine Fever Virus , Classical Swine Fever , Oryza , Viral Vaccines , Animals , Swine , Rabbits , Mice , Classical Swine Fever/prevention & control , Antibodies, Viral , Viral Envelope Proteins , Immunity
3.
Biol Res ; 53(1): 8, 2020 Feb 18.
Article in English | MEDLINE | ID: mdl-32070437

ABSTRACT

BACKGROUND: Rice is the staple food of many people around the world. However, most rice varieties, especially widely grown indica varieties and hybrids, are sensitive to cold stress. In order to provide a basis for the utilization of a common wild rice (CWR, Oryza rufipogon Griff.) named 'Chaling' CWR in cold-tolerant rice breeding and deepen the understanding of rice cold tolerance, the cold tolerance of ratoon 'Chaling' CWR was studied under the stress of the natural low temperature in winter in Changsha, Hunan province, China, especially under the stress of abnormal natural low temperature in Changsha in 2008, taking other ratoon CWR accessions and ratoon cultivated rice phenotypes as control. RESULTS: The results showed that ratoon 'Chaling' CWR can safely overwinter under the natural conditions in Changsha (28° 22' N), Hunan province, China, which is a further and colder northern place than its habitat, even if it suffers a long-term low temperature stress with ice and snow. In 2008, an extremely cold winter appeared in Changsha, i.e., the average daily mean temperature of 22 consecutive days from January 13 to February 3 was - 1.0 °C, and the extreme low temperature was - 4.7 °C. After subjected to this long-term cold stress, the overwinter survival rate of ratoon 'Chaling' CWR was 100%, equals to that of ratoon 'Dongxiang' CWR which is northernmost distribution in the word among wild rice populations, higher than those of ratoon 'Fusui' CWR, ratoon 'Jiangyong' CWR, and ratoon 'Liujiang' CWR (63.55-83.5%) as well as those of ratoon 'Hainan' CWR, ratoon 'Hepu' CWR, and all the ratoon cultivated rice phenotypes including 3 japonica ones, 3 javanica ones, and 5 indica ones (0.0%). CONCLUSIONS: The results indicate that ratoon 'Chaling' CWR possesses strong cold tolerance and certain freezing tolerance.


Subject(s)
Adaptation, Physiological/physiology , Cold Temperature , Cold-Shock Response/physiology , Oryza/physiology , Phenotype , Seasons
4.
Regul Toxicol Pharmacol ; 103: 292-300, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30738882

ABSTRACT

A 90-day subchronic toxicology screen of genetically modified (GM) rice Lac-3 expressing human lactoferrin (hLF) and its effects on the gut microbiota were studied in comparison to non-GM rice fed to Sprague-Dawley (SD) rats. Three different dietary concentrations (17.5%, 35% and 70%, w/w) of the GM rice or its corresponding non-GM rice were used. Additionally, the phylotypes of gut microbiota in the control group, the 70% GM rice diet group and the 70% non-GM rice diet group on day 90 were determined by 16S rRNA sequencing. The results of the 90-day subchronic feeding study demonstrated that the GM rice Lac-3 containing human lactoferrin (LF) gene is considered as safe as the non-GM rice. The results of bacterial 16S rRNA sequencing showed that the structure of gut microbiota in the 70% GM group slightly changed when compared with the control group and the 70% non-GM group. There were no significant differences in the microbiota diversity among the three groups.


Subject(s)
Gastrointestinal Microbiome/drug effects , Lactoferrin/toxicity , Oryza/genetics , Animals , Diet , Female , Humans , Lactoferrin/administration & dosage , Male , Rats , Rats, Sprague-Dawley
5.
Plant Cell Environ ; 41(3): 630-645, 2018 03.
Article in English | MEDLINE | ID: mdl-29314052

