Gene Cloning And Functional Analysis Of A Rice Semi-dwarf Mutant

Plant height is an important agronomic trait as a core component of rice plant architecture.The study on the regulation mechanism of rice plant height has important theory and application value for the improvement of rice plant architecture.In this study,the semi-dwarf mutant derived from radiation mutagenesis of indica rice R316 was identified for further study.The phenotype identification,gene mapping,transgenic verification,subcellular localization,and transcriptome sequencing assay were performed to preliminarily analyze gene function.The main findings of this research are as follows:1.Phenotype analysis of the mutants showed that there was no significant difference between the wild type and the mutant in the growth and development of the above-ground and below-ground parts in the early seedling stage.During the transplantation period,the wild type and mutant gradually showed differences in plant height and leaf color.At maturity stage,the plant height of mutant was significantly lower than that of wild type,the panicle was short,the yield was reduced,and the leaves of mutant were short and narrow with deep-shallow intervals.2.The results of tissue sections showed that the morphology of epidermal cells in mutant leaves was irregular.Larger mesophyll cells and smaller vascular tissue lead to shorter leaves.The mutant stem parenchyma cells became larger in volume,but the reduced number of layers resulted in thinner stem shortening.3.Mapping and cloning of mutant genes were completed.Genetic analysis showed that the mutant phenotype was controlled by a pair of recessive nuclear genes.Using BSA pool sequencing combined with gene fine mapping analysis,Os TMK1 was identified as a candidate gene,which encodes a transmembrane kinase.Loss of a base in the coding region of the mutant gene results in premature termination of translation.The function of candidate genes was further verified by gene knockout plants experiments.4.The results of subcellular localization showed that Os TMK1 was localized in the cell membrane.Its localization conforms to the localization characteristics of transmembrane kinase proteins.5.The tissue expression pattern of Os TMK1 was analyzed by fluorescence quantitative PCR.It was found that the gene was expressed in panicles,stems,floret,roots,leaves and leaf sheaths of rice plants.Among them,the highest expression was found in leaves and panicle.6.Transcriptome analysis revealed that the JAZ family genes Os JAZ6,Os JAZ 9,Os JAZ 10,Os JAZ 12,Os JAZ 13 downstream of jasmonic acid signaling pathway were downregulated.The differentially expressed genes in the transcriptome were verified by fluorescence quantitative PCR.7.The mutants were found to be insensitive to Me JA at seedling stage by hormone treatment with Me JA.The growth inhibitory effect of Me JA on mutants was weakened,suggesting that Os TMK1 may be involved in jasmonic acid signaling pathway to regulate rice development.