Soybean GmGolS2-2 improves drought resistance of transgenic tobacco / 生物工程学报

Galactinol synthase (GolS) genes play important roles in plant response to abiotic stress. In this research, the plant expression vector of soybean GmGolS2-2 gene was constructed and transformed into tobacco to study the drought tolerance of transgenic tobacco. A GmGolS2-2 gene with 975 bp coding sequence was cloned from soybean leaves by reverse transcription-polymerase chain reaction (RT-PCR). GmGolS2-2 was linked to the plant expression vector pRI101 by restriction enzyme sites Nde Ⅰ and EcoR Ⅰ, and transformed into tobacco by leaf disc method. Genomic DNA PCR and real-time PCR showed that three GmGolS2-2 transgenic tobacco plants were obtained. The growth status of GmGolS2-2 transgenic tobacco under drought stress was better than that of wild-type tobacco. After drought stress treatment, the electrolyte leakage and malondialdehyde content of transgenic tobacco were lower than those of wild-type tobacco, but the proline content and soluble sugar content were higher than those of wild-type tobacco. The results of real-time PCR showed that the heterologous expression of GmGolS2-2 increased the expression of stress-related genes NtERD10C and NtAQP1 in transgenic tobacco. The above results indicated that GmGolS2-2 improved drought resistance of transgenic tobacco.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Abstract Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one's (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Resumo A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaram entre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

Evaluation of the Chilli veinal mottle virus CP gene expressing transgenic Nicotiana benthamiana plants for disease resistance against the virus / Avaliação do gene CP do vírus Chilli veinal mottle que expressa plantas transgênicas Nicotiana benthamiana quanto à resistência a doenças contra o vírus

Vegetables are an important source of income and high-value crops for small farmers. Chilli (Capsicum spp.) is one of the most economically important vegetables of Pakistan and it is grown throughout the country. It is a rich source of nutrition especially vitamins A, B, C and E along with minerals as folic acid, manganese (Mn), potassium (K) and molybdenum (Mo). Chilli possesses seven times more amount of vitamin C than an orange. Vitamin A, C and beta carotenoids are strong antioxidants to scavenge the free radicals. Chilli production is restricted due to various biotic factors. Among these viruses, Chilli veinal mottle virus (ChiVMV) is one of the most destructive and menacing agents that inflicts heavy and colossal losses that accounted for 50% yield loss both in quality and quantity. Pathogen-Derived Resistance (PDR) approach is considered one of the effective approaches to manage plant viruses. In this study, ChiVMV was characterized on a molecular level, the coat protein (CP) gene of the virus was stably transformed into Nicotiana benthamiana plants using Agrobacterium tumefaciens. The transgenic plants were challenged with the virus to evaluate the level of resistance of plants against the virus. It was observed that the plants expressing CP gene have partial resistance against the virus in terms of symptoms' development and virus accumulation. Translation of this technique into elite chilli varieties will be resulted to mitigate the ChiVMV in the crop as well as an economic benefit to the farmers.

Vegetais são uma importante fonte de renda e culturas de alto valor para os pequenos agricultores. A pimenta-malagueta (Capsicum spp.) é uma das hortaliças mais importantes economicamente do Paquistão e é cultivada em todo o país. É uma rica fonte de nutrição, especialmente vitaminas A, B, C e E com minerais como ácido fólico, manganês (Mn), potássio (K) e molibdênio (Mo). O pimentão possui sete vezes mais vitamina C do que a laranja. Vitaminas A e C e betacarotenoides são antioxidantes fortes para eliminar os radicais livres. A produção de pimenta é restrita devido a vários fatores bióticos. Entre esses vírus, o ChiVMV é o agente mais destrutivo e ameaçador que inflige perdas pesadas e colossais que representam 50% da perda de rendimento, tanto em qualidade quanto em quantidade. A abordagem de resistência derivada de patógenos (PDR) é considerada uma das abordagens eficazes para gerenciar os vírus de plantas. Neste estudo, ChiVMV foi caracterizado em nível molecular e o gene CP do vírus foi transformado de forma estável em plantas Nicotiana benthamiana usando Agrobacterium tumefaciens. As plantas transgênicas foram desafiadas com o vírus para avaliar seu nível de resistência contra o vírus. Observou-se que as plantas que expressam o gene CP apresentam resistência parcial ao vírus em termos de desenvolvimento de sintomas e acúmulo de vírus. A tradução dessa técnica em variedades de pimenta de elite resultará na mitigação do ChiVMV na safra, bem como em benefícios econômicos para os agricultores em termos de melhor rendimento e baixo custo de produção.

