Cloning And Characterization Of ?-bisabolol Synthase Gene (MrBBS) From Matricaria Chamomilla L.

Chamomile(Matricaria recutita L.)is a kind of herb from Genus Matricaria in Asteraceae,which have anti-inflammatory,antibacterial,anti-oxidative,antihypertensive,lipidlowering,anti-tumor and calming effects.Its main medicinal active components are chamazulene and ?-bisabolol,which are often used as one of the evaluation criteria for the quality of chamomile.?-bisabolol is the part of sesquiterpenoids,and its biosynthetic pathway belongs to the isoprenoid metabolic pathway.On the one hand,we detected the content of volatile oil components and analyzed the content of ?-bisabolol from chamomile disc flowers(DF)and ray flowers(RF).On the other hand,we screened out ?-bisabolol synthase gene from transcriptional data of chamomile.And we described the cloning and functional analysis of the ?-bisabolol synthase genes from chamomile,which laid the foundation for improving the content of ?-bisabolol in the volatile oil of chamomile in molecular level.The main results were as follows:1.The components of volatile oil in DF and RF from chamomile were determined by gas chromatography-mass spectrometry(GC-MS)analyses.The results showed that 21 and 19 components were identified from the volatile oils in DF and RF,respectively,for a total of 30 components.The main contents of the volatile oils in DF and RF were ?-bisabolol oxide A,?-bisabolol oxide B,chamazulene and ?-bisabolol.And the content of ?-bisabolol in DF was higher than that in RF.2.In our study,the ?-bisabolol synthase genes were screened and identified from chamomile through the analysis of transcriptional data and named Mr BBS.The homology of the Mr BBS was compared and analyzed to confirm the open reading frame(ORF).We cloned the sequence of Mr BBS from chamomile by PCR amplification with specific primers.The ORF of Mr BBS1 gene of chamomile was 1,719 bp,which encoded 572 amino acids.The deduced amino acid molecular weight was 66.82 k Da and isoelectric point(p I)was 5.42.The length of Mr BBS2 gene ORF was 1,701 bp,which encoded 566 amino acids.The protein molecular weight was 66.60 k Da and p I was 5.48.We predicted the structure of Mr BBS protein through bioinformatic method.3.We successfully constructed the prokaryotic expression of chamomile Mr BBS gene in vitro,and optimized the protein expression conditions to obtain the recombinant protein.The recombinant proteins of Mr BBS1 and Mr BBS2 were 83 KDa and 81 KDa,respectively.The enzymatic reaction in vitro demonstrated that the Mr BBS1 protein can catalyze FPP substrate into ?-bisabolol,and inferred it has ?-bisabolol synthase activity.4.The levels of expression of Mr BBS from DF,RF,root,stem and leave in chamomile during peak flowering stage were analyzed by q PCR.The results showed that the level of Mr BBS expression was the highest in roots and the lowest in stem.The expression levels from high to low were roots,disc flowers,ray flowers,leaves,stems.5.The recombinant eukaryotic expression vector was constructed,and transformed into Agrobacterium tumefaciens EHA105 by electric shock,which was then injected into Nicotiana benthamiana L.cells for transient expression.Result of confocal laser scanning microscope showed that both Mr BBS1 and Mr BBS2 proteins were located on the chloroplast.