Analysis Of Flanking Sequences Of T-DNA Activation-tagging Population In Rice (Oryza Sativa L.)

Rice (Oryza sativa) is one of the most important food crops in the world.It is amodel plant for the grasses and has important syntenic relationship with other cerealspecies.On December 2002, International Rice Genome Sequencing Project(IRGSP)completed the sequencing of rice genome. So research about the function of ricegenome becomes more and more urgent. Mutants are an essential approach forfunctional genomics study. Mutant library can be established by T-DNA and transposontagging, which is an important way to found saturated mutant population. In this paper,amplifying rice flanking sequence with PCR walking on the basis of plenty ofactivation-tagging population mutant library of rice gene was improved and increasedefficiently. The flanking sequence of activation-tagging population and activatedretrotransposon Tos17 produced by tissue culture in rice genome were analyzed.Through analyzing RT-PCR of activation-tagging it was proved that theactivation-tagging vector PER38 play an important role in rice function genomics.Significant results had been achieved as described as follows:1. Amplifying rice flanking sequence with PCR-walking became more perfect. Thedigestion and ligation were done in same process. Such had no any effect on theefficiency of flanking sequence amplification but saved time, changed somenucleotides of adapter (ADA1), decreased the homology between adapter andsequences in rice genome, and reduced the probability of primer dimmers.Adjusting the second PCR reaction system from 20μL to 40μL ensured amplifyefficiency and was better for reclaiming of DNA, the highest amplify efficiencywas 87.39%, which made the foundation for amplifying and separating the massflanking sequence.2. We got some regularity of tagging insertion rice genome by studying the pattern oftagging insert. T-DNA and Tos17 tended to insert bigger chromosome, for example,chromosome 1. Genic region of the rice genome were hot spots for T-DNA andTos17 insertion while repeat region were cold spots. In the distribution of functiongene, ratios of cell Metabolion in Tos17 and T-DNA insertion were the highest, 34.20% and 44.66% respectively. Resistance gene was the second. Transcriptionfactor was the lest. The analysis of T-DNA insert-mutant in rice has crucial meaningfor the study of functional genomics, and analysising of flanking sequences ofmutants can be used to separate genes related to agricultural characters.3. RT-PCR of 10 individual activation-tagging genes showed that expression levels of6 genes, which ranged from 1.0Kb to 4.0Kb upstream or downstream to the T-DNAinsertion site, were significant higher than that of the wild type. And it alsoconfirmed that the CaMV 35S enhancers in the activation-tagging vectors activatedthe expression of gene indeed. PER38 vector system has a series character ofactivation-tagging, and it is very important to the study of functional genomics.