重瓣铁线莲鲨烯合酶<i>SQS</i>基因的克隆及表达分析

何舒澜; 杨谨; 黄娴; 陈观水

doi:10.19303/j.issn.1008-0384.2022.011.008

重瓣铁线莲鲨烯合酶SQS基因的克隆及表达分析

doi: 10.19303/j.issn.1008-0384.2022.011.008

何舒澜^1,,
杨谨²,
黄娴²,
陈观水^2, ,

1.
福建卫生职业技术学院，福建　福州　350101
2.
福建农林大学生命科学学院，福建　福州　350002

基金项目: 福建省中青年教师教育科研项目职业院校专项（JZ180548）；福建省大学生创新创业项目（S202110389044）

详细信息

作者简介:
何舒澜（1980−），男，硕士，副教授，研究方向：中草药栽培与鉴定（E-mail：181329113@qq.com）

通讯作者:
陈观水（1979−），男，博士，副教授，研究方向：药用植物次生代谢（E-mail：gshchenfafu@163.com）

中图分类号: S 567
计量
- 文章访问数: 490
- HTML全文浏览量: 200
- PDF下载量: 41
- 被引次数: 0
出版历程
- 收稿日期: 2022-05-27
- 修回日期: 2022-09-28
- 网络出版日期: 2022-12-28
- 刊出日期: 2022-11-28

Cloning and Expression of Squalene Synthase Gene from Clematis florida Thunb. var. plena D. Don

1.
Department of Pharmacy, Fujian Health College, Fuzhou, Fujian　350101, China
2.
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的鲨烯合酶是三萜类物质生物合成途径中的限速酶之一。克隆重瓣铁线莲的鲨烯合酶基因（Clematis florida squalene synthase，CfSQS），为利用基因工程技术提高重瓣铁线莲的三萜类次生代谢物的含量、提升重瓣铁线莲的药用价值奠定理论基础。方法根据重瓣铁线莲转录组数据并参考其他植物SQS基因设计引物，提取重瓣铁线莲叶片总RNA为模板，利用RT-PCR法扩增并克隆获得CfSQS基因全长cDNA序列，并对其进行生物信息学分析和时空表达分析。结果 CfSQS ORF序列长度为1227 bp，编码408个氨基酸，预测编码蛋白相对分子量为46.69 kDa，为不稳定、无信号肽的跨膜疏水蛋白，二级结构以α-螺旋（69.61%）和不规则卷曲（22.06%）为主；CfSQS 含有鲨烯合酶家族高度保守的氨基酸残基，包括含有4个高度保守且典型的17~23个氨基酸长度的结构域（Ⅰ~Ⅳ）和1个高度差异的疏水区（Ⅴ）。系统进化分析表明，CfSQS与黑种草（Nigella sativa L.）等毛茛科植物SQS聚在一类，说明其亲缘关系最近。时空表达分析表明，CfSQS在重瓣铁线莲的根、茎和叶器官中都有表达。结论首次克隆了重瓣铁线莲三萜类皂苷上游合成的关键酶基因CfSQS 的全长编码序列，并对其进行了生物信息学预测和组织表达分析。研究结果可为进一步研究重瓣铁线莲三萜类皂苷生物合成途径提供参考。
- 重瓣铁线莲 /
- 三萜皂苷 /
- 鲨烯合酶 /
- 生物信息学分析
Abstract: Objective The squalene synthase (SQS), the first key rate-limited enzyme in the triterpenoids biosynthetic pathway, from Clematis florida Thunb. var. plena D. Don was cloned, and its expression analyzed in preparation for an investigation to increase the medicinal value of the plant by means of genetic engineering. Method Primers were designed based on the transcriptome data on the SQS genes in C. florida and in other plants. Total RNA was extracted from C. florida leaves, and full-length cDNA obtained by RT-PCR. Bioinformatics and spatiotemporal expression of the gene were analyzed. Result The ORF of the SQS, designated as CfSQS, was 1 227 bp encoded 408 amino acids. It was an unstable hydrophobic protein comprised 69.61% of α helix and 22.06% of random coil. The deduced amino acid sequence predicted the presence of 4 highly conserved domains with 17–23 amino acids residues (I–IV) and one variable hydrophobic region (V). The predicted 3D structure of CfSQS by various programs was similar to those of SQSs from other species. It had a close phylogenetic relationship and in the same clade with the SQS of Nigella sativa. A spatiotemporal expression analysis indicated the gene to be expressed in all tissues. Conclusion The full-length cDNA sequence and bioinformatics of the squalene synthase gene CfSQS of C. florida were determined for the first time. The information helped to further clarify the synthesis of the saponin compounds and regulatory mechanism that would pave the way for future studies on the biosynthetic pathways of triterpene saponins in C. florida.
- Clematis florida /
- triterpene saponins /
- squalene synthase /
- bioinformatics analysis

HTML全文

图 1 重瓣铁线莲CfSQS基因的RT-PCR扩增

M：DL2000 DNA Marker；1、2： cDNA扩增产物。

Figure 1. RT-PCR of CfSQS in C. florida

M: DL2000; 1,2: amplification product of cDNA.

