Chinese Bulletin of Botany ›› 2019, Vol. 54 ›› Issue (2): 217-226.DOI: 10.11983/CBB18222
Special Issue: 逆境生物学专辑
• EXPERIMENTAL COMMUNICATIONS • Previous Articles Next Articles
Wenping Hua1,2,Chen Chen3,4,Yuan Zhi2,Li Liu1,Zhezhi Wang2,Cuiqin Li2,*()
Received:
2018-10-22
Accepted:
2019-02-19
Online:
2019-03-10
Published:
2019-09-01
Contact:
Cuiqin Li
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.
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 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 |
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 |
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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