ABSTRACT

The circadian clock enables organisms to rapidly adapt to the ever-changing environmental conditions that are caused by daily light/dark cycles. Circadian clock genes universally affect key agricultural traits, particularly flowering time. Here, we show that OsPRR37, a circadian clock gene, delays rice flowering time in an expression level-dependent manner. Using high-throughput mRNA sequencing on an OsPRR37 overexpressing transgenic line (OsPRR37-OE5) and the recipient parent Guangluai4 that contains the loss-of-function Osprr37, we identify 14,992 genes that display diurnal rhythms, which account for 52.9% of the transcriptome. Overexpressing OsPRR37 weakens the transcriptomic rhythms and alters the phases of rhythmic genes. In total, 3,210 differentially expressed genes (DEGs) are identified, among which 1,863 rhythmic DEGs show a correlation between the change of absolute amplitudes and the mean expression levels. We further reveal that OsPRR37 functions as a transcriptional repressor to repress the expression levels and amplitudes of day-phased clock genes. More importantly, OsPRR37 confers expanded regulation on the evening-phased rhythmic DEGs by repressing the morning-phased rhythmic DEGs. Further study shows that OsPRR37 expands its regulation on flowering pathways by repressing Ehd1. Thus, our results demonstrate an expanded regulation mechanism of the circadian clock on the diurnal rhythms of the transcriptome.


Subject(s)
Circadian Rhythm/physiology , Flowers/physiology , Gene Expression Regulation, Plant , Oryza/physiology , Plant Proteins/genetics , Circadian Clocks/genetics , Flowers/genetics , Oryza/genetics , Photoperiod , Plant Proteins/metabolism , Plants, Genetically Modified , Transcriptome
6.
New Phytol ; 210(3): 934-45, 2016 May.
Article in English | MEDLINE | ID: mdl-26781807

ABSTRACT

Cytoplasmic male sterility (CMS) and restoration of fertility (Rf) are widely distributed in plant species utilized by humans. RF5 and GRP162 are subunits of the restoration of fertility complex (RFC) in Hong-Lian rice. Despite the fact that the RFC is 400-500 kDa in size, the other proteins or factors in the complex still remain unknown. Here, we identified RFC subunit 3, which encodes a DUF1620-containing and WD40-like repeat protein (RFC3) that is present in all tissues but highly expressed in leaves. We established that RFC3 interacts with both RF5 and GRP162 in vitro and in vivo, and is transported into the mitochondria as a membrane protein. Furthermore, CMS RNA (atp6-orfH79) and CMS cytotoxic protein (ORFH79) accumulate when RFC3 is silenced in restorer lines. We presented the analysis with blue-native polyacrylamide gel electrophoresis, indicating that RFC is disrupted in the RNAi line. We concluded that RCF3 is indispensable as a scaffold protein for the assembly of the RFC complex. We unveil a new molecular player of the RFC in the Rf pathway in rice and propose the model of RFC based on these data.


Subject(s)
Oryza/physiology , Plant Proteins/chemistry , Plant Proteins/metabolism , Repetitive Sequences, Amino Acid , Amino Acid Sequence , Conserved Sequence , Fertility , Gene Expression Regulation, Plant , Gene Silencing , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mitochondrial Membranes/metabolism , Oryza/genetics , Plant Infertility/genetics , Plants, Genetically Modified , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment
7.
BMC Genomics ; 16: 232, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25886904

ABSTRACT

BACKGROUND: For heterozygous genes, alleles on the chromatin from two different parents exhibit histone modification variations known as allele-specific histone modifications (ASHMs). The regulation of allele-specific gene expression (ASE) by ASHMs has been reported in animals. However, to date, the regulation of ASE by ASHM genes remains poorly understood in higher plants. RESULTS: We used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to investigate the global ASHM profiles of trimethylation on histone H3 lysine 27 (H3K27me3) and histone H3 lysine 36 (H3K36me3) in two rice F1 hybrids. A total of 522 to 550 allele-specific H3K27me3 genes and 428 to 494 allele-specific H3K36me3 genes were detected in GL × 93-11 and GL × TQ, accounting for 11.09% and 26.13% of the total analyzed genes, respectively. The epialleles between parents were highly related to ASHMs. Further analysis indicated that 52.48% to 70.40% of the epialleles were faithfully inherited by the F1 hybrid and contributed to 33.18% to 46.55% of the ASHM genes. Importantly, 66.67% to 82.69% of monoallelic expression genes contained the H3K36me3 modification. Further studies demonstrated a significant positive correlation of ASE with allele-specific H3K36me3 but not with H3K27me3, indicating that ASHM-H3K36me3 primarily regulates ASE in this study. CONCLUSIONS: Our results demonstrate that epialleles from parents can be inherited by the F1 to produce ASHMs in the F1 hybrid. Our findings indicate that ASHM-H3K36me3, rather than H3K27me3, mainly regulates ASE in hybrid rice.