下载: 全尺寸图片幻灯片

图 2 重瓣铁线莲CfSQS ORF核苷酸和氨基酸序列

Figure 2. Nucleic acid and deduced amino acid sequences of CfSQS

下载: 全尺寸图片幻灯片

图 3 CfSQS跨膜区预测结果

Figure 3. Predicted transmembrane region in CfSQS

下载: 全尺寸图片幻灯片

图 4 CfSQS的二级结构预测分析

蓝色：α-螺旋；紫色：无规则卷曲；红色：延伸链；绿色：β-转角。

Figure 4. Predicted secondary structure of CfSQS

Blue represents alpha helix; purple, random coil; red, extended strand; green, beta turn.

下载: 全尺寸图片幻灯片

图 5 CfSQS蛋白质三级结构预测

Figure 5. Predicted tertiary structure of CfSQS

下载: 全尺寸图片幻灯片

图 6 重瓣铁线莲CfSQS氨基酸序列与其他物种SQS氨基酸序列多重比较

箭头标出的是12个鲨烯合酶的特异氨基酸活性位点；方框所示为2个富含天冬氨酸（DXXXD）的保守区；下划线所示为鲨烯合成酶的4个高度保守的结构域和1个高度差异的疏水区结构域（Ⅴ）。

Figure 6. Multi-alignment of deduced amino acid sequence of SQSs from C. florida and other plants

Specific amino acids required for SQS activity indicated by red arrows; two aspartate-rich regions (DXXXD) appeared in square boxes; and five domains were underlined and marked as I, II, III, IV, and V, facilitating FPP binding through a magnesium ion shown as region V.

下载: 全尺寸图片幻灯片

图 7 重瓣铁线莲CfSQS与其他物种SQS氨基酸序列系统发育树

Figure 7. Phylogenic tree of deduced amino acid sequences of SQSs from C. florida and other plants

下载: 全尺寸图片幻灯片

图 8 CfSQS基因在不同组织的RT-PCR检测

M：DL2000 DNA Marker；1:根; 2：茎；3：叶。

Figure 8. RT-PCR detection of CfSQS in different tissues

M: DL2000; 1: root; 2: stem; 3: leaf.

下载: 全尺寸图片幻灯片

参考文献(17)

