Cloning And Expression Analysis Of SA Binding Protein 2 Gene In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge?S.miltiorrhiza?is the herbal medicines whose dry root and steam is known as traditional Chinese medicine Danshen.It was widely used to treat cardio-cerebrovascular disease.Salicylic acid binding protein?SABP?,which was initially found in tobacco,belongs to the alpha/beta folding hydrolase family.SABP specifically bindes with salicylic acid?SA?,plays an important role in the SA signaling pathways.In this study,the SABP2 was cloned from S.miltiorrhiza suspension culture cells.The cDNA and amino acid sequences of SABP2 were analyzed by bioinformatics software.The over-expression and prokaryotic expression vectors of SABP2 were successfully constructed.After IPTG inducing in vitro,the SmSABP2 protein were acquired,and the esterase activity of SABP was tested.Using qPCR method,the relative expression level of SABP2 under SA induction were dected.Achived the following main results:1.The first cDNA encoding SABP was cloned and named SmSABP2.The full-length cDNA sequences were 1221 bp with an 780 bp open reading frame which encodes 259 amino acids.2.Bioinformatics analysis showed that SmSABP2 in S.miltiorrhiza had a 28.81 kDa molecular weight and 5.48 PI value,which belonged to hydrophobic protein with no signal peptide and transmembrane structure found.Secondary structure prediction show that the SmSABP2 by the 44.79% of the alpha helix,20.85% of the extension chain,28.19% of the random curl and 6.18% of the beta of folding composition.Structure domain analysis results show that the function of the SmSABP2 belongs to the family of esterase,containing a Abhydrolase₆ structure domain and a nucleophilic elbow structure domain3.The SmSABP2 prokaryotic expression vector were constructed.After IPTG inducing in vitro,the SmSABP2 protein were acquired,which showed high esterase activity.The expression level of SmSABP2 was increased after SA induction,which indicated that SmSABP2 was induced by SA.4.The over-expression vectors of SABP2 was successfully constructed in Salvia miltiorrhiza,which will provide help for futher functional study of SABP2 in plant secondary metabolism.