Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins

doi:10.3969/j.issn.1004-1524.2022.03.02

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (3): 428-436.DOI: 10.3969/j.issn.1004-1524.2022.03.02

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Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins

CHEN Qin¹^,²(), LIU Meijia¹^,², LIU Diqiu¹^,², QU Yuan¹^,², CUI Xiuming¹^,², GE Feng¹^,²^,^*()

1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
2. Key Laboratory of Panax Notoginseng Resources Sustainable Development and Utilization,Kunming 650500, China

Received:2021-07-26 Online:2022-03-25 Published:2022-03-30
Contact: GE Feng

Abstract

Abstract:

Farnesyl pyrophosphate synthase (PjFPS) and β-amyrin synthase (Pjβ-AS)are key enzymes in the biosynthetic pathway of Panax japonicus saponins (PJS). In this study, effects of PjFPS and PjβAS on PJS biosynthesis were investigated by overexpressing PjFPS and Pjβ-AS simultaneously in P. japonicus cells. The results showed that PjFPS and Pjβ-AS were key enzyme genes in PJS biosynthetic pathway and had regulatory effects on biosynthesis of P. japonicus. The cell lines overexpressing PjFPS and Pjβ-AS showed differential increases in the expression levels of several key enzyme genes related to the PJS synthesis pathway, and the highest levels of PJS were up to 2.4 times higher than those of the normal cell lines. Although overexpression of PjFPS alone also increased PJS synthesis, dual genes (PjFPS and Pjβ-AS) synergistically regulated PJS synthesis more effectively.

Key words: triterpenoid saponins, biosynthesis, Panax japonicus, farnesyl pyrophosphate synthase, β-amyrin synthase

CLC Number:

S567.5

CHEN Qin, LIU Meijia, LIU Diqiu, QU Yuan, CUI Xiuming, GE Feng. Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins[J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 428-436.

Figures/Tables 7

Table 1 Primers used for PCR

基因	上游引物Forward primer(5'→3')	下游引物Reverse primer (5'→3')	产物长度Product length/bp
Pjβ-AS	ATGTGGAGGCTAATGACAGCCAAGGG	TCAGACGCTTTTAGGTGGTAATCGAACA	2 384
PjFPS	ACAGACAACAACTTCCCCTCCAT	AGAATGAGCGATCTGAAGACGAG	1 136
nptⅡ	CTCTGATGCCGCCGTGTT	CCCTGATGCTCTTCGTCCA	450
hptⅡ	GAAGTGCTTGACATTGGGGAAT	AGATGTTGGCGACCTCGTATT	450

Table 2 Primers used for qRT-PCR

基因	上游引物Forward primer (5'→3')	下游引物Reverse primer (5'→3')	产物长度Product length/bp
Pjβ-AS	ATGTGGAGGCTAATGACAGCCAAGGG	TCAGACGCTTTTAGGTGGTAATCGAACA	174
PjFPS	ACAGACAACAACTTCCCCTCCAT	AGAATGAGCGATCTGAAGACGAG	168
18S RNA	AACCATAAACGATGCCGACCAG	TTCAGCCTTGCGACCATACTCC	162
PjDS	TGGGAGTTTCAGCCCGATG	GGGGAGGTGTATAAAGTAAAGAGCC	141
PjHMGR	GACATTTTGAACACCCTTGGACA	CTCTTAATTTTGATACACTGGCCGT	172
PjSE	TAGGTGAACTTCTACAACCAGGAGG	CAACCAGAGATGTAACAGTCCCC	178
PjSS	CGAGCACTTGACACTGTTGAGGAT	CTATTGCCTCCTGGTAACCGTTTC	185

Fig.1 PCR analysis of nptⅡ and hpt Ⅱ in transgenic P. japonicus cells A, PCR result of nptⅡ; B, PCR result of hptⅡ; M, DL2000 DNA marker; 1, Cell lines of control; 2, Transgenic cell lines containing empty vector of hptⅡ gene; 3-6, PjFPS/β-AS-transgenic cells.

Fig.2 Relative expression levels of key enzyme genes inP. japonicus cells FPS, Farnesyl pyrophosphate synthase gene; SS, squalene synthase gene; β-AS, β-amyrin synthase gene; HMGR, 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene; SE, squalene cyclooxygenase gene; DS, damacendiol synthase gene. WT, wild-type cell line; T1, T2, T3 and T4, four PjFPS/β-AS overexpression cell lines; T5, trans-empty vector transgenic cell line. * and ** meant significant differences between control (non-transgenic cells) and transgenic cell lines at the level of P<0.05 andP<0.01. The same as below.

Fig.3 Activities of FPS and β-AS in P. japonicus cell lines WT, Wild-type cell line; F1, F2, F3 and F4, PjFPS transgenic cell lines; F5, Trans-empty vector cell lines.The same as below.

Fig.4 Content of total saponins in transgenic P. japonicus cells

Fig.5 Contents of monomer saponins in P. japonicus cell lines

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Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins

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