Establishment Of An Arabidopsis Thaliana Cytokinin Transport Mutant Screening System And Gene Cloning

Cytokinin is a phytohormone which has an important role in regulating plant growth,development,leaf senescence and stress responses.In the past half century,remarkable progresses have been made in the study of physiological functions of cytokinin,but the study of its transport mechanism has been relatively slow.AtABCG14 was identified as a long-distance transporter for cytokinin acropetal translocation.As a half-size ABC transporter,AtABCG14 has to works with other ABC transporters to transport cytokinin acropetally.The interacted proteins of AtABCG14 have not yet been characterized.Moreover,more cytokinin transporters in plants remain to be identified to complehensively know the process of cytokinin translocation.Therefore,we screened mutants defected in cytokinin transport by forward genetic approach.Our study focuses on establishing a screening system for Arabidopsis cytokinin transporter or other proteins related to cytokinin translocation.The research results are as follows:(1)Generation-of a mutant library which is easy to be screened by the GFP observation.We generated the transgenic Arabidopsis of ARR5::eGFP which was used for EMS mutagenesis to obtain a mutant library for cytokinin transporter screening.(2)The screening of root shining(rs)mutants by the GFP observation.Taking advantage of GFP signal in the root,we screened the library described previously and found several mutants which have changed expression of ARR5 in roots.(3)The genetic and phenotypic analysis of rs mutants.We found that rs1 to 3 lost the normal distribution of cytokinin in tissues indicated by that the root and shoot showed opposite levels of cytokinin distribution.Similar to atabcg14 mutant,the spraying of trans-zeatin onto the shoot of rs1-3 partially recover the cytokinin-deficient mutant phenotypes.Therefore,we conclude that rs1 to 3 are mutants defected in cytokinin acropetal translocation.The root of rs4 lost the GFP signal in the meristem and elongation zone and it is likely that the cytokinin transport in this area was abnormal.(4)Genes cloning of rs mutant.Using map-based cloning combined with DNA sequencing,we identified rsl and rs2 were both alleles of atabcg14,which clearly proved the feasibility of the screening system.For rs4,we have obtained 7 candidate genes by whole genome resequencing and map-based cloning and further work will be pursued to clone the gene reponsible for the rs4 phenotype.In summary,we established a forward genetic approach for screening Arabidopsis cytokinin transport defected mutants.Using the system,we screened several mutants including atabcg14 alleles which defected in cytokinin transport.We also cloned genes of rs to identify cytokinin transport related proteins and to perform functional studies.The screening system and the genes cloned will contribute to the research of cytokinin long-distance translocation.