Map-based Cloning And Functional Analysis Of PLS2,a Premature Leaf Senescence Gene In Rice(Oryza Sativa L.)

Premature Leaf Senescence?PLS?usually causes the prematurely degradation of chlorophyll and some macromolecules,as well as over-accumulation of reactive oxygen species,and finally results in the reduction in yield and rice quality.In this dissertation,we provided the results about PSL2 gene isolation by the map-based cloning method to a space radiation mutant pls2,and preliminary studies in the PLS2 function.?1?At the heading stage,most leaves of pls2 displayed as senescence,accompanying with decrease in chlorophyll content and increase in starch granules.SAGs?SGR,NYC3,PAO,and Osl85?expression levels were higher in pls2 than WT,together with more hydrogen peroxide?H₂O₂?and malonaldehyde?MDA?accumulation and higher level of SOD activity and upregulated expression amounts of ROS scavenging genes.In addition,the net photosynthetic rate,stomatal conductance,and transpiration rate mutant were lower in pls2 than in WT.?2?Genetic analysis revealed that the phenotype of pls2 was determined by one pair of a recessive gene.We finally located the PLS2 gene into a 90kb between C-2 and SL-1-9 on Chr.3 by the method of map-based cloning.The candidate region contains fifteen ORFs.The LOC_Os03g15840,a putative glycosyltransferase,was considered as the candidate gene because there is C to T subtitution on the nine exon,resulted in the amino acid alternation from arginine?R?to cysteine?C?.Over expression of PLS2cDNA amplfied from the wild type and knock out the PLS2 by Crispr strageties also verified the identification of the LOC_Os03g15840 as PLS2.?3?qRT-PCR and GUS assays showed that PLS2 was ubiquitively expressed in organs and tissues including root,leaf sheath,stem,young leaf,and panicle.In situ hybridization?ISH?assay was carried out and we found the intense signals in leaf mesophyll cells.Subcellular results revealed the PLS2-GFP signal was merged with endoplasmic reticulum?ER?marker.?4?At heading stage,much more glucose,frucose,and sucrose accumulated in pls2 leaf than in WT.Less sucrose was in recovered transgenic plants than in the control,and more sucrose in knock out plants than in the control.?5?ABA content in the pls2 was lower than in the WT.Expression levels of genes related to ABA receptor,ABA signalling,and ABA synthesis and degradation significantly decreased,suggesting the mutation of PLS2 might affect ABA synthesis or signal transduction in pls2 plant.