Identification And Analysis Of Genes Preferentially Expressed In Stamen Of Wheat(Triticum Aestivum L.)

Stamens are the organs where male gametes are produced and developed,directly related to plant reproduction and seed formation.Study of genes preferentially expressed in stamen and investigating their functions in stamen development and fertility regulation showed significant importance in the value of theoretical and practical knowledge.In order to discover stamen-predominant genes,expression profiling was performed with the combination of expressed sequence tags(ESTs)and RNA-seq data by comparing transcriptomics.After confirmation by sqRT-PCR and qRT-PCR experiments,170 genes from 59 homoeologous loci were obtained for their predominant expression in wheat inflorescence,including 80 genes mainly expressed in stamens.Functional differentiation of homoeoalleles has occurred among nearly one-third of the identified gene sets.Annotated genes were involved in floral organ identity specification,anther and pollen development,and stamen-pistil interaction.Particularly,lipid metabolic pathways,including ?-hydroxylation of fatty acid,synthesis of alkanes and fatty alcohols,and metabolism of glycerophospholipid,played important roles in the development of stamens.Eighteen overrepresented decamer elements were isolated from upstream regulatory regions of 80 stamen-predominant genes,including the core sequences of pollen-specific cis-elements,hormone response elements,and transcription factor binding sites.IDG024 is one of the stamen-predominant genes.This gene was located on wheat homoeologous group 3 chromosomes and showed peak expression at the heading stage.It encodes a plant thionin family protein precursor-like peptide,which is thought to be Gramineae-specific.The encoded signal peptide structure at the N-terminus and subsequent cysteine residue sites were conserved.Homologous genes in barley and rice were also predominantly expressed in stamens.IDG024 was speculated to be related to fertility based on co-expression analysis.To investigate the promoter characteristics of IDG024,a 2369 bp fragment upstream from the start codon of IDG024-3B was analyzed for its activity.In stable transformation experiment and transient expression assay,reporter genes driven by this sequence expressed preferentially in wheat stamens,but not in Arabidopsis.Transient expression analysis showed that the 264 bp fragment upstream from ATG codon is the regulatory region for flower-specific expression,which is located within the 5'-UTR.Upstream sequence in the absence of the 264bp fragment could not drive the expression of reporter gene.Sequences corresponding to the above 264bp region in three IDG024 homoeoalleles were highly conserved with more than 90%similarity,and contained identical regulatory elements.Deletion analysis of cis-element in the 264bp region showed that the TGCGTACACGCA element located at-128 to-117 upstream of ATG codon is related to expression specificity of reporter gene.This element contains the GTAC core motif of SPL transcription factor binding site.