Abstract:

Insertion mutagenesis with T-DNA or transposable elements is currently one of the most powerful tools for screening various types of mutations in higher plants, and for analyzing functional information of genes related to those mutations. By using the Ac/Ds transposable elements system in rice, a great deal of Ds-inserted mutants may be efficiently generated, and used for functional identification of genes that influence important agronomic traits, by which the researches in rice functional genomics have been greatly promoted. As a key agronomic trait related to grain production in rice and other grass species and an important property parallel to branching in dicotyledonous plants, tillering in rice has attracted more research interests. In the present study, a rice tillering mutant was isolated from a collection of Ds-inserted mutant lines, and was designated dt1 (double tillers mutant) according to its tillering phenotype of a major tiller and a minor tiller developed at the same tillering node. Both tillers were found to be productive with the ears normally emerged from each of them. The Ds-flanking sequence was cloned from this mutant by using TAIL-PCR amplification technique. The sequenced Ds-flanking sequence was used as a query to perform an online search for homologous sequence against nucleotide sequence database by NCBI-BLAST, the results indicated that the Ds-flanking sequence showed 100% identity at the nucleotide level to the sequence of clone OJ1345H02 (gi|21281466) of rice chromosome 3. The structure of the Ds-inserted gene was predicted by using software of FGENESH and GeneMark, respectively, and gotten highly similar in the number, size and location of exons within the gene. The results also showed that the Ds element was inserted into the region between transcription start site and the first exon, at 173 bp upstream of the first exon. Moreover, the product encoded by the gene was predicted by NCBI Entrez server and Pfam. It was revealed that the deduced product was a rice formin-like protein containing a highly conserved FH2-domain. Given the fact that the Ds element as big as 5.9 kb in size was inserted immediately into upstream of the first exon, it is suggested that Ds insertion probably make the gene loss it’s function, and thereby generate the rice mutant dt1. In addition, the genotyping for Ds insertion in the progeny plants of dt1 mutant indicated that these progeny plants segregated into homozygous and heterozygous types for Ds insertion and Ds free plants, which suggested that the dt1 mutant was heterozygous for Ds insertion.

Key words: Oryza sativa L., Ds insertion mutagenesis, Tillering mutant