Analysis Of MiRNAs Related To Salt Tolerance In Wild Barley And Cloning Of HbLAC Gene And Construction Of Expression Vector

In this study,salt tolerance-related miRNAs excavated from high-quality pasture wild barley(Hordeum brevisubulatum)endophytic fungal symbiosis were studied.Through the prediction and function analysis of the target genes of 9 miRNAs,the temporal and spatial expression profile of miR397,the expression analysis under salt stress and evolution analysis of miR397,the bioinformatics analysis of miR397 target HvLACs,the cloning of HbLAC gene and the regulatory analysis in tobacco transient system and so on,provided the direction and basis for the further research of miR397 and HbLAC gene.The main results obtained are as follows:1.Predict the mined target genes of miRNAs and analyze the biological functions related to the target genes.Through miRanda,psRobot and psRNATarget three target prediction software,miR156,miR159,miR166,miR168-5p,miR171-3p,miR397,miR399 were predicted 15,11,3,1,1,7,3 target genes,respectively,but miR168-3p and miR171-5p did not get the target gene after taking the intersection.The protein domain analysis of the target genes in the CDS region located target sites was obtained.Same as mainstream targeted regulation,miR156 mainly targets the SPL family genes,miR168-5p targets the AGO,miR397 targets the LAC genes,and miR399 is in the 5’UTR region,but miR399 targets regulation of ubiquitination related genes in 5′UTR region.2.Wild barley miR397 is evolutionarily conservative,has tissue timing specificity,and participates in salt stress response.The analysis of miR397’s spatiotemporal expression profile found that its relative expression level was highest in leaves,followed by stems,and lowest in roots;the relative expression levels at seedling,jointing,and maturation stage showed a trend of first decrease and then increase,that is,the lowest expression levels during jointing.Under NaCl(0,150,300,and 450 mM)stress conditions,miR397 was down-regulated,both with and without endophytic fungi.In addition,through structural prediction analysis of miR397 precursors and base conservativeness and evolution analysis of miR397 family,wild barley miR397 has a stable structure and is highly conservative in evolution.3.The HvLACs genes participate in various stresses and the cloned HbLAC gene is associated with wild barley lignification.The 2000 bp region upstream of the CDS region of the HvLACs genes have a variety of hormonal and stress-responsive cis-acting elements,suggesting that the HvLACs promoters can participate in a wide range of stresses through binding to transcription factors.Using homologous recombination cloning method to clone wild barley laccase gene HbLAC and construct HbLAC expression vector pCAMBIA2300-GFP-LAC and miR397 overexpression vector pCXSN-FLAG-397.Sequence alignment and evolutionary analysis of 17 LACs proteins from Arabidopsis thaliana and HbLAC suggested the HbLAC is most likely to participate in the lignification process of wild barley.4.The regulatory relationship between wild barley miR397 and tobacco NtLACs was studied in a tobacco transient expression system,and it was found that miR397 can negatively regulate NtLAC4,NtLAC17 and NtLAC12.