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1.
Electron. j. biotechnol ; 51: 95-109, May. 2021. tab, ilus
Article in English | LILACS | ID: biblio-1343466

ABSTRACT

Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology's high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.


Subject(s)
Transformation, Genetic , Biological Products , Chloroplasts , Crops, Agricultural , Biotechnology , Recombinant Proteins/biosynthesis , Plants, Genetically Modified
2.
Electron J Biotechnol ; 49: 42-49, Jan. 2021. tab, graf, ilus
Article in English | LILACS | ID: biblio-1291646

ABSTRACT

BACKGROUND: Late embryogenesis abundant (LEA) proteins were reported to be related to adversity stress and drought tolerance. Lea-3 from Arachis hypogaea L. (AhLea-3) was previously found to be related to salt tolerance according to the result of transcriptome profiling and digital gene expression analysis. So, AhLea-3 was cloned and the salt tolerance was validated by transgenic peanut plants. RESULTS: AhLea-3 was isolated from M34, a salt-resistant mutant of peanut, with its cDNA as the template. AhLea-3 contains one intron and two extrons, and the full-length cDNA sequence contains 303 bp. AhLea3 was ligated to pCAMBIA1301 to obtain the overexpression vector pCAMBIA1301-AhLea-3, which was then transferred into peanut variety Huayu23. The expression level of AhLea-3, as determined by qRTPCR analysis, was >10 times higher in transgenic than in non-transgenic plants. Five days after they were irrigated with 250 mM NaCl, the transgenic plants showed less severe leaf wilting, higher activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), and lower malonic dialdehyde content than non-transgenic plants. Relative to non-transgenic plants, the transgenic plants had a higher photosynthetic net rate, stomatal conductance, and transpiration rate, and a lower intercellular CO2 concentration after salt stress treatment (250 mM NaCl). CONCLUSIONS: These results indicate that overexpression of AhLea-3 increased the salt tolerance of transgenic peanut plants. AhLea-3 might become a useful gene resource for the variety breeding of salinity tolerance in peanut.


Subject(s)
Arachis/metabolism , Plant Proteins/metabolism , Plants, Genetically Modified/metabolism , Salt Tolerance , Arachis/genetics , Plant Proteins/isolation & purification , Transformation, Genetic
3.
Braz. arch. biol. technol ; 64: e21200316, 2021. tab, graf
Article in English | LILACS | ID: biblio-1278451

ABSTRACT

Abstract To discover and isolate a glyphosate-resistant gene from Fragaria vesca through gene mining. An open reading frame (ORF) of 1563 bp encoding EPSPSwas amplified from Fragaria vesca (FvEPSPS). FvEPSPS (Genebank: XP004306932.1) encodes a polypeptide of 520 amino acids and it has hightly homologous with EPSPS from other plants. qRT-PCR analysis showed that the FvEPSPS was expressed extensively in all tissues including leaves, roots and stems, with higher expression in leaves. Furthermore, transgenic Arabidopsis Thaliana exhibited 10 mM glyphosate to resistance. Therefore, this research offers a new glyphosate-resistant gene for development of transgenic crops.


Subject(s)
Plants, Genetically Modified , Arabidopsis , Fragaria , Herbicides/adverse effects
4.
Electron. j. biotechnol ; 47: 72-82, sept. 2020. tab, ilus, graf
Article in English | LILACS | ID: biblio-1253093

ABSTRACT

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.


Subject(s)
Aphids/pathogenicity , Pest Control, Biological , Pinellia/chemistry , Plant Viruses , Tobacco , Blotting, Southern , Polymerase Chain Reaction , Promoter Regions, Genetic , Plants, Genetically Modified , Plant Leaves/chemistry , Transgenes , Disease Resistance , Crop Protection
5.
Electron. j. biotechnol ; 45: 1-9, May 15, 2020. ilus
Article in English | LILACS | ID: biblio-1177370

ABSTRACT

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.


