Functional Analysis Of LbVHA-d2 And LbVHA-a3 Genes In Lycium Barbarum L. Under Salt Stress

The vacuolar H+-ATPase(V-H+-ATPase),a key enzyme in ion regionalization,has an important regulatory role in ensuring normal cellular life activities under adversity conditions.Hence,the study of the function of V-H+-ATPase in Lycium barbarum L.is of reference significance for exploring the mechanism of salt tolerance in wolfberry.Furthermore,V-H+-ATPase is encoded by subunits,of which,the V-H+-ATPase d2 subunit(VHA-d2)is involved in the control of reversible dissociation or proton transport coupled with ATP hydrolysis;the V-H+-ATPase a3 subunit(VHA-a3)is essential for proton transport.Based on this,this paper uses the Lycium barbarum L.as material.We measured the physiological indicators under different salt stresses,cloned Lb VHA-d2 and Lb VHA-a3 genes,and carried out expression determination and functional identification around their genes.The results are as follows:1.By density gradient centrifugation and atomic spectral index method to analyze the leaves and roots of H+-ATPase activity,Na+ and K+content changes.Salt stress 1 d,with the increase of the concentration of salt stress,the leaf PM-H+-ATPase and V-H+-ATPase activity were higher than contrast,but no significant change in the roots.Salt stress 7 d,the leaves and roots activity of tendency in 200 mmol·L-1 highest NaCl treatment.Additionally,leaves and roots activity reduced in 15 d salt stress.At the same time,Na+content was increased,the content of K+was reduced after the first increase trend and Na+/K+ratio first decreases after increasing in leaves and roots at different time under salt stress.2.Based on the V-H+-ATPase activity and Na+and K+ content,the relationship between the adoption of TA cloning Lb VHA-d2 and Lb VHA-a3 genes bioinformatics analyses.The results show that LbVHAd2 gene codes 351 amino acids,it was an unstable acid hydrophilic protein and had not membrane structure.In addition to this,LbVHA-a3 gene codes 819 amino acids,it was an unstable acidic hydrophobicity protein and had six transmembrane regions.Their secondary and tertiary structures were mainly composed of alpha helix,and had a high degree of homology and affinity with plants of the Solanaceae family such as mandarins.Their secondary structures mainly located in the plasma membrane.Quantitative real-time PCR analysis showed that salt stress 1 d,LbVHA-d2 gene expression were upregulated in leaves and roots,and Lb VH4-a3 gene expression was down-regulated then up-regulated.Salt stress 7 d,LbVHA-d2 gene expression was up-regulated in leaves and down-regulated in root under 300 mmol·L-1 NaCl than control.LbVHA-a3 gene expression was all down-regulated.Salt stress 15 d,LbVHAd2 gene expression both highest in leaves and roots under 100 mmol·L-1 NaCl,and LbVH4-a3 gene expression both were down-regulated in leaves and roots.3.Overexpression of LbVHA-d2 and LbVHA-a3 genes in tobacco and functional analysis of transgenic tobacco.The results show that LbVHA-d2 gene expression of OLbVHA-d2 tobacco and Lb VHA-a3 gene expression of OLb VHA-a3 tobacco and PM-H+-ATPase and V-H+-ATPase activity were increased under salt stress(PM-H+-ATPase and V-H+-ATPase activity were decreased under salt stress 1 d).The content of Na+ decreased in leaves and there was no significant change Na+ content in root.The K+content and Na+/K+ ratio in leaves and roots were increased.Correlation analysis showed that Lb VHAd2 and Lb VHA-a3 expression were positively correlated with PM-H+-ATPase and V-H+-ATPase activity and negatively correlated with Na+ content,K+ content and Na+/K+ ratio under salt stress,except for LbVHA-a3 expression was negatively correlated with PM-H+-ATPase and positively correlated with Na+/K+ ratio in OLbVHA-a3 roots.The results above show that the increase of PM-H+-ATPase and V-H+-ATPase activity and LbVHAd2 and LbVHA-a3 genes expression provided the impetus for the Na+ exclusion of cytoplasm,the compartmentalization of tonoplast to Na+and maintained the Na+/K+ homeostasis in cytoplasm.Longterm salt stress decreased the PM-H+-ATPase and V-H+-ATPase activities and inhibited gene expression,which reduced the Na+transport rate across plasma membrane and resulted in the accumulation of Na+and damage.Increased expression of LvVHA-d2 and genes in tobacco promotes plasma membrane and vesicle membrane H+-ATPase activity and increases ion transport rate,which in turn increases salt resistance in Lycium barbarum L..The results can provide reference for the study of V-H+ATPase function and subunits of Lycium barbarum L.under salt stress.