Identification And Analysis Of Interaction Between SmMYB36 And SmCSN5 Protein In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is a perennial herb in the labiaceae family.It is a traditional Chinese herbal medicine.It’s medicinal value is determined by its root and stem,and It’s secondary metabolites,tanshinones and salvianolic acid.Research records show that S.miltiorrhiza is mainly used in the treatment of promoting blood circulation to remove blood stasis,reducing inflammation and eliminating bacteria,dilating blood vessels and so on,and has significant curative effect on cardiovascular and cerebrovascular diseases,pulmonary hypertension,atherosclerosis and other diseases.This study focused on the transcription factor SmMYB36,a key regulatory factor in the secondary metabolite synthesis pathway of S.miltiorrhiza.SmMYB36 was used as bait protein,and the proteins interacting with it were screened through yeast two hybrid,which laid a foundation for further study on the indirect role of post-translational modifications(PTMs)of transcription factor proteins in regulating the synthesis of tanshinones and phenolic acids:The results of this study are as follows:1.pGBKT7-SmMYB36 bait vector was constructed with SmMYB36 as bait protein.A total of 236 clones were obtained by yeast two-hybrid screening of AD library.After sequencing and alignmenting of these clones,27 significant interaction proteins were obtained,and 2 phosphorylated related proteins,2 transcription related proteins,5ubiquitination related proteins,and several other enzymatic proteins were found by sorting.2.Basic bioinformatics analysis was performed on the screened interaction proteins to clarify their related functions.One of the ubiquitination related proteins,numbered190413-2-2,was identified as SmCSN5 protein by BLASTx sequence alignment,local blast and phylogenetic tree analysis and so on.3.The interaction between SmMYB36 and SmCSN5 protein was verified by Y2 H,Bi FC,LCI and Pull-down and other in vitro and in vivo experiments,which confirmed that there was indeed an interaction between SmMYB36 and SmCSN5.SmCSN5 interacts with SmMYB36 in the amino acid region 1-99(JAMM motif)of SmCSN5 and in the amino acid region 112-153 of SmMYB364.The effect of SmCSN5 on the functional localization of SmMYB36 was analyzed,the subcellular co-localization of SmMYB36 and SmCSN5 was carried out by Aturobacterium-mediated tobacco transient expression system,the results showed that SmMYB36 interacts with SmCSN5 and functions mainly in the nucleus.