[1]	周春权, 赵立, 蒋畅, 等. 畲药“十二时辰”醇提液皮肤用药的毒性实验研究 [J]. 中国医药科学, 2020, 10(19):69−71,100. doi: 10.3969/j.issn.2095-0616.2020.19.016 ZHOU C Q, ZHAO L, JIANG C, et al. Study on the toxicity of ethanolic extracts from Clematis florida Thunb. var. Plena DE. Don Ointment by skin administration [J]. China Medicine and Pharmacy, 2020, 10(19): 69−71,100.(in Chinese) doi: 10.3969/j.issn.2095-0616.2020.19.016
[2]	沈廷明, 黄春情, 吴军军, 等. GC-MS法分析畲药十二时辰花中挥发油成分 [J]. 中草药, 2020, 51(24):6362−6366. doi: 10.7501/j.issn.0253-2670.2020.24.027 SHEN T M, HUANG C Q, WU J J, et al. GC-MS analysis of volatile oil from traditional She medicine Clematis florida var. plena flower [J]. Chinese Traditional and Herbal Drugs, 2020, 51(24): 6362−6366.(in Chinese) doi: 10.7501/j.issn.0253-2670.2020.24.027
[3]	朱扶蓉, 滕春福, 李清禄. 响应面法优化畲药十二时辰总皂苷超声提取工艺的研究 [J]. 时珍国医国药, 2016, 27(12):2874−2878. ZHU F R, TENG C F, LI Q L. Optimization of ultrasonic extraction process of total saponins from She medicine Clematis florida var. plena by response surface methodology [J]. Lishizhen Medicine and Materia Medica Research, 2016, 27(12): 2874−2878.(in Chinese)
[4]	YANG N N, ZHANG Y F, ZHANG H T, et al. The in vitro and in vivo anti-inflammatory activities of triterpene saponins from Clematis florida [J]. Natural Product Research, 2021, 35(24): 6180−6183. doi: 10.1080/14786419.2020.1833203
[5]	JIANG Y F, QIAN R J, ZHANG W B, et al. Composition and biosynthesis of scent compounds from sterile flowers of an ornamental plant Clematis florida cv. ‘Kaiser’ [J]. Molecules (Basel, Switzerland), 2020, 25(7): 1711. doi: 10.3390/molecules25071711
[6]	黄泽豪, 张月玲, 沈贤娟. 畲药十二时辰的生药鉴定 [J]. 亚热带植物科学, 2013, 42(2):104−108. doi: 10.3969/j.issn.1009-7791.2013.02.003 HUANG Z H, ZHANG Y L, SHEN X J. Pharmacognostic identification of Clematis florida var. plena [J]. Subtropical Plant Science, 2013, 42(2): 104−108.(in Chinese) doi: 10.3969/j.issn.1009-7791.2013.02.003
[7]	王琴波, 王雨晴, 杨谨, 等. 20种中药基原植物鲨烯合酶基因的生物信息学分析 [J]. 中草药, 2021, 52(3):838−844. doi: 10.7501/j.issn.0253-2670.2021.03.027 WANG Q B, WANG Y Q, YANG J, et al. Bioinformatics analysis of squalene synthase gene from 20 Chinese herbal plants [J]. Chinese Traditional and Herbal Drugs, 2021, 52(3): 838−844.(in Chinese) doi: 10.7501/j.issn.0253-2670.2021.03.027
[8]	何焱, 李忠玥, 李卿, 等. 藏药翼首草角鲨烯合成酶的生物信息学分析 [J]. 基因组学与应用生物学, 2020, 39(7):3139−3150. doi: 10.13417/j.gab.039.003139 HE Y, LI Z Y, LI Q, et al. Bioinformatics analysis of squalene synthase in Pterocephalus hookeri(C. B. Clarke) Höeck [J]. Genomics and Applied Biology, 2020, 39(7): 3139−3150.(in Chinese) doi: 10.13417/j.gab.039.003139
[9]	AUGUSTIN J M, KUZINA V, B. ANDERSEN S, et al. Molecular activities, biosynthesis and evolution of triterpenoid saponins [J]. Phytochemistry, 2011, 72(6): 435−457. doi: 10.1016/j.phytochem.2011.01.015
[10]	ZHAO Y J, CHENG Q Q, SU P, et al. Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants [J]. Applied Microbiology and Biotechnology, 2014, 98(6): 2371−2383. doi: 10.1007/s00253-013-5496-3
[11]	JFAB C, GLAB C, MEI Y, et al. Isolation and functional analysis of squalene synthase gene in tea plant Camellia sinensis [J]. Plant Physiology and Biochemistry, 2019, 142: 53−58. doi: 10.1016/j.plaphy.2019.06.030
[12]	陶晨陈, 马成通, 吴耀生, 等. 红花栝楼鲨烯合酶基因的克隆及其序列分析 [J]. 中草药, 2015, 46(7):1034−1041. doi: 10.7501/j.issn.0253-2670.2015.07.018 TAO C C, MA C T, WU Y S, et al. Cloning and sequence analysis of squalene synthase gene from Trichosanthes rubriflos [J]. Chinese Traditional and Herbal Drugs, 2015, 46(7): 1034−1041.(in Chinese) doi: 10.7501/j.issn.0253-2670.2015.07.018
[13]	WANG J R, LIN J F, GUO L Q, et al. Cloning and characterization of squalene synthase gene from Poria cocos and its up-regulation by methyl jasmonate [J]. World Journal of Microbiology and Biotechnology, 2014, 30(2): 613−620. doi: 10.1007/s11274-013-1477-z
[14]	RONG Q X, JIANG D, CHEN Y J, et al. Molecular cloning and functional analysis of squalene synthase 2(SQS2) in Salvia miltiorrhiza bunge [J]. Frontiers in Plant Science, 2016, 7: 1274.
[15]	LIU M, LI L N, PAN Y T, et al. cDNA isolation and functional characterization of squalene synthase gene from Ornithogalum caudatum [J]. Protein Expression and Purification, 2017, 130: 63−72. doi: 10.1016/j.pep.2016.10.002
[16]	ZHA L P, LIU S, SU P, et al. Cloning, prokaryotic expression and functional analysis of squalene synthase (SQS) in Magnolia officinalis [J]. Protein Expression and Purification, 2016, 120: 28−34. doi: 10.1016/j.pep.2015.12.008
[17]	JIANG D, RONG Q, CHEN Y, et al. Molecular cloning and functional analysis of squalene synthase (SS) in Panax notoginseng [J]. International Journal of Biological Macromolecules, 2017, 95: 658−666. doi: 10.1016/j.ijbiomac.2016.11.070