Development of a sensitive monoclonal antibody-based sandwich ELISA to detect Vip3Aa in genetically modified crops.

OBJECTIVES: To develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to detect Vip3Aa in genetically modified (GM) crops and their products. RESULTS: Vegetative insecticidal proteins (Vips) are secreted by Bacillus thuringiensis (Bt) and are known to be toxic to Lepidoptera species. Vip3Aa family proteins, Vip3Aa19 and Vip3Aa20, were successfully applied in GM crops to confer an effective and persistent insecticidal resistance. A sensitive monoclonal antibody-based sandwich ELISA was developed to detect Vip3Aa in GM crops and their products. Two monoclonal antibodies were raised against the overexpressed and purified His-Vip3Aa20, were purified from mouse ascites and characterized. A sandwich ELISA method was developed using the 2G3-1D7 monoclonal antibody for capture and the biotin-labeled 1F9-1F5 monoclonal antibody for detection of Vip3Aa20. The linear detection range of the method was found to be approximately 31.25-500 pg/ml, with a sensitivity of 10.24 pg/ml. CONCLUSIONS: The established ELISA was effective for detecting Vip3Aa family proteins other than Vip3Aa8, and was successfully applied in the detection of Vip3Aa20 and Vip3Aa19 expressed in transgenic maize and cotton.

iTRAQ-based quantitative proteomic analysis of two transgenic soybean lines and the corresponding non-genetically modified isogenic variety.

To investigate the unintended effects of genetically modified (GM) crops, an isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic analysis was performed with seed cotyledons of two GM soybean lines, MON87705 and MON87701×MON89788, and the corresponding non-transgenic isogenic variety A3525. Thirty-five differentially abundant proteins (DAPs) were identified in MON87705/A3525, 27 of which were upregulated and 8 downregulated. Thirty-eight DAPs were identified from the MON87701×MON89788/A3525 sample, including 29 upregulated proteins and 9 downregulated proteins. Pathway analysis showed that most of these DAPs participate in protein processing in endoplasmic reticulum and in metabolic pathways. Protein-protein interaction analysis of these DAPs demonstrated that the main interacting proteins are associated with post-translational modification, protein turnover, chaperones and signal transduction mechanisms. Nevertheless, these DAPs were not identified as new unintended toxins or allergens and only showed changes in abundance. All these results suggest that the seed cotyledon proteomic profiles of the two GM soybean lines studied were not dramatically altered compared with that of their natural isogenic control.

Ruthenium-catalyzed remote C5-sulfonation of N-alkyl-8-aminoquinolines.

There is an increasing demand for the methodologies of positionally selective remote C-H functionalizations. We herein report the ruthenium-catalyzed remote C5-selective sulfonation of 8-aminoquinoline derivatives. Moderate to high yields were obtained with different substituted substrates. The catalytic system exhibited a high regio-selectivity, as well as good functional group tolerance. Mechanistic study supported a radical pathway. A bifunctional ruthenium-catalytic cycle is proposed.

Newly identified CSP41b gene localized in chloroplasts affects leaf color in rice.

A rice mutant with light-green leaves was discovered from a transgenic line of Oryza sativa. The mutant has reduced chlorophyll content and abnormal chloroplast morphology throughout its life cycle. Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation, here designated as lgl1. To isolate the lgl1 gene, a high-resolution physical map of the chromosomal region around the lgl1 gene was made using a mapping population consisting of 1984 mutant individuals. The lgl1 gene was mapped in the 76.5kb region between marker YG4 and marker YG5 on chromosome 12. Sequence analysis revealed that there was a 39bp deletion within the fourth exon of the candidate gene Os12g0420200 (TIGR locus Os12g23180) encoding a chloroplast stem-loop-binding protein of 41kDa b (CSP41b). The lgl1 mutation was rescued by transformation with the wild type CSP41b gene. Accordingly, the CSP41b gene is identified as the LGL1 gene. CSP41b was transcribed in various tissues and was mainly expressed in leaves. Expression of CSP41b-GFP fusion protein indicated that CSP41b is localized in chloroplasts. The expression levels of some key genes involved in chlorophyll biosynthesis and photosynthesis, such as ChlD, ChlI, Hema1, Ygl1, POR, Cab1R, Cab2R, PsaA, and rbcL, was significantly changed in the lgl1 mutant. Our results demonstrate that CSP41b is a novel gene required for normal leaf color and chloroplast morphology in rice.