Analysis Of Gene Expression Profiles During Dedifferentiating Mature Wheat Embryos

Wheat is one of the most important crops in the world.The utilization of biotechnology in its cultivar improvements,however,is rather retarded comparison with that in other crops.The bottleneck of the problem is the immaturation of wheat tissue culture technique.The immature embryos are current targets in wheat transformation.But acquirement of immature embryos is strictly limited by seasons and consistency of development stages is different to judge which not only increases the labour constraints but also hampers experimental flexibility of wheat genetic transformation heavily.Mature wheat embryos have a lot of advantages in their availability throughout the year and consistency of development stages.Using mature wheat embryos as explants is also not practice at present due to lower regeneration rate in vitro culture.Therefore it is of significance to improve ability of differentiation and regeneration in mature embryo culture and to reveal molecular mechanism at the level of gene expression.We observed changes of mature wheat embryos which inducted different time by 2,4-D using optical microscope.Using an Affymetrix GeneChip(?)Wheat Genome Arrays representing approximately 55000 wheat genes, we studied gene expression differences between mature Wheat embryos cultured for 24 h in MS medium containing 2mg/L 2,4-D and without 2,4-D and studied comprehensive transcript profiles of mature wheat embryos induced for different time (0-2-6-12-24-72 h).We analysized the relevance of gene expression differences between dedifferentiation and germination of wheat mature embryos and acquired genes of exhibited meaningful changes during dedifferentiating mature wheat embryos induced by 2,4-D,Hierarchical clustering with the average linkage method and K-means method were employed by the software of cluster3.0,we chose specific express difference genes from clustered results which referred to as candidate genes associated with dedifferentiation.Results are as follows:1)The process of dedifferentiation of this part of cells was finished about 24 h.The four stages were divided apparently according to changes of histological structure in dedifferentiation.The first(0～2h)was the stage of signal transduction(SST)in which the perception and transduction the stimulation of auxin signal were occurred in cells.The second(2～12 h)was stage of signal response(SSR)in which the cells quickly responded the stimulation and a series of biological changes occurred in the cell wall,cell membrane and cytoplasm,so that the cell changed in morphology.The third(12～24h)was stage of cell remodification(SCR)in which the cell structures were remodeled based on the changes occurred in SSR and prepared for cell division and formation of new cell.The fourth(about 24h)was stage of calli formation(SCF). The main missions of this stage were formation of calli.2)There were 2427 transcripts between dedifferentiation and germination of wheat mature embryos.Among of these transcripts,there were 341 transcripts containing 150 reported genes and 191 ests which had strongly similies to rice or Arabidopsis thaliana.171 transcripts were up and 170 transcripts down.3)we studied gene expression differences between mature Wheat embryos cultured for 24 h in MS medium containing 2mg/L 2,4-D and without 2,4-D,we analysized the relevance of gene expression differences between dedifferentiation and germination of wheat mature embryo.Among of 341 meaningful genes,82 genes were all up-regulated,43 genes down,compare with control experiment.4)The 15000 transcripts have altered meaningful changes by more than 2-fold at least at one time point.(different treatment for 0 h,2 h,6 h,12 h,24 h,72 h),Gene annotations were obtained through batch BLASTs(BLASTx;E-value＜e-10)using the Affymetrix probe set sequences in tandem with sourcing annotations directly from the Affymetrix website(www.Affymetrix.com),there are only 2143 transcripts of them have detectable similarities to entries in sequence databases,those reported genes were categorized into 22 groups based on the functions and there are respectively involved in Membrane and Substance Transportion,Signal Transduction, cytoskeleton,cell wall,protein degradation,cell division,chromosome structure,lipid Metabolites et al.5)Hierarchical clustering with the average linkage method and K-means method were employed by the software of cluster3.0,we chose specific express difference genes from clustered results which referred to as candidate genes associated with dedifferentiation,we presuming functions and express patterns of those genes during dedifferentiating wheat mature embryos.