三七细胞中共超表达FPS、SS对皂苷合成的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0706

摘要/Abstract

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。

关键词: 法呢基焦磷酸合酶, 鲨烯合酶, 三七皂苷, 共超表达

Abstract:

This work aims to study the effect of FPS（farnesyl pyrophasphate synthase）and SS（squalene synthase）co-overexpression in Panax notoginseng cells on saponins synthesis. The pCAMBIA1300S-FPS overexpression vector was transferred into the P. notoginseng cells in which SS was overexpressed in advance by Agrobacterium tumefaciens EHA105. Transgenic cell lines were screened through double antibiotic culture and genomic PCR. Relative expressions of FPS and SS in the transgenic cell lines were analyzed by qRT-PCR. Contents of P. notoginseng saponins（PNS）and monomer saponins were detected by spectrophotometry and chromatography,respectively. In four transgenic cell lines with overexpression of both FPS and SS,relative expression levels of FPS were all higher than those of two control groups of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 3.26 times and 3.74 times of the above two control lines;relative expressions of SS showed no significant difference compared with the line in which only SS was overexpressed,but were higher than the non-transgenic line. Contents of PNS were all higher than those of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 2.46 times and 1.73 times of the above two control lines. Contents of monomer saponins Re,Rh1,Rd and Rg1 were mostly higher than those of the control lines. Overexpression of FPS and SS in P. notoginseng cells may enhance the accumulation of PNS and monomer saponins. In addition,co-overexpression of FPS and SS may further improve PNS content compared with the overexpression of only SS.

Key words: farnesyl pyrophasphate synthase, squalene synthase, Panax notoginseng saponins, co-overexpression

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.

图/表 11

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

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

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

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5 三七细胞中总皂苷含量

Fig. 5 Contents of PNS in P. notoginseng cells

图6 三七细胞中部分单体皂苷含量

Fig. 6 Contents of monomer saponins in P. notoginseng cells

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

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