Exploring Rice (Oryza Sativa L.)Gene Function By T-DNA Insertional Mutagenesis And RNA Interference Strategies

Rice (Oryza sativa L.) is one of the most important crops in the world, and a model plant for molecular biology studies of gramineous crops and monocotyledons. Now the sequencing of rice genome is nearly completed, and more than 40,000 putative genes have been identified from it. More than 32,000 full-length cDNA cloned have been collected from rice and sequenced. Exploring the functions of these genes is the next challenge. As rice is easy to transform using Agrobacterium tumefaciens mediated method, creation and functional analysis of transgene-induced mutants has become one of the major technical approaches for rice functional genomics research. In this study, T-DNA insertional mutagenesis and RNA interference (RNAi) strategies were used to create rice mutants, and functional analysis on corresponding genes and their expression patterns were carried out based on their mutation phenotypes.This work is sponsored by Hi-Tech Research and Development Program and National Natural Science Foundation of China. By cooperation with many colleagues, some research progresses have been achieved, and detailed descriptions are presented as follows:A rapid and efficient A. tumefaciens mediated rice transformation protocol was established for large-scale production of enhancer trap lines. Regeneration of transgenic plants could be as short as 90d starting from callus induction. The frequencies of hygromycin-resistant callus induction and plant regeneration were over 90 and 80%, respectively. PCR and Southern blot analysis showed that about 95% hygromycin resistant plants were transgenic. Key factors for transformation efficiency improvement included decreasing co-culture temperature to 19-22 ℃, and subculture selection for 4¹2d after selection culture for 10¹5d.The efficiency of GAL4/VP16-VAS-GUS enhancer trap system in rice was evaluated. Immature T₁ seeds of 9,120 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 T₁ seedlings. About 70% seed-positive lines showed seedling-positive GUS staining results. Using PCR walking method, 127 T-DNA flanking rice sequences were rescued from 212 samples. 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 10 candidate promoters were randomly selected for expression pattern analysis. 6 promoters showed the same GUS expression patterns as those of the original lines, which indicated that GAL4/VP16-VAS-GUS enhancer trap system was effective for gene regulatory element identification and gene expression pattern analysis in rice.Functional analysis on putative rice receptor-like kinase (RLK) genes was carried out using transgene-induced RNAi method, and a novel rice blast resistance related gene (coded as RK41) was identified. T₀ RK41-RNAi transformant with single-copy T-DNA insertion and well-suppressed gene expression was selected out by Southern and Northern blots. RT-PCR detection was carried out on its T₁...