Study On The Regulation Of The Transcription Factor MYC2 Related To Triterpenoid Saponins In Psammosilene Tunicoides

Psammosilene tunicoides is a single species of Caryophyllaceae,which is the national level two protection plant,and one of the main ingredients in "yunnan baiyao".Its main component is the triterpenoid saponins.It is necessary to find a new way to synthesize the Psammosilene tunicoides triterpenoid saponins,since it is challenging to integrate artificially owing to its complex structure,and it has limited resources.There may be a new way to obtain plenty of triterpenoid saponins in the Psammosilene tunicoides in the future,which research the key enzyme genes regulation in the biosynthetic pathway of the Psammosilene tunicoides and the synthesis of the Tri-saponin of Psammosilene tunicoides by genetic engineering technology.This experiment intent to validate the relationship between transcription factor MYC2 and three key enzymes in the synthesis pathway of Psammosilene tunicoides by Yeast Two-Hybrid.TA cloning,direct construction of fusion expression vector and in-fusion cloning are used to construct The recombinant plasmid.While the technique of Real-time PCR was used to detect Pt?-AS,PtSE1,PtSE2,Pt MYC2 Changes of gene fold expression in respectively in 0h,2h,12 h and 24 h after treatment with methyl jasmonate.Based on the information of transcription group obtained in the previous period,this experiment screens gene annotated as ?-AS,SE1,SE2,and MYC2 from a large number of transcripts,and analyze the transcription and actual sequences in the light of the construction requirements of Yeast Two-Hybrid Bait protein.Simultaneously,the experiment designs the full-length amplification primer of each gene according to the transcription sequence.Firstly,the experiment obtains the fusion expression vectors of two pGADT7-Pt?-AS and pGADT7-PtSE1 by TA cloning,and analyze the Pt?-AS and PtSE1 of the Clones,as well as get the conclusion: the full length of ?-AS is 2715 bp,it has complete ORF,2283 bp,Non-transmembrane protein,non-secretory protein,not suitable for the construction of Bait protein,the SE1 gene is 1855 bp,complete ORF,1467 bp,non-transmembrane protein,non-secretory protein,can be used to construct Bait protein.Two bait fusion expression vectors(the p GBKT7-PtSE1 and pGBKT7-PtSE2)and one fusion expression vector of the prey(pGADT7-PtSE2)were obtained by direct constructing the fusion expression vector.Afterwards,the PtSE2 of the clone was analyzed and concluded that the length of SE2 is 1931 bp,and it has intact ORF,the 1566 bp protein which is not the transmembrane protein.Therefore,it does not meet the requirement of constructing Bait protein.The two fusion expression vectors involving pGBKT7-PtMYC2 and pGADT7-PtMYC2 were obtained using the method ofin-fusion cloning,and PtMYC2 was first cloned from Psammosilene tunicoides.The research concluded that the PtMYC2 gene of the clone is 1932 bp in length,with a complete ORF 1716 bp and encoded 571 amino acids,not the transmembrane protein,non-secretory protein,and it can be used to construct Bait protein after analyzing.The Real-time PCR was used to detect the changes of gene fold expression of PtMYC2,PtSE1,PtSE2 and Pt?-ASgene.Afterwards,getting the result that the relative expression amount in each gene decreased after 2h treatment with MeJA;however,all expression increased in different degree after 12 h Pt?-AS,PtSE1 and PtSE2,only the PtMYC2 was still declining.Finally,the relative expression of four genes increased in varying degrees after 24 h.In conclusion,the relationship between PtSE1 and PtMYC2 may not exist by the experiment of Yeast Two-Hybrid.