Effect of SmGGPPS2 Expression on Tanshinones Biosynthesis in Salvia miltiorrhiza

doi:10.11983/CBB18222

Abstract

Abstract: Geranylgerany pyrophosphate synthase (GGPPS) is an important regulator in the plant diterpenoid biosynthesis pathway. The GGPPS gene family plays a critical role in the development of the medicinal model plant Salvia miltiorrhiza. However, the biological function of SmGGPPS2, especially in the biosynthesis of tanshinone or other active ingredients, is still unclear. For functional investigation, SmGGPPS2 expression was up- or down-regulated in S. miltiorrhiza plants via overexpression or RNA interference, respectively. Then we detected the content of tanshinones, the expression of genes related to tanshinone biosynthesis, and the physiological indexes of transgenic S. miltiorrhiza plants. The content of fat-soluble components, such as tanshinone IIA and ferruginol, was increased significantly in SmGG- PPS2-overexpressed lines compared with the wild type, and the content of fat soluble-components was lower in SmGGPPS2-RNAi lines than those in the wild type lines. With the regulation of SmGGPPS2, the expression of key enzyme genes related to tanshinone biosynthesis in S. miltiorrhiza, such as SmHMGR1 and SmCPS1, was changed. In addition, the regulation of SmGGPPS2 expression also affected the resistance of S. miltiorrhiza. Our results indicate that SmGGPPS2 plays an important regulatory role in tanshinone biosynthesis.

Key words: Salvia miltiorrhiza, GGPPS, tanshinone, gene expression

Wenping Hua,Chen Chen,Yuan Zhi,Li Liu,Zhezhi Wang,Cuiqin Li. Effect of SmGGPPS2 Expression on Tanshinones Biosynthesis in Salvia miltiorrhiza[J]. Chinese Bulletin of Botany, 2019, 54(2): 217-226.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18222

https://www.chinbullbotany.com/EN/Y2019/V54/I2/217

Figures/Tables 8

Table 1 Primers used for vector construction and detection in this study

Primer name	Sequence (5′→3′)
OG2S (KpnI)	GGGGTACCGCGAAGAGCGTGGAAGC- AGA
OG2R (BamHI)	CGGGATCC GGCAATAGCAATCAAGGGAGCA
G2i1S (EcoRI)	CGGAATTC CTCCTCTCAACCTCTGTCAAAACTC
G2i1R (KpnI)	GGGGTACCTCATCTGGACCACTGCC- TCC
G2i2S (BamHI)	CGGGATCC CTCCTCTCAACCTCTGTCAAAACTC
G2i2R (HindIII)	CCCAAGCTTTCATCTGGACCACTGCC- TCC
35SF	GCCGTGAAGACTGGCGAACA
35SR	AGGAAGGGTCTTGCGAAGGATAGT

Table 2 Primers used for qPCR in this study

Primer name	Sequence (5′→3′)	Primer name	Sequence (5′→3′)
ActinS	AGGAACCACCGATCCAGACA	SmDXS1S	TGAGAGCGACTACGACTGCTTTGG
ActinR	GGTGCCCTGAGGTCCTGTT	SmDXS1R	CCCATCCAGATTGGCAGTAGGC
SmHMGR1S	GCAACCATCTACTCTCGTCCCA	SmDXS2S	GGTCGAGGAACTGGAGGGATTG
SmHMGR1R	GTGCTCCATGAGCTGCATCAG	SmDXS2R	CGTCAGGATTTCGTGCGGATA
SmHMGR2S	GGGTTCAACTACGAGGCCATACTG	SmDXS3S	CACGAATGGGCTGCCAAAAT
SmHMGR2R	TGTTTGTGCTCGCCACCAGG	SmDXS3R	CCATCGAATCCAATGAAGCCAC
SmHMGR3S	AGTCTCGTGATGTCCCTGCTCG	SmGGPPS1S	GGGGCTATTTTGGGAGGTGGAA
SmHMGR3R	GCCTCAACCTGCTTGGCGTA	SmGGPPS1R	CAGCAGCTTGGGATACGTGGTC
SmIPI1S	AGCGTGCATCCAAATCCAGAC	SmGGPPS2S	CGGTCTCCTCTCAACCTCTGTCAA
SmIPI1R	GATAGCTTCAAGCCCCCCTCA	SmGGPPS2R	CTCCTTCATCTGGACCACTGCCT
SmCPS1S	ATGGATGGGCAGCAGCAGTAAA	SmGGPPS3S	GGCCAGTGCTCTGCTGTCTGTG
SmCPS1R	CGTCCTCAACAACGTCCTGATGTATT	SmGGPPS3R	TCGGCCACCTCCATCGCTT

Figure 1 Phenotypes and PCR-screening of the transgenic lines of Salvia miltiorrhiza (A) SmGGPPS2-overexpression transgenic line (OG2-3), SmGGPPS2-RNAi transgenic line (IG2-6), and wild type (Bar= 2 cm); (B) SmGGPPS2-overexpression transgenic lines (1-13); (B) SmGGPPS2-RNAi transgenic lines (1-20). M: DNA marker DL2000; WT: Wild type; c: Negative control; p: Positive control

Figure 2 Expression of SmGGPPS2 in overexpression (A) and RNAi (B) Salvia miltiorrhiza transgenic linesWT: Wild type. * Significant differences (P?0.05); ** Significant differences (P?0.01); *** Significant differences (P? 0.001). The data are normalized.

Figure 3 GC-MS chromatogram of Salvia miltiorrhiza transgenic linesA: Phthalic acid diisobutyl ester; B: 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione; C: Ferruginol; D: b-monopalmitin; E: Diisooctyl phthalate; F: 7β-hydroxytotarol; G: Tanshinone IIA; H: Cryptotanshinone; I: Campesterol; J: Stigmasterol; K: β- sitosterol

Figure 4 Contents of different ingredients from fat-soluble extract in transgenic lines (A) and contents oftanshinone IIA in different transgenic lines (B) of Salvia miltiorrhizaA–K see Figure 3. WT: Wild type. *** Significant differences (P<0.001)

Figure 5 Relative expression levels of genes involved in tanshinones biosynthesis of Salvia miltiorrhiza transgenic lines WT: Wild type. * Significant differences (P<0.05); ** Significant differences (P<0.01); *** Significant differences (P<0.001)

Figure 6 The resistance physiological indexes of different Salvia miltiorrhiza transgenic linesSOD: Superoxide dismutase; MDA: Malondialdehyde; POD: Peroxidase; WT: Wild type. * Significant differences (P<0.05); ** Significant differences (P<0.01)

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