Subject(s)
Gene Expression Regulation, Plant , Histones/metabolism , Oryza/genetics , Protein Processing, Post-Translational , Alleles , Chimera/genetics , Epigenomics , Oryza/classification
8.
Plant Mol Biol ; 87(1-2): 17-30, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25307286

ABSTRACT

Plant architecture directly affects biomass in higher plants, especially grain yields in agricultural crops. In this study, we characterized a recessive mutant, plant architecture determinant (pad), derived from the Oryza sativa ssp. indica cultivar MH86. The mutant exhibited severe dwarf phenotypes, including shorter and stunted leaves, fewer secondary branches during both the vegetative and reproductive growth stages. Cytological studies revealed that pad mutant growth defects are primarily due to the inhibition of cell expansion. The PAD gene was isolated using a map-based cloning strategy. It encodes a plasma membrane protein OsMCA1 and a SNP responsible for a single amino acid change was found in the mutant. PAD was universally expressed in rice tissues from the vegetative to reproductive growth stages, especially in seedlings, nodes and rachillae. Quantitative real-time PCR analysis revealed that the most of the genes responding to gibberellin (GA) metabolism were up-regulated in pad mutant internodes. The endogenous GA content measurement revealed that the levels of GA1 were significantly decreased in the third internode of pad mutants. Moreover, a GA response assay suggested that OsMCA1/PAD might be involved in the regulation of GA metabolism and signal transduction. Our results revealed the pad is a loss-of-function mutant of the OsMCA1/PAD, leading to upregulation of genes related to GA deactivation, which decreased bioactive GA levels.


Subject(s)
Genes, Plant , Gibberellins/metabolism , Oryza/genetics , Plant Proteins/genetics , Polymorphism, Single Nucleotide , Cloning, Molecular , Genetic Complementation Test , Phylogeny , Plant Roots/metabolism , Plant Stems/metabolism , Polymerase Chain Reaction
9.
BMC Plant Biol ; 15: 55, 2015 Feb 17.
Article in English | MEDLINE | ID: mdl-25848690

ABSTRACT

BACKGROUND: Sec18p/N-ethylmaleimide-sensitive factor (NSF) is a conserved eukaryotic ATPase, which primarily functions in vesicle membrane fusion from yeast to human. However, the function of the OsSec18 gene, a homologue of NSF in rice, remains unknown. RESULTS: In the present study, we investigated the function of OsSec18 in rice and found that OsSec18 complements the temperature-sensitive phenotype and interferes with vacuolar morphogenesis in yeast. Overexpression of OsSec18 in rice decreased the plant height and 1000-grain weight and altered the morphology of the protein bodies. Further examination revealed that OsSec18 presented as a 290-kDa complex in rice endosperm cells. Moreover, Os60sP0 was identified a component of this complex, demonstrating that the OsSec18 complex contains another complex of P0(P1-P2)2 in rice endosperm cells. Furthermore, we determined that the N-terminus of OsSec18 can interact with the N- and C-termini of Os60sP0, whereas the C-terminus of OsSec18 can only interact with the C-terminus of Os60sP0. CONCLUSION: Our results revealed that the OsSec18 regulates vacuolar morphology in both yeast and rice endosperm cell and the OsSec18 interacts with P0(P1-P2)2 complex in rice endosperm cell.


Subject(s)
Endosperm/cytology , Endosperm/metabolism , Multiprotein Complexes/metabolism , Oryza/metabolism , Plant Proteins/metabolism , Vacuoles/metabolism , Adenosine Triphosphatases , Endosperm/ultrastructure , Gene Expression Profiling , Gene Expression Regulation, Plant , Genetic Complementation Test , Membrane Fusion , Molecular Weight , Mutation/genetics , Organ Specificity/genetics , Oryza/cytology , Oryza/genetics , Phenotype , Plants, Genetically Modified , Protein Binding , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins , Vacuoles/ultrastructure , Vesicular Transport Proteins
10.
Plant Cell ; 24(1): 109-22, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22247252

ABSTRACT

The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene-mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS-Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration.


