Isolation Of Flanking Sequences From A Rice T-DNA Insertional Mutant Library And Function Study Of OsBC1L Family Genes

With the finish of the rice genome sequencing project, the study on rice genomics has stepped into the era of functional genomics. In order to analyze the function of rice genes on the genome scale, a best way is to generate lots of mutants by which people can analyze the function of genes by forward genetics or reverse genetics strategies. For the cloning of T-DNA or Tos17 inserted genes, the isolation of flanking sequence is necessary. Using the flanking sequence database, people can find easily the mutant of interested gene and clone it.In this study, I finished the PCR positive test of 12,432 T0 T-DNA inserted lines in our mutant library. By TAIL-PCR, I isolated 5,082 flanking sequences of T-DNA. COBRA-like proteins play important roles in cellulose synthesis and cell expansion, so I studied the biological function of two COBRA-like genes, OsBC1L4 and OsBC1L5, by both the forward genetics and reverse genetics methods in this research. In addition, I also cloned a gene which affects the tiller number of rice and analyze its function.The detail results in this study were summarized as follows:1. I finished the PCR tests of 12,432 T0 T-DNA inserted lines and found 10,770 (86.6%) lines were T-DNA positive.2. Employing TAIL-PCR method, I isolated 5,082 flanking sequences of T-DNA,2,644 (52%) of which can be located onto the rice genome (E-value<10-5).3. By screening the tiller mutants in the field, we obtained two cosegregation lines, 04Z11EM13 (OsBC1L4) and LTW1.4. I analyzed the molecular characterization, chromosomal location, phylogenetic relationships and expression patterns of rice OsBC1L family members. In addition, I obtained 5 OsBC1L inserted lines and found that OsBC1L5 was involved in the development of rice pollen.5. In the 04Z11EM13 line, T-DNA was inserted into the fourth exon of OsBC1L4 gene and caused the mutant phenotype of less tiller and dwarf. We cloned this gene by cosegregation and complementation experiments. The mutation of OsBC1L4 results in abnormal cell expansion, decreased secondary cell wall thickness, and increased starch accumulation. By measuring the content of crystal cellulose, I found a 24% reduction of cellulose in osbc1l4 mutants. I analyzed the expression pattern of OsBC1L4 by RT-PCR and in situ hybridization and found that OsBC1L4 was expressed in both the sclerenchyma and parenchyma cells. OsBC1L4 protein is mainly located in the cell wall and plasma membrane. Correlation coefficient analysis indicated that the expression of OsBC1L4 was highly correlated to that of several primary wall-forming cellulose synthase genes (CESAs). Moreover, the expression level of several cellulose-related genes is increased in osbc1l4 mutants, which suggests that a feedback mechanism may exist to compensate for the reduced cellulose production in osbc1l4 mutants by increasing the expression of other cellulose-related genes during cellulose synthesis.6. In the OsBC1L5 line, T-DNA was inserted into the single exon of OsBC1L5 gene. This line has no homozygous plant of OsBC1L5 insertion. I performed reciprocal cross between OsBC1L5/osbcll5 heterozygous and the wild type plants and found the osbcll5 male gametophyte had a severe defect. By in vitro pollen germination, I found OsBC1L5 gene might be involved in the germination of rice pollen. I investigated the phenotype of OsBC1L5 RNAi plants and found OsBC1L5 gene might also affect the development of pollen wall in rice.7. In the LTW1 line, T-DNA was inserted into the single exon of LTW1 gene and caused a less tiller and wilted mutant phenotype. I cloned this gene by cosegregation and complementation experiments. In addition, I obtained an allelic mutant from POSTECH mutant library, which had the same mutant phenotype with LTW1 and also showed the cosegregation of the mutant phenotype and T-DNA insertion. By GUS staining of the POSTECH promoter trap line, I investigated the expression pattern of LTW1 gene.