The Screening,Cloning And Functional Research Of Arabidopsis Thaliana Development Defect Mutants

Screening mutants with specific developmental phenotypes is an important prerequisite for the study of plant gene function.Our research took Arabidopsis thaliana as our research object,screened a series of developmental defect mutants that mutated by EMS.We did systemic classification and phenotypic description targeted to the mutants,and did research on some of the mutants.In addition,identified the T-DNA inserted mutant of CDC5 and prepared the polyclonal antibody of CDC5 protein which provides support to research on the function research of CDC5.The following results were obtained.?1?There were four types developmental defect mutants were screened: late flowering mutants?3?,leaf color mutants?16?,leaf shape mutants?12?and trichome abnormal mutants?5?,and backcross the single gene mutant with WT to purify it's background.?2?We identified the mutated gene of F14-46 V,F10-25 No.3,F12-12 S and F14-50 by the way of genetics and molecular biology.Among them,VAR2 was mutated in F14-46 V,it encoded a subunit of the FtsH protein complex and participated in the degradation of light-destroied D1 protein.EGY1 was mutated in F10-25 No.3,it encoded a ATP-independent chloroplast thylakoid membrane metalloproteinases which are essential for the accumulation of chlorophyll and chlorophyll a/b binding?CAB?proteins in chloroplast membranes.TUB4 was mutated in F12-12 S,which encoded a beta-microtubu protein which constitutes the cytoskeleton together with ?-tubulin.KTN1 was mutated in F14-50,it encoded a microtubule-cleavable protein that precisely controls the number of microtubules,the speed of their assembly,and disassembly speed by regulating dynamic microtubule changes.Then we found the changed amino acid were well conserved by amino acid sequence homology alignment and it was located in the conserved domain.?3?Identified GK₂78B09 which is the loss-of-function mutant of CDC5,found the mutant of CDC5 resulted in the inhibited development of main root by comparing the root length of WT and GK₂78B09.In addition,the growth rate and length of the main root of GK₂78B09 was lower than WT.In addition,we induced-Expressing the N terminal 144 amino acid and purified the protein by nickel column affinity purification,then immunized rabbits by the purified protein to achieve polyclonal antibody targeted to CDC5.We tested 100 ng of CDC5 antigen by the polyclonal antibody.In summary,this study screened four major types of Arabidopsis thaliana developmental defect mutants and cloned the mutated genes in four of them,then counted the main root developmental defect phenotypes of the CDC5 T-DNA insertion loss-of-function mutant.Polyclonal antibodies against CDC5 were prepared.