Subject(s)
Oryza/metabolism , Oryza/physiology , Plant Infertility/physiology , Plant Proteins/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Molecular Sequence Data , Oryza/genetics , Plant Infertility/genetics , Plant Proteins/genetics
11.
Biochim Biophys Acta ; 1830(12): 5515-25, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23644036

ABSTRACT

BACKGROUND: As the most abundant protein in the blood, human serum albumin (HSA) plays an important role in maintaining plasma oncotic pressure and fluid balance between the body's compartments. HSA is thus widely used in the clinic to treat diseases. However, the shortage of and safety issues arising from using plasma HSA (pHSA) underscore the importance of recombinant HSA (rHSA) as a promising substitute for pHSA. SCOPE OF REVIEW: Here, we review the production of rHSA, from expression to downstream processing, and highlight the scalability and cost-effectiveness of the two main expression platforms. We also discuss the biosafety of commercially available pharmaceutical rHSA with respect to impurities and contaminants, followed by an analysis of recent progress in preclinical and clinical trials. We emphasise the challenges of producing pharmaceutical-grade rHSA. MAJOR CONCLUSIONS: rHSA can be highly expressed in various hosts and seems to be identical to pHSA. rHSA generated from yeast appears to be as efficient and safe as pHSA in a series of preclinical and clinical trials, whereas rHSA from rice seeds exhibits great potential for more cost-effective production. Cost-effective products with no adverse effects will likely play a vital role in future human therapeutics. GENERAL SIGNIFICANCE: Our understanding of pharmaceutical-grade rHSA production has improved with respect to expression hosts, biochemical properties, downstream processing, and the detection and removal of impurities. However, due to the large dosages required for clinical applications, the production of sufficient quantities of rHSA still presents challenges. This article is part of a Special Issue entitled Serum Albumin.


Subject(s)
DNA, Recombinant/genetics , Serum Albumin/genetics , Animals , Animals, Genetically Modified , Clinical Trials as Topic , Humans , Plants, Genetically Modified , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , Serum Albumin/biosynthesis , Serum Albumin/therapeutic use
12.
BMC Genomics ; 15: 297, 2014 Apr 22.
Article in English | MEDLINE | ID: mdl-24755044

ABSTRACT

BACKGROUND: Sprague and Tatum (1942) introduced the concepts of general combining ability (GCA) and specific combining ability (SCA) to evaluate the breeding parents and F1 hybrid performance, respectively. Since then, the GCA was widely used in cross breeding for elite parent selection. However, the molecular basis of GCA remains to unknown. RESULTS: We studied the transcriptomes of three varieties and three F1 hybrids using RNA-Sequencing. Transcriptome sequence analysis revealed that the transcriptome profiles of the F1s were similar to the positive GCA-effect parent. Moreover, the expression levels of most differentially expressed genes (DEGs) were equal to the parent with a positive GCA effect. Analysis of the gene expression patterns of gibberellic acid (GA) and flowering time pathways that determine plant height and flowering time in rice validated the preferential transcriptome expression of the parents with positive GCA effect. Furthermore, H3K36me3 modification bias in the Pseudo-Response Regulators (PRR) gene family was observed in the positive GCA effect parents and demonstrated that the phenotype and transcriptome bias in the positive GCA effect parents have been epigenetically regulated by either global modification or specific signaling pathways in rice. CONCLUSIONS: The results revealed that the transcriptome profiles and DEGs in the F1s were highly related to phenotype bias to the positive GCA-effect parent. The transcriptome bias toward high GCA parents in F1 hybrids attributed to H3K36me3 modification both on global modification level and specific signaling pathways. Our results indicated the transcriptome profile and epigenetic modification level bias to high GCA parents could be the molecular basis of GCA.


Subject(s)
Genotype , Hybridization, Genetic , Oryza/genetics , Phenotype , Transcriptome , Epigenesis, Genetic , Flowers , Gene Expression Profiling , Gene Expression Regulation, Plant , Histones/metabolism , Metabolic Networks and Pathways , Oryza/metabolism , Quantitative Trait, Heritable , Signal Transduction
13.
Plant Mol Biol ; 84(3): 315-27, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24104862

