Screening And Verification Of Polyphenol Oxidase Interaction Protein From Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is a traditional Chinese herbal medicine.It is widely used in the treatment of cardiovascular diseases,liver protection,antibacterial and anti-inflammatory.It is widely used in clinical treatment at home and abroad.It's Secondary metabolite are mainly divided into phenolic compounds and tanshinones.Among them,salvianolic acid B is a representative of phenolic acid components,mainly synthesized by the phenylalanine pathway and the tyrosine pathway.At the same time,polyphenol oxidase is a family gene,and the research on the polyphenol oxidase gene family of Salvia miltiorrhiza is incomplete.Previous studies by our group showed that Salvia polyphenol oxidase(SmPPO,GenBank accession number KF712274)can positively regulate the accumulation of salvianolic acid B and respond to drought stress,but the response mechanism is not clear.In this paper,the polyphenol oxidase gene family of Salviamiltiorrhiza was analyzed by bioinformatics method,and the SmPPO interaction protein wasexplored by molecular biology method.The relationship between SmPPO gene andsalvianolic acid B synthesis pathway was explored.The following results were obtained:1.Searching for the polyphenol oxidase gene family members from the Chinese traditional medicine database and the Kunming University of Science and Technology published by the HMM Search method,and finally determined that the Salvia miltiorrhiza has 26 polyphenol oxidase gene family members.Intrafamily evolution analysis and interspecies evolution analysis of the SmPPO gene family showed that the evolution within the family was divided into three clusters,and the SmPPO was in the third cluster,and the three clusters were significantly different in number;the polyphenol oxidase family between species In evolution,Salvia miltiorrhiza has a close relationship with the evolution of potato and spinach.The polyphenol oxidase phylogenetic tree between species is arranged in the order of monocotyledons,mites and dicots.2.RNA was extracted from the roots,stems,leaves and flowers of Salvia miltiorrhiza.The tissue-specific expression analysis of SmPPO was carried out.The results showed that the polyphenol oxidase of Salvia miltiorrhiza was mainly expressed in roots and stems,and expressed in a small amount in flowers.Almost no expression in leaves;SmPPO sequence was used as a template to amplify the sequence,and a recombinant 35S-1301-GFP subcellular localization vector was constructed.After transfecting tobacco with Agrobacterium,the fluorescence was observed under bio-laser confocal microscopy to determine the location of SmPPO.On the nucleus,cell membrane and chloroplast.3.The pGADT7 vector was constructed by using the key enzymes in the salvianolic acid B synthesis pathway and the pre-laboratory yeast two-hybrid screening of the positive protein that may interact with SmPPO as a template,respectively,and then with pGBKT7-SmPPO.Co-transformation verification showed that tyrosine aminotransferase(GenBank accession number DQ334606.1,TAT)and phenylalanine ammonia lyase(GenBank accession number EF462460.1,PAL1)interacted with SmPPO;The recombinant p SPYNE(R)173 vector and p SPYCE(M)vector were constructed by coding regions of SmPPO,TAT and PAL1 genes,and the bimolecular fluorescence detection(BiFC)test was carried out by tobacco injection.The results showed that only TAT interacted with SmPPO.PAL1 does not interact with SmPPO.4.The amino acid sequences of SmPPO and TAT were analyzed,and the amino acid sequences of SmPPO and TAT were truncated according to their domains.The bait vector and the prey vector were constructed and tested for co-transformation.The results showed that the SmPPO and TAT interaction sites were SmPPO N.The front part is 126 aa;the truncated TAT domain part does not interact with the full length of SmPPO,indicating that only the complete TAT can interact with SmPPO.5.Subcellular localization analysis of TAT,PPO-N(pre-165aa)and PPO de-N-terminal(166aa-589aa)showed that TAT was localized on the cell membrane;PPO-N(pre-165aa)was located at on the nucleus,cell membrane,and chloroplast;the N-terminal portion of PPO(166aa-589aa)is localized on the chloroplast,and is hardly expressed in the cell membrane and not in the nucleus.