生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 50-58.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0706
杨延1(), 于龙凤1, 王绍梅1, 李卫娜2, 葛锋3()
收稿日期:
2021-06-02
出版日期:
2022-03-26
发布日期:
2022-04-06
作者简介:
杨延,女,硕士研究生,研究方向:药用植物生物技术;E-mail: 基金资助:
YANG Yan1(), YU Long-feng1, WANG Shao-mei1, LI Wei-na2, GE Feng3()
Received:
2021-06-02
Published:
2022-03-26
Online:
2022-04-06
摘要:
研究三七细胞中共超表达法呢基焦磷酸合酶(farnesyl pyrophasphate synthase,FPS)基因、鲨烯合酶(squalene synthase,SS)基因对三七细胞皂苷合成的影响。利用农杆菌EHA105将pCAMBIA1300S-FPS超表达载体转入已超表达SS的三七细胞内,双抗生素培养及基因组PCR筛选转双基因阳性株系、qRT-PCR测阳性株系中FPS、SS表达量,分光光度法及色谱法分别测总皂苷和单体皂苷量。在4株转FPS、SS细胞中,FPS的相对表达量均比两对照(未转基因细胞和仅转SS细胞)高,最高分别是未转基因对照的3.26倍和仅转SS对照的3.74倍;SS的相对表达量与仅转SS对照相比无显著差异,但均比转基因对照高;总皂苷含量均比两对照高,最高分别是未转基因对照的2.46倍和仅转SS对照的1.73倍;Re、Rh1、Rd和Rg1 单体皂苷量较两对照大多有不同幅度增加。在三七细胞中共超表达FPS、SS,可有效提高细胞中的总皂苷和部分单体皂苷量;此外,与仅超表达SS比较,共超表达FPS、SS能够进一步提高皂苷含量。
杨延, 于龙凤, 王绍梅, 李卫娜, 葛锋. 三七细胞中共超表达FPS、SS对皂苷合成的影响[J]. 生物技术通报, 2022, 38(3): 50-58.
YANG Yan, YU Long-feng, WANG Shao-mei, LI Wei-na, GE Feng. Effects of FPS and SS Co-overexpression in Panax notoginseng Cells on Saponins Synthesis[J]. Biotechnology Bulletin, 2022, 38(3): 50-58.
培养基类型 Culture medium type | 配方 Formula |
---|---|
预培养基/共培养基 Pre-culture medium/Co-culture medium | MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+40 mg/L乙酰丁香酮 |
除菌培养基 Sterilizing culture medium | MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+400 g/L Cef |
筛选培养基 Secreening culture medium | MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+50 g/L Kan+25 g/L Hyg B |
表1 三七细胞培养基配方
Table 1 Formulas of P. notoginseng culture media
培养基类型 Culture medium type | 配方 Formula |
---|---|
预培养基/共培养基 Pre-culture medium/Co-culture medium | MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+40 mg/L乙酰丁香酮 |
除菌培养基 Sterilizing culture medium | MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+400 g/L Cef |
筛选培养基 Secreening culture medium | MS+2.0 g/L 2,4-D+1.0 g/L KT+3%蔗糖+7.8 g/L 琼脂+50 g/L Kan+25 g/L Hyg B |
基因 Gene | 上游引物 Upstream primer(5'-3') | 下游引物 Downstream primer(5'-3') |
---|---|---|
npt II | CTCTGATGCCGCCGTGTT | CCCTGATGCTCTTCGTCCA |
hpt II | GAAGTGCTTGACATTGGGGAAT | AGATGTTGGCGACCTCGTATT |
表2 基因组PCR引物序列
Table 2 Primers used for genomic PCR
基因 Gene | 上游引物 Upstream primer(5'-3') | 下游引物 Downstream primer(5'-3') |
---|---|---|
npt II | CTCTGATGCCGCCGTGTT | CCCTGATGCTCTTCGTCCA |
hpt II | GAAGTGCTTGACATTGGGGAAT | AGATGTTGGCGACCTCGTATT |
基因 Gene | 上游引物 Upstream primer(5'-3') | 下游引物 Downstream primer(5'-3') |
---|---|---|
SS | GCAGGACTTGTTGGATTAGGGT | AACATGCGTGACTTTGGTATCTC |
FPS | ATCCTCATCTCACCGCTCTTT | AAAGAGCGGTGAGATGAGGAT |
GAPDH | CTACCAACTGTCTTGCTCCCCT | TGATGCAGCTCTTCCACCTCTC |
