Study On The Function Of Grape VvMAPK9 In Response To Salt Stress

Grape is an important cash crop in China,its cultivation area and yield are in the forefront of the world.However,the unfavorable ecological environment in some planting areas has brought many problems to grape production,among which soil salinization is the main limiting factor.Selecting salt-tolerant grape rootstock for grafting culture is an effective way to solve this problem.Compared with conventional breeding,the genetic engineering technology is timesaving and efficient.Therefore,it is of great significance to identify salt-tolerant genes and study their mechanisms.The mitogen-activated protein kinases(MAPKs)pathway plays an important role in plant resistance to various adverse environments.In our previous research,we discovered a gene VvMAPKK3 which response to salt stress highly,further research found that VvMAPK9 interacts with it and is induced by salt stress,but the molecular mechanism is not clear.Therefore,in this study,the VvMAPK9 gene was cloned from grape rootstock A35,and the sequence characteristic expression pattern and salt-resistant function of this gene were analyzed.The main results of this study are as follows:(1)Bioinformatics analysis of VvMAPK9 gene.The length of VvMAPK9 open reading frame is 1128 bp,it encoding a polypeptide containing 375 amino acid residues with a predicted molecular weight of 42.615 kDa and an isoelectric point of 6.24.Subcellular localization analysis revealed that VvMAPK9 was located in the nucleus and cytoplasm.Phylogenetic analysis revealed that VvMAPK9 belongs to group B of MAPK.Multiple sequence alignment revealed it contains the conserved phosphorylation site TEY and CD domain.(2)Expression pattern analysis of grape VvMAPK9.qRT-PCR results showed that VvMAPK9 was mainly expressed in young leaves,mature leaves and roots of grape rootstock,but the expression in petioles and stems was relatively low.In addition,the expression of VvMAPK9 gene was induced by abiotic stresses and ABA.(3)The overexpression of VvMAPK9 improved the salt tolerance of Arabidopsis thaliana.Under salt stress,the germination rate of transgenic plants was significantly higher,and the root length was also significantly longer than wild-type plants.1 week old Arabidopsis were watered with 200 mM NaCl.After 2 weeks,it was found that leaves of the wild-type plants significantly turned yellow and withered,while the transgenic plants grew well.This indicates that the overexpression of VvMAPK9 can improve the salt tolerance of Arabidopsis thaliana.(4)The overexpression of VvMAPK9 improved the salt tolerance of grape callus.Under normal condition,the growth of the transgenic grape callus and the wild type callus were basically the same.However,on the medium containing 150 and 200 mM NaCl,the wild-type grape callus grew slowly and turned black,while the transgenic grape callus was yellow with better activity and was significantly larger than the wild-type.We determined the relative conductivity and found that the cell membrane permeability of transgenic callus under salt stress was smaller.These results suggested that the overexpression of VvMAPK9 increased the salt tolerance of grape callus.(5)The improvement of grape’s salt-tolerance by VvMAPK9 may be related to the activation of antioxidant enzyme system and the expression of ion transport gene.The anti-superoxide anion activity and the activity of SOD and POD in the VvMAPK9-overexpressing grape callus was significantly higher than the wild type callus under salt stress.The overexpression of VvMAPK9 increased the expression levels of ROS scavenging enzyme genes(VvSOD,VvPOD)and ion transporter genes(VvNHXP,VvHKT1,VvHKT2)in grape callus under salt stress.The result showed that VvMAPK9 may can activate the antioxidant system and regulate the expression of ion transporter gene,thus improving the salt tolerance of grape callus.