Characterization Of Three Chlorophyll Deficient Mutants In Rice

Mutants are important resources for functional genomics. More than 20000 independent transgenic mutant lines of japonica variety Nipponbare were obtained by Agrobacterium tumefaciens mediated rice transformation method in our laboratory. Three chlorophyll deficient mutants including low temperature sensitive chlorophyll deficient mutant Itc, chlorophyll b less mutant cbl and high temperature sensitive chlorophyll deficient mutant were isolated from T₁ lines of the T-DNA insertion mutant population. In this research, we investigated phenotype and genetic analysis of the three mutants, cloning and primary functional analysis of genes responsible for these mutations. The results are summarized as follows:1.The ltc mutant displayed low temperature sensitive chlorophyll deficiency. When grown under 23℃ or lower temperature (restrictive temperature), the mutants developed pale yellow or albino phenotypes with significant decrease in total chlorophyll and carotenoid contents. However, mutants and wild type plants grown under high temperature(32℃) had similar chlorophyll content. Genetic analysis revealed that a single nuclear-encoded recessive gene was responsible for the mutation.2. PCR and hygromycin resistance assay showed that the mutation was co-segregated with the T-DNA insertion. The sequence flanking T-DNA was obtained by TAIL-PCR. The integrated T-DNA was located in the first exon of a putative plasma membrane proton ATPase gene. To suppress the expression of the ltc gene, a RNA interference vector was constructed. Some T₀ transgenic RNAi plants displayed chlorophyll deficient phenotype similar to the phenotype of the ltc mutant under the same temperature.3.The cbl mutant was characterized with chlorophyll b less, yellow leaf, semi-dwarf and reduced tillers. The mutation was controlled by a single recessive gene and was not caused by T-DNA insertion.4. A high-resolution physical map of the chromosomal region around the cbl gene was made by a large number of markers and mapping population. The cbl genewas mapped within 23kb region between marker IDJ9 ^Q marker RM496 on chromosome 10. A chlorophyll a oxygenase gene within the region was considered as the candidate of the cbl gene. RNA interference vector was constructed for suppressing the expression of the chlorophyll a oxygenase gene. To RNA interference transgenic plants with yellow green leaf phenotype were produced.5. The cdel(t) gene displayed normal phenotype under 23 °C or lower temperature (nonrestrictive temperature). However, when grown under 26°Cor higher (restrictive temperature) the plant exhibited an abnormal phenotype characterized by yellow green leaf. Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation. PCR analysis and hygromycin resistance assay indicated the mutation was not caused by T-DNA insertion.6. To isolate the cdel(t) gene, a map-based cloning strategy was employed. A high-resolution physical map of the chromosomal region around the cdel(t) gene was made. Finally, the cdel(t) gene was mapped in 7.5kb region between marker ID10 and marker ID 11 on chromosome 2. Sequence analysis revealed only one candidate gene, glutamyl-tRNA synthetase gene (OsGluRS), in the 7.5kb region.