表3 qRT-PCR引物序列
Table 3 Primers used for qRT-PCR
基因 Gene | 上游引物 Upstream primer(5'-3') | 下游引物 Downstream primer(5'-3') |
---|---|---|
SS | GCAGGACTTGTTGGATTAGGGT | AACATGCGTGACTTTGGTATCTC |
FPS | ATCCTCATCTCACCGCTCTTT | AAAGAGCGGTGAGATGAGGAT |
GAPDH | CTACCAACTGTCTTGCTCCCCT | TGATGCAGCTCTTCCACCTCTC |
时间 Time/min | 乙腈Acetonitrile/% | 水water/% |
---|---|---|
0-30 | 20 | 80 |
30-60 | 20→45 | 80→55 |
60-78 | 45→75 | 55→25 |
78-80 | 75→100 | 25→0 |
80 | 20 | 80 |
表4 乙腈:水梯度洗脱表
Table 4 Elution gradient of acetonitrile to water
时间 Time/min | 乙腈Acetonitrile/% | 水water/% |
---|---|---|
0-30 | 20 | 80 |
30-60 | 20→45 | 80→55 |
60-78 | 45→75 | 55→25 |
78-80 | 75→100 | 25→0 |
80 | 20 | 80 |
图1 转基因细胞系中npt II基因的基因组PCR检测 M:Trans2K® DNA marker;1-7:转基因细胞系;8:阳性对照;9:阴性对照
Fig. 1 Genomic PCR detection of npt II in transgenic cell lines M:Trans2K® DNA marker. 1-7:Transgenic cells lines. 8:Positive control. 9:Negative control
图2 转基因细胞系中hpt II基因的基因组PCR检测 M:Trans2K® DNA marker;1-7:转基因细胞系;8:阳性对照;9:阴性对照
Fig. 2 Genomic PCR detection of hpt II in transgenic cell lines M:Trans2K® DNA marker. 1-7:Transgenic cells lines. 8:Positive control. 9:Negative control
图3 三七细胞 C:未转基因三七细胞;T-SS:仅转SS基因三七细胞;T-1-T-4:转双基因细胞系
Fig. 3 P. notoginseng cells C:Non-transgenic P. notoginseng cells. T-SS:The P. notoginseng cells which were transferred by SS. T-1-T-4:The P. notoginseng cells which were transferred by SS and FPS
图4 三七细胞中FPS和SS基因的相对表达量 C:未转基因三七细胞;T-SS:仅转SS基因三七细胞;T-1-T-4:转双基因细胞系;*:与C相比,P<0.05;**:与C相比,P<0.01。下同
Fig. 4 Relative transcription levels of FPS and SS in P. notoginseng cells C:Non-transgenic P. notoginseng cells. T-SS:The P. notoginseng cells which were transferred by SS. T-1-T-4:The P. notoginseng cells which were transferred by SS and FPS. *P< 0.05 compared to the C(control);** P< 0.01 compared to the C(control). The same below
图7 三七总皂苷生物合成途径 AATC:乙酰辅酶A酰基转移酶;HMGS:3-羟基-3-甲基戊二酰辅酶A合酶;HMGR:HMG-CoA还原酶;GPPS:牻牛儿基焦磷酸合酶;FPS:法呢基焦磷酸合酶;SS:鲨烯合酶;SE:鲨烯环氧酶;DS:达玛烯二醇合酶;P450:P450单加氧酶;CAS:环阿屯醇合酶。虚线表示多步酶促反应
Fig. 7 Biosynthetic pathway of PNS in P. notoginseng AATC:Acetoacetyl-CoA acyltransferase;HMGS:3-Hydroxy-3-methylglutaryl-CoA synthase;HMGR:3-hydroxy-3-methylglutaryl-CoA reducetase;GPPS:ggeranyl pyrophosphate synthase;FPS:farnesyl pyrophosphate synthase;SS:squalene synthase;SE:squalene epoxidase;DS:dammarenediol-Ⅱ synthase;P450:P450-monooxygenase;CAS:cycloartenol synthase. Dotted lines in indicate multi-step enzymatic reaction
[1] |
He NW, Zhao Y, Guo L, et al. Antioxidant, antiproliferative, and pro-apoptotic activities of a saponin extract derived from the roots of Panax notoginseng(Burk. )F. H. Chen[J]. J Med Food, 2012, 15(4):350-359.
