Cloning and expression analysis of U6 promoters in Panax quinquefolius / 中国中药杂志

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

Interações populacionais mediadas por quorum sensing na microbiota do soro-fermento utilizado na produção do queijo Canastra / Population interactions mediated by quorum sensing in the microbiota of the endogenous culture used in the production of canasta cheese

Bactérias regulam a expressão de diversos fenótipos de acordo com a sua densidade populacional, em um comportamento conhecido como quorum sensing. Em micro-organismos de origem alimentar, o quorum sensing pode influenciar na formação de biofilmes, produção de toxinas e de enzimas hidrolíticas. Em bactérias Gram-negativas a sinalização é normalmente mediada por moléculas de N-acilhomoserina lactona (AHLs), conhecidas por autoindutor 1 (AI-1). Estudos revelam a inibição do quorum sensing nestas bactérias por enzimas que degradam as AHLS, em um processo denominado quorum quenching. Tipicamente brasileiro, o queijo Canastra é um produto artesanal maturado, produzido a partir de leite cru e do pingo, um tipo de soro-fermento coletado e utilizado diariamente na produção. A composição microbiana do pingo é diversificada e característica da região produtora. Essa combinação de bactérias, única em cada queijaria, resulta em aroma e textura típicos. Enquanto a microbiota Gram-positiva contribui para o desenvolvimento de sabor, textura e aroma no produto, bactérias Gram-negativas nesses queijos são geralmente associadas à formação de olhaduras, aromas desagradáveis, má coagulação da massa e até à patogenicidade. Este trabalho visou analisar a interação entre a microbiota Gram-positiva e Gram-negativa presente no pingo pela detecção dos sistemas de quorum sensing e quorum quenching nas amostras. A presença de AHLs foi avaliada em 45 amostras de pingo, a partir da extração em acetato de etila acidificado e da avaliação dos extratos por meio de bioensaios com Agrobacterium tumefaciens WCF47(pCF218)(pCF372) e KYC55(pJZ410)(pJZ372)(pJZ384), resultando em apenas uma amostra positiva. Em seguida, 350 isolados foram obtidos a partir de 11 amostras de pingo, sendo 200 isolados classificados como Gram-positivos e 150 Gram-negativos. Os Gramnegativos foram avaliados quanto à produção de AHLs in vitro através de ensaio em placa utilizando as estirpes biossensoras A. tumefaciens WCF47(pCF218)(pCF372), Chromobacterium violaceum CV026 e Escherichia coli pSB403, resultando em 39 isolados produtores de AHLs, provenientes de 10 pingos diferentes. Já os isolados Gram-positivos foram analisados quanto à capacidade de inibição do QS utilizando as estirpes biossensoras C. violaceum CV026 e A. tumefaciens WCF47(pCF218)(pCF372), em meio suplementado com C6-HSL ou 3-oxo-C12-HSL. Foi detectada a inibição total da resposta ao quórum por 78 isolados testados, enquanto a inibição parcial foi provocada por outros 63. A inibição do crescimento das estirpes biossensoras também foi observada para 24 isolados. Os isolados promotores de inibição parcial foram recultivados em meio mínimo com C6-HSL ou 3-oxo-C12-HSL como únicas fontes de carbono. Foram recuperados 28 isolados, e a ação desses sobre diferentes substratos foi avaliada, resultando em 22 isolados produtores de lactonases e 6 produtores de acilase. Os 39 isolados Gram-negativos e os 28 isolados Gram-positivos finais foram identificados por MALDI-TOF MS, resultando, segundo o conhecimento do autor, no primeiro relato de produção de AHLs por Pseudomonas fulva, Enterobacter xiangfangensis e Lelliottia amnigena, bem como a produção de lactonases por Staphylococcus xylosus e a produção de acilase por S. aureus, Microbacterium maritypicum e Rothia kristinae. Este trabalho mostrou que interações populacionais mediadas por quorum sensing dependente de AHLs na microbiota do soro-fermento são possíveis. Porém, essas interações estão propensas a serem inibidas por meio de lactonases e acilases produzidas por parte das bactérias Gram-positivas

