Spinach PTI1-3 Gene Cloning And Function Analysis Of High Temperature Response

The“greenhouse effect”causes global warming,which seriously affects plant growth and development,resulting in the reduction of crop yields.Study on the molecular mechanism of plant high temperature response is one of the hot issues in the world.Spinach(Spinicia oleracea L.)is an important green leafy vegetable in China.Study on the Molecular regulation mechanism of spinach heat-responsive is of great significance for using molecular marker-assisted breeding to cultivate a new variety of heat-resistant and disease-resistant spinach.Pto-interacting 1(PTI1)gene encodes serine/threonine kinase,which is involved in the regulation of ROS balance and MAPK signaling pathway,as well as lipid metabolism and other processes.The study of PTI1 on high temperature response regulation will provide important information for further analysis of plant high temperature response molecular mechanism.We found 4 genes encoding the PTI1 protein in the spinach genome with a protein homology similarity of 68.38%.We cloned the Spo PTI1-3 gene with a maximum open reading frame of 1224 bp encoding a total of 407 amino acids with a theoretical molecular mass of 45.19 k Da.Spo PTI1-3 protein has high homology with PTI1 protein of various plants,and the kinase domain,phosphorylation functional site and cysteine site of PTI1 protein are conserved among many different species.Transient expression of tobacco indicated that the Spo PTI1-3 protein was localized in the cytoplasm and nucleus.In the different tissues and organs of spinach,the Spo PTI1-3 gene had the highest gene expression in the fibrous roots,and the lowest in the first pair of true leaves.The Spo PTI1-3 gene was expressed in the new stems in the different tissues and organs of spinach during the twitching stage,and the expression level was the lowest in the old leaves.Fluorescence quantitative PCR analysis showed that the expression of Spo PTI1-3 gene in spinach heat-resistant Sp75 leaves was decreased compared with the control spinach heat sensitive material Sp73 after treatment at 37 °C,indicating that Spo PTI1-3 under high temperature stress.The expression of the gene is inhibited.However,under the treatment of 200 m M Na Cl,100 m M Na2CO3,200 m M Na HCO3 and 200 ?M Cd Cl2,the expression level of Spo PTI1-3 gene increased,indicating that Spo PTI1-3 responded to salt stress and heavy metal stress.The high temperature response expression pattern of Spo PTI1-3 gene showed that the growth of wild-type yeast strain and Spo PTI1-3 overexpressing yeast strain was inhibited under high temperature treatment at 37 °C,but overexpressing yeast strain was more high temperature than wild type.Sensitive,this indicates that the Spo PTI1-3 gene plays a negative regulatory role under high temperature stress.However,the results of the expression pattern of Spo PTI1-3 gene under different salt treatments showed that under different salt stress,the growth of yeast decreased with the increase of treatment concentration.Wild type and overexpressing yeast strains More sensitive than salt.This indicates that the Spo PTI1-3 protein is able to respond to salt stress.Yeast two-hybrid assay showed no interaction between Spo PTI1-3 protein and Spo OXI1-1 protein.After overexpressing the Spo PTI1-3 gene,the root length of Arabidopsis thaliana was inhibited,but the number of root hairs increased significantly,especially in the mutant gene to complement the Arabidopsis phenotype.Moreover,the overexpression of Spo PTI1-3 Arabidopsis thaliana and the genetically replenished Arabidopsis thaliana were earlier than the control group.This suggests that the Spo PTI1-3 protein may be more potent than the At PTI1-3 protein.