Construction And Analysis Of Rice T-DNA Insertional Mutants

Rice (Ory2a sattva ) is one of the most important grain crops in the wotld. Sinceits smaller genomic size (430 Mb), amenable to manipulations in vitro and efficientregeneration system for several varieties, it has been considered as a model formolecular study of cereals. With the compIetion of rice genome sequencing, largeinformation will be available which may play important roles in the research of ricefunctional genomics.This project 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 viaAgrobacterium-mediated transfOrmation and make some analysis of important mutants.The major results obtained are as follows:l. By using an activation vector, pSKI0l5, An Agrobacterium-mediatedtransformation protocol fOr japonica rice cultivar "zhonghua l1" has beenestablished. As a result, a number of T-DNA insertional tagging lines have beenproduced.2. On the basis of pSKI0l5, we developed a new activation tagging vector whichconfers resistance to both herbicide Basta and antibiotics hygromycin. They canbe alternatively used in seedling stage and in vitro selection of ricetransformants.3. Experiment data show that co-cultivation of calli derived from scutella of ricemature seeds with Agrobacterium tumefaciens can produce resistant calli withan efficiency of 45 %. On the shoot regeneration medium supplemented with2 mg/L of 6-BA and 0.1 mg/L of zeitin, 50 % of resistant calli are able toregenerate shoots. Meanwhile, the treatment of partial desiccation and sorbitolstress can promote shoot regeneration.4. Rice immature inflorescences were inoculated with Agrobacterium tumefaciensstrain LBA4404 carrying pCAMBlA-l30l with application of vacuumm m-infiltration. Based on the total number of co-cultivated inflorescences, the highest frequency of GUS transient expression is nearly 28.0 % for inflorescences bearing flowers 2 to 3 mm in length. Vacuum infiltration-facilitated inflorescence transformation can save nearly half of time in tissue culture than transformation of scutella calli derived from mature seeds.5. Tagging lines were screened through Basta spray application in field. The optimal concentration of Basta was determined to be 500 mg/L. Statistics showed that the segregation ratio in TI generation was 3:1, 15:1 and 63:1 respectively. Based on above data, the percentage of tagging lines with one, two, or three loci insertion was 29.3 %, 40 % and 19 % respectively. An average number of insert loci number was 1.88.6. T-DNA flanking sequences from rice tagging lines were obtained by TAIL-PCR and plasmid rescue. Homologous search was done by BLASTn and BLASTx with the data in GenBank. The chromosome location of flanking sequences and possible encoding proteins were presumed.7. Various agronomically important phenotypes of mutants, such as dwarf, sterile and low vigor etc. in rice T-DNA tagging lines have been characterized. Co-segregation of Basta resistance and particular phenotype are under further investigation.