Positional Cloning And Functional Analysis Of Chlorophyll Fluorescence Decaying Gene In Arabidopsis Thaliana

Plant senescence,which is a series of declining processes that controlled by internal factors and affected by external factors,can cause natural death of plants.The beginning time and speed of the senescence can determine the life of a plant organ or organism directly and can affect the yields and qualities of crops in the future.Therefore,the research on the mechanism of plant senescence not only has important significance in developmental biology,but also improves the ornamental value of landscape plants,which means the research has both social and economic benefits.The cell death in the process of leaf senescence,is a kind of programmed cell death.In this process,the nutrients in the leaf can be transported to the flowering,seed,or other tissues reused.As the main photosynthetic organs,leaves accumulate a large number of nutrients,it is very important for the health of plants to select and reuse the nutrients in the leaf accumulated in the photosynthetic stage.There is a series of changes of the cell structure in the senescent leaves,and the most remarkable feature is the disintegration of the organelles in the cell.The first change is the change in the structure and content of chloroplast and grana.The fluorescence released from the excited state to the ground state is chlorophyll fluorescence.In 1931,Kautsky found that the chlorophyll fluorescence and linked chlorophyll fluorescence to the photosynthesis,and then,the research of chlorophyll fluorescence phenomenon has been studied widely and deeply,and gradually formed the chlorophyll fluorescence induction theory.Chlorophyll fluorescence kinetic analysis method,as a sensitive probe to study the function of chloroplast,is widely used in the study of photosynthesis,which provides a lot of valuable information for the function and structure of photosynthetic related membrane.Changes of chloroplast structure and content affect the chlorophyll fluorescence parameters in leaf senescence.The earliest structural changes in the chloroplast are the grana structure and content change,so the chlorophyll fluorescence parameters can be an indicator of aging mutants,then gene cloning,and clarify the relationship between photosynthetic membrane function and senescence.Laboratory constructs the Arabidopsis thaliana mutant library by EMS mutagenesis.Screening the abnormal mutants of chlorophyll fluorescence parameters Fv/Fm by chlorophyll fluorescence imaging system when 10 d and 25 d after germination stage.In this study,E61 is one of the stable genetic mutants with a phenotype of lower chlorophyll fluorescence parameters Fv/Fm.Genetic analysis revealed that the mutant E61 was a single gene recessive mutation,and we located the mutant gene on the terminal end of chromosome ? near the SSLP molecular marker apply positional cloning technique.The mutation gene was AT1G73470 and the 413 rd bit base of the gene was changed from G to A,and its encoded amino acid was changed from alanine(GCU)to threonine(ACU).When we clone the mutated gene,we constructed the complementation vector,over expression vector,tissue localization vector and subcellular localization vector,the get the transgenic Arabidopsis thaliana plants of those vectors.The results of the complementation experimental showed that the phenotype of lower chlorophyll fluorescence parameters Fv/Fm was indeed caused by the mutation of gene AT1G73470.The results of GUS staining showed that the expression of the gene was higher in the young stem and leaf in Arabidopsis thaliana,but not express in root,flower and senescence leaves.The subcellular localization showed that target protein and GFP fusion protein in sporadic distribution in mesophyll cells.The subcellular localization of GFP gene to construct the fusion vector of protoplast transient into mesophyll cells of Arabidopsis thaliana,under confocal laser scanning microscope showed that target protein and GFP fusion protein in sporadic distribution in mesophyll cells,but not expressed in mitochondria,and the specific subcellular localization needs further study.The experiment of physiological and biochemical,found that E61 had resistance to ABA in germination.E61 is slower than that of WT in develop;flowering and senescence were later than WT;and the growth period is longer than WT.the content of chlorophyll a and chlorophyll b E61 was higher than WT.E61 has a lower content of soluble sugar and a higher content of MDA.The enescence of dark inducing shows that chlorophyll degradation speed of E61 is faster than WT.In summary,the phenotype of chlorophyll fluorescence decay in E61 is caused by the mutation of gene AT1G73470,and gene AT1G73470 is involved in the process of chlorophyll catabolism,which affects the growth process of flowering and senescence.