Identification And Abiotic Stress Analysis Of GS Gene Family In Wheat(Triticum Aestivum L.)

Glutamine synthase(GS)is not only the key enzyme of plant nitrogen metabolism,but also involved in the synthesis of osmotic regulator proline and antioxidant glutathione,which plays an important role in regulating plant growth and development and response to stress.At present,the research on GS gene family in Arabidopsis thaliana,rice and other model crops is thorough,while wheat,as an allohexaploid crop,has a huge genome.The functional research and regulation mechanism of GS gene family in wheat are not completely clear.In this paper,the members of GS family of hexaploid wheat were identified comprehensively by bioinformatics method,and their sequence characteristics,evolution ways and expression patterns under various abiotic stresses were systematically analyzed.The regulatory mechanism and protein interaction network of TaGS were further studied,which provided theoretical basis for analyzing the biological functions of TaGS and the research of wheat genetic improvement.The main results are as follows:1.A total of 12 GS genes were identified in the whole genome of wheat by BLASTP tool,and 6,5,5,4 and 6 GS gene members were identified in the genomes of five representative species Arabidopsis thaliana,kidney bean,alfalfa,rice and maize,respectively.The structure of GS among different species was highly conserved.Through phylogenetic tree analysis,the identified GS proteins of Gramineae plants can be divided into four subfamilies: GS1-1,GS1-2,GS1-3 and GS2.According to chromosome location analysis and homologous relationship,the TaGS genes were renamed as TaGS1-1-6A/6B/6D,TaGS1-2-4A/4B/4D,TaGS1-3-4A/4B/4D,TaGS2-2A/2B/2D.All TaGS proteins have a p I of < 7,showing weak acidity.The amino acid number,molecular weight MW and isoelectric point PI of TaGS2 are higher than those of TaGS1.The analysis of gene structure,domain and conserved motif showed that GS gene had similar exon and intron structure in the same subgroup,and the differences of gene structure among different subgroups were obvious.Most GS contain two conserved domains of Gln_synth_C and Gln_synth_N and nine highly conserved Motif motifs.Co-linear analysis and Ka/Ks ratio showed that GS gene underwent purification selection in the evolution process,which maintained the conservation and stability of gene function.Whole genome replication event was the main mechanism of GS gene amplification in wheat.2.Expression profile analysis showed that TaGS gene participated in the whole growth and development period of wheat,had tissue-specific expression characteristics and responded to various abiotic stresses.TaGS1-1 and TaGS1-2 are mainly expressed in roots,and TaGS1-3 and TaGS2 are mainly expressed in grains and leaves.QRT-PCR was used to analyze the expression changes of twelve TaGS genes in wheat under cold,heat,salt and drought stress.It was found that TaGS homologous genes in different subgenomes had similar expression patterns: under four stress treatments,TaGS1-3 was induced to express in the leaves and roots of wheat seedlings,and its expression increased first and then decreased with the increase of stress treatment time;TaGS1-2was up-regulated in leaves,and the expression increased gradually with the treatment time,while the expression in roots was inhibited.TaGS2 was down-regulated in both roots and leaves,and the expression decreased gradually with the increase of stress treatment time.3.TaGS is finely regulated at multiple levels of transcription,post-transcription and post-translation.The transcription factor regulation network of TaGS gene was predicted,and it was found that TF families related to stress response,such as ERF,MYB,LBD and NAC,might interact with TaGS gene.TaGS promoter contains a variety of light,hormone,growth and development and stress response elements,and the types,numbers and arrangement of cis elements in different gene promoters are different.There are 19 mi RNAs targeting 12 TaGS genes,which indicates that mirnas play an important role in mediating the expression of TaGS genes.TaGS includes posttranslational modification sites such as phosphorylation,glycosylation,S-nitrosation,palmitoylation,ubiquitination and SUMO,among which phosphorylation sites and ubiquitination sites exist in all TaGS,indicating that TaGS are regulated by various post-translational modifications.4.TaGS proteins can interact with various proteins and participate in complex physiological and biochemical reactions in plant cells.210 and 133 TaGS candidate interacting proteins were obtained by yeast two-hybrid technique and IP-MS experiment,respectively.The results of GO enrichment analysis showed that interacting proteins were mainly involved in photorespiration,ion transport,stress response,metabolism and synthesis of organic matter,etc.Through the above two groups of experiments,a total of 19 overlapping candidate proteins were screened,including protein kinase domain protein,F-box domain protein,nucleoside diphosphate kinase and RING-finger E3 ubiquitin ligase which participated in ubiquitination modification.They were mainly located in cytoplasm and chloroplast and involved in multiple biological processes such as plant growth and development,cell signal transduction and stress response.