R2R3-MYB转录因子PnMYB1调控三七皂苷生物合成

doi:10.13560/j.cnki.biotech.bull.1985.2021-0981

摘要/Abstract

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。

关键词: 三七, 皂苷, 转录因子, 三萜类化合物

Abstract:

This work aims to clarify the regulation role of transcription factor PnMYB1 to Panax notoginseng saponins（PNS）biosynthesis. RACE technique was applied to obtain the full length of PnMYB1 gene,the phylogenetic tree analysis of PnMYB1 were carried out. The plant overexpression vector of PnMYB1 was constructed and infected P. notoginseng cells,and the contents of ginsenosides R1,Rg1,Re,Rb1 and Rd in the transgenic P. notoginseng cells were detected. For instant expression analysis,tobacco leaves were co-transfected with PnMYB1 and the gene promoters of some key enzymes involved in the biosynthetic pathway of PNS,including squalene synthase（PnSS）,squalene epoxidase（PnSE）,dammarenediol synthase（PnDS）and cycloartenol synthase（PnCAS）. The GUS expression system was employed to confirm whether PnMYB1 transcription factor interacted with the promoters of key enzyme genes in the biosynthetic pathway of PNS. The results showed that PnMYB1 transcription factors belonged to the R2R3-MYB family. In PnMYB1-overexpressed cells of P. notoginseng,5 important triterpenoid saponins increased to some extent in the transgenic cells. Further analysis confirmed that PnMYB1 significantly increased the expressions of PnSE and PnDS by activating the promoters of PnSE and PnDS,thus regulating the biosynthesis of PNS. In conclusion,PnMYB1 transcription factor may simultaneously regulate the expressions of two key enzyme genes involved in the biosynthesis of PNS and then affect its biosynthesis.

Key words: Panax notoginseng, saponins, transcription factor, triterpenoid

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.

