Map-Based Cloning And Functional Analysis Of A Rolled Leaf Mutant Zw235 In Rice

Leaf is the main place for plant photosynthesis. Fine leaf morphology is helpful to shape ideotype and improve the photosynthesis efficiency. Three-dimensional structure of plant leaf is crucial for photosynthesis, including light absorption, CO2 fixation and gas exchange in photosynthesis process. Appropriate leaf morphology is an important ideotype characteristic of super rice. Slightly rolled Leaf can keep leaf erect, contribute to increase the light transmittance and light saturation point, but not affect the light compensation point, resulting improvement the whole plant photosynthesis efficiency. In addition, the appropriate leaf curl can reduce the radiation of the sun to leaves, lower the transpiration of leaf under the condition of water shortage, reduce the plant body moisture loss, enhance plant resistance and increase the accumulation of photosynthetic products, so as to boost crop yield. Therefore breeders always have focused on the improvement of plant leaf shape.We have screened a roll-leaf mutant zw235 from rice T-DNA library in order to study mechanism of leaf shape building. With compare to wild type(wt), the mutant grow tightly and show straight and upright leaf of which two edges curl inward with dwarf and light green character. Furthermore, chlorophyll content and photosynthetic efficiency of the mutant zw235 notably is ferior to wt. Through observe leaf microstructure, we find the number and area of mutant’s bulliform cells are smaller. Geneticaly analysis suggested that zw235 mutant characters are controlled by a pair of recessive nuclear genes. By building F2 mapping population and applying map-based gene cloning method, we precisely locate locus site within 23.5 kb interval on No.5 chromosome. We find single base mutation in CDS of one of candidate genes sequenced, which induce the gene(ZW235) expression ended up in advance and corresponding function lost. The gene complementarity test and CRISPR-Cas9 test proved that ZW235 function deficiency indeed result in plant blade roll phenotype. It indicated that gene ZW235 is nuclear localization protein after establishing ZW235-GFP fusion protein expression vector and doing rice protoplast subcellular localization. According to the result of yeast two-hybrid and one-hybrid and relevant literature,we preliminary work out that gene ZW235 is interacted with KNOX familyⅠOSH6 and OSH71 expression protein and then combine with YAB1 promoter region binding element. What’s more, gene ZW235 through inhibite gene YAB1 expression, change the number and area of bulliform cell in mutant’s leaf, leading to the mutant zw235 leaf rolling.The inheritance and phenotype characteristics of rolled leaf mutant zw235 were defined in this study, and we have separated and verified the mutant gene ZW235, preliminary interpretation the function of the gene, which provides a basis for further researching the rice leaf development and achieving rice ideotype.