Map-based Cloning And Function Analysis Of A Leaf Early Senescence Gene OsLED In Rice

Rice is one of the most important cereals in the world, and more than half people around the world feed on it. For China, grain output especially rice yield is so important that even affects Chinese food security.For rice plants, leaves are main organs for photosynthesis. Presenesced and poor-developed rice leaves would lead to low setting percentage and weak cereal quality, which directly decides crop yield and deible value. Studying molecular mechanisms of rice leaf senescence would not only make sense to help us understand the process of plant development, regulation, and senescence, but also offers hints for molecular breeding. In our lab, a leaf early senescence mutant osled from Rice Variety9311was obtained by60Co y-radiation. Through map-based cloning technology and other related physiological and cytological methods, we succefully separated tarket gene OsLED and studied potential gene functions. Main results are as follows:1. Main agronic traits of mutant osled and analysis of drought resistanceIn field, mutant osled senesces quite early even at tillering stage(approximately4to5leaves). When compared with wild type93-11, mutant osled exhibits increased growth period but decreased plant height, efficient panicles, TKW(Thousand Kernel Weight), ripening rate. Simulated drought stress (7.5%PEG6000) and natural soil drought experiment show that mutant were more sensitive to drought.2. Physiological, biochemical and histochemical staining analysis of mutant osledAnalysis in physiology and biochemistry showed the content of malondialdehyde (MDA), activities of superoxide dismutase (SOD) and peroxidase (POD) were basically unchanged from flag leaf to third-top leaf in wild type, while significantly increased, and dramatically higher in the second-top and third-top leaves of mutant. Contents of soluble protein and chlorophyll, and catalase (CAT) activity all gradually reduced in top three leaves of both the mutant and wild type, however, contents in second-top and third-top leaves of the mutant were quite lower than these in wild type. Histochemical staining represented an incomplete system of membrane or cell death, O2-and H2O2were seriously accumulated.3.OsLED responses to phytohormones to regulate plant developmentResults suggest that growth of mutant osled were seriously restrained after treatment with plant hormones. Plant height and root length of mutant osled were decreased and were obviously shorter than wild type. After treatment with1.5mM SA, root length and plant height were reduced by77.1%and63.9%respectively. When treated with12.5U.M and20.0μM JA for6days, plant height of mutant osled dropped by64.6%and91.2%correspondingly. Besides, After treated with H2O2for6days, plant height of osled decreased by93.9%.4. SEM(Scanning Electron Microscope) analysis of mutant osled under normal conditions and that treated with hormonesWhen compared with wild type plants, mutant osled plants show more plant hairs on the surface of top-first leaf. It represents that the density and number of macro hairs and micro hairs get increased. SEM analysis results suggest that paclobutrazol extreamly suppressed plant hairs development of mutant osled.Number of macrohairs on adaxial epidermis of osled plants dropped by28.6after treatment with1.5mM SA. However, number of microhairs increased by18.6%. Besides, results suggest that treatment with20.0μM JA resulted into the obvious growth of macrohairs and microhairs on adaxial epidermis of osled.5.Gene mapping and sequence analysis of OsLEDGenetic analysis indicated that OsLED was a recessive gene. We fine mapped this gene into an interval of164Kb on the long arm of rice chromosome3. Through selection and sequence analysis of candidate genes, a gene AK111569(LSD1-liked gene) was found two AA bases lost in CDS. This change leads fram shift mutation. Besides, we further confirmed this results after sequencing target genes of the wild type and mutant plant from F2.6. Phenotypes of transgenic rice plantWe constructed pTCK03-RNAi-OsLED Vector and transferred it into an indica variety kasalath using rice tissuetransformation system. Compared with osled mutant, OsLED-RNAi transgenic rice plants senesced earlier even than osled mutant.Besides, we have also cloned pC1300-OsLED and transferred it into osled mutant plants to observe whether it can rescue mutant phenotype. Results show that pC1300-OsLED transgenic plants didn’t show senescence phenotype while osled mutant plants senesced seriously.