Functional Analysis Of CSE And DHN In Larix Kaempferi

Caffeoyl shikimate esterase?CSE?has been reported to be involved in lignin biosynthesis and may respond to abiotic stress.Dehydrin?DHN?is a hydrophilic protein that is overexpressed during dehydration stress and involved in stress resistance,which is related to the protection mechanism of plant dehydration.This paper studies the function of CSE and DHN genes of Larix kaempferi.According to the transcriptome data of Larix laricina,one CSE gene and four DHN genes were cloned from Larix kaempferi stems,which named with Lk CSE,Lk DHN1,Lk DHN2,Lk DHN3 and Lk DHN4.The homologous sequence alignment and phylogenetic analyses from different species indicated that Lk CSE is likely to have catalytic activity and CSE appears before the divergence of angiosperms and gymnosperms,but CSE in most monocots may get lost during evolution,and Lk DHN belong to the SK₃type dehydrin.The subcellular localization of Lk CSE was observed to be endoplasmic reticulum and plasma membrane,and the subcellular localization of Lk DHN was located in the nucleus.The CSE and DHN of Larix kaempferi were induced by prokaryotic expression and purified by affinity chromatography,and finally Lk CSE,Lk DHN,Lk DHN?K and Lk DHN?S were obtained.In vitro assays with the recombinant enzyme validated the catalytic activity of Lk CSE,indicating its function in converting caffeoyl shikimate into caffeate and shikimate.Additionally,the optimum reaction p H and temperature of Lk CSE were determined to be 6.0 and 30°C,respectively.The values of K_m and V_max of Lk CSE for caffeoyl shikimate were 98.11?M and 14.44 n M min^-1,respectively.The result of electrophoretic mobility shift assay indicated that the Lk DHN can non-specifically bind to DNA,and may have the function of protecting or repairing DNA.The binding ability of Lk DHN?K and DNA almost disappeared,indicating that K segment plays an important role in the interaction between DHN and DNA.This paper provides a reference for the study on the stress resistance of CSE and DHN of Larix kaempferi.