Functional analysis of GUS expression patterns and T-DNA integration characteristics in rice enhancer trap lines

Abstract

Enhancer trapping has been regarded as a powerful tool for functional analysis of novel genes and regulatory elements. In order to evaluate its efficiency in rice genome, we carried out comprehensive analysis on rice T-DNA insertional mutagenesis lines harboring GAL4 /VP16-UAS- GUS enhancer trap system. T 1 seeds of 9120 independent enhancer trap lines were collected for GUS expression pattern analysis on embryo, endosperm and seed capsule. About 40% lines showed positive staining results in one part of the seeds at least, and 212 samples were randomly selected from these lines for further GUS assay on leaves and roots of T 1 seedlings. About 70% seed-positive lines showed seedling-positive GUS staining results. One hundred and twenty-seven T-DNA flanking sequences containing rice homologous sequences were also rescued from 212 selected lines using PCR walking method. Insert locations and integration patterns of the T-DNA insertions were analyzed in detail. Putative genes and promoters were predicted from rice genomic sequences flanking the T-DNA insertion sites, and expression patterns of several candidate promoters were analyzed and compared with those of the original enhancer trap lines. The results confirmed the effectiveness of GAL4 /VP16-UAS- GUS enhancer trap system in rice. Our work provided useful resources for functional analysis of rice enhancer trap lines generated by T-DNA insertion, and indicated that enhancer trapping was an effective strategy for rice functional genomics research.