Bacteria regulate the expression of different phenotypes according to their population density, in a behavior known as quorum sensing. In food-borne microorganisms, quorum sensing can influence the formation of biofilms, production of toxins and hydrolytic enzymes. In Gram-negative bacteria, signaling is normally mediated by Nacyl homoserine lactone molecules (AHLs), known as autoinducer 1 (AI-1). Studies reveal the inhibition of quorum sensing in these bacteria by enzymes that degrade AHLS, in a process called quorum quenching. Typically Brazilian, Canastra cheese is a matured artisanal product, produced from raw milk and pingo, a type of endogenous culture collected and used daily in production. The microbial composition of pingo is diverse and characteristic of the producing region. This combination of bacteria, unique in each cheese factory, results in a typical aroma and texture. While the Gram-positive microbiota contributes to the development of flavor, texture and aroma in the product, Gram-negative bacteria in these cheeses are generally associated with the formation of eyes, off-flavors, poor curd coagulation and even pathogenicity. Thus, this work aimed to analyze the interaction between the Gram-positive and Gram-negative microbiota present in this culture by detecting quorum sensing and quorum quenching systems in the samples. The presence of AHLs was evaluated in 45 samples of pingo, with extraction with acidified ethyl acetate and the evaluation of the extracts through bioassays with Agrobacterium tumefaciens WCF47(pCF218)(pCF372) and KYC55(pJZ410)(pJZ372)(pJZ384 ), resulting in only one positive sample. Then, 350 isolates were obtained from 11 endogenous culture samples, with 200 being classified as Gram-positive and 150 Gram-negative. Gram-negatives were evaluated for the production of AHLs in vitro by plaque assay using the biosensor strains A. tumefaciens WCF47(pCF218)(pCF372), Chromobacterium violaceum CV026 and Escherichia coli pSB403, resulting in 39 AHL-producing isolates from 10 different samples. Gram-positive isolates were analyzed for their ability to inhibit quorum sensing using biosensor strains C. violaceum CV026 and A. tumefaciens WCF47(pCF218)(pCF372), in medium supplemented with N-hexanoyl-L-homoserine lactone or 3-oxo-dodecanoyl-Lhomoserine lactone. Total inhibition of the quorum response was detected by 78 tested isolates, while partial inhibition was caused by 63. Growth inhibition of biosensor strains was also observed for 24 isolates. Partial inhibition promoter isolates were recultured on minimal medium with C6-HSL or 3-oxo-C12-HSL as sole carbon sources. Twenty-eight isolates were recovered, and the action of these isolates on different substrates was evaluated, resulting in 22 lactonase producers and 6 acylase producers. The 39 Gram-negative isolates and the final 28 Grampositive isolates were identified by MALDI-TOF MS, resulting, to the best of the author's knowledge, in the first report of AHL production by Pseudomonas fulva, Enterobacter xiangfangensis and Lelliottia amnigena, as well as the lactonase production by Staphylococcus xylosus and acylase production by S. aureus, Microbacterium maritypicum and Rothia kristinae. This work demonstrated that population interactions mediated by AHLs-dependent quorum sensing in Canastra cheese endogenous culture microbiota are possible. However, these interactions are prone to inhibition by lactonases and acylases produced by Gram-positive bacteria

Herbicide management in glyphosate and sulfonylurea-tolerant soybeans / Manejo de herbicidas em soja tolerante a glifosato e sulfonilureias

There is little information on the efficacy and selectivity of sulfonylureas, isolated and in association with glyphosate, in glyphosate and sulfonylurea-tolerant soybeans. Thus, the present study aims to evaluate the efficacy of weed control and selectivity of sulfonylureas, isolated and in association with glyphosate, at post-emergence (V4) of RR2/STS soybean. The experiments were conducted in the in areas located in Piracicaba City, São Paulo State (SP), Brazil (experiment I) and Palotina City, Paraná State (PR), Brazil (experiment II). Treatments were composed of application of the herbicides sulfometuron, chlorimuron, halosulfuron, ethoxysulfuron and glyphosate, isolated and in association, in the BMX Garra RR2/STS cultivar. Experiment I was conducted focusing on the evaluation of the efficacy of weed control; whereas experiment II focused mainly on the evaluation of herbicide selectivity. The experimental design was the randomized complete block, with four replications. Crop injury, weed control, and variables related to agronomic performance were evaluated. Data were submitted to analysis of variance, and the means of the treatments were compared with the Tukey test. Sulfonylureas in association with glyphosate were effective in weed control and selective for the BMX Garra RR2/STS soybean cultivar. The sulfometuron + chlorimuron + glyphosate association presented phytotoxic potential for the BMX Garra RR2/STS cultivar.(AU)

