Map-based Cloning And Functional Analyses Of Rolling Leaf 14(RL14) And Temperature-impressible Leaf Color1(tlc1) Genes In Rice (Oryza Sativa L.ssp.indica)

1. RL14 gene map base clone and functional researchAs an important agronomic trait, leaf rolling in rice(Oryza sativa L.) has attracted much attention from plant biologists and breeders. However, the relevant molecular mechanism for some kind of leaf rolling mutant remains unclear. Here, we use histological analysis, SEM and other basic method to research rl14-1 mutants various characteristic and map-based cloning of the gene RL14, and through qRT-PCR, in situ hybridization analysis RL14 genes expression pattern.1.1. Phenotypic characterization of rl14-1 mutantsHistological analysis found that rl14-1 mutant plants had incurved leaves due to the shrinkage of bulliform cells on the adaxial side. Moreover, rl14-1 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, compared with the wild type.1.2. Map-based cloning and expression pattern analysis of RL14The RL14 locus was localized further to a 24.5-kb region between markers SID2 and SW24. Amplification of relevant DNA fragments and sequence comparison revealed that rl14-1 and rl14-2 resulted from a single base deletion in the 517 bp upstream of the start codon and a single nucleotide mutation at codon 190 (T/C) in the second exon, respectively. The identity of rl14 was subsequently confirmed by genetic complementation experiments. The shape of leaves and bulliform were all restored to levels of wild-type plants upon transformation with the RL14 gene.Our results reveal RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature.1.3. RL14 affected formation of the secondary cell wallExpression of genes related to secondary cell wall formation was affected in rl14-1 mutants, cellulose and lignin content was altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results to water deficiency, which is the major reason for the abnormal shape of the bulliform cells.2. TLC1 gene map base clone and functional researchChloroplasts are essential for the unique photoautotrophic and sessile existence of higher plants. Chloroplasts account for 50% of the total soluble protein in leaves, and these proteins are encoded by both nuclear and chloroplast genomes. Plastid development from proplastids to photosynthetically active chloroplasts is one of the most important metabolic processes during plant growth and is coordinately regulated by both plastid and nuclear genes. A number of chlorophyll (Ch1) and chloroplast associated mutations that affect leaf coloration have been identified and are referred to as virescent (v), stripe (st), albino, chlorina, zebra, and yellow variegated depending on their diverse phenotypes. In this study, we describe a leaf color mutant temperature-impressible leaf color1 (tlcl)(tlc1) from the EMS mutant library. The mutant plant exhibits a yellow-green leaf phenotype, decreased level of Chl, and delayed chloroplast development at 20℃. Fine mapped tlcl locus and identified tlcl gene by its expression pattern.2.1. Phenotypic characterization of tlcl mutantsunder paddy field conditions, tlcl plants largely exhibit a yellow and pale green leaves, while it displayed yellow at 20℃and pale at 30℃.The chlorophyll content decreased but the H2O2 content increased in tlcl mutant leaves. TEM observation found the development of chloroplast in mutant leaves was delayed. Photosynthetic capacity was significantly lower in tlcl mutant leaves; assembly of photosynthetic complexes was impaired in tlcl mutant.2.2. Fine map of tlcl locus and tlcl mutation analysis.Fine mapped the tlcl locus between RM1533O and Y10 from a segregating population of 560 recessive individuals. The tlcl gene was narrowed down to a 68.4kb genomic DNA region between the SSR markers RM15330 and Y10, and cosegregated with Y7. There were 13 hypothetical and expressed genes in this region that were already annotated and we identified tlcl candidate gene by its expression trait. 2.3. Transcription analysis of chloroplast development-related genes in mutant plants.We analyze the expression of genes associated with Chl biosynthesis, photosynthesis, or chloroplast development by qRTPCR. The result show transcript levels of chloroplast code gene were down regulated in mutant.Together we conclude TLC1 gene functioned for chloroplast code gene expression and chloroplast differentiation and development.