Gene Cloning And Catalytic Function Study Of Steroidal Saponin 26-O-β-glucosidase From Anemarrhena Asphodeloide

Objective Anemarrhena asphodeloides Bunge(Zhimu)is an important traditional Chinese medicine herb that contains more than 50 steroidal saponins,and exhibits hemolytic,cytotoxic,anti-inflammatory properties.Zhimu accumulates two types of steroidal saponins:the spirostanol and furostanol derivatives,the aglycone F-ring is a six-membered pyran ring or five-membered furan ring respectively.In contrast to spirostanol glycosides,furostanol glycosides do not show typical saponin characteristics.Upon cell damage,plant releases a key enzyme to immediately transform furostanol glycosides into more aggressive antifungal active spirostanol glycosides for plant against fungal attack.The enzyme responsible for this conversion is called furostanol glycoside 26-O-β-glucosidase(F26G),and it is the key gene involve the biosynthesis pathway of steroidal saponins.Secondly,Zhimu is mainly planted in the north,and the real estate area is Yixian,Hebei.Geographical ecological environment is the main factor that produces th e authenticity of medicinal materials,while water and temperature are the main differences between north and south.Therefore,we designed drought stress by single factor to verify whether water is related to gene expression and accumu lation of major components of Zhimu,Methods(1)The candidate gene with high expression,furostanol glycoside 26-O-β-glucosidase(F26G),was screened out by transcriptome data of rhizomes and aboveground parts of Zhimu.And then Designing AaF16G1 gene homologous arm primer for gene cloning.The cloned gene was ligated into the vector pCold I,which was transformed into E.coli BL21(pGro7)for protein expression.The purified AaF26G1 enzymes are used to react with Steroid saponins and different skeleton triterpenoids in vitro,which was used for substrate testing.(2)Predict the key catalytic sites of AaF26G1 by homology modeling with other types of β-glucosidases of known crystal structure.And then verify the catalytic activity of the enzyme by site-directed mutagenesis.(3)Design in vitro antifungal experiment to investigate the antifungal effect of Timosaponin BⅡ and Timosaponin AⅢ.(4)Designing drought stress experiment to detect and analyze the metabolites and transcriptomes of Zhimu,and to investigate the gene expression level and metabolites accumulation of Zhimu.Results(1)The complete open reading frame(ORF)sequence of AaF26G1 was 1650 bp.AaF26G 1 is belong to β-glucosidase.The enzyme has high substrate specificity in the biotransformation of furostanol glycosides to spirostanol glycosides,and is an ideal glucosidase for biotransformation of steroidal saponins.(2)At present,the glutamic acid such as the single mutation at positions 240,455,and 511 is alanine,and the catalytic function of the enzyme is weakened or even disappeared.The activity of 504,512,520 aromatic amino acids was weakened,and the activity of 311 and 313 amino acids with free hydroxyl groups was weakened.(3)The antifungal effect of steroidal sapon ins indicates that Timosaponin AⅢis better than Timosaponin BⅡ.And Timosaponin AⅢ is better than Validamycin in antifungal.(4)There were 41 peaks,originating from Zhimu leaves and rhizome of metabolomic analysis,which had significant difference.At this stage,a preliminary analysis of the transcriptome data was carried out,in which found a total of 5333 Unigenin differentially expressed genes.Conclusion In this study,we functional characterized a new AaF26G1 genes that coding aβ-glucosidases from the transcriptome of Zhimu.In vitro assays AaF26G1 could catalyze the cleavage of the C-26 glucosidic bond of furostanol glycosides efficiently.AaF26G1 showed a relative higher specificity,only 4 similar furostanol glycosides could be used as the suitable substrate from 12 structurally diverse steroid saponins,triterpernoids and flavonoids.In addition,the catalytic mechanism of AaF26Gl confirmed by site-directed mutagenesis that the key amino acid residue of AaF26G1 was 240,455 glutamic acid.The results of in vitro antibacterial experiments indicate that AaF26G1 is involved in insect resistance.The accumulation of timosaponin BⅡ in the roots of drought group was more likely to be related to the syn thesis mechanism of steroidal saponin.