ABSTRACT

Transcriptomic data for Sorghum propinquum, the wild-type sorghum, are limited in public databases. S. propinquum has a subterranean rhizome and transcriptome data will help in understanding the molecular mechanisms underlying rhizome formation. We sequenced the transcriptome of S. propinquum aerial-shoot and rhizome using an Illumina platform. More than 70 % of the genes in the S. propinquum genome were expressed in aerial-shoot and rhizome. The expression patterns of 1963 and 599 genes, including transcription factors, were specific or enriched in aerial-shoot and rhizome respectively, indicating their possible roles in physiological processes in these tissues. Comparative analysis revealed several cis-elements, ACGT box, GCCAC, GATC and TGACG box, which showed significantly higher abundance in aerial-shoot-specific genes. In rhizome-specific genes MYB and ROOTMOTIFTAPOX1 motifs, and 10 promoter and cytokinin-responsive elements were highly enriched. Of the S. propinquum genes, 27.9 % were identified as alternatively spliced and about 60 % of the alternative splicing (AS) events were tissue-specific, suggesting that AS played a crucial role in determining tissue-specific cellular function. The transcriptome data, especially the co-localized rhizome-enriched expressed transcripts that mapped to the publicly available rhizome-related quantitative trait loci, will contribute to gene discovery in S. propinquum and to functional studies of the sorghum genome. Deep transcriptome sequencing revealed a clear difference in the expression patterns of genes between aerial-shoot and rhizome in S. propinquum. This data set provides essential information for future studies into the molecular genetic mechanisms involved in rhizome formation.


Subject(s)
Genes, Plant , Plant Components, Aerial/metabolism , Rhizome/metabolism , Sorghum/genetics , Transcriptome , Gene Expression Regulation, Plant , RNA, Plant/isolation & purification , Real-Time Polymerase Chain Reaction
14.
Anal Biochem ; 450: 4-10, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24388867

ABSTRACT

Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.


Subject(s)
DNA, Plant/analysis , DNA, Plant/genetics , Drug Contamination , Oryza/genetics , Real-Time Polymerase Chain Reaction/methods , Recombinant Proteins/chemistry , Serum Albumin/chemistry , Benzothiazoles , Calibration , DNA Primers/genetics , DNA, Plant/chemistry , Diamines , Genome, Plant/genetics , Humans , Limit of Detection , Organic Chemicals/chemistry , Quinolines , RNA, Plant/genetics , RNA, Ribosomal, 5S/genetics , Recombinant Proteins/genetics , Reproducibility of Results , Serum Albumin/genetics , Taq Polymerase/metabolism
15.
Proc Natl Acad Sci U S A ; 108(47): 19078-83, 2011 Nov 22.
Article in English | MEDLINE | ID: mdl-22042856

ABSTRACT

Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin.


Subject(s)
Bioreactors , Biotechnology/methods , Models, Molecular , Oryza/metabolism , Seeds/metabolism , Serum Albumin/biosynthesis , Animals , Humans , Plants, Genetically Modified , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Serum Albumin/chemistry
16.
Plant Mol Biol ; 83(1-2): 153-61, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23564402

ABSTRACT

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.


Subject(s)
Endoplasmic Reticulum Stress , Gene Expression Regulation, Plant , Seeds/growth & development , Serpins/metabolism , Cell Death , Genes, Plant , Organ Size , Oryza/genetics , Oryza/metabolism , Plant Cells/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Seed Storage Proteins/genetics , Seed Storage Proteins/metabolism , Serpins/genetics , Transcriptome
17.
BMC Plant Biol ; 13: 221, 2013 Dec 21.
Article in English | MEDLINE | ID: mdl-24358981

ABSTRACT

BACKGROUND: Extensive studies on heterosis in plants using transcriptome analysis have identified differentially expressed genes (DEGs) in F1 hybrids. However, it is not clear why yield in heterozygotes is superior to that of the homozygous parents or how DEGs are produced. Global allele-specific expression analysis in hybrid rice has the potential to answer these questions. RESULTS: We report a genome-wide allele-specific expression analysis using RNA-sequencing technology of 3,637-3,824 genes from three rice F1 hybrids. Of the expressed genes, 3.7% exhibited an unexpected type of monoallelic expression and 23.8% showed preferential allelic expression that was genotype-dependent in reciprocal crosses. Those genes exhibiting allele-specific expression comprised 42.4% of the genes differentially expressed between F1 hybrids and their parents. Allele-specific expression accounted for 79.8% of the genes displaying more than a 10-fold expression level difference between an F1 and its parents, and almost all (97.3%) of the genes expressed in F1, but non-expressed in one parent. Significant allelic complementary effects were detected in the F1 hybrids of rice. CONCLUSIONS: Analysis of the allelic expression profiles of genes at the critical stage for highest biomass production from the leaves of three different rice F1 hybrids identified genotype-dependent allele-specific expression genes. A cis-regulatory mechanism was identified that contributes to allele-specific expression, leading to differential gene expression and allelic complementary effects in F1 hybrids.


