生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 74-83.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0981
雷君1,2(), 陈勤1,2, 邓兵1,2, 张金渝3, 刘迪秋1,2, 崔秀明1,2, 葛锋1,2()
收稿日期:
2021-08-02
出版日期:
2022-05-26
发布日期:
2022-06-10
作者简介:
雷君,女,硕士研究生,研究方向:药用植物生物技术;E-mail: 基金资助:
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
Published:
2022-05-26
Online:
2022-06-10
摘要:
旨在明确PnMYB1转录因子对三七皂苷生物合成具有调控作用。利用RACE技术获得PnMYB1基因全长,对PnMYB1进行系统发育树分析;构建PnMYB1植物过表达载体并侵染三七细胞,检测转基因三七细胞中人参皂苷R1、Rg1、Re、Rb1和Rd的含量;将PnMYB1与鲨烯合酶(PnSS)、鲨烯环氧化酶(PnSE)、达玛烯二醇合成酶(PnDS)和环阿屯醇合成酶(PnCAS)等参与三七皂苷生物合成途径的关键酶的基因启动子共转染烟草叶片,进行瞬时表达分析,利用GUS表达系统验证PnMYB1转录因子能否与三七皂苷生物合成关键酶基因的启动子相互作用。结果显示,PnMYB1转录因子属于R2R3-MYB家族;在过表达PnMYB1的三七细胞中,五种重要三萜皂苷在转基因细胞中均有不同程度的增加,进一步分析证明PnMYB1转录因子通过激活PnSE和PnDS的启动子,促使PnSE和PnDS的表达水平显著升高,进而实现对三七皂苷生物合成的调控。PnMYB1转录因子可以同时调控三七皂苷生物合成途径中两个关键酶基因的表达,从而影响三七皂苷的生物合成。
雷君, 陈勤, 邓兵, 张金渝, 刘迪秋, 崔秀明, 葛锋. R2R3-MYB转录因子PnMYB1调控三七皂苷生物合成[J]. 生物技术通报, 2022, 38(5): 74-83.
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 |
表1 实时荧光定量PCR的引物
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 |
表2 启动子克隆的引物
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 |
表3 五种重要单体皂苷线性回归方程
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 |
图3 三七细胞系T1-T6的PnFPS(A)、PnDS(B)、PnSE(C)、PnSS(D)、PnMYB1(E)和PnCAS(F)基因相对表达量 WT:野生型三七细胞;对照组比较:**P<0.01;*P<0.05. 下同
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
图5 转PnMYB1三七细胞系的HPLC分析 A:标准品;B:野生型三七细胞;C:转PnMYB1 T3三七细胞株系
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
图6 三七细胞系T1-T6单体皂苷R1(A)、Rg1(B)、Re(C)、Rb1(D)和Rd(E)含量
Fig. 6 Contents of monomer saponins R1(A),Rg1(B),Re(C),Rb1(D)and Rd(E)in T1-T6 transgenic cell lines of P. notoginseng
图7 GUS荧光活性分析 PnDSP1、PnSEP1、PnSSP1和PnCASP1分别表示对应的启动子片段单独转染的烟草叶片;PnDSP1/PnMYB1、PnSEP1/PnMYB1、PnSSP1/PnMYB1和PnCASP1/PnMYB1分别表示PnDSP1、PnSEP1、PnSSP1和PnCASP1启动子片段分别与PnMYB1共同转染的烟草叶片;PBI 121:PBI 121转染的野生型烟草叶片;WT:野生烟草叶片(与PnDSP1比较:**P<0.01,与PnSEP1比较:##P <0.01)
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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