T-DNA Insertion Mutants Of Arabidopsis Thaliana

After seven consecutive years of growth, the estimated global area of transgenic crops for 2003 was 67700000 ha. Though no undesirable and harmful effects to human health and environment has been observed during the commercial plantation of transgenic crops, the safety issue of transgenic crops is still extensively concerned. For transgenic plants, unanticipated effects may additionally arise from the process of introducing foreign genes or as a result of the effects of environmental factors/genetic background on the expression of the transgene(s). The potential occurrence of unanticipated alterations in the composition of genetically modified crops as a result of the genetic modification process is one of the key elements of the safety assessment procedure.Unintended effects may be identified by an analysis of the agronomical/morphological characteristics of the new plant and an extensive chemical analysis of key nutrients, anti-nutrients and toxicants typical for the plant. Limitations of this analytical, comparative approach are the possible occurrence of unknown toxicants and anti-nutrients, in particular in food plant species with no history of (safe) use; and the availability of adequate detection methods. Several new strategies have been developed to identify unintended alterations in the genetically modified crops that may occur as a result of the genetic modification process. These include comparative chemical analysis of single compounds in GM crops and their conventional non-GM counterparts, and profiling methods such as DNA/RNA microarray technologies, proteomics and metabolite profiling. Thereby, we examine the potential of proteomics to perform comparative analyses of protein patterns may be of importance for transgenic plants safety assessment.In order to search for unintended changes due to the genetic modification, the comparisons of the same proteome in wild type and mutant Arabidopsis thaliana was undertaken. Arabidopsis plants, Columbia wild-type strain, were transformed with activation-tagging vector pSKI015 by floral dip. Sequences flanking T-DNA insertions were amplified using a thermal asymmetric interlaced polymerase chain reaction protocol (TAIL-PCR) and sequenced and then subjected to BLAST searches of the public databases to identify the T-DNA insertion site.A total of 12 mutants represent 5 different T-DNA insertion site types include intergenic, in intron, in exon, upstream of a gene and downstream of a gene were selected in this study. The whole plants of T3 generation of ppt herbicide-resistant Arabidopsis mutant were harvested between the growth stage of flowing complete and senescent, and stored at -70C for the extraction of total protein.The 2-D PAGE of the same proteome in wild type and mutant Arabidopsis thaliana was undertaken. We chose a pH interval 3-10 in the first dimension electrophoresis for an overview of total protein distribution, and to monitor global changes that occur in the proteins due to genetic modification. Many protein spots were observed on a 2-D PAGE gel. Image analysis detected 400-500 protein spots on 2-D PAGE gel after staining with Coomassie Brilliant Blue.2-DE gels of wild-type and mutant Arabidopsis' proteome were run and 4 replicates were carriedout. With the use of PDQuest Ver. 7.2 software, quantitative variation of protein spots between wild-type and mutant Arabidopsis in 2-DE gels were compared. The results showed the natural variations in protein patterns of individual plant was obvious, this indicated that more work are needed to define the baseline of natural variations. It's the base for further analysis. On summary of the comparisons, distinct variations were sort into two categories: (i) higher accumulation level in the mutant plants than in the wild-type ones; (ii) lower accumulation level in the mutant plants than in the wild-type ones. A total of 102 spots were chosen for above two categories. Proteins were characterized after in-gel trypsin cleavage by mass spectrometry and peptide mass fingerprinting. 66 Proteins were identifi...