Há poucas informações sobre a eficácia e seletividade de sulfonilureias, isoladas e associadas ao glifosato, na soja tolerante ao glifosato e às sulfonilureias. Assim, o presente estudo teve como objetivo avaliar a eficácia no controle de plantas daninhas e seletividade de sulfonilureias, isoladas e em associação com o glifosato, em pós-emergência (V4) de soja RR2/STS. Os experimentos foram conduzidos em áreas localizadas nos municípios de Piracicaba, São Paulo (SP), Brasil (experimento I) e Palotina, Paraná (PR), Brasil (experimento II). Os tratamentos foram compostos pela aplicação dos herbicidas sulfometurom, clorimurom, halossulfurom, etoxissulfurom e glifosato, isolados e em associação, no cultivar BMX Garra RR2/STS. O experimento I foi realizado com o foco principal na avaliação da eficácia no controle de plantas daninhas; ao passo que o experimento II se concentrou principalmente na avaliação da seletividade dos herbicidas. O delineamento experimental foi o de blocos casualizados, com quatro repetições. Foram avaliados sintomas de injúria, controle de plantas daninhas e variáveis relacionadas ao desempenho agronômico. Os dados foram submetidos à análise de variância, e as médias dos tratamentos foram comparadas pelo teste de Tukey. Sulfonilureias associadas ao glifosato foram eficazes no controle de plantas daninhas e seletivas para o cultivar de soja BMX Garra RR2/STS. A associação sulfometurom + clorimurom + glifosato apresentou potencial fitotóxico para o cultivar BMX Garra RR2/STS.(AU)

Advances in Agrobacterium tumefaciens-mediated transgenic cucumber / 生物工程学报

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.

Construction and application of actin fluorescent marker in Verticillium dahliae Kleb / 生物工程学报

Actin filaments play an important role in fungal life processes such as growth, development and cytokinesis. The expression vector pSULPH-Lifeact-mCherry of fluorescent mCherry-labeled actin was transferred into Verticillium dahliae Kleb. wild type V592 by the genetic transformation system mediated by Agrobacterium tumefaciens to obtain the stable fluorescent labeled actin strain V592/Lifeact-mCherry. Then we detected its biological phenotype and the dynamic changes of actin fluorescence during the process of spore germination, mycelial growth and development. There was no significant difference in the colony morphology, colonial growth rate, sporulation and germination rate between the fluorescent labeled actin strain and the wild type. The actin fluorescence signal was observed at the tip of the conidia and hyphae and the septum clearly. Actin participated in the formation of the contractile actomyosin ring (CAR) during cytokinesis by observing the dynamic behavior of the actin in the process of hyphal septum formation. The fluorescent labeled actin strain can be used to study the dynamics of actin in fungal development to provide theoretical and practical support for further study of the mechanism of actin in fungal development and pathogenesis.

Agrobacterium-mediated transformation of the wild orchid Cattieya maxima Lindi / Transformación mediada por Agrobacterium de la orquídea silvestre Cattleya maxima Lindl / Transformação da orquídea silvestre Cattleya maxima Lindl mediada por Agrobacterium

Abstract Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80%) than untransformed controls (45 - 57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis.

Resumen Los protocormos son estructuras anatómicas únicas: son similares a los rizoides y se forman por vástagos jóvenes de orquídeas bajo condiciones fisiológicas. Los tejidos explantados de orquídeas producen estructuras llamadas Cuerpos Similares a Protocormos (PLBs) cuando están expuestos a condiciones apropiadas de crecimiento in vitro. Tanto la naturaleza propagativa de los PLBs como la facilidad con que se generan, hacen de estas estructuras una alternativa atractiva, frente a la mediada por semillas, para la producción de gran número de plantas en crecimiento. Para aumentar la embriogénesis somática y optimizar el procedimiento, se transformaron PLBs de Cattleya maxima usando el método de Agrobacterium tumefaciens. El T-DNA portaba un gen de resistencia a la Higromicina, un marcador visible (GFP5-GUSA) y un gen de arroz que codificaba para el receptor tipo quinasa de embriogénesis somática (SERK), considerado importante en la embriogénesis somática. Los PLBs tratados generaron embriones somáticos y desarrollaron plántulas resistentes a la Higromicina. La inserción del T-DNA se confirmó por PCR, y la expresión de GFP se observó usando un estereomicroscopio fluorescente. Los PLBs transformados de Cattleya maxima fueron más eficientes en desarrollar embriones somáticos (60-80%) que los controles no transformados (45-57%) y este contraste se maximizó en medio Murashige y Skoog (MS) libre de hormonas (80% de las plantas transformadas en comparación con 57% de las no transformadas). Estos hallazgos apoyan la noción de que SERK juega un papel importante en la embriogénesis de orquídeas.

