Large-scale Construction And Primary Analysis Of Rice T-DNA Insertional Mutant Pool

Rice(Oryza sativa)has been considered as a model for genomic study of cereals because of its colinearity with the other cereal crops,its smaller genomic size (4.3(108 bp), amenable to manipulations in vitro and efficient regeneration system for several varieties, Rice(Oryza sativa)genome sequencing has been finished and it now is the time to carry out the research of rice functional genomics.This research was sponsored by The National High-Tech Program(863 Program). The aim of this thesis is to generate a population of T-DNA insertional mutants via Agrobacterium-mediated transformation and make some analysis of important mutants to supply technique and material to the research of rice functional genomics. The major results obtained are as follows:By using a binary tagging vector, pFX-E24.2-15R ,An Agrobacterium-mediated transformation protocol for japonica rice cultivar "Nipponbare" has been established. As a result, a number of T-DNA insertional tagging lines have been produced. Experiment data showed that embryogenic calli derived from scutella of rice mature seeds sterilized for 30 minutes on the MS medium supplemented with 300 mg/L Cassamino acid + 4mg/L 2,4-D + 2.8g/L proline were bestThe induced calli were subcultured on the MS medium supplemented with 300 mg/L Cassamino acid + 2mg/L 2,4-D + 2.8g/L proline for 6～7 days in the dark and became amenable to transformation. The transformation frequency was 95% and 10,000 transformants were produced.The optimum bacterium concentration was OD600 0.14～0.19 ,co-culture temperature was 19～20℃, and coculture time was3~4 days. The results showed that the calli were cultured on the NB medium supplemented with 1mg/L NAA+3mg/L 6-BA(6-Benzyladenine)+5mg/L ABA for 7 days in the dark and with 2 mg/L 6-BA(6-Benzyladenine)+ 0.5mg/L NAA under continuous cool light (5000lux),which enhanced the differentiation frequencies . After 15 days , the calli were subculture in time .Meanwhile,the differentiation medium was without Hygormycin.The PCR and Southern blot analysis of the transformants indicated that the positive frequency was 95%.The anti-hygormycin test of transformants showed 40% one loci insertion.the Southern blot analysis determined the percentage of tagging lines with one, two, threeor more than three loci insertion was 43 %, 30 % and 27 % respectively. An average number of insert loci number was 1.7 The phenotype of T110,000 transformant lines was investigated and an important phenotype database was established.Various agronomically important phenotypes of mutants, such as dwarf,no tillering,low vigor and mimic macula in T1 transformants have been characterized.T-DNA flanking sequences from some rice tagging lines were obtained by PCR-Walking, and an effective method of amplifying the flanking sequences was set up.