Construction And Analysis Of Cotton Enhancer Trap Population

As the most important economic crop in the world, Cotton plays an important role in the national economy, but its functional genomics research is relatively backward. To keep up with the strategies of the high-throughputed of plant functional genome research, building a cotton mutant library turns out to be urgent. In the abstract, we can interpret the function of gene recur to gain one of the mutant gene in the genome.At present, the method of gene's function is to create the mutant of gene, to look the gene's mutation phenotype to explain the function of gene.Enhancer trap system is a powerful approach for identifying functional gene and it is based on checking the expression pattern of a reporter gene that randomly integrated into the genome of plant to disentomb the new gene and its' modulatory sequences.This research aimed to establish a cotton Enhancer trap mutant library by T-DNA insertion. The main results of our research are in the following:1. A high-efficiency genetic transformation system mediated by Agrobacterium of Cotton variety "YZ1" was established. 70%-80% of the total co-cultured calli was resistant calli, and 60% of the resistant calli could regenerat into plants. The high efficiency of transformation and regeneration made it reachable to obtain large amount of T-DNA inserted mutagenesis.2. 84 independent transformations were obtained, 82% of the Regenerations was positive in PCR test.3. This study found that the GUS reporter gene expression patterns of different cell lines regeneration varied greatly, this phenomenon supportted the hypothesis that the T-DNA inserted into the plants' DNA was random events. Many GUS gene in the PCR test is positive, for the plants have specific expression, and frequencies in the callus tissue,in the embryo-like bodies,in the flowers,roots are respective 63.1%,47.6%,28.6%,31%.4. TAIL-PCR and PCR-Walking were done to amplify sequences flanking the T-DNA insertion site in Enhancer trap lines. The result is as follows: Only the former two TAIL-PCR steps have smear; 600 bp and 800 bp fragments were obtained by PCR-Walking.