Construction Of Rice T-DNA Insertion Mutant Library And Screening And Analysis Of Short-root Mutants

Rice (Oryza sativa.L) is one of the most important crops, which is widely cultivated in the world. Compared with other monocotyledon, the rice's genome is smaller, so it has been considered as the model plant to study monocotyledon. Root, one of the three major organs in plant, is very important to the plant growth and development. The length of root is an important index that stands for the plant development level. Several experiments were done to elucidate the molecular genetics mechanism that effects the growth of rice root. 1) A mutant library of 100,000 independent lines caused by T-DNA insertion in rice was established through Agrobacterium tumefaciens-mediated transformation. Then 1,000 mutant lines were detected by GUS-staining and PCR analysis. The result showed that the positive ratio of the mutant library was about 90%. The numbers of the T-DNA inserted in the mutant library examined by Realtime-PCR analysis was 1.6 in average and the average of TOS copies was 3.0, which was one copy more than that in the wild type. The T-DNA insertion mutant library was the foundation for screening different varieties of mutant and seperating the valuable gene in the future. 2) The T-DNA insertion mutant library was subjected to a lager-scale screening by the way of sand culture. Finally five mutant lines, named albino/etiolation leaf, double seedling, small seedling, angustifoliate and short root mutant, were obtained. 3) The effects of IAA and NAA on elongation of shoot and root and the number of root per plant were studied during the seedling stage. It was found that both could promote the elongation of root and shoot at low concentration levels (<0.1mg/L), but the effects on shoot elongation was inferior to that on root. At higher concentration levels (>10mg/L), both inhibited the elongation of root and shoot. The number of root per plant was also inhibited by higher concentration of IAA and NAA (>10mg/L). 4) The phenotype and development of the short root mutants was mainly researched. The flanking sequences of the T-DNA insertion site in 200 short root mutants was PCR-amplified and then analyzed by PCR-walking analysis. Finally, 100 flanking sequence were obtained and the alignment analysis found that 50% of them were homologous with the putative rice genes. Most of the genes are related to root development and signaling transduction. Future work is planed to confirm whether those mutants were caused by T-DNA insertion or not with the co-segregation analysis, and to carry out the functional complementation analysis.