Subject(s)
Tobacco/metabolism , Chloroplasts/genetics , Chloroplasts/metabolism , Zea mays/genetics , Green Fluorescent Proteins/metabolism , Transformation, Genetic , Biotechnology , Polymerase Chain Reaction , Plants, Genetically Modified , Plastids/genetics , Green Fluorescent Proteins/genetics , Escherichia coli , Genome, Chloroplast
6.
Braz. j. med. biol. res ; 53(2): e8749, 2020. tab, graf
Article in English | LILACS | ID: biblio-1055490

ABSTRACT

For a long time, vaccines have been the main mode of defense and protection against several bacterial, viral, and parasitic diseases. However, the process of production and purification makes them expensive and unaffordable to many developing nations. An edible vaccine is when the antigen is expressed in the edible part of the plant. This reduces the cost of production of the vaccine because of ease of culturing. In this article, various types of edible vaccines that include algal and probiotics in addition to plants are discussed. Various diseases against which research has been carried out are also reviewed. This article focused on the conception of edible vaccines highlighting the various ways by which vaccines can be delivered.


Subject(s)
Humans , Communicable Disease Control/methods , Vaccines, Edible/administration & dosage , Vaccines, Edible/immunology , Vaccines, Edible/chemistry , Biotechnology/methods , Plants, Genetically Modified/genetics
7.
Braz. arch. biol. technol ; 63: e20180428, 2020. tab, graf
Article in English | LILACS | ID: biblio-1132198

ABSTRACT

Abstract Development of transgenic Bt crops with stable and high level of Bt protein expression over generations under different environmental conditions is critical for successful deployment at field level. In the present study, progenies of transgenic cotton Coker310 event, CH12 expressing novel cry2AX1 gene were evaluated in T3 generation for stable integration, expression and resistance against cotton bollworm, Helicoverpa armigera. The cry2AX1 gene showed stable inheritance and integration in the T3 progeny plants as revealed by PCR and Southern blot hybridization. The expression of Cry2AX1 protein on 90 days after sowing (DAS) was in the range of 1.055 to 1.5 µg/g of fresh leaf tissue except one plant which showed 0.806 µg/g of fresh leaf tissue and after 30 days (i.e., on 120 DAS) three plants recorded in between 0.69 to 0.82 µg/g and other plants are in range of 0.918 to 1.058 µg/g of fresh leaf tissue. Detached leaf bit bioassay in T3 progeny on 110 DAS recorded mortality of 73.33 to 93.33 per cent against H. armigera and severe growth retardation in surviving larvae. These results indicate that the expression of chimeric cry2AX1 is stable and exhibits insecticidal activity against H. armigera in T3 progeny of CH12 event of transgenic cotton.


Subject(s)
Animals , Bacillus thuringiensis/pathogenicity , Pest Control, Biological/methods , Gossypium/genetics , Endotoxins/genetics , Moths , Plant Diseases/prevention & control , Plants, Toxic , Biological Assay , Plants, Genetically Modified
8.
Chinese Journal of Biotechnology ; (12): 643-651, 2020.
Article in Chinese | WPRIM | ID: wpr-827004

ABSTRACT

Cucumber (Cucumis sativus) is an important vegetable crop in the world. Agrobacterium-mediated transgenic technology is an important way to study plant gene functions and improve varieties. In order to further accelerate the transgenic research and breeding process of cucumber, we described the progress and problems of Agrobacterium tumefaciens-mediated transgenic cucumber, from the influencing factors of cucumber regeneration ability, genetic transformation conditions and various additives in the process. We prospected for improving the genetic transformation efficiency and safety selection markers of cucumber, and hoped to provide reference for the research of cucumber resistance breeding and quality improvement.


Subject(s)
Agrobacterium tumefaciens , Metabolism , Breeding , Cucumis sativus , Genetics , Microbiology , Plants, Genetically Modified , Microbiology , Research , Transformation, Genetic
9.
Chinese Journal of Biotechnology ; (12): 700-706, 2020.
Article in Chinese | WPRIM | ID: wpr-826906

