Isolation Of Arabidopsis Photoperiod Regulating Flower Mutants And Identification Of The Mutant Genes And Proteome Analysis

In recent years,the flowering of plants has been the research focus.4 pathways that regulate flowering time were discovered:photoperiod pathway,jarovization pathway,self-determination pathway and GA pathway.Photoperiod is one of the major factors which control the flowering time.The CRY1,CRY2 that located on the upstream of photoperiod pathway take the light signal directly,induce the biological clock factors and expression of transcription factors to accelerate the flowering.The result showed mutant cry1 took more time to come into flower than the col-4 under several illumination conditions;the flowering time of mutant cry2 was delayed with the treatment of longtime illumination;the phenotype of mutant cry1cry2 was especially obviously.In order to further study the mechanism which cyptochrome regulate the photoperiod for flowering,the mutant cry1cry2 were used as the material in this article. A library of Arabidopsis mutants was constructed by activation tagging method, which several mutants that related to flowering time were obtained from. SCC106-D,SCC60-D was late flowering mutant and SCC105-4-D,SCC147-3-D was early flowering mutant.IPCR and TAIL-PCR were used to identify the flanking genomic sequences of mutated target genes.After the identification of the T-DNA insertion site,the semi-quantitative RT-PCR was used to analyse expression of the mutant genes as the first step towards the function of mutant genes,the results indicated that the expression of the flanking genomic sequences was enhanced compared with the col-4 and cry1cry2 mutant,for instance,the At3g57760 gene in mutant SCC106-D,the AT4G27240 and AT4G27260 genes in mutant SCC60-D,the AT5G19950 gene in mutant SCC105-4-D,the AT2G34160,AT4G18197,AT4G18205 and AT4G18210 genes in mutant SCC147-3-D. some candidate genes related with flowering were obtained.By measuring and analyzing the length of hypocotyl and root under different wavelengths of light we found that mutant SCC106-D was not hypersensitive to different wavelengths of light,but mutant SCC60-D was hypersensitive to red light. These results showed the mutant SCC106-D was constitutively photomorphogenic. The protein of T-DNA insertion mutant SCC106-D and mutant cry1cry2 was separated by two-dimensional electrophoresis,different spots were obtained and identified.Thirty seven protein spots,which were differentially expressed in the cry1cry2 and SCC106-D,were identified by the method:MALDI-TOF-TOF-MS peptide fingerprint analysis of the protein spots and protein database searching. Twenty two of them were identified succesfully.By identification of these spots,some protein related with energy metabolism,secondary metabolism and regulation were obtained.The result showed in the paper laid the foundation for further study of photoperiod regulation by cyptochrome.