Functional Analysis Of TaRSR1 Transcription Factor In Grain Starch Synthesis In Bread Wheat (Triticum Aestivum L.)

Our previous study indicated that transcription levels of wheat starch regulator 1(Ta RSR1)were significantly negatively correlated with those of 11 starch synthesis-related enzyme genes(Ta AGPS1-a,Ta AGPL1,Ta GBSSI,Ta SSI,Ta SSIIa,Ta SSIV,Ta BEI,Ta BEIIb,Ta PUL,Ta PHOL,and Ta DPE1)during the entire grain-filling period(35 days)of bread wheat.Thus,we speculated that Ta RSR1 transcription factor might negatively regulate the expression of these 11 starch synthesis-related enzyme genes.To confirm the above speculation,in the present study,the function of Ta RSR1 in regulating the synthesis of grain storage starch was identified using the barley stripe mosaic virus-virus induced gene-silencing(BSMV-VIGS)technique in field experiments.The main results were summarized as following:(1)In this study,the Ta RSR1 transcription factor 5′ c DNA sequence was isolated using the rapid amplification of c DNA ends(RACE)method from bread wheat(Triticum aestivum L.).The open reading frame(ORF)of the Ta RSR1 transcription factor was 1485 bp,and it encoded a predicated protein of 484 amino acids.There are 8 exons and 7 intros in Ta RSR1 transcription factor genome sequences.Quantitative real-time PCR(RT-q PCR)was used to measure the transcription level of Ta RSR1 transcription factor three copies.The results indicate that the highest expression of three copies is D.(2)In the present study,the chosen c DNA fragment(284 bp)for silencing had highest sililarities in three copies of Ta RSR1 transcription factor.We constructed BSMV-Ta RSR1 vector and BSMV-VIGS virus was inoculated on the wheat spikes at heading stage under field conditions.BSMV-GFP(green fluorescent protein)virus was also inoculated on wheat spikes,which was used as control.Both the grain starch contents,one thousand kernel weights,grain length and width of BSMV-VIGS-Ta RSR1-silenced wheat plants markedly increased.RT-q PCR was used to measure the expression of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-Ta RSR1-infected wheat plants at 20,27,and 31 days after anthesis.The results suggested that the transcription levels of 7 starch synthesis-related enzyme genes were significantly induced at three time points: Ta SSI,Ta SSIV,Ta BEIII,Ta ISA1,Ta ISA3,Ta PHOL,and Ta DPE1 genes.These results indicate that Ta RSR1 acts as a negative transcription factor and plays a key role in grain starch synthesis in wheat grains by regulating the expression of above 7 starch synthesis-related enzyme genes.