Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

Cloning and characterization of the proximal promoter region of the mouse glutamate-L-cysteine ligase regulatory subunit gene.

We describe upregulation of the mRNA for the mouse glutamate-cysteine ligase regulatory subunit gene (Glcl-r) in Hepa-1 cells treated with beta-napthoflavone (BNF) and tert-butylhydroquinone (tBHQ). A 2-kb fragment of the proximal promoter region of the gene was cloned and sequenced, and sequence analysis reveals a high degree of homology when compared to the human glutamate-cysteine ligase regulatory subunit gene promoter. Primer extension analysis indicates a major transcription start site 218 bp upstream of the translation start codon in a CpG-rich region, suggesting that transcription is Sp1 mediated. Reporter constructs containing nested deletion fragments of the Glcl-r promoter demonstrate that regulatory elements sufficient for basal and tBHQ-inducible expression lie between -273 and -787 bp relative to the translation start codon and that the distal promoter may contain negative regulatory elements.

Molecular cloning and sequencing of the cDNA encoding mouse glutamate-cysteine ligase regulatory subunit.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify and clone the regulatory subunit of mouse glutamate-cysteine ligase (Glclr) using primers adapted from the published rat Glclr cDNA sequence, and from mouse genomic DNA. Amplified cDNA was cloned into a plasmid vector, and additional RT-PCR reactions coupled with 3' RACE were used to amplify and sequence 3' regions covered by the rat primer. Comparison of the mouse Glclr cDNA sequence and predicted protein sequence with that of rat Glclr and human GLCLR revealed extensive homology in cDNA and amino acid sequences among these species.

Probenicid inhibition of fluorescence extrusion after MCB-staining of rat-1 fibroblasts.

The intracellular fluorescence level of cells stained continuously with monochlorobimane was monitored by flow cytometry in order to assess the initial rate of glutatione to monochlorobimane conjugation as a measure of glutathione S-transferase activity. In addition to a rapid initial increase and a plateau level, a decline in fluorescence intensity was found upon prolonged flow cytometric monitoring. Exposure to probenicid, an inhibitor of an ATP-dependent organic anion pump, prevented this decrease. Incubation with vanadate and verapamil was without effect. Thus, extrusion of fluorescentglutathione-conjugate perturbs the proportionality between initial glutathione level and monochlorobimane-dependent fluorescence intensity. Monitoring by flow cytometry the decrease in monochlorobimane-dependent fluorescence may be useful to detect multidrug resistant cells.