Cloning And Functional Analysis Of SOS Gene From Desert Plant Karelinia Caspia

Due to geography,environment and climate,and abiotic stress is one of the important reasons for the decline in crop yield.Vegetation loss due to abiotic stress reaches 6%?20%,and the global salinized soil accounts for 5%of the land area.Saline alkali land is a part of ecosystem.Its unique physical and biological characteristics often lead to the abnormal circulation of ecosystem material and energy,resulting in the waste of ecological resources,fragile ecological environment,and serious economic and secondary disasters.The signal pathway of salt hypersensitivity(SOS)stress is related to ion stability and salt tolerance.SOS signaling pathway consists of three main proteins:SOS1,SOS2 and SOS3.Through the cloning of SOS pathway genes,this study provides gene resources and research basis for the application of functional genes.Karelinia caspia less is a desert plant widely distributed in the Tarim Basin of Xinjiang.Because it often grows in deserts and other natural environments,firewood has strong drought resistance,high temperature resistance,salt resistance and other stress resistance.It is one of the most important plant resources to study the stress resistance of desert plants,and provides gene resources for crops such as cotton.This paper aims to clone the genes related to the SOS pathway of firewood and analyze its gene function.The main results are as follows:(1)The SOS pathway related genes SOS2 and SOS3 genes were successfully cloned and analyzed by bioinformatics.The sequencing sequence of SOS2 was analyzed.The results showed that its length was1404 bp and encoded 467 amino acids.It was named Kc SOS2.The sequencing sequence of SOS3 was analyzed.The results showed that its length was 651 bp and contained 216 amino acids.Therefore,it was named Kc SOS3.NCBI online comparison showed that the similarity of amino acid sequences between Kc SOS2 and sunflower SOS2 was the highest,up to 90.83%;Kc SOS3 and sunflower SOS3 had the highest amino acid sequence consistency,up to 90.32%.(2)The gene expression patterns of Kc SOS2 and Kc SOS3 in Karelinia caspia were analyzed under the four abiotic stress conditions of 300 mmol/L Na Cl and 100 mmol/L Na₂CO₃,natural drought for 6 days,45?high temperature and 4?low temperature,and 15%PEG osmotic stress.The results showed that the expression of SOS3 was similar in untreated roots,stems and leaves,but higher in roots than in stems and leaves.It indicates that Kc SOS3 is expressed in tissue-specific manner.The expression of Kc SOS3gene in roots and stems showed a low high low trend under saline alkali stress;The expression in leaves showed a downward trend.The expression of Kc SOS2 in roots,stems and leaves decreased.When Kc SOS3gene was treated under drought conditions,the expression of Kc SOS3 gene in roots,stems and leaves showed a high low trend.The expression of Kc SOS2 decreased in roots,stems and leaves.At 45?,the expression of Kc SOS3 gene showed a high low high trend in roots and a low high low trend in stems;The expression in leaves showed a downward trend.The expression of Kc SOS2 increased in the stem;The expression in leaves showed a high low high trend.When Kc SOS3 gene was treated with low temperature at 4?,the expression of Kc SOS3 gene in roots showed a low high trend;The expression in the stem showed a downward trend.When Kc SOS2 gene was treated at low temperature at 4?,the expression of Kc SOS2 in roots,stems and leaves showed a downward trend.When Kc SOS3 gene was treated with 15%PEG,the expression of Kc SOS3 gene in roots,stems and leaves showed a low high trend.The expression of Kc SOS2 in roots,stems and leaves showed a downward trend.(3)Prokaryotic overexpression vectors p ET-28a-Kc SOS2 and p ET-28a-Kc SOS3 were successfully constructed for prokaryotic expression analysis.Using the designed primers containing double enzyme digestion sites,Kc SOS2 and Kc SOS3 plasmids with enzyme digestion sites were obtained and recombined with p ET-28a in E.coli DH5?save in.The expression of Kc SOS2 and Kc SOS3 was induced in E.coli BL21,and the protein bands were about 50k Da and 24k Da,indicating that Kc SOS2 and Kc SOS3 genes can be expressed in prokaryotic.(4)Eukaryotic overexpression vectors p K2GW7-Kc SOS2 and p K2GW7-Kc SOS3 were successfully constructed,and transgenic tobacco,cotton and arabidopsis were transformed.The obtained firewood genes SOS2 and SOS3 were constructed on p K2GW7 plant expression vector by gene recombination(Gateway)technology.The plant expression vectors p K2GW7-Kc SOS2 and p K2GW7-Kc SOS3 were obtained,and the obtained vectors were introduced into Agrobacterium GV3101 strain.The hypocotyls of tobacco and cotton were transformed by Agrobacterium mediated leaf disc method and Arabidopsis was transformed by flower dipping method.