RESUMO
Chromosome microdissection followed by microcloning is an efficient tool combining cytogenetics and molecular genetics that can be used for the construction of the high density molecular marker linkage map and fine physical map, the generation of probes for chromosome painting, and the localization and cloning of important genes. Here, we describe a modified technique to microdissect a single chromosome, paint individual chromosomes, and construct single-chromosome DNA libraries.
Assuntos
Cromossomos de Plantas , Clonagem Molecular/métodos , Biblioteca Gênica , Microdissecção/métodos , Secale/genética , Coloração Cromossômica/métodos , Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase/métodosRESUMO
∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants.
Assuntos
Evolução Molecular , Genes de Plantas , Oxirredutases/genética , Plantas/enzimologia , Plantas/genética , Alumínio/toxicidade , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Oxirredutases/metabolismo , Filogenia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transformação Genética/efeitos dos fármacosRESUMO
Δ8-sphingolipid desaturase and Δ6-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ6-fatty acid desaturase is derived from Δ8-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ6-fatty acid desaturase and Δ8-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ6-fatty acid desaturase and a Δ8-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ6- and Δ8-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114-174, 206-257, and 258-276 played important roles in Δ6-substrate recognition, and the last two regions were crucial for Δ8-substrate recognition; and (3) amino acid residues 114-276 of Δ6-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ8-sphingolipid desaturase) and acyl-PC (a substrate of Δ6-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ6-desaturase activity in the fusion protein but a loss in Δ8-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ6-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Oxirredutases/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Ácidos Graxos Dessaturases/genética , Modelos Moleculares , Mutagênese , Oxirredutases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Defensins are small cationic peptides that could be used as the potential substitute for antibiotics. However, there is no efficient method for producing defensins. In this study, we developed a new strategy to produce defensin in nitrate reductase (NR)-deficient C. ellipsoidea (nrm-4). We constructed a plant expression vector carrying mutated NP-1 gene (mNP-1), a mature α-defensin NP-1 gene from rabbit with an additional initiator codon in the 5'-terminus, in which the selection markers were NptII and NR genes. We transferred mNP-1 into nrm-4 using electroporation and obtained many transgenic lines with high efficiency under selection chemicals G418 and NaNO(3). The mNP-1 was characterized using N-terminal sequencing after being isolated from transgenic lines. Excitingly, mNP-1 was produced at high levels (approximately 11.42 mg/l) even after 15 generations of continuous fermentation. In addition, mNP-1 had strong activity against Escherichia coli at 5 µg/ml. This research developed a new method for producing defensins using genetic engineering.
Assuntos
Antibacterianos/biossíntese , Chlorella/enzimologia , Chlorella/metabolismo , Mutação , Nitrato Redutase/deficiência , alfa-Defensinas/biossíntese , alfa-Defensinas/genética , Animais , Antibacterianos/farmacologia , Chlorella/genética , Códon de Iniciação/genética , Escherichia coli/efeitos dos fármacos , Fermentação , Vetores Genéticos/genética , Plantas Geneticamente Modificadas , Coelhos , Transformação Genética , alfa-Defensinas/farmacologiaRESUMO
Δ6-fatty acid desaturase is an important enzyme in the catalytic synthesis of polyunsaturated fatty acids. Using domain swapping and a site-directed mutagenesis strategy, we found that the region of the C-terminal 67 amino acid residues of Δ6-fatty acid desaturase RnD6C from blackcurrant was essential for its catalytic activity and that seven different residues between RnD6C and RnD8A in that region were involved in the desaturase activity. Compared with RnD6C, the activity of the following mutations, V394A, K395I, F411L, S436P, VK3945AI and IS4356VP, was significantly decreased, whereas the activity of I417T was significantly increased. The amino acids N, T and Y in the last four residues also play a certain role in the desaturase activity.
