Molecular Genetic Regulation Mechanism Of Key Gene Q In Wheat Panicle Development

The number of spikes per unit area,the number of grains per spike,and the grain weight are the three key factors controlling wheat yield.The spike architecture of wheat directly affects wheat yield and is an important agronomic trait in wheat domestication and selective breeding.Therefore,studies on the genetic regulation of wheat spike development have important theoretical significance and application value.There have been more studies on panicle architecture in maize and rice,but studies on the molecular mechanisms of wheat spike architecture are still very limited.The Q gene is an important gene related to the domestication of wheat spike traits.However,the molecular mechanism of how the Q gene regulates wheat spike architecture remains unclear.In this study,from the wheat miR172 gene as an starting point,the functional regulation of Q gene was studied in depth,and the molecular genetic regulation of wheat spike formation was explored.The tae-miR172 precursor from wheat cultivar(Triticum aestivum)Kenong 199(KN199)was identified by gene prediction and miRNA secondary structure analysis.The tae-miR172 precursor was overexpressed driven by the maize ubiquitin promoter,transformed into the wheat cultivar KN199 and a series of stable transgenic wheat lines were obtained.Phenotypic analysis of transgenic wheat plants showed that overexpression of tae-miR172 caused a speltoid spike phenotype,reminiscent of the wheat with the q gene.Real-time PCR analysis indicated that the expression levels of tae-miR172 in tae-miR172 transgenic wheat was significantly increased,and the expression levels of Q gene was significantly decreased.Through rapid amplification of cDNA 5' ends(5' RACE)method and transient expression assays in Nicotiana benthamiana leaves showed that the domestication gene Q is targeted by tae-miR172,and tae-miR172 could inhibit Q protein accumulation by post-transcriptional cleavage.Subcellular localization and transcriptional activity analysis showed that the Q protein is a nuclear transcription factor with transcriptional inhibitory activity.Structural analysis of Q protein revealed that the Q protein contains an ethylene-responsive element binding factor-associated amphiphilic repression(EAR)motif that mediates transcriptional repression activity,which may be involved in the recruitment of the wheat transcriptional co-repressor TOFLESS(TaTPL).Transcriptional expression analyses revealed that the transcriptional expression patterns of Q and TaTPL were quite similar.their transcripts were predominantly accumulated in flag leaves and spikes,with highest levels in early stage of spikes,and the expression levels gradually decreased with the development of spike.Yeast two-hybrid(Y2H)assay,bimolecular fluorescence complementation(BiFC)experiment and firefly luciferase complementation(LCI)experiments showed that Q and TaTPL directly interacted.LCI experiments indicating that the N-terminal atypical EAR motif(LDLNVE)of Q protein is essential for the physical interaction with TaTPL,but not the C-terminal typical EAR motif(LDLDLR);the CTLH motif of TaTPL is essentially required for the physical interaction with Q protein.Transcriptional activity analysis indicated that the N-terminal EAR motif of Q is essentially required for its transcriptional repression activity.Taken together,these results reveal the functional regulation of Q protein by tae-miR172 and transcriptional co-repressor TaTPL in controlling the spike architecture of bread wheat,providing new insight into understanding of the genetic regulatory network of wheat spike development.