Cloning And Expression Analysis Of Polyphenol Oxidase Gene In Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge is a traditional Chinese medicinal materials in the mint family.It displays in most parts of our country, but mainly in Shaanxi, Henan and Anhui province. Asmore advanced exploration of effective component and deep understanding of the synthesispathway in Salvia miltiorrhiza Bunge, the study of its related functional genes is urgent andnecessary.The enzyme polyphenol oxidase (PPO) was wide spread in Kingdom Plantae, someresearches showed it can response to drought stress and had tightly relationship with thecontent of the total phenolic acids. But whether PPO can regulate the bio-synthesis of thephenolic acids still need to be further addressed.Hairy root of Salvia miltiorrhiza Bungeemploy was employed as the material. The firststrain of cDNA encoding PPO from Salvia miltiorrhiza hairy roots was cloned andcharacterized. Multiple sequence alignments of PPO genes and proteins in Tubiflorae werecompared by bioinformatics analysis. Process Salvia miltiorrhiza hairy roots by ascorbic acid,yeast extract, Ag+and L-cysteine, expression analysis of PPO gene was quantified byreal-time PCR and their effects on the accumulation of salvianolic acid B were evaluated byhigh performance liquid chromatography (HPLC) method, we get the following results:1、The first cDNA encoding PPO was cloned. Gene bank accession number isKF712274. The full-length cDNA of PPO is1930bp with a1769bp open reading frameencoding589amino acids. Sequence alignments of PPO genes and proteins in Tubifloraerevealed that SmPPO showed homology to other PPOs.2、Bioinformatics analysis showed that PPO in Salvia miltiorrhiza Bunge had a65.8KDmolecular weight and6.56kD PI value, which belonged to hydrophilic protein but no signalpeptide found. PPO exhibiting tryosinase family characteristics, which has PPO1_DWL motifand PPO1_KFDV motif.8α-helix and7β-sheet were found spreading in the wholepolypeptide chain. Two highly conserved N-myristoylation sites were firstly found at theC-terminus of thylakoid transfer domain, and a casein kinase II phosphorylation site wasfound in the N-terminus of DWL motif.3、Processing Salvia miltiorrhiza hairy roots by different reagents, expression analysisof real-time PCR indicated that PPO transcriptional levels significantly increased in yeast extract (YE)-elicited hairy roots, whereas it significantly decreased with treatments of Ag+,ascorbic acid and cysteine treatments.4、Processing Salvia miltiorrhiza hairy roots by different reagents, expression analysisof high performance liquid chromatography (HPLC) method indicated that accumulation ofsalvianolic acid B significantly increased in yeast extract (YE)-elicited hairy roots, whereas itsignificantly decreased with treatments of Ag+, ascorbic acid and cysteine treatments.