图/表 10

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

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

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1 氨基酸序列比对

Fig. 1 Amino acid sequence alignment

图2 PnMYB1系统进化树分析

Fig. 2 Phylogenetic tree analysis of PnMYB1

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4 三七细胞系总皂苷含量

Fig. 4 Content of total saponins in the cell lines of P. notoginseng

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）

参考文献 31

[1]	Ng TB. Pharmacological activity of Sanchi ginseng(Panax notoginseng)[J]. J Pharm Pharmacol, 2010, 58(8):1007-1019. doi: 10.1211/jpp.58.8.0001 URL
[2]	Kuzuyama T. Mevalonate and nonmevalonate pathways for the biosynthesis of isoprene units[J]. Biosci Biotechnol Biochem, 2002, 66(8):1619-1627. doi: 10.1271/bbb.66.1619 URL
[3]	Kim DH. Chemical Diversity of Panax ginseng, Panax quinquifolium, and Panax notoginseng[J]. J Ginseng Res, 2012, 36(1):1-15. doi: 10.5142/jgr.2012.36.1.1 URL
[4]	Zhang JZ. Overexpression analysis of plant transcription factors[J]. Curr Opin Plant Biol, 2003, 6(5):430-440. doi: 10.1016/S1369-5266(03)00081-5 URL
[5]	Heim MA, Jakoby M, Werber M, et al. The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity[J]. Mol Biol Evol, 2003, 20(5):735-747. doi: 10.1093/molbev/msg088 URL
[6]	Ma D, Pu G, Lei C, et al. Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the Amorpha-4, 11-diene synthase gene, a key gene of artemisinin biosynthesis[J]. Plant Cell Physiol, 2009, 50(12):2146-2161. doi: 10.1093/pcp/pcp149 URL
[7]	Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis[J]. Trends Plant Sci, 2010, 15(10):573-581. doi: 10.1016/j.tplants.2010.06.005 URL
[8]	Bedon F, Bomal C, Caron S, et al. Subgroup 4 R2R3-MYBs in conifer trees:gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses[J]. J Exp Bot, 2010, 61(14):3847-3864. doi: 10.1093/jxb/erq196 URL
[9]	Li CY, Leopold AL, Sander GW, et al. CrBPF₁ overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes[J]. Front Plant Sci, 2015, 6:818.
[10]	徐俊雄, 覃建兵, 王岩岩, 等. 虎杖MYB转录因子PcMYB1的表达特性和功能研究[J]. 中草药, 2020, 51(3):726-732.
	Xu JX, Qin JB, Wang YY, et al. Expression and function analysis of transcription factor PcMYB₁ from Polygonum cuspidatum[J]. Chin Tradit Herb Drugs, 2020, 51(3):726-732.
[11]	应宇翔, 何凤明, 张云峰, 等. 灯盏花MYB基因克隆及其荧光表达载体的构建[J]. 中草药, 2017, 48(20):4306-4315.
	Ying YX, He FM, Zhang YF, et al. Cloning of MYB gene and construction of greenfluorescent protein expression vector in Erigeron breviscapus[J]. Chin Tradit Herb Drugs, 2017, 48(20):4306-4315.
[12]	Puissant C, Houdebine LM. An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[J]. BioTechniques, 1990, 8(2):148-149. pmid: 1690559
[13]	Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[J]. Anal Biochem, 1987, 162(1):156-159. doi: 10.1006/abio.1987.9999 pmid: 2440339
[14]	Dai Y, Qin Q, Dai D, et al. Isolation and characterization of a novel cDNA encoding methyl jasmonate-responsive transcription factor TcAP2 from Taxus cuspidata[J]. Biotechnol Lett, 2009, 31(11):1801-1809. doi: 10.1007/s10529-009-0068-4 URL
[15]	Höfgen R, Willmitzer L. Storage of competent cells for Agrobacterium transformation[J]. Nucleic Acids Res, 1988, 16(20):9877. pmid: 3186459
[16]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[17]	Wako T, Kimura S, Chikagawa Y, et al. Characterization of MYB proteins acting as transcriptional regulatory factors for carrot phenylalanine ammonia-lyase gene(DcPAL3)[J]. Plant Biotechnol, 2010, 27(2):131-139. doi: 10.5511/plantbiotechnology.27.131 URL
[18]	Wang N, Xu H, Jiang S, et al. MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple(Malus sieversii f. niedzwetzkyana)[J]. Plant J, 2017, 90(2):276-292. doi: 10.1111/tpj.13487 URL
[19]	Gonzalez A, Zhao M, Leavitt JM, et al. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings[J]. Plant J, 2008, 53(5):814-827. doi: 10.1111/j.1365-313X.2007.03373.x URL
[20]	Dai X, Xu Y, Ma Q, et al. Overexpression of an R1R2R3 MYB gene, OsMYB3R-2, increases tolerance to freezing, drought, and salt stress in transgenic Arabidopsis[J]. Plant Physiol, 2007, 143(4):1739-1751. doi: 10.1104/pp.106.094532 URL
[21]	Elomaa P, Uimari A, Mehto M, et al. Activation of anthocyanin biosynthesis in Gerbera hybrida(Asteraceae)suggests conserved protein-protein and protein-promoter interactions between the anciently diverged monocots and eudicots[J]. Plant Physiol, 2003, 133(4):1831-1842. pmid: 14605235
[22]	Deluc L, Bogs J, Walker AR, et al. The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries[J]. Plant Physiol, 2008, 147(4):2041-2053. doi: 10.1104/pp.108.118919 URL
[23]	Li CH, Qiu J, Yang GS, et al. Isolation and characterization of a R2R3-MYB transcription factor gene related to anthocyanin biosynthesis in the spathes of Anthurium andraeanum(Hort. )[J]. Plant Cell Rep, 2016, 35(10):2151-2165. doi: 10.1007/s00299-016-2025-8 URL
[24]	Lin WK, Bolitho K, Grafton K, et al. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae[J]. BMC Plant Biol, 2010, 10:50. doi: 10.1186/1471-2229-10-50 pmid: 20302676
[25]	Lee MH, Jeong JH, Seo JW, et al. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene[J]. Plant Cell Physiol, 2004, 45(8):976-984. doi: 10.1093/pcp/pch126 URL
[26]	Deng B, Zhang P, Ge F, et al. Enhancement of triterpenoid saponins biosynthesis in Panax notoginseng cells by co-overexpressions of 3-hydroxy-3-methylglutaryl CoA reductase and squalene synthase genes[J]. Biochem Eng J, 2017, 122:38-46. doi: 10.1016/j.bej.2017.03.001 URL
[27]	Laitinen RA, Ainasoja M, Broholm SK, et al. Identification of target genes for a MYB-type anthocyanin regulator in Gerbera hybrida[J]. J Exp Bot, 2008, 59(13):3691-3703. doi: 10.1093/jxb/ern216 pmid: 18725377
[28]	Leung KW, Wong AS. Pharmacology of ginsenosides:a literature review[J]. Chin Med, 2010, 5:20. doi: 10.1186/1749-8546-5-20 URL
[29]	Lee JJ, Kwon HK, Jung IH, et al. Anti-cancer activities of ginseng extract fermented with Phellinus linteus[J]. Mycobiology, 2009, 37(1):21-27. doi: 10.4489/MYCO.2009.37.1.021 URL
[30]	Ni N, Liu Q, Ren H, et al. Ginsenoside Rb1 protects rat neural progenitor cells against oxidative injury[J]. Molecules, 2014, 19(3):3012-3024. doi: 10.3390/molecules19033012 URL
[31]	陈勤, 雷君, 葛锋, 等. 人参属三萜皂苷骨架修饰的研究进展[J]. 中药材, 2020, 11:2831-2837.
	Chen Q, Lei J, Ge F, et al. The research progress of ginseng triterpenoid saponin skeleton modifying enzyme[J]. Journal of Chinese Medicinal Materials, 2020, 11:2831-2837.