Subject(s)
Gene Expression Regulation, Plant/genetics , Oryza/genetics , Sequence Analysis, RNA , Alleles , Genotype , Hybridization, Genetic/genetics
18.
Plant Biotechnol J ; 11(9): 1029-33, 2013 12.
Article in English | MEDLINE | ID: mdl-24119183

ABSTRACT

Molecular Pharming, the production of recombinant pharmaceuticals through plant biotechnology, has the potential to transform the biologics sector of the pharmaceutical industry. More fascinating however, is how it might be used to improve access to modern medicines, and improve health of the poor in developing countries and emerging economies. Although improving global health through molecular pharming has been discussed for at least two decades, little progress has actually been made. In this manuscript, a four point plan is described to maximise the opportunity for molecular pharming to provide solutions. These are (i) to identify and prioritise important drug targets that are relevant to the poor; (ii) to support research and development partners in low to middle income countries to develop local expertise, transfer technology and build capacity; (iii) to increase collaboration between regulatory bodies to enable national regulatory frameworks to be developed in low to middle income countries; and (iv) to promote intellectual property management approaches that include socially responsible licensing. An existing case study is described to illustrate how this might be achieved.


Subject(s)
Biotechnology/methods , Molecular Farming/economics , Plants/genetics , Biological Products/metabolism , Biotechnology/economics , Developing Countries , Drug Design , Drug Industry/economics , Global Health , Humans , Intellectual Property , Molecular Farming/methods , Plants/metabolism , Plants, Genetically Modified , Research , Technology Transfer , Technology, Pharmaceutical/economics
19.
Int J Mol Sci ; 14(2): 3556-67, 2013 Feb 07.
Article in English | MEDLINE | ID: mdl-23434658

ABSTRACT

Basic fibroblast growth factor (FGF-2) is an important member of the FGF gene family. It is widely used in clinical applications for scald and wound healing in order to stimulate cell proliferation. Further it is applied for inhibiting stem cell differentiation in cultures. Due to a shortage of plasma and low expression levels of recombinant rbFGF in conventional gene expression systems, we explored the production of recombinant rbFGF in rice grains (Oryza sativa bFGF, OsrbFGF). An expression level of up to 185.66 mg/kg in brown rice was obtained. A simple purification protocol was established with final recovery of 4.49% and resulting in a yield of OsrbFGF reaching up to 8.33 mg/kg OsrbFGF. The functional assay of OsrbFGF indicated that the stimulating cell proliferation activity on NIH/3T3 was the same as with commercialized rbFGF. Wound healing in vivo of OsrbFGF is equivalent to commercialized rbFGF. Our results indicate that rice endosperm is capable of expressing small molecular mass proteins, such as bFGF. This again demonstrates that rice endosperm is a promising system to express various biopharmaceutical proteins.

20.
Transgenic Res ; 21(3): 499-510, 2012 Jun.
Article in English | MEDLINE | ID: mdl-21910016

ABSTRACT

Genetically modified plants expressing disease resistance traits offer new treatment strategies for human diseases, but at the same time present a challenge in terms of food safety assessment. The present 90-day feeding study was designed to assess the safety of transgenic rice expressing the recombinant human insulin-like growth factor-1 (rhIGF-1) compared to its parental wild rice. Male and female C57BL/6J rats were given a nutritionally balanced purified diet with 20% transgenic rhIGF-1 rice or 20% parental rice for 90 days. This corresponds to a mean daily rhIGF-1 protein intake of approximately 217.6 mg/kg body weight based on the average feed consumption. In the animal study a range of biological, biochemical, clinical, microbiological and pathological parameters were examined and several significant differences were observed between groups, but none of the effects were considered to be adverse. In conclusion, no adverse or toxic effects on C57BL/6J rats were observed in the design used in this 90-day study. These results will provide valuable information for the safety assessment of genetically modified food crops.


Subject(s)
Food Safety/methods , Food, Genetically Modified/toxicity , Insulin-Like Growth Factor I/metabolism , Oryza/toxicity , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/toxicity , Animals , Bacterial Load , Blood Chemical Analysis , Blood Glucose/metabolism , Body Weight , Feces/microbiology , Female , Humans , Insulin-Like Growth Factor I/genetics , Male , Mice , Mice, Inbred C57BL , Oryza/genetics , Plants, Genetically Modified/genetics , Quality Control , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Time Factors
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