ABSTRACT

The responsibility of root is absorbing water and nutrients, it is an important plant tissue, but easily to be affected by biotic and abiotic stresses, affecting crop growth and yield. The design of a synthetic root-specific promoter provides candidate promoters for the functional analysis and efficient expression of stress-related genes in crop roots. In this study, a synthetic root-specific module (pro-SRS) was designed using tandem four-copies of root specific cis-acting elements (OSE1ROOTNODULE, OSE2ROOTNODULE, SP8BFIBSP8AIB, and ROOTMOTIFAPOX1), and fused with minimal promoter from the CaMV 35S promoter to synthesize an artificially synthetic SRSP promoter. The SRSP promoter was cloned in pCAMBIA2300.1 by replacing CaMV 35S promoter so as to drive GUS expression. The constructs with SRSP promoter were transformed in tobacco by Agrobacterium-mediated method. SRSP promoter conferred root-specific expression in transgenic tobacco plants through Real-time PCR (RT-PCR) analysis and GUS histochemical staining analysis. It is indicated that the repeated arrangement of cis-acting elements can realize the expected function of the promoter. This study laid a theoretical foundation for the rational design of tissue-specific promoters.


Subject(s)
Agrobacterium , Genetics , Cloning, Molecular , Gene Expression Regulation, Plant , Plant Roots , Genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Genetics , Stress, Physiological , Tobacco , Genetics , Transformation, Genetic
10.
Chinese Journal of Biotechnology ; (12): 707-715, 2020.
Article in Chinese | WPRIM | ID: wpr-826905

ABSTRACT

OsRhoGDI2 was isolated as a putative partner of Rho protein family member OsRacD from rice panicles by yeast two-hybrid, but its function remains unknown. In order to identify the function of OsRhoGDI2, OsRhoGDI2 knockout mutants were created by CRISPR/Cas9 technology. The results showed that two different homozygous mutants were obtained in T0 generation, and eight kinds homozygous mutants were identified in T1 generation. Sequence analysis revealed that the base substitution or base deletion occurred near the editing targets of the gene in knockout rice, and it could be expected that the truncated OsRhoGDI2 proteins lacking the RhoGDI conserved domain would be generated. Phenotype analysis showed that the OsRhoGDI2 knockout rice plants were significantly lower than the control plants. Statistical analysis confirmed that the significant decrease of plant height was due to the shortening of the second and third internodes, suggesting that OsRhoGDI2 gene may be related with rice height control.


Subject(s)
CRISPR-Cas Systems , Genes, Plant , Genetics , Oryza , Genetics , Plants, Genetically Modified , rho Guanine Nucleotide Dissociation Inhibitor beta , Genetics
11.
Biosci. j. (Online) ; 35(6): 1886-1898, nov./dec. 2019. tab
Article in English | LILACS | ID: biblio-1049154

ABSTRACT

The objective of this study was to produce silage from intercropped RR glyphosate tolerant corn (RR - Roundup Ready) and RR soybeans under Brazilian Cerrado conditions. The research was carried out in off-season between February and June 2015, in Rio Verde, Goiás (Brazil). In the experimental period the rainfall was 865 mm and the average temperature of 23.7°C. Two trials were performed. The first had corn rows spaced in 1 m with a row of soybeans in the interrow. The second had double corn rows interspersed with one row of soybeans, all spaced in 0.50 m. In both trials, we used a randomized block design, in a 2x2+3 factorial scheme, with four replications. Primary factor consisted of two types of pre-sowing fertilization: one only in corn rows and the other over the total area. The secondary factor corresponded to two soybean varieties: medium and late cycle variety. In addition, three control treatments were made: one of corn and two of soybeans. The corn cutoff point was at the hard farinaceous stage when the milk line reaches half the grain, and in the sole crop soybean at the R5.5 final phase of grain filling. Silage was stored in PVC experimental silos for 60 days. The intercropped treatments, in both spatial arrangements, promoted increases in neutral detergent fiber and acid detergent fiber contents. Soybean monoculture presented high values of crude protein and ethereal extract. There were higher effluent losses in medium-cycle soybean variety silage when it was grown in single cropping. However, the lowest effluent losses were recorded for silages of medium-cycle soybean intercropped under a double row pre-sowing corn fertilization system. The soybeans mixed silages produced together with corn under off-season conditions did not yield satisfactory results.


