Transformation Screening And Identification Of DS Gene Of Panax Ginseng In Physcomitrella Paten

Ginseng as a traditional Chinese medicine(TCM)has a wide range of pharmacological effects and clinical efficacy.It has the reputation of the "king of the drug".Modern pharmacological studies have shown that the main effect component of ginseng is ginsenoside.Dammarane type ginsenoside is the main type of ginsenosides.However,as the main source of ginsenosides,the utilization rate of ginseng and American ginseng is very low which with high cost.At present,studies have been tried to improve the yield of ginsenosides by tissue culture and biotransformation.However,there are still restrictions on the expansion of industrial production.With the discovery of the biosynthetic pathway of ginsenoside in ginseng,it is possible to produce ginsenosides by combinatorial biosynthesis.Moreover,the studies had shown that the expression of ginsenosides in Escherichia coli and yeast has been realized.However,the production of protein in E.coli and yeast still has some problems,such as it is easy to form inclusion bodies,and the produced protein cannot be glycosylated modification.Physcomitrella patens is a new model organism,which has been studied for a long time.And it has its own unique advantages:a relatively simple developmental pattern and short growth cycle,The moss Physcomitrella patens offers unique properties as a contained system for protein production.It is grown in the predominant haploid gametophytic stage as tissue suspension cultures in photobioreactors.Efficient secretory signals and a transient transfection system allow the secretion of freshly synthesized proteins to the surrounding medium.The key advantage of Physcomitrella patens compared to other plant systems is the feasibility of targeted gene replacements.What’s more,it has the highest rate of homologous recombination in plants,which could be equal to yeast,enjoy the "green yeast"reputation.It can be grown in a photo reactor,which is easy to be controlled manually.It also has the terpenoid metabolic pathway,which is easy to be modified.It can also be used to modify and express the heterology gene.So far,we have been able to produce the non-allergenic human proteins such as human vascular endothelial growth factor(VEGF),antibody IgG1,IgG4 and erythropoietin,etc..The darmmarenediol-Ⅱ synthase(DS)can catalyze the of 2,3-oxidosqualene to form darmmarenediol-Ⅱ,which is the first key enzyme of dammarane type ginsenoside biosynthesis.In this study,the DS gene was transferred into Physcomitrella patens to express and construct the transgenic plant expressing DS.Part 1:The construction of expression vector phsp-DS-NPTObjective:Construction of the expression vector phsp-DS-NPT of ginseng DS gene in Physcomitrella patensMethods:First,the cloning vector containing pgDDS gene was obtained from Dai Zhubo Teacher in Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences.This thesis use DS to show this gene.The plasmid was amplified and then the DS gene with His tag was amplified,and the Sma Ⅰ and Sac Ⅰ cleavage sites were added upstream and downstream respectively.First,the cloning vector was constructed using TA clones with pEASY Blunt Zero,After PCR,double enzyme digestion and sequencing analysis,the correct clones were cloned and named pEASY DS Zero.After that,we constructed the expression vector phsp-DS-NPT of ginseng DS gene in Physcomitrella patens using the double enzyme digestion with phsp-HB-NPT preserved in our laboratory.Conclusion:We successfully amplified DS gene contains His tags,the total length of amplified fragment is 2341bp.The Blast aligning analysis with DS gene(accession No.GU183405.1)sequences alignment similarity is 100%,prove the sequence was correct.Then construct the expression vector using double enzyme digestion connection with phsp-HB-NPT.Then extract the plasmid using Sma Ⅰ and Sac Ⅰ digested.Agarose gel electrophoresis showed that there are two single bright bands in 2-3kb and more than 10kb,which is consistent with the predicted 2341bp and 10kb,this proved that the expression vector phsp-DS-NPT of ginseng DS gene in Physcomitrella patens was constructed successfully..Part 2:The transformation and selection of PEG mediated Physcomitrella patensObjective:The preparation of protoplast,and PEG mediated transformation was used,then screen out G418 resistant Physcomitrella patens.Methods:In the medium of PPNH4 culture,2g/L ammonium tartrate was added to induce the formation of protonema,and protoplasts were prepared by digestion with 2%driselase.Before transformation,the vector was linearized by Sac Ⅱ,and the linearized phsp-DS-NPT plasmid was transferred into the protoplasts by PEG mediated method.The protoplast concentration was 1.2×106/ml.The initial conversion system is as follows:100μl 0.25μg/μl linear DNA,250μl protoplasts,and 350μl PEG solution.After incubation at room temperature,3ml protoplasts were centrifuged and then regenerated by suspension.Cultured in a 3cm culture dish and incubated overnight at 25℃ dark.Then cultured for 10 days under normal conditions,lml regenerated protoplasts were transferred into a solid medium plate coated with cellophane and cultured for 3 days.The cellophane with regenerated protoplasts was transferred onto a solid medium containing 4mg/L G418 for 2 weeks.Transfer to normal medium for 2 weeks.Finally,we obtained the Physcomitrella patens which were resistant to G418.Conclusion:After the digestion of 2%digestion about 45min,the protoplasts formed were intact and sufficient in number.The average regeneration rate of protoplasts was 0.99%,and the average survival rate was 57.76%after antibiotics screening.Further identification is needed for plants resistant to G418.Part 3:The identification of transgenic Physcomitrella patensObjective:Identify the transgenic Physcomitrella patens plants which could express DS.Methods:For the continued identification of the Physcomitrella patens plants which were resistant to G418,the primers were designed according to the cDNA sequence DS gene,the amplified fragment size was 886bp,located in 411-1297bp.according to the Physcomitrella patens homology arm upstream sequence to design primers,the amplified fragment was 593bp,according to the Physcomitrella patens GAPDH sequence to design primers,the amplified fragment was 206bp.The expression protein size of DS gene is 85.5kDa.Genomic DNA,RNA and total protein were extracted by kit to PCR,RT-PCR and Western blot analysis respectively.Wish to obtain the transgenic transgenic Physcomitrella patens could expressing DS gene.Conclusion:PCR and RT-PCR agarose gel electrophoresis showed that the transgenic plants can amplify single bright band in 800-900bp,while the wild type in the same position did not amplify bands.For the amplification for homologous arm sequences and GAPDH sequences,both wild-type and transgenic plants can display a single bright band between 500-600bp and 200bp,respectively.For Western blot the transgenic plants appeared a clear and bright band in 70-90kDa,wild type and empty vector control showed weak bands,the above results showed that we finally acquired transgenic Physcomitrella patens,which could express DS gene.