Resumo Os protocormos são estruturas anatômicas únicas: são similares aos rizoides e se formam por hastes jovens de orquídeas sob condições fisiológicas. Os tecidos explantados de orquídeas produzem estruturas chamadas Corpos Similares a Protocormos (PLBs) quando estão expostos a condições apropriadas de crescimento in vitro. Tanto a natureza propagativa dos PLBs como a facilidade com que se generam, fazem com que estas estruturas sejam uma alternativa atrativa, comparativamente a mediada por sementes, para a produção de grandes números de plantas em crescimento. Para aumentar a embriogênesis somática e otimizar o procedimento, se transformaram PLBs de Cattleya maxima utilizando o método de Agrobacterium tumefaciens. O T-DNA carregava um gen de resistencia a Higromicina, um marcador visível (GFP5-GUSA) e um gen de arroz que codificava para o receptor tipo quinasa de embriogênesis somática (SERK), considerado importante na embriogênesis somática. Os PLBs tratados geraram embriões somáticos e desenvolveram plântulas resistentes a Higromicina. A inserção do T-DNA se confirmou por PCR, e a expressão de GFP se observou utilizando um estereomicroscópio de fluorescência. Os PLBs transformados de Cattleya maxima foram mais eficientes em desenvolver embriões somáticos (60-80%) que os controles não transformados (45-57%) e este contraste se potencializou em meio Murashige y Skoog (MS) livre de hormônios (80% das plantas transformadas em comparação com 57% das não-transformadas). Estes resultados apoiam a noção de que SERK desempenha um papel importante na embriogênesis de orquídeas.

Cloning and heterologous expression of a hydrophobin gene Ltr. hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis

Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.

Efficient regeneration and genetic transformation platform applicable to five Musa varieties

Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380­456, 310­372, 200­240, 130­156, and 100­130 well-developed shoots in only 240­270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.

Agrobacterium-Mediated Co-transformation of Multiple Genes in Metarhizium robertsii

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.

Expression of Hemagglutinin-Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum) plant by Agrobacterium-mediated transformation. Results: Putative transgenic plants were screened in a selection medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

Antitumor and anti-angiogenic potentials of isolated crude saponins and various fractions of Rumexhastatus D. Don

BACKGROUND: Cancer, being the foremost challenge of the modern era and the focus of world-class investigators, gargantuan research is in progress worldwide to explore novel therapeutic for its management. The exploitation of natural sources has been proven to be an excellent approach to treat or minify the excessive angiogenesis and proliferation of cells. Similarly, based the ethnomedicinal uses and literature survey, the current study is designed to explore the anti-tumor and anti-angiogenic potentials of Rumex hastatus. Anti-tumor and anti-angiogenic activities were carried out using potato-disc model and chorioallantoic membrane (CAM) assay respectively. Moreover, R. hastatus was also assessed for antibacterial activity against Agrobacterium tumefaciens (tumor causing bacterial strain). The positive controls used in anti-tumor, anti-angiogenic and antibacterial activities were vincristine sulphate, dexamethasone and cefotaxime respectively. RESULTS: The crude saponins (Rh.Sp), methanolic extract (Rh.Cr) and other solvent extracts like n-hexane (Rh.Hex), chloroform (Rh.Chf), ethylacetate (Rh.EtAc) and aqueous fraction (Rh.Aq) exhibited notable anti-tumor and anti-angiogenic activities. In potato tumor assay, the chloroform and saponin fractions were observed to be the most effective showing 86.7 and 93.3 % tumor inhibition at 1000 µg/ml with IC50 values 31.6 and 18.1 µg/ml respectively. Similarly, these two samples i.e., chloroform and saponins also excelled among the entire test samples in anti-angiogenic evaluation exhibiting 81.6 % (IC50 = 17.9 µg/ml) and 78.9 % (IC50 = 64.9 µg/ml) at 1000 µg/ml respectively. In contrast, the antibacterial investigations revealed a negligible potential against A. tumefaciens. CONCLUSION: Based on our results we can claim that R. hastatus possesses both anti-tumor and anti-angiogenic potentials. In all of the solvent fractions, Rh.Chf and Rh.Sp were most effective against tumor and angiogenesis while having negligible activity against A. tumefaciens. It can be concluded that Rh.Chf and Rh.Sp might be potential targets in the isolation of natural product having anti-neoplastic action.

Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.

Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato

Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding beta-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction.

