Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (5): 74-83.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0981
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LEI Jun1,2(), CHEN Qin1,2, DENG Bing1,2, ZHANG Jin-yu3, LIU Di-qiu1,2, CUI Xiu-ming1,2, GE Feng1,2()
Received:
2021-08-02
Online:
2022-05-26
Published:
2022-06-10
Contact:
GE Feng
E-mail:353597247@qq.com;panaxcordyceps@163.com
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.
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 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 |
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 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 |
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 |
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. 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. 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. 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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