Functional Analysis Of Ethylene Response Factor Gene TaERF7 In Thermo-Photo-Sensitive Male Sterile Of BNS In Wheat

The fertility of photo-thermo-sensitive male sterile line wheat is controlled by temperature and light,which can realize the utilization of wheat heterosis using this feature.Bainong sterile line(BNS),as a good material of photo-thermo-sensitive wheat,had great potential in the utilization of wheat heterosis.However,the key genes of male sterility and the abortion mechanism of BNS are still unclear.Based on the microarray data of anther gene expression profile of sterile line and fertile line,we found that an ethylene response factor(ERF)gene was differentially expressed in the anthers of the two lines,which was named TaERF7.Through bioinformatics,q RT-PCR and genetic transformation technology,we analyzed the sequence of TaERF7,expression and functional verification.In addition,subcellular localization,prokaryotic expression and yeast single hybrid technology were carried out to study TaERF7.The research results were as follows:1.The TaERF7 protein was a transcription factor containing 219 amino acids.Analyzing the protein sequence,TaERF7 contained the AP2 domain and two amphiphilic suppression(EAR)motifs,which was a transcriptional repressor.Yeast self-activation test found that TaERF7 had no self-activation activity,and the mutant of EAR motif could not be changed the feature.TaERF7 was located in the nucleus,and had the characteristics of transcription factors.The results of prokaryotic expression showed that TaERF7 fusion protein had good solubility.The optimal IPTG concentration for inducing fusion protein expression was 0.5 mmol/L,and the optimal concentration of imidazole buffer used for purification of fusion protein was 50 mmol/L.2.Analyzing the sequence of TaERF7 promoter region,we found that it contained multiple elements of low temperature and light response.Further research showed that TaERF7 in BNS was up-regulated by low temperature and short-day sunshine,while down-regulated in high-temperature and long-day sunshine.In anthers of BNS at medicinal spikelets and meiosis stages at different sowing dates,the expression level of TaERF7 gradually decreased with different sowing dates.At the same time,TaERF7 was expressed in different tissues and organs of BNS.3.The yeast single hybrid experiment indicated that TaTMS5 was the downstream target gene of TaERF7.In the young spikes of BNS at medicinal spikelets stage at different sowing dates,the expression levels of TaTMS5 and TaERF7 showed opposite trends,and had a significant negative correlation,which indicated that the expression of TaTMS5 was inhibited by TaERF7.In the young spikes of BNS at medicinal spikelets stage at early sowing date,the expression of TaERF7 was up-regulated by low temperature and short daylight,which inhibited the expression of TaTMS5.This might be an important reason for BNS male sterility.However,in the young spikes of of BNS at medicinal spikelets stage at late sowing date,the external environment changed to high temperature and long daylight,and TaERF7 expression decreased,which led to reduce the inhibitory effect on TaTMS5.BNS fertility was restored.4.Through transgenic experiments in Arabidopsis thaliana,it was found that overexpression of TaERF7 had an inhibitory effect on the growth of plants,while had a positive effect on plants responding to low temperature stress,which could improve the resistance of plants to cold stress.The mutation of the EAR motif would change the function of TaERF7.Firstly,the mutation changeed the inhibitory function of TaERF7 on plant growth into a promotion function,which made the dates of bolting and flowering of transgenic plants earlier.Secondly,the mutation of a single EAR motif did not affect the function of TaERF7 in improving the freeze resistance of plants,while the mutation of two EAR motifs would make this function missing.