Cloning And Functional Analysis Of The Panax Ginseng Glycoside Hydrolase Genes

Objective The Panax ginseng Glycoside Hydrolase（PgGH）Genes from Ginseng was cloned from adventitious roots of ginseng and studied for its function.To confirm the catalytic function of PgGH.Methods（1）q PCR was used to screen PgGH gene fragments screened in the laboratory in the early stage,and then PgGH gene full length was cloned by RT-PCR.（2）Obtained PgGH Genes was analyzed by bioinformatics.（3）PgGH gene was expressed prokaryotic and cell-free protein expression to obtain PgGH protein.（4）Ginsenoside were transformed by PgGH protein and exogenous glycosidase and their transformation types and products were compared.Results（1）Good quality RNA was extracted from the young adventitious roots of ginseng,and four PgGH Genes were cloned by reverse transcription and PCR,which were PgGH-1,PgGH-2,PgGH-3,PgGH-4.（2）Bioinformatics analysis results are as follows.PgGH-1 gene was 1866 bp in length and encoded 621 amino acids;PgGH-2gene was 1857 bp in length and encoded 618 amino acids;PgGH-3 gene was 1899 bp in length and encoded 632 amino acids;PgGH-4 gene was 1884 bp in length and encoded 627 amino acids.The molecular weights of the four PgGH proteins ranged from 67-70 k D;the instability index were all less than 40,it is inferred that the four proteins are stable;the theoretical p I of proteins are all between 6.10 and 8.90;PgGH-1 protein and PgGH-2 have approximately 20 aa transmembrane domains,and PgGH-3 protein and PgGH-4 do not contain transmembrane domains;the secondary structures of the four PgGH proteins are composed of a certain proportion ofα-helix,β-sheet,β-turn and random coils,among whichα-helix and random coils are the main structures of the four glycosidase proteins.The phylogenetic tree results are as follows,the four PgGH proteins were divided into two branches,among which PgGH-1 and PgGH-2 were closely related to malic glycosidase,and PgGH-3 and PgGH-4 were closely related to sesame glycosidase（3）Using E.coli competent cells for prokaryotic expression,and changing the DE3 strain,IPTG concentration,induction temperature and other conditions did not get the target protein.However,the target protein was obtained by adding linear template DNA to the wheat germ cell-free protein expression system and reacting at 25°C for 2 hours.（4）The enzyme activities of different glycosidases were determined,the enzyme activities of each glycosidase were as follows:commercialβ-glycosidase,Lactobacillus plantarum,Pediococcus lactis,PgGH-4,PgGH-2,PgGH-3,PgGH-4.PgGH and the glycosidases produced by liquid fermentation of two lactic acid bacteria and commercial glycosidases respectively hydrolyze ginsenosides Rb₁,Rd,Rg₁at 37°C,the results showed that PgGH can convert Rb₁into Rd alone,but not Rg₁and Rd;glycosidases obtained by fermentation of lactic acid bacteria can transform Rb₁and Rd to produce Rg₃and CK,and Rg₁into Rb1 and Rg₃;commercial glycosidases can transform Rb₁into Rd,and continue to transform Rd into other products,but cannot transform Rg_1.Conclusions The four PgGH genes cloned from the adventitious roots of ginseng can induce the expression of PgGH proteins,and the PgGH proteins can transform ginsenoside Rb₁into Rd alone.The ginseng glycosidase gene was preliminarily analyzed to enrich and perfect the research of ginsenoside metabolism pathway.