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Gene Cloning And Functional Analysis Of Early Leaf Senescence 3 In Arabidopsis Thaliana

Posted on:2019-06-20Degree:MasterType:Thesis
Country:ChinaCandidate:Y J LiFull Text:PDF
GTID:2370330548988617Subject:Genetics
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Leaf senescence is the final stage of leaf development,it shows gradual yellowing of leaves and decrease in photosynthesis.The process of leaf senescence is affected by aging rate and environmental factors,and phytohormone has an important role in the mediating process among these factors.Leaf senescence is also a key element which determines crop yield and quality.Therefore,it is crucial to explore the regulatory mechanism of phytohormone during leaf senescence for elucidating the mechanisms and improving the crop yield.In the dissertation,we generated an Arabidopsis mutant library by EMS mutagenesis and screened a mutant with early senescence phenotype,thus we named it els3(early leaf senescence 3).We isolated gene ELS3 by the map-based cloning and whole genome resequencing,and combined that with phenotypic analysis,RNA-sequencing,genetic and molecular biology method to investigate the molecular mechanism of leaf senescence regulated by ELS3.The results are as follows:(1)Phenotypic analysises shows that els3 mutant has early leaf senescence phenotype such as reduced content of chlorophyll,accumulating reactive oxygen species(ROS)and increased cell death in leaves at different stages.It also exhibited pleiotropic phenotypes including the defects in inflorescence and root development.(2)RNA-sequencing combined with Real-time PCR shows that senescence-related genes such as SAG 12 and SAG 13 were both up-regulated up to ten-fold in els3 mutant.Similarly,the key genes involved in salicylic acid(SA)metabolic pathway and signaling pathway were up-regulated dramatically in els3 mutant at 10 DAG(day after germination)compared to WT.The transcriptional factors such as WRKY75,ANAC019,SARD1,CBP60g responsible for the expression of ICS1/SID2,were all up-regulated from four-fold to ten-fold.(3)We measured the SA,and its derivatives in els3 mutant and WT showed the SA and 2,5-DHBA(2,5-dihydroxybenzoic acid)which is the hydroxyl compound of SA were all increased significantly.Thus we gave a hypothesis that the early senescence phenotype of els3 mutant was caused by the disturbed homeostasis of SA.(4)The AtTPST was characterized to be the gene responsible for the pheonotype of els3 mutation by map-based cloning combined with the whole genome resequencing.AtTPST is a transmembrane protein which mediates posttranslational modification localized in cis-Golgi.(5)We treated els3 mutant with synthetic PSK-a and found that PSK-a can recover the early senescence phenotype of els3 mutant.It indicated that the leaf senescence phenotype of els3 was caused by the absence of the PSK-a due to TPST mutation.(6)The double mutants of els3 with known SA mutants related to SA synthesis,signaling and metabolic pathway suppressed the early senescence of els3 mutant,suggesting that TPST participates in the regulation of leaf senescence by effecting SA synthesis and signaling pathway.In summary,we screened a leaf early senescence mutant els3 and characterized the gene ELS3(AtTPST).We further demonstrated that ELS3(AtTPST)plays an essential role in regulating leaf senescence through the SA pathway by catalyzing the substrate PSK.
Keywords/Search Tags:Arabidopsis thaliana, Leaf Senescence, Salicylic acid, Plant secret peptides
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