Assembly And Functional Identification Of Hydrophilic Short Peptide Gene NLEA1

Salt stress imposing physiological drought and ion toxicity on crops severely restricts seed germination and seedling growth,so that significantly reduce the economic yield and quality of crops.Enhancing crops salt toleranceappears to bethe key issue to mitigate salt stress,whereas,the identification of salt tolerance genes is one of the key means.Synthetic biology is an emerging field which is a new way to combine traditional engineering and modern biotechnology.Synthetic biology not only can create new biological organisms or species,but also can assemble complex biological systems in a simple way.Previously,we identified high-hydrophilic short peptides from plant LEAsproteins(Late Embryogenesis Abundant,LEA)database,and optimized the combinations of the peptides.A novel potential salt-tolerant gene,NLEA1(NovelLEA1),was synthesized and proved to possesshigh salt tolerancein yeast,Arabidopsisand wheat tranformants.Preliminary study has beenconductedtoclarifythe salt tolerance mechanism of NLEA1 through ABA and ROS measurements and transcriptome sequencing(RNA_seq).The main results listas below.1.Six hydrophilic peptides sequences have been assembled through filtrating hydrophilic LEA protein database with corresponding genes synthesized after the codon optimization.have synthesizedgene and finding the potential with strong hydrophilic peptide.In order to verify the functions of these candidategenes,a series of pYES2-NLEA vectors was constructed and transformed intoyeast cells(Saccharomycescerevisiae).After salt and cold treatments,NLEA1,NLEA4,genes were found to improve the salt tolerance of yeastsignificantly,and among them NLEA1 is the most powerful gene in resisting salt stress.2.In order to further verify whether NLEA1 can improve plant resistance,we transformed the NLEA1 gene into Arabidopsis and wheat for functional identification.The plant expression vectors pGWB14-NLEA1 and pWMB122-NLEA1 were constructed and tranformed into Arabidopsis and wheat,respectively.The results showed that NLEA1 gene can significantly enhanced the ability of salt tolerance in Arabidopsis and wheat.3.To clarify the mechanism of NLEA1,the contents of ABA and ROS were measured using transgenic Arabidopsis plants.The results demonstrated that NLEA1 transgenic plantsproduced much more ABA but less active oxygen.Transcriptomic sequencing identified 97 differentially expressed genes including some transcript factors genes responding to high osmotic pressure.Therefore,NLEA1 gene might mediate ABA and responsive genes to maintain ROS in low level in response to salt stress.In summary,wesynthesized the hydrophilic short NLEA1 gene and elucidated the mechanism underlying the salt tolerance,and explore a new pathway for the identification of salt tolerance genes,and create salt-tolerant transgenic wheat materials for deleting the selective marker gene.Hence,this study has important theoretical and appliedsignificance.