Effects of FPS and SS Co-overexpression in Panax notoginseng Cells on Saponins Synthesis

doi:10.13560/j.cnki.biotech.bull.1985.2021-0706

Abstract

Abstract:

This work aims to study the effect of FPS（farnesyl pyrophasphate synthase）and SS（squalene synthase）co-overexpression in Panax notoginseng cells on saponins synthesis. The pCAMBIA1300S-FPS overexpression vector was transferred into the P. notoginseng cells in which SS was overexpressed in advance by Agrobacterium tumefaciens EHA105. Transgenic cell lines were screened through double antibiotic culture and genomic PCR. Relative expressions of FPS and SS in the transgenic cell lines were analyzed by qRT-PCR. Contents of P. notoginseng saponins（PNS）and monomer saponins were detected by spectrophotometry and chromatography,respectively. In four transgenic cell lines with overexpression of both FPS and SS,relative expression levels of FPS were all higher than those of two control groups of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 3.26 times and 3.74 times of the above two control lines;relative expressions of SS showed no significant difference compared with the line in which only SS was overexpressed,but were higher than the non-transgenic line. Contents of PNS were all higher than those of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 2.46 times and 1.73 times of the above two control lines. Contents of monomer saponins Re,Rh1,Rd and Rg1 were mostly higher than those of the control lines. Overexpression of FPS and SS in P. notoginseng cells may enhance the accumulation of PNS and monomer saponins. In addition,co-overexpression of FPS and SS may further improve PNS content compared with the overexpression of only SS.

Key words: farnesyl pyrophasphate synthase, squalene synthase, Panax notoginseng saponins, co-overexpression

YANG Yan, YU Long-feng, WANG Shao-mei, LI Wei-na, GE Feng. Effects of FPS and SS Co-overexpression in Panax notoginseng Cells on Saponins Synthesis[J]. Biotechnology Bulletin, 2022, 38(3): 50-58.

Figures/Tables 11

Table 1 Formulas of P. notoginseng culture media

培养基类型 Culture medium type	配方 Formula
预培养基/共培养基 Pre-culture medium/Co-culture medium	MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+40 mg/L乙酰丁香酮
除菌培养基 Sterilizing culture medium	MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+400 g/L Cef
筛选培养基 Secreening culture medium	MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+50 g/L Kan+25 g/L Hyg B

Table 2 Primers used for genomic PCR

基因 Gene	上游引物 Upstream primer（5'-3'）	下游引物 Downstream primer（5'-3'）
npt II	CTCTGATGCCGCCGTGTT	CCCTGATGCTCTTCGTCCA
hpt II	GAAGTGCTTGACATTGGGGAAT	AGATGTTGGCGACCTCGTATT

Table 3 Primers used for qRT-PCR

基因 Gene	上游引物 Upstream primer（5'-3'）	下游引物 Downstream primer（5'-3'）
SS	GCAGGACTTGTTGGATTAGGGT	AACATGCGTGACTTTGGTATCTC
FPS	ATCCTCATCTCACCGCTCTTT	AAAGAGCGGTGAGATGAGGAT
GAPDH	CTACCAACTGTCTTGCTCCCCT	TGATGCAGCTCTTCCACCTCTC

Table 4 Elution gradient of acetonitrile to water

时间 Time/min	乙腈Acetonitrile/%	水water/%
0-30	20	80
30-60	20→45	80→55
60-78	45→75	55→25
78-80	75→100	25→0
80	20	80

Fig. 1 Genomic PCR detection of npt II in transgenic cell lines M：Trans2K® DNA marker. 1-7：Transgenic cells lines. 8：Positive control. 9：Negative control

Fig. 2 Genomic PCR detection of hpt II in transgenic cell lines M：Trans2K® DNA marker. 1-7：Transgenic cells lines. 8：Positive control. 9：Negative control

Fig. 3 P. notoginseng cells C：Non-transgenic P. notoginseng cells. T-SS：The P. notoginseng cells which were transferred by SS. T-1-T-4：The P. notoginseng cells which were transferred by SS and FPS

Fig. 4 Relative transcription levels of FPS and SS in P. notoginseng cells C：Non-transgenic P. notoginseng cells. T-SS：The P. notoginseng cells which were transferred by SS. T-1-T-4：The P. notoginseng cells which were transferred by SS and FPS. *P< 0.05 compared to the C（control）;** P< 0.01 compared to the C（control）. The same below

Fig. 5 Contents of PNS in P. notoginseng cells

Fig. 6 Contents of monomer saponins in P. notoginseng cells

Fig. 7 Biosynthetic pathway of PNS in P. notoginseng AATC：Acetoacetyl-CoA acyltransferase;HMGS：3-Hydroxy-3-methylglutaryl-CoA synthase;HMGR：3-hydroxy-3-methylglutaryl-CoA reducetase;GPPS：ggeranyl pyrophosphate synthase;FPS：farnesyl pyrophosphate synthase;SS：squalene synthase;SE：squalene epoxidase;DS：dammarenediol-Ⅱ synthase;P450：P450-monooxygenase;CAS：cycloartenol synthase. Dotted lines in indicate multi-step enzymatic reaction

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