doi: 10.1089/jmf.2011.1801 URL |
[2] |
Xia W, Sun C, Zhao Y, et al. Hypolipidemic and antioxidant activities of Sanchi(Radix notoginseng)in rats fed with a high fat diet[J]. Phytomedicine, 2011, 18(6):516-520.
doi: 10.1016/j.phymed.2010.09.007 URL |
[3] | Qian M, Yi L, Song-Lin L, et al. Chemical profiles and anticancer effects of saponin fractions of different polarity from the leaves of Panax notoginseng[J]. Chin J Nat Med, 2014, 12(1):30-37. |
[4] |
Yang CY, Wang J, Zhao Y, et al. Anti-diabetic effects of Panax notoginseng saponins and its major anti-hyperglycemic components[J]. J Ethnopharmacol, 2010, 130(2):231-236.
doi: 10.1016/j.jep.2010.04.039 URL |
[5] |
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 |
[6] | Luo H, Sun C, Sun Y, et al. Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers[J]. BMC Genomics, 2011, 12(Suppl 5):S5. |
[7] |
Niu Y, Luo H, Sun C, et al. Expression profiling of the triterpene saponin biosynjournal genes FPS SS SE, and DS in the medicinal plant Panax notoginseng[J]. Gene, 2014, 533(1):295-303.
doi: 10.1016/j.gene.2013.09.045 URL |
[8] |
Deng B, Zhang P, Ge F, et al. Enhancement of triterpenoid saponins biosynjournal 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 |
[9] | 杨延, 刘迪秋, 葛锋, 等. 三七细胞中过表达FPS基因对皂苷合成的影响[J]. 现代食品科技, 2015, 31(8):59-64. |
Yang Y, Liu DQ, Ge F, et al. Effect of over-expressing farnesyl pyrophosphate synthase(FPS)gene of Panax notoginseng cell on saponin synjournal[J]. Mod Food Sci Technol, 2015, 31(8):59-64. | |
[10] | 孙颖, 赵恒伟, 葛锋, 等. 三七中SS基因超表达载体的构建及其遗传转化[J]. 药学学报, 2013, 48(1):138-143. |
Sun Y, Zhao HW, Ge F, et al. The construction of over-expression vector for Panax notoginseng SS gene and its transformation[J]. Acta Pharm Sin, 2013, 48(1):138-143. | |
[11] | 杨延. 三七细胞中过表达FPS和干扰CAS表达对三七皂苷合成的影响[D]. 昆明:昆明理工大学, 2015. |
Yang Y. The effects of FPS overexpression and CAS expression interfered on the biosynthesis of saponins in Panax notoginseng cells[D]. Kunming:Kunming University of Science and Technology, 2015. | |
[12] | 陈旭, 党晓芳, 曹飒丽, 等. 三七中总皂苷含量测定的对照品筛选[J]. 中国实验方剂学杂志, 2013, 19(7):66-68. |
Chen X, Dang XF, Cao SL, et al. Screening of reference substances for content determination of total saponins in panpax notoginseng[J]. Chin J Exp Tradit Med Formulae, 2013, 19(7):66-68. | |
[13] | 杨林林, 杨利民, 马秀杰, 等. 人参法尼基焦磷酸合成酶基因的表达及其与皂苷含量的关系[J]. 吉林农业大学学报, 2017, 39(6):695-702, 708. |
Yang LL, Yang LM, Ma XJ, et al. Correlation between Panax ginseng farnesyl diphosphate synthase gene expression and ginsenoside content[J]. J Jilin Agric Univ, 2017, 39(6):695-702, 708. | |
[14] |
Kim YK, Kim YB, Uddin MR, et al. Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase[J]. ACS Synth Biol, 2014, 3(10):773-779.
