| Products/Services Used | Details | Operation |
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| Peptide Synthesis | A quantitative indirect ELISA was performed in microplates using anti-Cry10Aa polyclonal antibody produced in rabbit by GenScript© (New Jersey, USA) from a synthetic peptide (VSSDSKIVKGPGHT), secondary goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody (1706515, Bio-Rad® Laboratories, Hercules, California, USA) and 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate (T8768, Sigma-Aldrich, ST. Louis, Missouri, USA). Protein crude extracts were obtained from fresh and young leaves, flower buds, or tissue-specific from flower buds according to Ribeiro et al. (2017). Total protein samples were previously quantified using Bradford reagent (BioRad® Laboratories, Hercules, California, USA) according to Bradford (1976). Recombinant Cry10Aa protein expressed and purified from the E. coli BL21(DE3) strain was used to obtain a standard curve ranging from 0 to 300 µg of Cry10Aa protein. Protein extracts from non-transgenic plants were used as a negative control. ELISA assays were carried out in biological and technical triplicate. The absorbance of the developed color was measured at 450 nm. The standard curve was used to estimate the protein concentration in cotton tissues. In addition, Cry10Aa protein accumulation in flower buds was also evaluated by Western blotassay according to Ribeiro et al. (2017). | Get A Quote |
The cotton boll weevil (CBW, Anthonomus grandis) is the most destructive cotton insect pest affecting cotton crops. To overcome this problem, CBW-resistant genetically modified cotton plants overexpressing Bacillus thuringiensisentomotoxins were successfully obtained. Previous results showed that the overexpression of Cry10Aa protoxin led to high mortality of the CBW larvae in greenhouse conditions. In this study, we advanced three more generations (T2 to T4), with several cotton events constitutively overexpressing the Cry10Aa protoxin, and the transgene stability and agronomic performance were investigated. In addition, stable transgenic cotton overexpressing the Cry10Aa active (Cry10Aa protoxin lacking... More