Assuntos
Linoleoil-CoA Desaturase/química , Proteínas de Plantas/química , Ribes/enzimologia , Sequência de Aminoácidos , Linoleoil-CoA Desaturase/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas de Plantas/genética , Estrutura Terciária de ProteínaRESUMO
Δ(8)-Sphingolipid desaturase is the key enzyme that catalyses desaturation at the C8 position of the long-chain base of sphingolipids in higher plants. There have been no previous studies on the genes encoding Δ(8)-sphingolipid desaturases in Brassica rapa. In this study, four genes encoding Δ(8)-sphingolipid desaturases from B. rapa were isolated and characterised. Phylogenetic analyses indicated that these genes could be divided into two groups: BrD8A, BrD8C and BrD8D in group I, and BrD8B in group II. The two groups of genes diverged before the separation of Arabidopsis and Brassica. Though the four genes shared a high sequence similarity, and their coding desaturases all located in endoplasmic reticulum, they exhibited distinct expression patterns. Heterologous expression in Saccharomyces cerevisiae revealed that BrD8A/B/C/D were functionally diverse Δ(8)-sphingolipid desaturases that catalyse different ratios of the two products 8(Z)- and 8(E)-C18-phytosphingenine. The aluminium tolerance of transgenic yeasts expressing BrD8A/B/C/D was enhanced compared with that of control cells. Expression of BrD8A in Arabidopsis changed the ratio of 8(Z):8(E)-C18-phytosphingenine in transgenic plants. The information reported here provides new insights into the biochemical functional diversity and evolutionary relationship of Δ(8)-sphingolipid desaturase in plants and lays a foundation for further investigation of the mechanism of 8(Z)- and 8(E)-C18-phytosphingenine biosynthesis.
Assuntos
Brassica rapa/enzimologia , Brassica rapa/genética , Oxirredutases/genética , Alumínio/toxicidade , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Biocatálise , Evolução Molecular , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Espaço Intracelular/enzimologia , Dados de Sequência Molecular , Oxirredutases/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Transporte Proteico , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Nicotiana/citologia , Nicotiana/genéticaRESUMO
We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose- and time-dependent reduction in viability, and the agonist-treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca(2+) levels, resulting in Ca(2+) uptake by the mitochondria and subsequent H(2) O(2) accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, essential for the activation of caspase-like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca(2+) concentration but plays a negligible role in eATP-stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.
Assuntos
Trifosfato de Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Populus/efeitos dos fármacos , Populus/fisiologia , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Cálcio/análise , Sobrevivência Celular , Células Cultivadas , Citocromos c/metabolismo , Escuridão , Espaço Extracelular/metabolismo , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Luz , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Óxido Nítrico/metabolismo , Permeabilidade/efeitos dos fármacos , Brotos de Planta , Populus/efeitos da radiação , Populus/ultraestrutura , Receptores Purinérgicos/metabolismo , Suramina/farmacologiaRESUMO
Sodium/proton exchangers (NHX antiporters) play important roles in plant responses to salt stress. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na(+)/H(+) transporting activity. In this study, CkNHX1 were isolated from Caragana korshinskii, a pea shrub with high tolerance to salt, drought, and cold stresses. Transcripts of CkNHX1 were detected predominantly in roots, and were significantly induced by NaCl stress in stems. Transgenic yeast and Arabidopsisthalianasos3-1 (Atsos3-1) mutant over-expressing CkNHX1 and its hydrophilic C terminus-truncated derivative, CkNHX1-ΔC, were generated and subjected to NaCl and LiCl stresses. Expression of CkNHX1 significantly enhanced the resistance to NaCl and LiCl stresses in yeast and Atsos3-1 mutant. Whereas, compared with expression of CkNHX1, the expression of CkNHX1-ΔC had much less effect on NaCl tolerance in Atsos3-1 and LiCl tolerance in yeast and Atsos3-1. All together, these results suggest that the predominant expression of CkNHX1 in roots might contribute to keep C. korshinskii adapting to the high salt condition in this plant's living environment; CkNHX1 could recover the phenotype of Atsos3-1 mutant; and the hydrophilic C-terminal region of CkNHX1 should be required for Na(+)/H(+) and Li(+)/H(+) exchanging activity of CkNHX1.
Assuntos
Caragana/fisiologia , Cloreto de Lítio/metabolismo , Tolerância ao Sal/genética , Cloreto de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/biossíntese , Estresse Fisiológico/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Caragana/genética , Interações Hidrofóbicas e Hidrofílicas , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
In order to identify amino acid residues crucial for the enzymatic activity of Δ(8)-sphingolipid desaturases, a sequence comparison was performed among Δ(8)-sphingolipid desaturases and Δ(6)-fatty acid desaturases from various plants. In addition to the known conserved cytb(5) (cytochrome b(5)) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ(8)-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ(8)-sphingolipid desaturase activity and aids in our understanding of the structure-function relationships of the membrane-bound desaturases.