Evaluation of the Chilli veinal mottle virus CP gene expressing transgenic Nicotiana benthamiana plants for disease resistance against the virus / Avaliação do gene CP do vírus Chilli veinal mottle que expressa plantas transgênicas Nicotiana benthamiana quanto à resistência a doenças contra o vírus

Vegetables are an important source of income and high-value crops for small farmers. Chilli (Capsicum spp.) is one of the most economically important vegetables of Pakistan and it is grown throughout the country. It is a rich source of nutrition especially vitamins A, B, C and E along with minerals as folic acid, manganese (Mn), potassium (K) and molybdenum (Mo). Chilli possesses seven times more amount of vitamin C than an orange. Vitamin A, C and betacarotenoids are strong antioxidants to scavenge the free radicals. Chilli production is restricted due to various biotic factors. Among these viruses, Chilli veinal mottle virus (ChiVMV) is one of the most destructive and menacing agents that inflicts heavy and colossal losses that accounted for 50% yield loss both in quality and quantity. Pathogen-Derived Resistance (PDR) approach is considered one of the effective approaches to manage plant viruses. In this study, ChiVMV was characterized on a molecular level, the coat protein (CP) gene of the virus was stably transformed into Nicotiana benthamiana plants using Agrobacterium tumefaciens. The transgenic plants were challenged with the virus to evaluate the level of resistance of plants against the virus. It was observed that the plants expressing CP gene have partial resistance against the virus in terms of symptoms' development and virus accumulation. Translation of this technique into elite chilli varieties will be resulted to mitigate the ChiVMV in the crop as well as an economic benefit to the farmers.

Vegetais são uma importante fonte de renda e culturas de alto valor para os pequenos agricultores. A pimenta-malagueta (Capsicum spp.) é uma das hortaliças mais importantes economicamente do Paquistão e é cultivada em todo o país. É uma rica fonte de nutrição, especialmente vitaminas A, B, C e E com minerais como ácido fólico, manganês (Mn), potássio (K) e molibdênio (Mo). O pimentão possui sete vezes mais vitamina C do que a laranja. Vitaminas A e C e betacarotenoides são antioxidantes fortes para eliminar os radicais livres. A produção de pimenta é restrita devido a vários fatores bióticos. Entre esses vírus, o ChiVMV é o agente mais destrutivo e ameaçador que inflige perdas pesadas e colossais que representam 50% da perda de rendimento, tanto em qualidade quanto em quantidade. A abordagem de resistência derivada de patógenos (PDR) é considerada uma das abordagens eficazes para gerenciar os vírus de plantas. Neste estudo, ChiVMV foi caracterizado em nível molecular e o gene CP do vírus foi transformado de forma estável em plantas Nicotiana benthamiana usando Agrobacterium tumefaciens. As plantas transgênicas foram desafiadas com o vírus para avaliar seu nível de resistência contra o vírus. Observou-se que as plantas que expressam o gene CP apresentam resistência parcial ao vírus em termos de desenvolvimento de sintomas e acúmulo de vírus. A tradução dessa técnica em variedades de pimenta de elite resultará na mitigação do ChiVMV na safra, bem como em benefícios econômicos para os agricultores em termos de melhor rendimento e baixo custo de produção.

In silico cloning, expression and bioinformatics analysis of NtODB from common tobacco / 生物工程学报

It has been reported that ODB genes play an important role in homologous recombination-directed DNA repair, suggesting their potential applications in plant breeding. To analyze the expression characteristics of tobacco NtODB gene, the cDNA sequence of NtODB was obtained using in silico cloning technique. The physicochemical properties, signal peptide, and advanced structures of the predicted protein were analyzed using bioinformatics tools. The results showed that the NtODB gene has a 579-bp open reading frame which encodes a protein with 192 amino acid residues. The protein NtODB is predicted to be alkaline and hydrophilic. Real-time quantitative PCR showed that NtODB was constitutively expressed in different tissues. Subcellular localization showed that NtODB was mainly expressed in cell membrane and chloroplast. These results may help us to better understand and elucidate the roles of ODB genes in the homologous recombination-directed DNA repair.

JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas, influences plant development in transgenic tobacco

Background: Jatropha curcas L., as an important strategic biofuel resource with considerable economic potential, has attracted worldwide attention. However, J. curcas has yet to be domesticated. Plant height, an important agronomic trait of J. curcas, has not been sufficiently improved, and the genetic regulation of this trait in J. curcas is not fully understood. Zinc finger proteins (ZFPs), a class of transcription factors, have previously been shown to play critical roles in regulating multiple aspects of plant growth and development and may accordingly be implicated in the genetic regulation of plant height in J. curcas. Results: In this study, we cloned JcZFP8, a C2H2 ZFP gene in J. curcas. We found that the JcZFP8 protein was localized in the nucleus and contained a conserved QALGGH motif in its C2H2 structure. Furthermore, ectopic expression of JcZFP8 under the control of the 35S promoter in transgenic tobacco resulted in dwarf plants with malformed leaves. However, when JcZFP8 was knocked out, the transgenic tobacco did not show the dwarf phenotype. After treatment with the gibberellic acid (GA) biosynthesis inhibitor paclobutrazol (PAC), the dwarf phenotype was more severe than plants that did not receive the PAC treatment, whereas application of exogenous gibberellin3 (GA3) reduced the dwarf phenotype in transgenic plants. Conclusions: The results of this study indicate that JcZFP8 may play a role in J. curcas plant phenotype through GA-related pathways. Our findings may help us to understand the genetic regulation of plant development in J. curcas and to accelerate breeding progress through engineering of the GA metabolic pathway in this plant. How to cite: Shi X,Wu Y, Dai T, et al. JcZFP8, a C2H2 zinc-finger protein gene from Jatropha curcas, influences plant development in transgenic tobacco.

The antioxidative defense system is involved in the premature senescence in transgenic tobacco (Nicotiana tabacum NC89)

BACKGROUND: α-Farnesene is a volatile sesquiterpene synthesized by the plant mevalonate (MVA) pathway through the action of α-farnesene synthase. The α-farnesene synthase 1 (MdAFS1) gene was isolated from apple peel (var. white winterpearmain), and transformed into tobacco (Nicotiana tabacum NC89). The transgenic plants had faster stem elongation during vegetative growth and earlier flowering than wild type (WT). Our studies focused on the transgenic tobacco phenotype. RESULTS: The levels of chlorophyll and soluble protein decreased and a lower seed biomass and reduced net photosynthetic rate (Pn) in transgenic plants. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radicals (O2._) had higher levels in transgenics compared to controls. Transgenic plants also had enhanced sensitivity to oxidative stress. The transcriptome of 8-week-old plants was studied to detect molecular changes. Differentially expressed unigene analysis showed that ubiquitin-mediated proteolysis, cell growth, and death unigenes were upregulated. Unigenes related to photosynthesis, antioxidant activity, and nitrogen metabolism were downregulated. Combined with the expression analysis of senescence marker genes, these results indicate that senescence started in the leaves of the transgenic plants at the vegetative growth stage. CONCLUSIONS: The antioxidative defense system was compromised and the accumulation of reactive oxygen species (ROS) played an important role in the premature aging of transgenic plants.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

A choline monooxygenase gene promoter from Salicornia europaea increases expression of the -glucuronidase gene under abiotic stresses in tobacco (Nicotiana tabacum L.).

A 1312 bp 5' flanking region of Salicornia europaea choline monooxygenase (SeCMO) gene was isolated using the anchored PCR. To investigate the mechanism of regulation for this stress-induced gene, the SeCMO promoter--glucuronidase (GUS) chimeric gene constructs containing five deletions F1, F2, F3, F4 and F5 were introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. The functional properties of each promoter fragment were examined by assaying GUS activity in the leaves of transgenic tobacco treated with abiotic stresses (NaCl, PEG6000 and low temperature). The GUS activity in transgenic tobacco with F2 (-1056 to +8) construct showed highest increase under all the three abiotic stresses. Thus, the study provided a potential promoter induced by the salt, dehydration and cold for the plant genetic manipulation.

Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere / Diversidade genética de bactérias de rizosfera de tabaco produtoras de sideróforos

The genetic diversity of siderophore-producing bacteria of tobacco rhizosphere was studied by amplifiedribosomal DNA restriction analysis (ARDRA), 16S rRNA sequence homology and phylogenetics analysis methods. Studies demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium. A total of 28 ARDRA patterns were identified among the 299 siderophore-producing bacterial isolates.The 28 ARDRA patterns represented bacteria of 14 different genera belonging to six bacterial divisions, namely β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli, and Actinobacteria. Especially, γ- Proteobacteria consisting of Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia and Stenotrophomonas genus encountered 18 different ARDRA groups. Results also showed a greater siderophore-producing bacterial diversity than previous researches. For example, Sphingobacterium (isolates G-2-21-1 and G-2-27-2), Pseudomonas poae (isolate G-2-1-1), Enterobacter endosymbiont (isolates G-2-10-2 and N-5-10), Delftia acidovorans (isolate G-1-15), and Achromobacter xylosoxidans (isolates N-46-11HH and N-5-20) were reported to be able to produce siderophores under low-iron conditions for the first time. Gram-negative isolates were more frequently encountered, with more than 95% total frequency. For Gram-positive bacteria, the Bacillus and Rhodococcus were the only two genera, with 1.7% total frequency. Furthermore, the Pseudomonas and Enterobacter were dominant in this environment, with 44.5% and 24.7% total frequency, respectively. It was also found that 75 percent of the isolates that had the high percentages of siderophore units (% between 40 and 60) belonged to Pseudomonas. Pseudomonas sp. G-229-21 screened out in this study may have potential to apply to low-iron soil to prevent plant soil-borne fungal pathogen diseases.

A diversidade genética de bactérias de rizosfera de tabaco produtoras de sideróforos foi estudada por meio da técnica de análise de restrição do DNA ribossomal amplificado (ARDRA), homologia de seqüência de 16s rRNA e métodos de análise filogenética. Observou-se que 85% do total de 354 isolados produziram sideróforos em meio liquido com restrição de ferro.Entre os 299 isolados produtores de sideróforos identificou-se 28 padrões ARDRA, que representaram 14 gêneros bacterianos diferentes, pertencentes a seis divisões bacterianas: β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli e Actinobacteria. γ- Proteobacteria, consistindo de Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia e Stenotrophomonas, pertenceram a 18 grupos ARDRA. Os resultados também mostraram uma diversidade maior de bactérias produtoras de sideróforos doque a relatada em outros estudos. Por exemplo, Sphingobacterium (isolados G-2-21-1 e G-2-27-2), Pseudomonaspoae (isolado G-2-1-1), Enterobacter endosymbiont (isolados G-2-10-2 e N-5-10), Delftia acidovorans (isolado G-1-15) e Achromobacter xylosoxidans (isolados N-46-1HH e N-5-20), capazes de produzir sideróforos em condições de baixa disponibilidade de ferro, foram relatados pela primeira vez. Isolados Gram negativos foram encontrados com maior freqüência, correspondendo a mais de 95% da freqüência total. Entre as bactérias Gram positivas, foram encontrados apenas os gêneros Bacillus e Rhodococcus, com 1,7% da freqüência total. Além disso, neste ambiente houve predominância de Pseudomonas e Enterobacter, com 44,5% e 24,7% da freqüência total, respectivamente. Verificou-se também que 75% dos isolados com alta porcentagem de unidades de sideróforos (% entre 40 e 60) pertenceram a Pseudomonas. Pseudomonas sp. G- 229-21, selecionado neste estudo, apresenta potencial de aplicação em solos com baixo teor de ferro para prevenção dedoenças fúngicas em plantas.

A comparative analysis of green fluorescent protein and beta-glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters.

The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, beta-glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp ) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

Characterization of the nuclear ribosomal DNA unit in Oxalis tuberosa (Oxalidacea) and related species

Oxalis tuberosa is an octoploid Andean tuber crop called oca that belongs to the worldwide distributed genus Oxalis. The genus is very heterogeneous and its systematics is still problematic. It has been proposed that O. tuberosa evolved by polyploidization of a still not defined ancestor that belongs to an alliance of species sharing the same basic chromosome number (x = 8). Nuclear rDNA units of O. tuberosa and a selected group of four related diploid species were characterised by RFLP using different restriction endonucleases and southern hybridization probes to produce a restriction map for EcoRI and BamHI. The major rDNA unit length in O. tuberosa was estimated at 10.7 kbp. As expected, restriction site variation was observed mainly in the intergenic spacer (IGS), but was also detected in coding regions. Restriction site mapping organization of the transcribed rDNA unit of O. tuberosa is very similar to O. oblongiformis. Nucleotide sequencing of a region of O. peduncularis IGS generated a complex organization pattern of repeats and subrepeats. Diploid species O. peduncularis, O. tabaconasensis and O. aff. villosula exhibited a ladder pattern that is a consequence of a 170 bp subrepeat unit indicating that these species share organization similarity and sequence homology. The variation pattern provided information to compare among diploid species, although it did not help to clarify taxonomic relationships between O. tuberosa and the putative diploid ancestors analysed in this study. Nonetheless, the RFLP pattern exhibited by O. tuberosa for the IGS region was quite unique and will be a useful tool to prospect in other related species.

Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana.

Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum.

A RING zinc finger ankyrin protein gene,designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE.Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4- type RING finger domain was found at the C-terminal region of the AdZFP1 protein,and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869.Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root,stem and leaf of the plant.Semi-quantitative reverse- transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity,cold and heat to some extent.Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter.

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.

Molecular studies of E. coli mercuric reductase gene (merA) and its impact on human health.

Bacterial plasmids encode resistance systems for toxic metal ions including Hg2+ functioning by energy-dependent efflux of toxic ions. The inducible mercury resistance (mer) operon encodes both a mercuric ion uptake and a detoxification enzymes. In Gram-negative bacteria especially in E. coli, a periplasmic protein, MerP, an inner- membrane transport protein, MerT, and a cytoplasmic enzyme, mercuric reductase (the MerA protein), are responsible for the transport of mercuric ions into cell and their reduction to elemental mercury, Hg0. Phytoremediation involves the use of plants to extract, detoxify and/or sequester environmental pollutants from soil and water. Transgenic plants cleave mercury ions from methyl-mercury complexes; reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve less toxic elemental mercury. PCR were performed to detect 1695 bp of mercuric reductase gene (merA), which is mainly responsible for the conversion of mercuric (Hg+2) and mercurous (Hg+1) ions into non-toxic elemental mercury. PCR products of putative merA genes from environmental E. coli strains were purified and cloned into a plant expression vector pRT100. The construct will be transformed in calli of Nicotiana plants.

Overexpression of GbERF confers alteration of ethylene-responsive gene expression and enhanced resistance to Pseudomonas syringae in transgenic tobacco.

GbERF belongs to the ERF (ethylene responsive factor) family of transcription factors and regulates the GCC-box containing pathogen-related (PR) genes in the ethylene signal transduction pathway. To study the function of GbERF in the process of biotic stress, transgenic tobacco plants expressing GbERF were generated. Overexpression of GbERF did not change transgenic plant's phenotype and endogenous ethylene level. However, the expression profile of some ethylene-inducible GCC-box and non-GCC-box containing genes was altered, such as PR1b, PR2, PR3, PR4, Osmotin, CHN50, ACC oxidase and ACC synthase genes. These data indicate that the cotton GbERF could act as a transcriptional activator or repressor to regulate the differential expression of ethylene-inducible genes via GCC and non-GCC cis-elements. Moreover, the constitutive expression of GbERF in transgenic tobacco enhanced the plant's resistance to Pseudomonas syringae pv tabaci infection. In conclusion, GbERF mediates the expression of a wide array of PR and ethylene-responsive genes and plays an important role in the plant's response to biotic stress.

Expression of the crucifer-infecting TMV-Cg movement protein in tobacco plants complements in trans a TMV-U1 trafficking-deficient mutant

Tobamovirus movement proteins play a determinant role in the establishment of infections in plants, allowing the local movement of viral RNA genome through plasmodesmatas. We expressed the movement protein (MP) of the crucifer- and garlic-infecting Tobacco Mosaic Virus strain Cg (TMV-Cg) in both resistant Xanthi NN and sensitive Xanthi nn Nicotiana tabacum plants. MP-Cg function was assayed by inoculating transgenic plants with a trafficking-deficient mutant of TMV strain U1. Following infection, local necrotic lesions were developed in resistant transgenic plants, and a systemic infection was produced in sensitive tobaccos. Thus, movement function of the mutant virus was complemented in trans by MP-Cg expressed in transgenic plants, causing the same symptoms as wild-type strain. We demonstrated that the function of MP-U1 could be replaced efficiently by MP-Cg, even though these proteins share only 36% of identity. Similar hydrophobic patterns of MP-Cg and MP-U1 suggests structure and function conservations of both proteins. This work is an example of how two tobamoviruses differing in their host range help to understand viral movement mechanism during the infection.

Transgenic tobacco plants expressing BoRS1 gene from Brassica oleracea var. acephala show enhanced tolerance to water stress.

Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the Brassica oleracea var. acephala BoRS1 gene was transferred into tobacco through Agrobacterium-mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows that BoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.