Development Of A Tos17 Mutant Database For Functional Gene Research In Rice

Mutant for cloning novel gene is an important strategy for functional gene research. A lot of notable genes had been isolated by mutants created by various methods. In this study, we generated a Tos17 insertional mutant library using Zhonghua 11 (japonica cv.) as a recipint. Tosl 7 is a'copy-paste'mode retrotransposon which is activated by tissue culture, becoming inactive in regenerated plants. T1 Tos17 mutants had been screened by forward genetic strategy to identify the mutant which phenotype co-segregate with Tos17 insertion sites and provide a base for gene isolation and cloning. The flanking sequences of Tos17 were isolated by Adapter-PCR and TAIL-PCR. Through the analysis the flanking sequence of Tos17, we can characterize the Tos17 distribution pattern in rice genome and carry out reverse genetic study of the corresponding genes.The main results are described as follows:1. A total of 15,543 independent Tos17 mutants had been created in Zhonghua 11. Our Southern blot result showes that there are four native copies in Zhonghua 11. After 3 months cultivation, each To line had 1.4 new copy numbers in the generation plants on average. The total insertion site was evaluated about 21,760 in the whole genome.2. Employing Adapter-PCR and TAIL-PCR methods,601 Tos17 flanging sequences had been isolated which can be well mapped onto the rice genome. And 524 Tos17 flanking sequences are unique.3. The analysis of 524 independent flanking sequences shows that Tos17 insertions were biased towards large chromosome. The insertion sites are highly correlated with the chromosome size (r=0.717, P<0.01). Within chromosomes, Tos17 strongly favored the genic sequences (SR=10.85) and strongly disfavored the intergenic (SR=-8.88) and TE-related (SR=-3.68) sequences. In rice genes, strong bias towards coding sequences (SR=4.55), but not towards the 5'upstream (SR=-4.62) and 3'downstream regions(SR=-3.25). In the coding region, Tosl7 strongly favored the exon region (SR=2.48).4.1,881 T1 Tos17 insertional lines had been screened morphologically under normal growth condition in 2008, the result showed that the mutant frequency was 5.69%. The main mutant phenotypes were:dwarf, sterility, albino leaf, yellow leaf, delayed heading, et al.12 insertion sites from 10 mutant lines had been test by PCR and 10 insertion sites were correct.