Objetivou-se neste trabalho adequar o sistema de consórcio entre milho com tolerância ao herbicida glifosato (RR - Roundup Ready) e soja RR para obtenção de silagem de qualidade em condições do Cerrado brasileiro. A pesquisa foi conduzida na safrinha entre fevereiro e junho de 2015, em Rio Verde, Goiás (Brasil). A pluviosidade do período experimental foi de 865mm e a temperatura média de 23,7°C. Realizou-se dois ensaios, adequando formas de adubação de semeadura, arranjo de plantas e cultivares. Utilizou-se delineamento em blocos casualizados, em fatorial 2x2+3, com quatro repetições, sendo o fator primário duas modalidades de adubação de semeadura na linha: somente no milho, e em área total; o fator secundário correspondeu a duas variedades de soja: ciclo médio e tardio; mais três testemunhas: uma de milho e duas de soja. O ponto de corte do milho foi no estádio farináceo duro quando a linha do leite atinge metade do grão, e na soja em monocultivo no estádio fenológico R5.5, fase final de enchimento de grãos. Armazenou-se a silagem em silos experimentais de PVC por 60 dias. Os tratamentos de consórcio em ambos os arranjos aumentaram os teores de fibra em detergente neutro e fibra em detergente ácido. Os monocultivos de soja obtiveram valores elevados de proteína bruta e extrato etéreo. Ocorreu maior perda por efluente na silagem da variedade de soja de ciclo médio cultivada em monocultivo. No entanto, as menores perdas por efluente foram registradas para silagens de culturas de soja de ciclo médio consorciadas sob um sistema de fertilização pré-semeadura de linha de milho, no arranjo de linha dupla. A inserção de soja para produzir silagens mista juntamente com o milho em condições de safrinha não teve resultados satisfatórios.


Subject(s)
Silage , Soybeans , Zea mays , Plants, Genetically Modified
12.
Electron. j. biotechnol ; 41: 22-29, sept. 2019. ilus
Article in English | LILACS | ID: biblio-1087255

ABSTRACT

We highlight the importance of the mixed genetic approaches (classical and currents) to improve the social perception related to the GMOs acceptance. We pointed out that CRISPR/Cas9 events could carry DNA variability/rearrangements related to somaclonal variations or epigenetic changes that are independent from the editing per se. The transformation of single cells, followed by plant regeneration, is used to generate modified plants, transgenic or genome editing (CRISPR/Cas9). The incidence of undesirable somaclonal variations and/or epigenetic changes that might have occurred during in vitro multiplication and regeneration processes, must be carefully analyzed in replicates in field trials. One remarkable challenge is related to the time lapse that selects the modified elite genotypes, because these strategies may spend a variable amount of time before the results are commercialized, where in all the cases it should be take into account the genotype × environment interactions. Furthermore, this combination of techniques can create an encouraging bridge between the public opinion and the community of geneticists who are concerned with plant genetic improvement. In this context, either transgenesis or genomic editing strategies become complementary modern tools to facing the challenges of plant genetic improvement. Their applications will depend on case-by-case analysis, and when possible will necessary associate them to the schemes and bases of classic plant genetic improvement.


Subject(s)
Plants, Genetically Modified , Gene Transfer Techniques , CRISPR-Cas Systems , Gene Editing , Transformation, Genetic , Mutagenesis , DNA Methylation , Genetic Enhancement , Epigenesis, Genetic
13.
Braz. j. biol ; 79(2): 248-256, Apr.-June 2019. tab, graf
Article in English | LILACS | ID: biblio-989445

ABSTRACT

Abstract The use of GMO expressing Bt toxin in soybean production has increased significantly in the last years in Brazil in order to manage the damage caused by lepidopteran pests. In this study, we compared the richness and abundance of owlet moths (Noctuoidea) associated with Bt and non-Bt soybean. We determined the temporal variations as a function of phenology, and correlated the population variations of the most common species with meteorological variables. The research was conducted at the experimental area of Embrapa Cerrados. The collection method used was differentiated being suppressive and absolute. A total of 13 species were collected, of which eight occurred on Bt soybeans. The most representative taxa were Chrysodeixis includens (72.87%), Anticarsia gemmatalis (18.17%) and Spodoptera spp (5.22%). The number of larvae belonging to species targeted by the Bt technology was 10 times lower on Bt than on non-Bt soybeans. Utetheisa ornatrix and Elaphria deltoides were recorded on soybean for the first time, observing larvae of both species in non-Bt soybean and those of U. ornatrix also in Bt soybean. Only A. gemmatalis larvae correlated (p <0.05) negatively with precipitation. This study provided field information on the abundance and species richness of owlet moths on non-Bt soybeans, associated with the effects of Bt soybean. When considering the different levels of infestation between cultivars as a criterion, larvae monitoring is of substantial importance in order to develop the lost control program.


