Identification Of SiLEA＿2 Gene Family And Functional Analysis Of SiLEA14 In Setaria Italica Stress Response

Millet(Setaria italic L.),a gramineous genus of Setaria,is one of the important food crops in northern China.It has the characteristics of strong drought resistance and rich nutrition,which is of great significance for ensuring food security.Smut is a kind of fungal disease in millet production,which seriously restricts the improvement of millet yield.It was reported that Late embryogenesis abundant protein(LEA)SiLEA14 was involved in the response of millet to drought and salt stress.Our previous study found that the expression of SiLEA14 gene was significantly up-regulated in Jigu 20,a smut resistant variety infected by smut fungus.Therefore,in this study,the millet and the model plant Arabidopsis thaliana were used as experimental materials,and the methods of molecular biology,physiology and bioinformatics were used to identify the members of the LEA＿2 gene family in which the millet SiLEA14 belongs,and the phylogenetic evolution and gene structure of the family members were analyzed.In addition,by constructing Arabidopsis thaliana heterologous overexpressed plants of SiLEA14,this study explored the mechanism of SiLEA14 gene’s involvement in millet resistance to stress,especially biotic stress,in order to cultivate new and long-term effective varieties of millet resistance through genetic engineering technology.The experimental results are as follows:1.In this study,65 members of LEA＿2 gene family of millet were screened and named SiLEA1-SiLEA65 according to their positions on chromosomes.Bioinformatic analysis was conducted on their protein physicochemical properties,phylogenetic tree,domain,subcellular localization and secondary structure prediction.Through phylogenetic analysis,we divided these SiLEA proteins into 9 groups.Among these proteins,the maximum number of SiLEA proteins in Group IX is 14.In Group Ⅲ and Ⅵ,the LEA＿2 gene family members of millet and maize have high similarity.It is speculated that these members are closely related in evolution and may have similar biological functions.Collinearity analysis showed that tandem repetition and large chromosome replication played an important role in the expansion of LEA＿2 gene family in millet.Therefore,it was speculated that intron or exon insertion or loss,exon or pseudoexon occurred during the evolution of LEA＿2 gene family in millet,and the structural diversity may lead to the emergence of homologous genes with different functions.2.This paper explored the expression patterns of SiLEA members of different groups in the phylogenetic tree in response to salt stress and induction of plant defense hormone SA,Me JA,and found that under 250 mmol/L Na Cl and 10 mmol/L SA treatment,The relative expression levels of SiLEA14,SiLEA5,SiLEA6 and SiLEA21 genes all increased first and then decreased.The relative expression levels of SiLEA14,SiLEA6 and SiLEA21 genes increased first and then decreased under 100μmol/L Me JA treatment.These results suggest that SiLEA5,SiLEA6 and SiLEA21 may play a role in the response of millet to salt stress and biological stress.3.The promoter region of SiLEA14 gene was analyzed,and it was found that it contained cis-acting elements related to plant defense hormones such as SA and Me JA,suggesting that it might be involved in plant response to biological stress.Allogeneic overexpression analysis showed that Arabidopsis thaliana plants with SiLEA14 allogeneic overexpression increased resistance to Botrytis cinerea,and differentially expressed genes related to SA and Me JA signaling pathways.In conclusion,SiLEA14 contains 65 members of the LEA＿2 gene family,and tandem repetition and large fragment replication may be the potential driving force for the expansion of the family.The expression pattern analysis showed that some members of LEA＿2 gene family were responsive to salt stress and SA,Me JA treatment.Arabidopsis plants overexpressing SiLEA14 showed increased resistance to Botrytis cinerea.Further studies showed that SiLEA14 gene may be involved in plant response to Botrytis cinerea stress by regulating SA and Me JA signaling pathway.This study provides target genes for the future cultivation of new varieties of millet resistant to stress and lays a certain theoretical basis.