Identification And Characterization Of Wheat APYRASE Gene Family Members

Wheat(Triticum aestivum L.),which belongs to the Gramineae family,is one of the most important food crops worldwide.The yield and quality of the wheat still cannot meet the large demand from the increasing population.The climate change and human activities expose wheat to abiotic stresses,such as heavy metals,droughts and over salinities,which significantly limited the improvement of the wheat yield and quality.At the same time,diseases such as head blight and powdery mildew are becoming more and more serious,which also causes severe yield loss every year.At present,the methods in improving the stress tolerance and increasing the yield and quality,can be mainly divided into two aspects:one is to improve the soil environment and farming techniques;the other is to generate the stress-tolerant cultivars.This study seeks breakthroughs by using molecular genetic breeding to generate high stress-resistant wheat cultivars,and provide supports for molecular wheat breeding.APYRASEs(APYs),which belong to the GDA1-CD39 superfamily,are a group of phosphohydrolases.It was later confirmed to be widely existed in animals,bacteria and plants.Many studies in Arabidopsis,poplar and some other plants,showed that APYs were not only related to plant growth and development,but also regulated the plant stress.Studies has showed that Apyrase regulated the plant growth and resistance by regulating the extracellular ATP(eATP)in Arabidopsis to prevent the ROS boost.In poplar,overexpression of APY improved the plant growth and development under drought and salt stress.Therefore,it was speculated that APY is positively related to stress resistance in plants.The research on wheat APY may help us to obtain high stress-resistant wheat cultivars.Thus,focusing on TaAPY gene family,we carried out the following experiments:1.There are nine members of TaAPYs identified in the wheat genome.Analysis of the nucleic acid sequences,protein sequences and higher protein structures showed that they were highly conserved.According to phylogenetic and sequence analysis,nine members were divided into three groups,namely Group ?(including TaAPYl,TaAPY2,TaAPY3-1,TaAPY3-2,TaAPY3-3 and TaAPY3-4),Group ?(TaAPY5 and TaAPY6)and Group ? with only TaAPY7.Moreover,according to the newly published wheat genome,we found that all the nine genes have copies in all three A,B,D chromosome groups.2.This study also investigated the expression pattern of TaAPYs under stresses.Under abiotic stress,the expression level of all the wheat APYs was generally up-regulated in the aerial part,and some genes were stress-specific.Among them,TaAPY3-4 was particularly activated under various stress conditions.Under biotic stress,all genes are up-regulated at 24 h after pathogen inoculation.These results indicate that TaAPY is involved in the process of wheat resistance to both abiotic stress and biotic stresses.3.To further explore whether the wheat APY could degrade the eATP,we have selected the active gene TaAPY3-1 and purified the protein using the bacteria system.The results confirmed that the wheat APY encoded by TaAPY3-1 has the activity of degrading ATP in vitro,and could maintain high enzymatic activity over a wide temperature range.This trait probably gives the APY the ability to take its role in high temperature resistance.This results also provide strong evidences that the wheat APYs regulate plant stress tolerance by controlling the extracellular ATP degradation.4.Results of the AtAPY3 and TaAPY5 transgenic experiment showed that under NaCl,mannitol and ABA treatment,the seed germination rate of Arabidopsis lines was different.AtAPY3 over-expression line has higher germination rate than that of wild-type plants,and much higher than that of apy Arabidopsis mutant.With the constant increase of NaCl concentration,the time required for seed germination of each line was gradually extended,and the germination rate was decreased.However,compared to the wild type and apy3,the seed germination of AtAPY3 over-expression lines was less inhibited.For TaAPY5 transgenic experiment,the real-time qPCR showed that the expression of TaAPY5 in over-expression lines was extremely high.The overexpression lines had longer hypocotyl,weaker seedlings and retarded growth.The results also showed that under low salt stress,the root length of the over-expression lines was longer than wild-type.In this experiment,we also overexpressed the TaAPY5 in wheat,and already obtained some positive transgenic wheat plants.