Assuntos
Aminoácidos Essenciais/química , Brassica rapa/enzimologia , Oxirredutases/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Aminoácidos Essenciais/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredutases/classificação , Oxirredutases/genética , Linhagem , Proteínas de Plantas/genética , Conformação ProteicaRESUMO
Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric acetyl-CoA carboxylase (ACCase) that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin carboxyl carrier protein, and CO2 to form carboxybiotin carboxyl carrier protein. In this study, we cloned four genes encoding BC from Brassica napus L. (namely BnaC.BC.a, BnaC.BC.b, BnaA.BC.a, and BnaA.BC.b), and two were cloned from each of the two parental species Brassica rapa L. (BraA.BC.a and BraA.BC.b) and Brassica oleracea L. (BolC.BC.a and BolC.BC.b). Sequence analyses revealed that in B. napus the genes BnaC.BC.a and BnaC.BC.b were from the C genome of B. oleracea, whereas BnaA.BC.a and BnaA.BC.b were from the A genome of B. rapa. Comparative and cluster analysis indicated that these genes were divided into two major groups, BnaC.BC.a, BnaA.BC.a, BraA.BC.a, and BolC.BC.a in group-1 and BnaC.BC.b, BnaA.BC.b, BraA.BC.b, and BolC.BC.b in group-2. The divergence of group-1 and group-2 genes occurred in their common ancestor 13-17 million years ago (MYA), soon after the divergence of Arabidopsis and Brassica (15-20 MYA). This time of divergence is identical to the previously reported triplicated time of paralogous subgenomes of diploid Brassica species and the divergence date of group-1 and group-2 genes of α-carboxyltransferase, another subunit of heteromeric ACCase, in Brassica. Reverse transcription PCR revealed that the expression level of group-1 and group-2 genes varied in different organs, and the expression patterns of the two groups of genes were similar in different organs, except in flower. However, two paralogs of group-2 BC genes from B. napus could express differently in mature plants tested by generating BnaA.BC.b and BnaC.BC.b promoter-ß-glucuronidase (GUS) fusions. The amino acid sequences of proteins encoded by these genes were highly conserved, except the sequence encoding predicted plastid transit peptides. The plastid transit peptides on the BC precursors of Brassica (71-72 amino acid residues) were predicted based on AtBC protein, compared, and confirmed by fusion with green fluorescent protein. Our results will be helpful in elucidating the evolution and the regulation of ACCase in the genus Brassica.
Assuntos
Brassica napus/enzimologia , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Evolução Molecular , Genes de Plantas/genética , Filogenia , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Brassica napus/genética , Clonagem Molecular , Análise por Conglomerados , Biologia Computacional , Componentes do Gene , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Heteromeric acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme in fatty acid biosynthesis in dicots, is a multi-enzyme complex consisting of biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase (alpha-CT and beta-CT). In the present study, four genes encoding alpha-CT were cloned from Brassica napus, and two were cloned from each of the two parental species, B. rapa and B. oleracea. Comparative and cluster analyses indicated that these genes were divided into two major groups. The major divergence between group-1 and group-2 occurred in the second intron. Group-2 alpha-CT genes represented the ancestral form in the genus Brassica. The divergence of group-1 and group-2 genes occurred in their common ancestor 12.96-17.78 million years ago (MYA), soon after the divergence of Arabidopsis thaliana and Brassica (15-20 MYA). This time of divergence is identical to that reported for the paralogous subgenomes of diploid Brassica species (13-17 MYA). Real-time reverse transcription PCR revealed that the expression patterns of the two groups of genes were similar in different organs, except in leaves. To better understand the regulation and evolution of alpha-CT genes, promoter regions from two sets of orthologous gene copies from B. napus, B. rapa, and B. oleracea were cloned and compared. The function of the promoter of gene Bnalpha-CT-1-1 in group-1 and gene Bnalpha-CT-2-1 in group-2 was examined by assaying beta-glucuronidase activity in transgenic A. thaliana. Our results will be helpful in elucidating the evolution and regulation of ACCase in oilseed rape.