doi: 10.1021/sb400194g URL |
[15] | 刘美佳, 于怡琳, 姜森, 等. 珠子参中法尼基焦磷酸合酶(FPS)对皂苷生物合成的影响研究[J]. 植物研究, 2018, 38(4):611-618. |
Liu MJ, Yu YL, Jiang S, et al. Effect of farnesyl-pyrophosphate synthase(FPS)on the biosynjournal of saponins in Panax japonicus[J]. Bull Bot Res, 2018, 38(4):611-618. | |
[16] | 邢朝斌, 龙月红, 李非非, 等. 刺五加鲨烯合酶基因家族两成员的表达及其与皂苷含量的关系[J]. 西南农业学报, 2014, 27(3):1252-1255. |
Xing ZB, Long YH, Li FF, et al. Relationship between expression of two member of squalene synthase gene family from Eleutherococcus senticosus and saponins content[J]. Southwest China J Agric Sci, 2014, 27(3):1252-1255. | |
[17] | 杨延, 刘迪秋, 葛锋, 等. 三七细胞中SS、DS共超表达对皂苷合成的影响[J]. 现代食品科技, 2015, 31(2):7-13. |
Yang Y, Liu DQ, Ge F, et al. Effects of co-overexpression of squalene synthase and dammarenediol- II synthase in Panax notoginseng cells on saponin synjournal[J]. Mod Food Sci Technol, 2015, 31(2):7-13. | |
[18] | 朱华, 吴耀生. 实时荧光定量PCR检测三七SS基因表达的初步实践[J]. 广西植物, 2008, 28(5):703-707. |
Zhu H, Wu YS. Initial practice of Real Time PCR for the expression of SS gene in Panax notoginseng[J]. Guihaia, 2008, 28(5):703-707. | |
[19] |
Ruan CC, Zhang H, Zhang LX, et al. Biotransformation of ginsenoside Rf to Rh1 by recombinant β-glucosidase[J]. Molecules, 2009, 14(6):2043-2048.
doi: 10.3390/molecules14062043 URL |
[20] | 冯亮, 胡昌江, 余凌英. 人参皂苷Rgl及其代谢产物的药代动力学研究[J]. 药学学报, 2010, 45(5):636-640. |
Feng L, Hu CJ, Yu LY. Pharmacokinetics of ginsenosides Rg1 and its metabolites in rats[J]. Acta Pharm Sin, 2010, 45(5):636-640. | |
[21] | 卢丞文, 殷涌光. 人参皂苷Rd药理活性和制备方法研究进展[J]. 北方园艺, 2015(23):202-205. |
Lu CW, Yin YG. Research progress on pharmacological activity and preparation methods of ginsenoside rd[J]. North Hortic, 2015(23):202-205. | |
[22] |
Cho WC, Chung WS, Lee SK, et al. Ginsenoside Re of Panax ginseng possesses significant antioxidant and antihyperlipidemic efficacies in streptozotocin-induced diabetic rats[J]. Eur J Pharmacol, 2006, 550(1/2/3):173-179.
doi: 10.1016/j.ejphar.2006.08.056 URL |
[23] | Li Y, Tang J, Khatibi NH, et al. Treatment with ginsenoside rb1, a component of Panax ginseng, provides neuroprotection in rats subjected to subarachnoid hemorrhage-induced brain injury[J]. Acta Neurochir Suppl, 2011, 110(pt 2):75-79. |
[24] | 张萍. 三七皂苷生物合成途径HMGR基因的克隆及功能研究[D]. 昆明:昆明理工大学, 2015. |
Zhang P. Molecular cloning and functional study of PnHMGR gene in the biosynthetic pathway of Panax notoginseng saponins[D]. Kunming:Kunming University of Science and Technology, 2015. |
[1] | 龙月红;邢朝斌;王明艳;吴鹏;陈龙;梁能松;何闪;. 刺五加鲨烯合酶基因cDNA的克隆与序列分析[J]. , 2012, 0(02): 112-116. |
[2] | 周娟;赵瑞强;陈莉;吴耀生;. 三七法呢基焦磷酸合酶原核表达载体的构建及其表达[J]. , 2009, 0(09): 130-133. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||