Screening And Functional Identification Of Synthetic Hydrophilic Short Peptide Gene NLEAs

Saline-alkali lands restrict the growth and development of crops,reduce the yield of crops,and seriously challenge the sustainable growth of agricultural production.With the changes of the climate and environment,the land salinization will be deepened gradually.The one of the effective ways for crops to resist the salt damage of land is to improve crops genetically by means of genetic engineering and improve their salt resistance.In order to improve the salt tolerance of crops in the process of genetic improvement,the acquisition and application of salt tolerance genes is one of important pathways.Biosynthesis is based on molecular biology and bioinformatics,thoroughly analyzes the structure and function of biomacromolecules at the molecular level,so as to create a new biomacromolecular substance with biological activity by means of artificial synthesis.Synthetic biology provides a new approach to crop genetic improvement.Based on LEA protein,this study verified the salt tolerance of plant salt tolerance gene NLEA4 obtained by biosynthesis method,and screened a large number of synthetic hydrophilic short peptide genes.The main research results are as follows:1.In order to verify the biosynthetic NLEA4 hydrophilic short peptide gene,Arabidopsis expression vector was constructed by Gateway cloning technology and introduced into Arabidopsis using Agrobacterium transformation method for functional verification.The results showed that transgenic NLEA4 Arabidopsis had salt tolerance.Transgenic Arabidopsis showed resistance to sodium chloride stress and mannitol stress at seedling stage.The analysis in qRT-PCR of several salt tolerance marker genes showed that NLEA4 transgenic Arabidopsis may participate in the ABA-dependent signaling pathway and induce the expression of stress response genes downstream of ABA to improve the plant salt tolerance.We then analyzed some physiological indexes of transgenic Arabidopsis and quantitative analysis of related salt-tolerant genes.The results showed that transgenic Arabidopsis could cope with salt stress by regulating physiological metabolic activities such as stomatal opening and photosynthetic rate.2.In order to clone and verify the foreign gene expression in yeast with high throughput and efficiency,in this study,an efficient expression clone T vector was constructed with the yeast expression vector pYES2 as backbone.Yeast expression vector pYES2-T was constructed by inserting two Xcm ? restriction enzyme sites at MCS.After Xcm? enzyme digestion,T vector will produce two prominent T ends convenient for exogenous gene with A additon for subsequent TA cloning.The target gene was under control of GAL1 promoter that can be induced by galactose,so that the target gene expression could be induced by galactose after the recombinant was transformed into S.cerevisaes.This new vector facilitates the screen and detection of the target genes.3.In order to verify the feasibility of the synthetic NLEAs and to obtain more salt tolerant short peptide genes,10 hydrophilic amino acid as synthsis unit were selected and 142 NLEAs were synthesized.Yeast T expression vector was used for screening synthetic hydrophilic peptide gene pool in S.cerevisiae.The eight short peptide genes have proved the resistance to salt stress,and bioinformatics prediction to its working mechanism in salt tolerance ability was conducted.From the above,this study synthesized,screened and verified the hydrophilic short peptide gene NLEAs,indicating that the biosynthetic and molecular biological methods can have the feasibility and research value in the mining and application of salt-tolerant genes.