Establishment And Preliminary Analysis Of Activation-tagging Population In Rice(Oryza Sativa.L.)

With the completment of the whole genome sequencing in rice (Oryza sativa L.) to identify functional characterization of unknown or predicted genes becomes a major task in rice functional genomics. To confirm gene function by mutant analysis based on a saturated mutant population is a straight and efficient way. As rice is easy to transform using Agrobacterium tumefaciens mediated method, construction 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 strategies were used to establish rice mutants of activation-tagging;analysis of the T-DNA flanking sequences of activation- tagging and enhancer-trapping generated previously , RT-PCR analysis of activation- tagging were carried out and the important role of the activation-tagging vector PER38 for rice function genomics was also inquired.This work is part of Hi-Tech Research and Development Program (863) of China entitled " establishment and functional analysis of a lagre T-DNA insertional mutant population" supported by Ministry of Science and Technology. Significant progress has been achieved as described as follows:1. By cooperation with many colleagues, a rapid and efficient A. tumefaciens mediated rice transformation system was established and used for large-scale T-DNA mutant production. Regeneration of transgenic plants could be as short as 90days starting from callus induction using mature seeds. 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 from normally 28-30℃ to 19-22℃, and subculture selection for 4~12d after selection culture for 10~15d. A large-scale rice activation-tagging population consisting of about 50,000 individual transgenic lines has been generated by the proceture.2. Using PCR walking and Tail-PCR methods, 172 and 493 T-DNA flanking rice sequences were rescued from PER38 activation-tagging population and pFX-E24.2-15R enhancer trapping population generated previously in our lab,respectively. The efficiency of flanking sequence amplification and the structure as wll as distribution of T-DNA insertion sites of two populations were investigated and compared .RT-PCR analysis of activation-tagging were also carried out. Results showed that:(1) The efficiency of flanking sequence amplification with both vector and rice sequences is sigenificantly higier in activation-tagging population than that of enhancer-trapping population, 63.2% and 38.1%, respectively.(2) T-DNA distributions revealed by flanking sequence amplification in the genic regions and intergenic regions are quite similar in both activation-tagging and enhancer-trapping populations, T-DNA prefer to inserted in genic regions than intergenic regoins;the distribution of T-DNA insertion sites on chromsomes is similar to that of putitave expressed genes and expressed genes of Genbank rather than hypothetical genes.(3) RT-PCR analysis of 11 individual activation-tagging genes showed that expression levels of 6genes, ranged from 1.1Kb to 4.0Kb upstream or downstream to the T-DNA insertion site ,are siganificant higher than that of the wild type and confirmed that the CaMV 35S enhancers in the activation-tagging vectors truly levitates gene expression.In summary, we have generated a large-scale activation tagging population consisting of about 50,000 indivadual T-DNA insertional lines and the regenerated lines has a series character of activation-tagging which provided a valuable platform for rice functional genomics reserch .