Function Analysis Of DSG1 Encoding E3 Ubiquitin Ligase In Rice (Oryza Sativa L.)

Plant height and grain shape are the most important agricultural traits in rice.The basic research of plant height and grain development is considered as a important way to enhance the rice yield.A rice mutant with a stable inheritance,affecting the development of plant height and grain shape,was identified from EMS-treated Jinhui10.This mutant showed dwarf and short grain,and was termed as dwarf and short grain 1(dsg1).dsg1 was controlled by one recessive gene and mapped in a 70 Kb region on chromosome 3.By sequencing analysis,we selected the candidate gene which encoding a U-box domain,and invovling in the development of plant height and grain in rice.In this study,we will validate the candidate gene,then study the expression and regulation mode of DSG1 through expression analysis,overexpression analysis,promoter analysis,subcellular localization,phenotype analysis,cytological and hormone pathway analysis.1.Morphological and Agronomical Characters Analysis: Compared with wild type,in seedling stage,the root length,leaf sheath length and plant height decreased significantly of dsg1 mutant;In reproductive growth phase,the plant height,all internode length and grain length,spikelet length,seed setting rate were to reduce or decrease of dsg1 mutant;Besides,number of cells in a unit area increased,lowering the average cell length of spikelet;From the fall to the third,leaf width increased,and third leaf length is shortened and the lower leaf curled up severely.2.Genetic Analysis and Gene Location: All populations of F1 by hybridizing sterile lines Xinong 1A and dsg1 mutant showed normal phenotype,and separation ratio of F2 population is 3:1(normal: dwarf),which showed that the traits of dsg1 were controlled by a single recessive nuclear gene.Through gene location,DSG1 gene is located on a area of 190 kb between SSR3-28 and RM14645 on chromosome 3 and mutant gene of LOC_Os03g13010 finally was identified by sequencing analysis.3.Candidate Genes Validates of DSG1: By structuring wild-type DNA vector of LOC_Os03g13010 and transformation into dsg1 mutant,consequently,the mutation phenotype of positive transgenic plants has been fully restored.Therefore,it’s sure that LOC_Os03g13010 is the purpose gene of DSG1.4.Expression Pattern Analysis of DSG1: Expression level of DSG1 was detected in different organizations through the qRT-PCR and ProDSG1::GUS.DSG1 expression is better in some tissues,including root,stem,leaf,leaf sheath and in spikelet.Among them,the DSG1 expressed in leaf and spikelet is higher than other tissues.5.Subcellular Localization: To study the expressed location of DSG1,we incorporated the coding sequence of DSG1 to N end of GFP,and removed the termination codon,making DSG1 transient expression in rice protoplast under control of 35 s promoter.Finally,DSG1 protein was mainly located in the nucleus and cytoplasm.6.Over-expression Research: By building super expression vector,and transformation Zhonghua11.DSG1 expression quantity increased significantly in the over-expression plants.Compared with wild type,however,there were no significant differences in plant height and grain length of over-expression plants.7.Cytological Analysis: Through the asana mirror,paraffin section,scanning electron microscopy analysis,We understood the reasons leading to the phenotype of dsg1 mutant is the defect of cell division or elongation in different tissue.Meanwhile,DSG1 also participated in the leaf lateral growth.8.Hormones Pathway Analysis: There is a typical phenotype of BR detect in dsg1 mutant phenotype,including dwarf and compact of plant shape,width and erect of leaves,and tillers decreased.Through tilt experiments of leaf Angle showed that DSG1 participates in BR pathway,and belongs to the BR insensitive mutants.related genes of BR pathway,at the same time,expression quantity in mature leaf of dsg1 decreased than wild type,such as OsD1,OsGSK1,OsMADS55 and BZR1.Besides,we through the qRTPCR to research the change of expression quantity in DSG1 plant with treating by all sorts of exogenous hormones,including BR,GA,IAA,ETH,SA and ABA.The results showed that DSG1 is restrained by BR,ETH,IAA and SA.Which suggests DSG1 may participate in multiple hormones pathway.9.Activity measured of E3 Ubiquitin Ligase: DSG1 codes a U-box domain,which related to protein domain of E3 ligase activity.In order to check whether dsg1 associated with lack of E3 ligase activity,we measured the E3 ligase activity of wild type and dsg1 mutant plants in the seedling and early stages.E3 ligase activity of dsg1 mutant was significantly lower than the wild type,indicating that the reduction of E3 ligase activity associated with dsg1 phenotype.