Resumo O uso de OGM que expressam toxina Bt na produção de soja tem aumentado significativamente nos últimos anos no Brasil e são utilizados para conter os danos causados ​​pelos lepidópteros pragas. Neste estudo comparamos a riqueza e a abundância de Noctuoides (Noctuoidea) associados à soja Bt e não-Bt. Determinamos as variações temporais em função da fenologia e correlacionamos às variações populacionais das espécies mais comuns com variáveis ​​meteorológicas. A pesquisa foi conduzida na área experimental da Embrapa Cerrados. O método de coleta utilizado foi diferenciado sendo supressivo e absoluto. Um total de 13 espécies foram coletadas, das quais oito ocorreram em soja Bt. Os taxa mais representativos foram Chrysodeixis includens, Anticarsia gemmatalis e Spodoptera spp. O número de larvas pertencentes às espécies alvo da tecnologia Bt foram 10 vezes menores na soja Bt do que em soja não-Bt . Utetheisa ornatrix e Elaphria deltoides foram registradas na soja pela primeira vez, observando-se larvas de ambas espécies na soja não-Bt e as de U. ornatrix também na soja Bt. Somente as larvas de A. gemmatalis se correlacionaram (p <0,05) negativamente com a precipitação. Este estudo forneceu informações em campo sobre a abundância e riqueza de espécies na soja não- Bt, associada aos efeitos da soja Bt. A importância do monitoramento das lagartas é substancial, a fim de tomar a melhor decisão de controle, considerando-se os diferentes níveis de infestação entre cultivares como critério.


Subject(s)
Animals , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Endotoxins/genetics , Endotoxins/pharmacology , Hemolysin Proteins/genetics , Hemolysin Proteins/pharmacology , Soybeans/genetics , Soybeans/parasitology , Brazil , Pest Control, Biological , Plants, Genetically Modified/genetics , Plants, Genetically Modified/parasitology , Larva/drug effects , Moths/drug effects
14.
Demetra (Rio J.) ; 14: e33380, mar. 2019. tab, graf
Article in English, Portuguese | LILACS | ID: biblio-1099534

ABSTRACT

O milho é um dos cereais mais utilizados na produção de alimentos, sendo em sua maioria transgênico. Diante disso, objetivou-se desenvolver e avaliar formulações de biscoitos com farinha não transgênica de milho (FMNT) e farinha de semente de abóbora (FSA), adicionados de especiarias. Inicialmente, foram analisadas quimicamente três marcas de FMT (farinha de milho transgênica) e uma marca de FMNT. Paralelamente, realizou-se pesquisa com 80 consumidores sobre o biscoito proposto, sendo então elaboradas três formulações: F1 (sem FSA), F2 (com 25% FSA) e F3 (com 50% FSA). Cada formulação de biscoito foi avaliada quanto às características microbiológicas, sensoriais e químicas. No que se refere às características das farinhas de milho, a FMNT apresentou maior quantidade de proteínas e capacidade antioxidante. A pesquisa de mercado indicou que as formulações de biscoito propostas têm potencial comercial, e que deveriam ser adicionados orégano e manjericão. Todas as formulações de biscoito estavam seguras microbiologicamente, mas a formulação 2 foi a que apresentou melhor aceitação no que se refere a sabor e impressão global, assim como melhor intenção de compra, além de ser a preferida pelos provadores. O índice de aceitabilidade (IA) indicou a formulação 2 com potencial para consumo e comercialização. Em relação à composição centesimal e química, a formulação 3 apresentou características melhores por conter mais cinzas (7,73), proteínas (34,22), lipídios (28,77), energia (494,43), redução do ferro (23,34) e fenólicos totais (196,40). Diante do exposto, somente a formulação 2 apresenta potencial comercial, uma vez que contém um IA igual ao mínimo estabelecido para comercialização, enquanto que a formulação 3 apresentou melhores características químicas. (AU)


