Homoeologous Regulation And Functional Study Of A Grain Length Related Gene Ta GASR7 In Wheat(tritivum Aestivum L.)

In cereals, grain morphology such as grain length, width and thickness often has a significant impact on crop yields. In rice, a number of genes related to grain morphology have been identified. In wheat, however, genes involved in grain development are hardly known. Bread wheat is an allohexaploid. Of the six sets of chromosomes, two come from Triticum urartu(AA), two from Aegilops speltoides(BB), and the last two sets from Aegilops tauschii(DD). GASR7 is a member of Snakin/GASA gene family in higher plants. Although it is reported that Ta GASR7-A1 is mainly a genetic determinant of grain length in wheat with pleiotropic effects on grain weight and yield, little is known about divergence of its homoeolog expression and function in different grain development stages and during wheat allohexaploidization. Previously, we obtained all the three homoeologous sequences of Ta GASR7 and located this gene on wheat chromosome 7. Here, by BAC library screening we further obtained the promoter sequences of its three homologs and studied their genetic and epigenetic regulation in different development stages and during allohexaploidization through cis-element and DNA methylation analying. Moreover, we explored the effects of silencing or overexpressing the three homoeologs on grain length or other phynotypes by the virus induced gene silenicing(VIGS) technique. The main results are as follows:1. We found that the B homoeolog of GASR7 was the main contributor to the total expression level of this gene in both the maternal tetraploid progenitor and the hexaploid progeny, whereas the expression levels of the A and D homoeologs were much lower. To understand possible mechanisms regulating different GASR7 homoeologs, we firstly analyzed the promoter sequences of three homoeologous genes and found that all of them contained gibberellic acid(GA) response elements, with the Ta GASR7 B promoter(p Ta GASR7B) uniquely characterized by an additional predicted transcriptional enhancer. This was confirmed by the GA treatment of spikes where all three homoeologs were induced, with a much stronger response for Ta GASR7 B.2. Mcr BC enzyme assays showed that the methylation status at p Ta GASR7 D was increased during allohexaploidization, consistent with the repressed expression of Ta GASR7 D. For p Ta GASR7 A, the distribution of repetitive sequence-derived 24-nucleotide(nt) small interfering RNAs(si RNAs) were found. Such an observation suggests possible epigenetic regulation because 24-nt si RNAs are known to mediate RNA-dependent DNA methylation.3. We successfully established the virus-based silencing or overexpressing target gene system in wheat. Using this system, we achieved up- or down-regulation of target gene expressions in spike through heading date flag leaf inoculation.4. The down-regulation of Ta GASR7A/D during allohexaploidization was possibly related with their subfunctionalization. Plants transciently overexpressing Ta GASR7A/D by BSMV inoculation of flag leaves at heading date stage showed decreased chlorophyll content of spike(Ta GASR7A) anddecreased seed setting rate(Ta GASR7D) respectively, while plants transciently overexpressing Ta GASR7 B showed no any obvious phynotypes, suggesting that the divergence of biological function of Ta GASR7 A, B, and D. In newly synthesized allohexaploid wheat, the expression of Ta GASR7 A and Ta GASR7 D were significantly inhibited, suggesting that inhibiting the expression Ta GASR7A/D might contribute to growth vigor and fertility improvement of polyploid wheat.5. The effects of down regulation of Ta GASR7A/B/D on grain length trait. The preliminary data showed that down regulation of Ta GASR7 might have effects on grain length and 1000 grain weight, which need to be further confirmed.Taken together, our data demonstrate that genetic and epigenetic mechanisms may be involved in the divergence of GASR7 homoeolog expression in polyploid wheat. Moreover, the preliminary exploration of Ta GASR7 three homologous gene functions will faciliate the application of Ta GASR7 in future wheat molecular breeding.