Studies On A Rice T-DNA Insertion Mutant With Novel Type Lesion Mimic

Rice(Oryza sativa L)is the staple food for more than one third of the world population. Because of its small genome size, relatively well developed genetic maps and easily to be transferred, rice is a model plant for functional genomics research. With the completion of rice genome sequencing, large information will be available which may play important roles in rice functional genomics. Obtaining a mutant library by insertion is a powerful strategy for the research of rice functional genomics.Agrobacterium-mediated transformation techniques have made T-DNA insertional mutagenesis the most extensively applied method for approaching genomewide mutagenesis in functional genomics research. Due to low copies, 1.5 on average, of random insertion and the character of stable inheritability, T-DNA insertion has been successfully applied in rice and Arabidopsis functional genomics studies.In this experiment, On the basis of the collection of T-DNA tag lines which was established , lots of mutants with distinct phenotype variation have been found in T1 generation, including disease spot on the leaf, diversity of individual plant height, curling leaves, breaking leaves, weak capacity of tillow, and death of plant. We have performed genetics analysis on the novel type lesion mimic mutant AZT91, which has evident variety of mutant phenotype. Specially, we have performed the resistance detection of HYG on T0-generation seed, genetics analysis of T1-generation phenotype, the resistance detection of HYG on T1-generation leaves, and the PCR detection of T1-generation plant.Through the resistance detection of HYG on T0-generation seed(χ2 =0.0145<3.84=χ2 0.05),we have got the segregation ratio of 3:1, and we have elementarily confirmed that the mutant belongs to insertion with single copy. And by phenotype analyzing of the T1-generation, we have confirmed its mutant is a lesion mimic mutant and phenotype, such as delayed growth, dwarfism, brown spot on leaves, light green leaf color then growing yellow, as well as death of plant. Finally, the plants with mutant phenotype and wild phenotype in T1 generation fitted to 1:3, so we confirmed that the AZT 91 mutant is controlled by recessive gene.The resistance detection of HYG on T1-generation leaves confirmed that the mutant phenotype was co-segregated with T-DNA. And the PCR detection also showed that the co-segregation of AZT91 mutant was caused by T-DNA insertion. TAIL-PCR's amplification on the flanking sequence of T-DNA left border and its results has further confirmed that the AZT91 mutant phenotype is caused by T-DNA insertion with single copy.According to Liu Y G's TAIL-PCR condition and procedure with little change, we amplified the flanking sequence of T-DNA left border. A set up of the efficient technology of high-throughput thermal asymmetric interlaced PCR (TAIL-PCR) was established. The Homology comparing the 226bp flanking sequence acquired in our experiment with the GeneBank using BLASTn showed that the sequence locates in the No.3 chromosome of the rice (AP008209, AP006869), and the Homology comes to 100%. The analysis of GeneScan has showed that the mutant has a posses monooxygenase character gene near the 35S promoter. It is elementarily supposed that the AZT91 mutant phenotype is caused by 35S promoter's activating the overexpression of the posses monooxygenase character gene after T-DNA insertion, therefore, the organism metabolize with great disorder, then destroy the balance of the dynamic equilibrium of the cell, resulting in dominant in-of-function and ending in death.An elementarily bioinformatics analysis on this gene was performed, and its DNA sequence was amplified (2231bp). The research of its function is still undergoing.