Corn is one of the most used cereals in food production, being mostly transgenic. Therefore, the objective was to develop and evaluate formulations of biscuits with non-transgenic corn flour (NTCF) and pumpkin seed meal (PSM), added with spices. Initially, three brands of TCF (transgenic corn flour), and a brand of NTCF were chemically analyzed. In parallel, a survey was carried out with 80 consumers on the proposed biscuit. Three formulations were formulated: F1 (without PSM), F2 (with 25% PSM) and F3 (with 50% PSM). Each cookie formulation was evaluated for microbiological, sensory and chemical characteristics. Regarding the characteristics of corn flour, NTCF showed a higher amount of protein and antioxidant capacity. Market research has indicated that the proposed biscuit formulations have commercial potential, and that oregano and basil should be added. All biscuit formulations were microbiologically safe; however, formulation 2 was the one that showed the best acceptance for taste, overall impression, purchase intention, and was the preferred formulation by tasters. The acceptability index (IA) indicated formulation 2 with potential for consumption and commercialization. In relation to the centesimal and chemical composition, formulation 3 showed better characteristics as it contained more ash (7.73), proteins (34.22), lipids (28.77), energy (494.43), iron reduction (23.34) and total phenolics (196.40). Considering the above, only formulation 2 has commercial potential, since it contains an acceptance index equal to the minimum established for commercialization, while formulation 3 presented better chemical characteristics. (AU)


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Food, Formulated , Cookies , Edible Grain , Plants, Genetically Modified , Flour
15.
Article in Chinese | WPRIM | ID: wpr-772050

ABSTRACT

OBJECTIVE@#To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production.@*METHODS@#Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by sequence synthesis and subcloning. The two vectors were inoculated on either or leaves agroinfiltration. The expression of recombinant rhPA in leaves was examined using Western blotting and ELISA, and the fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA).@*RESULTS@#Five to nine days after infiltration with an inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated leaves was significantly higher than that in leaves ( < 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles.@*CONCLUSIONS@#Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.


Subject(s)
Humans , Plant Leaves , Plants, Genetically Modified , Plasminogen , Plasminogen Activators , Metabolism , Recombinant Proteins , Tobacco
16.
Chinese Journal of Biotechnology ; (12): 1511-1519, 2019.
Article in Chinese | WPRIM | ID: wpr-771778

ABSTRACT

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.


Subject(s)
Animals , Bacillus thuringiensis , Bacterial Proteins , Cryptochromes , Metabolism , Endotoxins , Hemolysin Proteins , Moths , Pest Control, Biological , Plants, Genetically Modified
17.
Chinese Journal of Biotechnology ; (12): 1676-1685, 2019.
Article in Chinese | WPRIM | ID: wpr-771763

ABSTRACT

Drought stress affects the growth and development of rice, resulting in severe loss in yield and quality. Ectopic expression of the bacterial RNA chaperone, cold shock protein (Csp), can improve rice drought tolerance. Archaeal TRAM (TRM2 and MiaB) proteins have similar structure and biochemical functions as bacterial Csp. Moreover, DNA replication, transcription and translation of archaea are more similar to those in eukaryotes. To test if archaeal RNA chaperones could confer plant drought tolerance, we selected two TRAM proteins, Mpsy_3066 and Mpsy_0643, from a cold-adaptive methanogenic archaea Methanolobus psychrophilus R15 to study. We overexpressed the TRAM proteins in rice and performed drought treatment at seedling and adult stage. The results showed that overexpression both TRAM proteins could significantly improve the tolerance of rice to drought stress. We further demonstrated in rice protoplasts that the TRAMs could abolish misfolded RNA secondary structure and improve translation efficiency, which might explain how TRAMs improve drought tolerance transgenic rice. Our work supports that ectopic expression of archaeal TRAMs effectively improve drought tolerance in rice.