Assuntos
Acetil-CoA Carboxilase/genética , Brassica napus/genética , DNA de Plantas/genética , Proteínas de Plantas/genética , Região 5'-Flanqueadora/genética , Acetil-CoA Carboxilase/classificação , Acetil-CoA Carboxilase/metabolismo , Brassica napus/enzimologia , Brassica rapa/enzimologia , Brassica rapa/genética , Clonagem Molecular , DNA de Plantas/química , Evolução Molecular , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Gamma-linolenic acid (gamma-linolenic acid, GLA; C18:3 Delta(6, 9, 12)) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Delta(6)-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Delta(6)-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Delta(6)-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Delta(6)-desaturases that could use both alpha-linolenic acids (ALA; C18:3 Delta(9,12,15)) and linoleic acid (LA; C18:2 Delta(9,12)) precursors in vivo, whereas RnD8A/B were Delta(8)-sphingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Delta(6)-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Delta(6,9,12,15)) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Ribes/enzimologia , Sequência de Aminoácidos , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/genética , Cromatografia Gasosa-Espectrometria de Massas , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ribes/genética , Ribes/metabolismo , Homologia de Sequência de Aminoácidos , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Using confocal microscopy, X-ray microanalysis and the scanning ion-selective electrode technique, we investigated the signalling of H(2)O(2), cytosolic Ca(2+) ([Ca(2+)](cyt)) and the PM H(+)-coupled transport system in K(+)/Na(+) homeostasis control in NaCl-stressed calluses of Populus euphratica. An obvious Na(+)/H(+) antiport was seen in salinized cells; however, NaCl stress caused a net K(+) efflux, because of the salt-induced membrane depolarization. H(2)O(2) levels, regulated upwards by salinity, contributed to ionic homeostasis, because H(2)O(2) restrictions by DPI or DMTU caused enhanced K(+) efflux and decreased Na(+)/H(+) antiport activity. NaCl induced a net Ca(2+) influx and a subsequent rise of [Ca(2+)](cyt), which is involved in H(2)O(2)-mediated K(+)/Na(+) homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na(+)/H(+) antiport system, the NaCl-induced elevation of H(2)O(2) and [Ca(2+)](cyt) was correspondingly restricted, leading to a greater K(+) efflux and a more pronounced reduction in Na(+)/H(+) antiport activity. Results suggest that the PM H(+)-coupled transport system mediates H(+) translocation and triggers the stress signalling of H(2)O(2) and Ca(2+), which results in a K(+)/Na(+) homeostasis via mediations of K(+) channels and the Na(+)/H(+) antiport system in the PM of NaCl-stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Citosol/metabolismo , Homeostase , Peróxido de Hidrogênio/metabolismo , Populus/citologia , Estresse Fisiológico/efeitos dos fármacos , Amilorida/farmacologia , Transporte Biológico/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Citosol/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Populus/efeitos dos fármacos , Populus/metabolismo , Potássio/metabolismo , Prótons , Protoplastos/citologia , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Vanadatos/farmacologiaRESUMO
The ability of a plant to maintain an ionic homeostasis is crucial in plant salt tolerance. Direct evidence based on data from the non-invasive measurement of ion fluxes would not only offer new insight about the function of the transporter but also provide a whole plant approach for dissecting salt adaptation mechanisms. Here, we review some reports using the ion-selective microelectrodes to characterize the net ion fluxes of tissues or cells.
Assuntos
Homeostase , Plantas/metabolismo , Cloreto de Sódio/farmacologia , Eletrodos Seletivos de Íons , Microeletrodos , Raízes de Plantas/metabolismo , Plantas/efeitos dos fármacosRESUMO
The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world's food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus: high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.
Assuntos
Cloroplastos/genética , Animais , Expressão Gênica , Humanos , Transformação Genética , TransgenesRESUMO
To map important ESTs to specific chromosomes and (or) chromosomal regions is difficult in hexaploid wheat because of its large genome size and serious interference of homoeologous sequences. Large-scale EST sequencing and subsequent chromosome localization are both laborious and time-consuming. The wheat alien addition line TAi-27 contains a pair of chromosomes of Thinopyrum intermedium (Host) Barkworth & D.R. Dewey that carry the resistance gene against barley yellow dwarf virus. In this research, we developed a modified technique based on chromosome microdissection and hybridization-specific amplification to isolate expressed sequences from the alien chromosome of TAi-27 by hybridization between the DNA of the microdissected alien chromosome and cDNA of Th. intermedium infected by barley yellow dwarf virus. Twelve clones were selected, sequenced, and analyzed. Three of them were unknown genes without any hit in the GenBank database and the other nine were highly homologous with ESTs of wheat, barley, and (or) other plants in Gramineae induced by abiotic or biotic stress. The method used in this research to isolate expressed sequences from a specific chromosome has the following advantages: (i) the obtained expressed sequences are larger in size and have 3' end information and (ii) the operation is less complicated. It would be an efficient improved method for genomics and functional genomics research of polyploid plants, especially for EST development and mapping. The obtained expressed sequence data are also informative in understanding the resistance genes on the alien chromosome of TAi-27.
