RESUMEN
The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3') was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer.
Asunto(s)
Arachis/genética , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas , Endotoxinas/genética , Regulación Bacteriana de la Expresión Génica , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/inmunología , Bioensayo , Southern Blotting , Células Cultivadas , Clonación Molecular , Endotoxinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Terapia Genética/métodos , Vectores Genéticos , Proteínas Hemolisinas , Higromicina B , Insectos , Mutagénesis Insercional , Proteínas de Plantas/inmunología , Plantas Modificadas Genéticamente , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Semillas/genética , Transformación GenéticaRESUMEN
Somatic embryos of jack, a Glycine max (L.) Merrill cultivar, were transformed using microprojectile bombardment with a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) driven by the 35S promoter and linked to the HPH gene. Approximately 10 g of tissue was bombarded, and three transgenic lines were selected on hygromycin-containing media and converted into plants. The recovered lines contained the HPH gene, but the Bt gene was lost in one line. The plasmid was rearranged in the second line, and the third line had two copies, one of which was rear-ranged. The CryIAc protein accumulated up to 46 ng mg-1 extractable protein. In detached-leaf bioassays, plants with an intact copy of the Bt gene, and to a lesser extent those with the rearranged copy, were protected from damage from corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvetbean caterpillar (Anticarsia gemmatalis). Corn earworm produced less than 3% defoliation on transgenic plants, compared with 20% on the lepidopteran-resistant breeding line GatIR81-296, and more than 40% on susceptible cultivars. Unlike previous reports of soybean transformation using this technique, all plants were fertile. To our knowledge, this is the first report of a soybean transgenic for a highly expressed insecticidal gene.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas , Endotoxinas/biosíntesis , Genes Sintéticos , Glycine max/fisiología , Transformación Genética , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Bioensayo , Cartilla de ADN , Endotoxinas/genética , Endotoxinas/toxicidad , Genes Bacterianos , Proteínas Hemolisinas , Lepidópteros , Control Biológico de Vectores , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Glycine max/genéticaRESUMEN
A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas , Endotoxinas , Protoplastos/metabolismo , Solanum tuberosum/genética , Secuencia de Aminoácidos , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Bacteriano , Escherichia coli , Exones , Genes Sintéticos , Proteínas Hemolisinas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
The complete nucleotide sequence of the asd gene of Streptococcus mutans encoding aspartate beta-semialdehyde dehydrogenase (EC 1.2.1.11), an enzyme comprised of 357 amino acids, having an Mr of 38,897 and active in the biosynthetic pathway of lysine, threonine, methionine, diaminopimelic acid, and isoleucine, has been determined. In addition we report the 276 nucleotides upstream of the structural gene which contain a highly efficient promoter identified by both RNA polymerase binding and in vitro transcription analysis. A leader transcript which terminates at a fixed point immediately preceding the asd promoter region was identified in the DNA sequence and confirmed by in vitro transcription analysis as well. The close proximity of this transcript and its p-independent transcriptional terminator to the asd coding sequence suggests involvement in a mechanism of regulation. Message stability experiments indicate the half-life of asd specific messages to be comparable to that of Escherichia coli messages. Conditions of varying concentrations of lysine, threonine, and methionine exert no apparent control over expression of the S. mutans asd gene in Escherichia coli suggesting the requirement of an accessory regulatory element specific for the S. mutans asd gene.
Asunto(s)
Aspartato-Semialdehído Deshidrogenasa/genética , Genes Bacterianos , Genes , Regiones Promotoras Genéticas , Streptococcus mutans/genética , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Bacteriano , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Lisina/farmacología , Metionina/farmacología , Hibridación de Ácido Nucleico , Plásmidos , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Treonina/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Transformation (i.e., DNase-sensitive genetic transfer) of strains of Streptococcus mutans representing serotypes c and e was accomplished by using chromosomal DNA from a Rifr Strr Spcr isolate of strain GS5 (UAB525) and a chimeric plasmid, pYA629. Shuttle plasmid pYA629 comprises the S. mutans plasmid pVA318, an inducible erythromycin resistance determinant originally isolated from a group A streptococcal strain, the tetracycline resistance gene and replication region of the Escherichia coli plasmid pBR322, and the promoter region of the S. mutans gene for aspartate beta-semialdehyde dehydrogenase. The strains examined for recipient ability included those known to lack a cryptic plasmid (GS5, UA130, UA159, and MT8148) and those known to contain a widely disseminated 5.8-kilobase cryptic plasmid (LM7, V318, UA101, UA174, and 3098791). The transformation frequencies in GS5 for GS5 chromosomal antibiotic resistance markers were comparable to those reported by others, but UA101, UA130, UA159 and UA174 were transformed with both chromosomal and plasmid markers at much higher efficiencies. In a larger strain survey, strains containing the 5.8-kilobase cryptic plasmid were more frequently transformable with both chromosomal and pYA629 DNAs than were strains lacking this cryptic plasmid. All plasmid-containing strains except LM7 lost their resident cryptic plasmids when transformed with pYA629. LM7 transformed with pYA629 retained pLM7. There are therefore at least two incompatibility groups among S. mutans cryptic plasmids. yPA629 DNA isolated from either E. coli or S. mutans transformed S. mutans with equal efficiency. pYA629 DNA isolated from S. mutans transformed both restriction-deficient and restriction-proficient E. coli recipients. Therefore, the strains of S. mutans used lack a restriction-modification system for pYA629 DNA sequences. S. mutans strains that are readily transformable, display maximal cariogenicity in gnotobiotic rats, and give high scores for in vitro measures of important virulence attributes have been identified to facilitate studies on the genetic basis and control of virulence.