Subject(s)
Droughts , Ectopic Gene Expression , Gene Expression Regulation, Plant , Oryza , Plant Proteins , Plants, Genetically Modified , Stress, Physiological
18.
Article in Chinese | WPRIM | ID: wpr-777439

ABSTRACT

DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.


Subject(s)
Abscisic Acid , Acetates , Cloning, Molecular , Cold Temperature , Cyclopentanes , Dendrobium , Genetics , Gene Expression Regulation, Plant , Oxylipins , Plant Proteins , Genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Response Elements , Stress, Physiological , Tobacco
19.
Chinese Journal of Biotechnology ; (12): 327-336, 2019.
Article in Chinese | WPRIM | ID: wpr-771373

ABSTRACT

Nowadays, available phosphorus (P) deficiency in soil and weed resistance to herbicides have emerged as two severe limiting factors for sustainable agriculture. Therefore, it is of urgent needs to improve plant absorption/utilization ability of the soil P, seek phosphate (Pi)-alternative P fertilizers, and develop new forms of weed control systems. Phosphite (Phi), as a P resource of relatively high amount only less than Pi in Earth, can be converted to utilizable Pi uniquely in some bacterial species by oxidization via its specific dehydrogenase (PTDH), but inhibits plant growth and development. This implies that Phi might rather become a suitable P fertilizer for plants if introducing a PTDH detoxifier from bacteria. Herein, we created the transgenic tobaccos harboring a Pseudomonas PTDH gene (PsPtx) amplified from the soil metagenome previously. RT-PCR showed that the exotic PsPtx gene could express similarly in root, stem and leaf tissues of all transgenic lines. PsPtx transgenic tobaccos could utilize Phi by oxidization as the sole Pi supply, and also outperformed wild-type tobacco with a remarkably dominant growth under Phi stress conditions. Moreover, the PsPtx gene was preliminarily evaluated with a notable quality as a potential candidate of the selection marker in plant genetic transformation. Conclusively, PsPtx and its encoded phosphite dehydrogenase might be applicable for developing a dual system of plant phosphorus utilization and weed control using Phi as P fertilizer and herbicide, and provide an effectual solution to some obstacles in the current crop transgenic studies.


Subject(s)
Oxidoreductases , Phosphites , Phosphorus , Plants, Genetically Modified , Weed Control
20.
Biol. Res ; 52: 14, 2019. graf
Article in English | LILACS | ID: biblio-1011416

ABSTRACT

BACKGROUND: Drought is a major abiotic stress affecting global wheat (Triticum aestivum L.) production. Exploration of drought-tolerant genes is essential for the genetic improvement of drought tolerance in wheat. Previous studies have shown that some histone encoding genes are involved in plant drought tolerance. However, whether the H2B family genes are involved in drought stress response remains unclear. METHODS: Here, we identified a wheat histone H2B family gene, TaH2B-7D, which was significantly up-regulated under drought stress conditions. Virus-induced gene silencing (VIGS) technology was used to further verify the function of TaH2B-7D in wheat drought tolerance. The phenotypic and physiological changes were examined in the TaH2B-7D knock-down plants. RESULTS: In the TaH2B-7D knock-down plants, relative electrolyte leakage rate and malonaldehyde (MDA) content significantly increased, while relative water content (RWC) and proline content significantly decreased compared with those in the non-knocked-down plants under drought stress conditions. TaH2B-7D knock-down plants exhibited severe sagging, wilting and dwarf phenotypes under drought stress conditions, but not in the non-knocked-down plants, suggesting that the former were more sensitive to drought stress. CONCLUSION: These results indicate that TaH2B-7D potentially plays a vital role in conferring drought tolerance in wheat.


Subject(s)
Plant Proteins/genetics , Stress, Physiological/genetics , Triticum/genetics , Gene Expression Regulation, Plant/genetics , Gene Silencing , Droughts , Phenotype , Plant Proteins/metabolism , Stress, Physiological/physiology , Triticum/metabolism , Plants, Genetically Modified/genetics , Plant Physiological Phenomena/genetics , Real-Time Polymerase Chain Reaction
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