Assuntos
Cromossomos de Plantas , Regulação da Expressão Gênica , Triticum/genética , Triticum/virologia , Mapeamento Cromossômico , Cromossomos/ultraestrutura , Clonagem Molecular , DNA Complementar/metabolismo , DNA de Plantas/metabolismo , Etiquetas de Sequências Expressas , Genes de Plantas , Genoma de Planta , Luteovirus/genética , Modelos Genéticos , Oligonucleotídeos/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: To obtain important expressed sequence tags (ESTs) located on specific chromosomes is currently difficult. Construction of single-chromosome EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes. In this research we developed a method to rapidly isolate ESTs from chromosome 1R of rye by combining the techniques of chromosome microdissection with hybrid specific amplification (HSA). RESULTS: Chromosome 1R was isolated by a glass needle and digested with proteinase K (PK). The DNA of chromosome 1R was amplified by two rounds of PCR using a degenerated oligonucleotide 6-MW sequence with a Sau3AI digestion site as the primer. The PCR product was digested with Sau3AI and linked with adaptor HSA1, then hybridized with the Sau3AI digested cDNA with adaptor HSA2 of rye leaves with and without salicylic acid (SA) treatment, respectively. The hybridized DNA fragments were recovered by the HSA method and cloned into pMD18-T vector. The cloned inserts were released by PCR using the partial sequences in HSA1 and HSA2 as the primers and then sequenced. Of the 94 ESTs obtained and analyzed, 6 were known sequences located on rye chromosome 1R or on homologous group 1 chromosomes of wheat; all of them were highly homologous with ESTs of wheat, barley and/or other plants in Gramineae, some of which were induced by abiotic or biotic stresses. Isolated in this research were 22 ESTs with unknown functions, probably representing some new genes on rye chromosome 1R. CONCLUSION: We developed a new method to rapidly clone chromosome-specific ESTs from chromosome 1R of rye. The information reported here should be useful for cloning and investigating the new genes found on chromosome 1R.
Assuntos
Cromossomos de Plantas/genética , Etiquetas de Sequências Expressas , Secale/genética , DNA Complementar/genética , DNA de Plantas/genética , ImmunoblottingRESUMO
Yeast complementation experiments were carried out to define the possible function of PeNhaD1, a Na(+)/H(+) antiporter gene from Populus euphratica Oliv., a salt resistant tree. PeNhaD1 was introduced to the Saccharomyces cerevisiae mutant strain ANT3 (Deltaena1-4::HIS3 Deltanha1::LEU2), which lacks the plasma membrane Na(+)/H(+) antiporter gene ScNHA1 or GX1 (Deltanhx1::TRP1), which lacks tonoplast Na(+)/H(+) antiporter gene ScNHX1. Our results showed that PeNhaD1 rescued the normal growth of ANT3 in the presence of high salt (80 mmol/L NaCl on solid medium or 400 mmol/L in liquid medium, pH 6.0), but not that of GX1, suggesting that PeNhaD1 may play a role in salt tolerance of Populus euphratica by maintaining the capacity for salt exclusion under saline condition.
Assuntos
Proteínas de Plantas/fisiologia , Populus/genética , Cloreto de Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/fisiologia , Expressão Gênica/efeitos dos fármacos , Teste de Complementação Genética , Mutação , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The technique of chromosome microdissection and microcloning has been developed for more than 20 years. As a bridge between cytogenetics and molecular genetics, it leads to a number of applications: chromosome painting probe isolation, genetic linkage map and physical map construction, and expressed sequence tags generation. During those 20 years, this technique has not only been benefited from other technological advances but also cross-fertilized with other techniques. Today, it becomes a practicality with extensive uses. The purpose of this article is to review the development of this technique and its application in the field of genomic research. Moreover, a new method of generating ESTs of specific chromosomes developed by our lab is introduced. By using this method, the technique of chromosome microdissection and microcloning would be more valuable in the advancement of genomic research.
RESUMO
The Bacillus subtilis Sac B gene with the vacuolar targeting signal sequence driven by 35S promotor was transferred into Lespedeza thunbergii by Agrobacterium mediated method. Total 62 Kan-resistant plants were obtained, of which 5 plants were proved to be transgenic plants. The transgenic plants were characterized by PCR amplification, PCR-Southern hybridization and RT-PCR. The physiological assay results showed that the transgenic plants were more tolerant to stress than the controls under the condition of 200mmol/L NaCl and 5% PEG, respectively, and that the content of soluble sugar in trnsgenic plants was significantly higher than that of controls in the period of tests (5-15 days) under salt and PEG stress.