Cloning And Expression Analysis Of MAPK Gene From Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is the herbal medicines whose dry root and steam is known as traditional Chinese medicine Danshen and salvianolic acid B is one of the main medicinal components. Mitogen activated protein kinase(MAPK) is a big family belonging serine/threonine protein kinase widely existing in animals and plants. MAPK can be induced by various biotic and abiotic stresses and involved in growth, development and the process of signal transduction of stress responses. We obtained the complete nucleotide sequences of a MAPK gene and cloned it from Salvia miltiorrhiza Bunge cell suspension culture using rapid-amplification of c DNA ends. And the c DNA and amino acid sequences were analyzed by bioinformatics software. We used quantitative reverse-transcription polymerase chain reaction(q RT-PCR) to monitor the expression of the MAPK after treatment with salicylic acid(SA), methyl jasmonate(Me JA), hydrogen peroxide(H2O2), sodium nitroprusside(SNP), calcium chloride(Ca Cl2), lack of calcium ion and sodium chloride(Na Cl) to Salvia miltiorrhiza Bunge cell suspension culture. Polyethylene glycol(PEG) was used to induce green fluorescent protein introduced into protoplast of S. miltiorrhiza for deceiving the fluorescence of fusion protein by laser scanning confocal microscope, so that we can make sure the location of the MAPK we cloned. In order to study the function of MAPK in the synthesis of salvianolic acid B, the contraction of over-expression and RNA interference vectors of Sm MAPK6 were introduced into S. miltiorrhiza by leaf disk method. The main results were as follows:1、The first c DNA encoding MAPK was cloned and named Sm MAPK6. The full-length c DNA sequences were 1467 bp with an 1122 bp open reading frame which encodes 373 amino acids, a 44 bp five prime untranslated region and a 301 bp three prime untranslated region.2、Bioinformatics analysis showed that Sm MAPK6 in Salvia miltiorrhiza Bunge had a 42.67 KD molecular weight and 5.67 PI value, which belonged to hydrophilic protein with no signal peptide and transmembrane structure found. It is a member of mitogen activated protein kinase family TEY subfamily that belongs to the serine/threonine protein kinase family, and contains activation site, ATP binding site,peptide substrate binding site A-loop and kinase docking site. Protein sequence alignment shows it is similar with Ga MAPK3 and Gh MAPK9 with the percent of 85%, and it has 11 basic motifs with TEY motif was included between Ⅶand Ⅷ. Phylogenetic analysis showed that it was a member of group A.3、q RT-PCR analysis exhibit a higher m RNA level expression in leave than in root and steam of S. miltiorrhiza, while SA, Me JA, H2O2, NO,Ca2+ and Na Cl are both improve the expression of Sm MAPK6 in m RNA level, and there is a more significant increase in the response to H2O2.4、Test of transient expression of green fluorescent protein gene transferred into protoplast of Salvia miltiorrhiza by PEG shows Sm MAPK6 located both in nucleus and cytoplasm.5、Over-expression and RNAi vectors of Sm MAPK6 were successfully constructed. This work is prepared for the fuether study of the function of Sm MAPK6 in the accumulation of salvianolic acid B in S. miltiorrhiza.