Biosynthesis of Panax notoginseng Saponins Regulated by R2R3-MYB Transcription Factor PnMYB1

doi:10.13560/j.cnki.biotech.bull.1985.2021-0981

Abstract

Abstract:

This work aims to clarify the regulation role of transcription factor PnMYB1 to Panax notoginseng saponins（PNS）biosynthesis. RACE technique was applied to obtain the full length of PnMYB1 gene,the phylogenetic tree analysis of PnMYB1 were carried out. The plant overexpression vector of PnMYB1 was constructed and infected P. notoginseng cells,and the contents of ginsenosides R1,Rg1,Re,Rb1 and Rd in the transgenic P. notoginseng cells were detected. For instant expression analysis,tobacco leaves were co-transfected with PnMYB1 and the gene promoters of some key enzymes involved in the biosynthetic pathway of PNS,including squalene synthase（PnSS）,squalene epoxidase（PnSE）,dammarenediol synthase（PnDS）and cycloartenol synthase（PnCAS）. The GUS expression system was employed to confirm whether PnMYB1 transcription factor interacted with the promoters of key enzyme genes in the biosynthetic pathway of PNS. The results showed that PnMYB1 transcription factors belonged to the R2R3-MYB family. In PnMYB1-overexpressed cells of P. notoginseng,5 important triterpenoid saponins increased to some extent in the transgenic cells. Further analysis confirmed that PnMYB1 significantly increased the expressions of PnSE and PnDS by activating the promoters of PnSE and PnDS,thus regulating the biosynthesis of PNS. In conclusion,PnMYB1 transcription factor may simultaneously regulate the expressions of two key enzyme genes involved in the biosynthesis of PNS and then affect its biosynthesis.

Key words: Panax notoginseng, saponins, transcription factor, triterpenoid

LEI Jun, CHEN Qin, DENG Bing, ZHANG Jin-yu, LIU Di-qiu, CUI Xiu-ming, GE Feng. Biosynthesis of Panax notoginseng Saponins Regulated by R2R3-MYB Transcription Factor PnMYB1[J]. Biotechnology Bulletin, 2022, 38(5): 74-83.

Figures/Tables 10

Table 1 Primers used for qRT-PCR

引物名称 Primer name	序列 Sequence（5'-3'）
PnGAPDH-F	CTTTGGTTTAAGGAACCCAGAGG
PnGAPDH-R	AAGGGGAGCAAGGCAGTTAGTAG
PnSS-F	CGAGCACTTGACACTGTTGAGGAT
PnSS-R	CTATTGCCTCCTGGTAACCGTTTC
PnDS-F	CAAGCACACGATGGTCACTGGC
PnDS-R	CATTTTGATGGTTGTAAACGAAGCG
PnSE-F	AGGTGAACTTCTACAACCAGGAGGC
PnSE-R	CTCAACCAGAGATGTAACAGTCCCC
PnCAS-F	GAAATTATACCCTGACCACCGT
PnCAS-R	CCAAACCTTTTACACCGAACC
PnMYB1-F	GCAGGTTTAAAGAGATGTGGGAAG
PnMYB1-R	CTGAAGCAGAGGAGGAGTGATTG

Table 2 Primers used for promoter clone

引物名称 Primer name	序列 Sequence（5'-3'）
PnCAS-GSP1	AGCTTTCTCGATGTCCGCAAGCTCTTC
PnCAS-GSP2	GGATTTCCTCCCTCTGCAATTTTAAGC
PnDS-GSP1	TCCCAAAATGCTTAAAGCCTCGATCCA
PnDS-GSP2	CTTGTAGGGCTTATTGTTATGCAGATTGTG
PnSS-GSP1	CATTTCATGTCCCGTTTTCCTGTAAGAAC
PnSS-GSP2	ACAGTGTTTCTTTCCAGCGAGGACTCC
PnDSP-F	ATCGATTCAATACCGTGTGCTACTATGCAAC
PnDSP-R	GGATCCTGGTTATGTGGTGTACATAGATGGC
PnCASP-F	AAGCTTTGAGGGGCCAAATTCGTTG
PnCASP-R	TCTAGACACTCTGCACACAAATTTAGCTCC
PnSEP-F	AAGCTTTTGTGGGTCAGATCAGATGGA
PnSEP-R	GGATCCGGTGTTGGTTGGACGTTCAC

Table 3 Linear regression equations for five major mono-mer saponins

人参皂苷标准品 Ginsenoside standards	回归方程 Regression	R²
Rg1	y = 3944.9x +69.409	0.9997
Re	y = 2646.8x+22.808	0.9999
Rb1	y = 3227.3x+49.436	0.9998
Rd	y = 2953.5x+41.408	0.9998
R1	y = 2705.6x+50.697	0.9996

Fig. 1 Amino acid sequence alignment

Fig. 2 Phylogenetic tree analysis of PnMYB1

Fig. 3 Relative expressions of gene PnFPS（A）,PnDS（B）,PnSE（C）,PnSS（D）,PnMYB1（E）and PnCAS（F）in the T1-T6 cell lines of P. notoginseng WT：Wild type cell of P. notoginseng;**P<0.01 vs control group;*P<0.05 vs control group. The same below

Fig. 4 Content of total saponins in the cell lines of P. notoginseng

Fig. 5 HPLC analysis of transformed PnMYB1 P. notogin-seng cell line A：Standard. B：Wild-type P. notoginseng cell. C：Transformed PnMYB1 T3 P. notoginseng cell line

Fig. 6 Contents of monomer saponins R1（A）,Rg1（B）,Re（C）,Rb1（D）and Rd（E）in T1-T6 transgenic cell lines of P. notoginseng

Fig. 7 GUS fluorescence activity analysis PnDSP1,PnSEP1,PnSSP1 and PnCASP1 refer to the tobacco leaves transgenic with PnDSP1,PnSEP1,PnSSP1 and PnCASP1 promoter fragments,respectively. PnSSP1/PnMYB1,PnSEP1/PnMYB1,PnDSP1/PnMYB1 and PnCASP1/PnMYB1 refers to the tobacco leaves that PnSSP1,PnSEP1,PnDSP1 and PnCASP1 promoter fragments co-transfected with PnMYB1,respectively. PBI 121：PBI 121-transfected wild-type tobacco leaves. WT：Wild tobacco leaves.（** P < 0.01 vs PnDSP1;and ## P <0.01 vs PnSEP1）

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