RESUMO
Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration.
Assuntos
Agrobacterium tumefaciens/genética , Nicotiana/fisiologia , Transformação Genética , Zeatina/metabolismo , Agrobacterium tumefaciens/fisiologia , Arginina/análogos & derivados , Arginina/metabolismo , DNA Bacteriano , DNA de Plantas/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Folhas de Planta/genética , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Brotos de Planta/genética , Brotos de Planta/microbiologia , Brotos de Planta/fisiologia , Plantas/genética , Plantas/microbiologia , Plantas Geneticamente Modificadas , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Regeneração , Especificidade da Espécie , Nicotiana/genética , Nicotiana/microbiologia , Virulência , Zeatina/análiseRESUMO
The eIF4A gene family codes for proteins which unwind secondary structures of mRNA during translational initiation. The tobacco eIF4A-10 promoter is one of a few of constitutive promoters found in plants. Research was conducted to identify the proximal promoter elements and to evaluate the potential application of the promoter for regulating transgene expression in a range of crop plants. A large intron (892 bp) in the leader sequence was found to be dispensable for constitutive promoter activity and did not contribute to the overall performance of the promoter. Deletion analysis showed that the upstream region between -151 bp and -73bp relative to the transcriptional start site was essential for the high level of expression and the constitutive activity. The data indicated that the elements in this region may coordinate and compensate each other for the high levels of promoter expression. The downstream leader sequence also contained a strong quantitative enhancer element that was essential for the full activity of the eIF4A-10 promoter. The eIF-4A10 promoter was found to be active in a wide range of plant species and tissues indicating that it will be useful for the constitutive expression of transgenes in plants.
Assuntos
Fator de Iniciação 4A em Eucariotos/genética , Nicotiana/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Transformação Genética , Sequência de Bases , Clonagem Molecular/métodos , DNA de Plantas , Regulação da Expressão Gênica de Plantas , Íntrons , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Análise de Sequência de DNARESUMO
Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for ß-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12-15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species.