Spinach SpoRaf28 Gene Cloning And Function Analysis Of High Temperature Response

Abiotic stress has a significant effect on plant growth,and high temperature stress seriously affects plant growth and yield.Studying the molecular mechanism of plant high temperature response is of great significance for crop improvement.Spinach?Spinacia oleracea L.?is a cold-tolerant vegetable that is widely grown in northern China.Spinach is not suitable for long-distance transportation,which leads to insufficient supply of spinach in the south in summer.It is of great significance to cultivate heat-resistant spinach varieties and promote planting in the south.We have cultivated spinach heat-resistant material Sp75 and began to study the high-temperature response mechanism of spinach by molecular biology.The mitogen-activated protein kinase?MAPK?cascade signaling pathway plays an important role in plant development and high temperature response.This pathway functions by sequential phosphorylation by the cascade-activated tertiary kinases MAP3 K,MAP2K and MAPK.There are 35 MAP3 K and 5 MAP2 K in the spinach genome,and the relationship between high temperature response pattern and mutual regulation is not clear.This study analyzed 35 members of the Spo MAP3 K family in a genome-wide manner,with Arabidopsis thaliana?Beta vulgaris L?Chenopodium quinoa Willd?Cucurbita moschata L?Cucurbita maxima Duch?Cucumis sativus L,and bamboo shoots.The Raf28 protein of plants such as Cucurbita maxima Duch.and Cucumis sativus L is highly homologous,and the kinase domain,phosphorylation functional site and cysteine site of the protein are relatively conservative.We cloned the Spo Raf28 gene in the Spo MAP3 K family,which has a maximum open reading frame of 1101 bp and encodes a total of 366 amino acids.Subcellular localization of Spo Raf28 using tobacco transient expression technology revealed that the protein was localized in the cytoplasm.Real-time PCR analysis showed that there was no significant difference in the expression level of Spo Raf28 gene in spinach heat sensitive material Sp73,while in the heat-resistant material Sp75,the expression level of Spo Raf28 gene increased significantly.The expression level of Spo Raf28 gene in spinach material Sp75 was significantly different in different tissues and organs during the six-leaf stage and the twitch stage,and the highest expression was observed in both leaves.After the spinach material Sp75 was treated at 37 ? for 72 h,the leaves showed obvious wilting,and the expression of Spo Raf28 gene increased.However,under the conditions of different saline-alkali?NaCl,Na₂CO₃,NaHCO₃?and heavy metal?CdCl₂?stress,the leaf phenotype was absent.Obviously,the expression level of Spo Raf28 gene decreased,indicating that Spo Raf28 was induced by high temperature,but was inhibited by salt and heavy metal stress.Under the high temperature treatment at 37 ?,the growth of both the yeast strain overexpressing the Spo Raf28 gene and Arabidopsis plants was promoted,indicating that Spo Raf28 plays a positive regulatory role in the high temperature response process.The results of the yeast two-hybrid assay indicated that Spo Raf28 did not interact with Spo MAP2 Ks.Our results indicate that the MAP3 Kfamily member Spo Raf28 plays a positive regulatory role in the spinach high-temperature response signaling pathway,providing new evidence for studying the molecular regulation mechanism of the MAPK cascade pathway.