Expression of modified Cry1Ac gene of Bacillus thuringiensis in transgenic tobacco plants. 2004

Lucyna Honorata Misztal, and Adrianna Mostowska, and Maria Skibinska, and Joanna Bajsa, and Wojciech Grzegorz Musial, and Artur Jarmolowski
Adam Mickiewicz University, Department of Biotechnology, 5 Miedzychodzka Street, 60-371 Poznan, Poland. lmiszatal@amu.edu.pl

Several mutations were introduced into the Cry1Ac toxin gene, resulting in four variants with altered sequences that were responsible for low expression of the toxin in transgenic plants. These variants were as follows: V1, with modified three A/T-rich regions, including the first signal of transcription termination; V2, with modified five putative polyadenylation signals (polyadenylation signals PAS) and the second signal of transcription termination; V3, with four initial AUUUA motifs; V4, with modification of six PASs, four AUUUA motifs, as well as the first and the second signals of transcription termination. The modified variants and the initial WT gene were cloned into the binary vector pBI121 and introduced into tobacco plants (Nicotiana tabacum) by Agrobacterium tumefaciens-mediated transformation. The presence of transgenes in the tobacco plants was confirmed by the polymerase chain reaction (PCR) method. The expression of particular variants of the Cry1Ac gene in tobacco was assayed using Western blotting with antibodies against the domain II of the Cry1Ac toxin. The average expression of WT was estimated to be 0.0025% of soluble proteins, and the expression levels of modified variants were 0.004%, 0.0098%, 0.0125%, and 0.0043% for V1, V2, V3, and V4, respectively. In this article we described the construction of a variant of the Cry1Ac gene (V3) with 12 point mutations leading to an average level of expression in transgenic plants five times higher than that observed in the case of the WT gene. Our results have shown for the first time that the modification of AUUUA sequences has a significant effect on the expression of the Cry1Ac gene in transgenic plants.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D004731 Endotoxins Toxins closely associated with the living cytoplasm or cell wall of certain microorganisms, which do not readily diffuse into the culture medium, but are released upon lysis of the cells. Endotoxin
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D006460 Hemolysin Proteins Proteins from BACTERIA and FUNGI that are soluble enough to be secreted to target ERYTHROCYTES and insert into the membrane to form beta-barrel pores. Biosynthesis may be regulated by HEMOLYSIN FACTORS. Hemolysin,Hemolysins,Hemalysins,Proteins, Hemolysin
D000083722 Bacillus thuringiensis Toxins Endotoxins produced by BACILLUS THURINGIENSIS used in transgenic plants and insecticides. When eaten by a susceptible insect they are protease activated in the insect midgut resulting in death from bacterial septicemia. B thuringiensis Toxins,B. thuringiensis Toxins,Bt Toxin,Bt Toxins,Toxin, Bt,Toxins, B thuringiensis,Toxins, B. thuringiensis
D001413 Bacillus thuringiensis A species of gram-positive bacteria which may be pathogenic for certain insects. It is used for the biological control of the Gypsy moth. Bacilan,Dipel,Thuricide
D001426 Bacterial Proteins Proteins found in any species of bacterium. Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
D001427 Bacterial Toxins Toxic substances formed in or elaborated by bacteria; they are usually proteins with high molecular weight and antigenicity; some are used as antibiotics and some to skin test for the presence of or susceptibility to certain diseases. Bacterial Toxin,Toxins, Bacterial,Toxin, Bacterial

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