Cloning Of Ginsenoside-?-rhamnosidase Gene

In this paper,the clone of ginsenoside-a-rhamnosidase's gene sequence from Absidia sp.G3g was studied.For this enzyme can particularity hydrolyze Ginsenoside Re into Ginsenoside Rg1.The proper method to isolate total RNA from Absidia sp.G3g and to improve TakaRa RNAiso Reagent was defined from several ways for total RNA abstracting.The RNA ed in this way was examed to be completed and was detected with clear 5S,18S and 28Scomplete subunits in agarose gel electrophoresis.The primers were designed according to the homologous N terminal amino acids sequence of the ginsenoside-a-rhamnosidase from microorganisms Absidia sp.G3g.The unknown part sequence of gene CDS was amplificated by RT-PCR.Then aim primes,designed by above sequece,was amplificated in RACE to produce the complete gene sequence of ginsenoside-a-rhamnosidase from Absidia sp.G3gThe complete gene in CDS of ginsenoside-a-rhamnosidase was firstly isolated,then ligated to pMD19-T Simple Vector and transformed into E.coli competent cell JM109.The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes.The results showed that clones were obtained which containing recombinant plasmids with the target fragment incorporated.It was found to have no homegeneity with a kown rhamnosidase through the homogeneity comparison with the DNA described in NCBI after obtained DNA sequencing.