Cloning and expression of TNF related apoptosis inducing ligand in Nicotiana tabacum

Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand [TRAIL] as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV [Cauliflower Mosaic Virus] helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay [Methylthiazol Tetrazolium Assay]. The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable

Construction of transgenic tobacco expressing tomato GGPS2 gene and analysis of its low light tolerance / 生物工程学报

To explore the influence of low light on the synthesis of carotenoids, chlorophyll and the adaptability of transgenic plants with tomato Solanum lycopersicon L. GGPS2 gene, we constructed a vector containing a GGPS2 gene with green fluorescent protein (GFP) as report gene under the control of a cauliflower mosaic virus 35S promoter and introduced it into tobacco Nicotiana tabacum L. cv. Wisconsin 38 by Agrobacterium tumefaciens-mediated transformation. PCR analysis of the DNA from kanamycin resistant tobacco indicated that the transgenic tobacco containing the nptII gene, SlaGGPS2 gene and without contamination of Agrobacterium. We also detected the root tip of kanamycin resistant tobacco showing characteristic fluorescence. The contents of carotenoid, chlorophyll and photosynthesis of transgenic tobacco increased in comparison with wild tobacco after low light treatment. In addition, leaf mass per unit area, total dry weight, ratio of root to shoot in transgenic tobacco were all higher than that of the wild tobacco, which proved that the transgenic tobacco could increase the accumulation of biomass and promote it transport to root. The transgenic tobacco with SlaGGPS2 gene can increase the contents of carotenoid, chlorophyll, enhance the photosynthetic rate, promote the biomass accumulation and its distribution to root. Hence, the transgenic tobacco with SlaGGPS2 gene had increased low light tolerance and the SlaGGPS2 gene maybe can be used in other crops.

Agrobacterium tumefaciens-mediated transformation of Aureobasidium pullulans and high-efficient screening for polymalic acid producing strain / 生物工程学报

To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.

Transient GUS gene expression in cassava (Manihot esculenta Crantz) using Agrobacterium tumefaciens leaf infiltration / Expresión transitoria del gen GUS en yuca (Manihot esculenta Crantz) utilizando infiltración con Agrobacterium tumefaciens

Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.

Objetivo. Evaluar la expresión transitoria del gen GUS en hojas de yuca (Manihot esculenta Crantz) por medio de infiltración con Agrobacterium tumefaciens. Materiales y métodos. Se utilizaron las cepas GV3101 y AGL1 de A. tumefaciens conteniendo el plásmido pCAMBIA1305.2, para evaluar la expresión transitoria del gen GUS. La infiltración de A. tumefaciens (agroinfiltración) se realizó tanto en hojas de plantas "in Vitro" como de plantas adultas de 1 mes. Las hojas se incubaron en tampón X-GLUC, se destiñeron y se fotografiaron para detectar la actividad de la enzima β-glucuronidasa (GUS). Resultados. Los ensayos de agroinfiltración en hoja muestraron la expresión transitoria del gen GUS en variedades cultivadas en la costa norte y en los llanos orientales, MCOL2215 (Venezolana) y CM6438-14 (Vergara) respectivamente, tanto en plantas "in Vitro" como en plantas adultas. La cepa hipervirulenta de A. tumefaciens AGL1 mostró una mayor eficiencia para la expresión transitoria en hojas de yuca. Conclusiones. Se recomienda utilizar la cepa AGL1 para evaluar la expresión transitoria de genes de interés por agroinfiltración en hojas de yuca.

Subcellular localization and resistance to Gibberella fujikuroi of AtELHYPRP2 in transgenic tobacco / 生物工程学报

The subcellular localization and the resistance to fungal pathogen Gibberella fujikuroi of the protein encoded by Arabidopsis AtELHYPRP2 (EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 2, AT4G12500) were investigated using transgenic tobacco plants. The coding sequence of AtELHYPRP2 was amplified from genomic DNA of Col-0 ecotype. After restriction digestion, the PCR fragment was ligated into pCAMBIA1302 to produce a fusion expression vector, pCAMBIA1302-AtELHYPRP2-GFP. Then the recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 and transgenic tobacco plants were regenerated and selected via leaf disc transformation method. RT-PCR and Western blotting analyses showed that AtELHYPRP2 expressed effectively in transgenic tobacco plants. Observation under laser confocal microscopy revealed that the green fluorescence of AtELHYPRP2-GFP fusion protein could overlap with the red fluorescence came from propidium iodide staining, indicating AtELHYPRP2 is localized to cell surface. Antimicrobial experiments exhibited that the constitutive expression of AtELHYPRP2 could enhance the resistance of tobacco to fungal pathogen G. fujikuroi and the infection sites could accumulate H2O2 obviously. The basal expression levels of PR1 and the systemic expression levels of PR1 and PR5 in transgenic tobacco plants were higher than that of the wild-type plants, suggesting AtELHYPRP2 may play a role in systemic acquired resistance.