Screening Of Rice Chlorophyll Deficient Mutants From T-DNA Insertion Pool And Isolation Of Flanking Sequences Of T-DNA

After a large scale rice T-DNA insertion mutant pool was constructed, the screening of chlorophyll deficient mutant was done in this study. The chlorophyll deficient mutants were the largest portion of rice mutants. Because the evident recognizable phenotypes, it was easy in distinguishing the mutants from wild phenotype. In these mutants,because the chlorophyll deficiency, the chloroplast development was affected and the photosynthesis was also damaged. So, these mutants were not only the good materials for studying the chlorophyll synthesis and decrease, but also the precious materials for the research of chloroplast development and photosynthesis. In this study, after screening the T-DNA insertion pool in seedling stage, many chlorophyll deficient mutants were obtained and the flanking sequences were rescued by PCR-walking method and the structures of the T-DNA left border were also identified after sequencing the flanking sequences. At last, the genes tagged by T-DNA insertion were decided by PCR cosegregation analysis and the gene transformation system for complement experiment was set up. The specific results were shown as following:1 Altogether 430 lines of chlorophyll deficient were got from about 10,000 T-DNA enhancer trapping insertion lines. After GUS staining for detecting the expression of report gene in mutant lines, the results of 34 lines were positive and in 9 lines, the mutant phenotypes indicated cosegregating with the report gene.2 The flanking sequences of T-DNA left border were rescued by PCR-walking method,and for more bands, the DraI, EcoRV and SspI restriction enzymes were used. 10 bands were amplified from 9 mutant lines, in which 2 bands were obtained from line A401. After sequencing and homology searching, 6 sequences were contained rice genomic DNA, other 4 sequences were vector backbone sequences for LB readthrough or unexpected sequencing termination.3 The sequence information of LB was acquired in 8 sequences from 10, other 2 were couldn't obtained the information because the unexpected sequencing termination. The deletion phenomena of LB were observed in 7 sequences and the deletion degrees changed from 3-63bp, but in 1 event the deletion degree reached 130bp. Apart from the deletion phenomena, 2 DNA filler events were also found in the 8 sequences. The LB readthrough and repeats were also observed in theses sequence.4 After homology searching, 4 T-DNA insertions were proven to be existed in genic regions of rice genomes, other 2 were in intergenic regions. The 4 tagged genes by T-DNA were experienced PCR cosegregation analyzed and 3 were proven cosegregated with their mutant phenotypes. The three genes were: the putative GTP-binding gene targeting to chloroplast and relating to the chloroplast ribosome construction; the putative serine hydroxymethyltransferase(SHMT) gene targeting to mitochondrion and relating to photorespiration; the putative Fe-SOD gene targeting to chloroplast and relating to water-water cycle in chloroplast.5 The G418 concentration was decide in transformation system for the gene complement experiment and proper transformation system was set up. Although this system was not high effective as the hygromycin screening system, it can satisfy the conditions for complement experiment by large scale transformation.