Cloning, Bioinformatics and Expression of SmSLS2 Gene in Swertia mussotii

doi:10.7525/j.issn.1673-5102.2019.03.013

Abstract

Abstract: Secologanin synthase(SLS) is the key enzyme of secoiridoid pathway. We obtained the SmSLS2 gene full-length sequence according to the transcriptome of Swertia mussotii. We cloned the gene, analyzed the bioinformation of SmSLS2, constructed the vectors and performed the gene expression. The results showed that SmSLS2 cDNA complete sequence had a length of 1 566 bp(GenBank:MH535904), encoding 521 amino acid residues, and the pI of SmSLS2 was 8.92. Domain analysis results showed that SmSLS2 had one transmembrane domain, and the protein secondary and tertiary structures were analyzed and forecasted. The gDNA sequence of SmSLS2 was 2 576 bp, contained five exons and four introns. The SmSLS2 protein shared high identity with other SLS proteins of plants. Prokaryotic expression vector pET-28a-sumo-SmSLS2 was constructed and transformed into Escherichia coli BL21(DE3) for expression under 37℃, and induced by 0.5 mmol·L^-1 IPTG. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. SmSLS2 gene was successfully cloned from S.mussotii, and it will provide a foundation for further functional research on SmSLS2 protein and increasing the product of iridoid compound by genetic engineering in S.mussotii.

Key words: Swertia mussotii Franch, secologanin synthase2, gene cloning, bioinformations analysis, prokaryotic expression

CLC Number:

Q949.776.4

LI Wen-Jing, FENG Yu, HOU Xiao-Qiang, Sun Yan-Xiang, Han Mei-Ling, LI Xiao-Xue, WANG Yong, XIANG Bei-Bei. Cloning, Bioinformatics and Expression of SmSLS2 Gene in Swertia mussotii[J]. Bulletin of Botanical Research, 2019, 39(3): 431-440.

References

1. 中国科学院中国植物志编辑委员会. 中国植物志[M]. 北京:科学出版社,1988:400. Editorial Committee of Flora of China,Chinese Academy of Sciences. Flora of China[M]. Beijing:Science Press,1988:400.
2. 舒光明. 川西獐牙菜的化学成分、药理作用和临床应用研究进展[J]. 现代药物与临床,2012,27(2):176-179. Shu G M. Research progress on chemical constituents,pharmacological actions,and clinical applications of Swertia mussotii[J]. Drugs & Clinic,2012,27(2):176-179.
3. 郭爱华. 龙胆科獐牙菜属药用植物化学成分和药理作用的研究进展[J]. 山西中医学院学报,2005,6(1):57-59. Guo A H. Research progress on chemical constituents and pharmacological actions of Swertia mussotii[J]. Journal of Shanxi College of Traditional Chinese Medicine,2005,6(1):57-59.
4. 罗翠婷,毛双双,陈河如,等. 川西獐芽菜化学成分研究[J]. 中草药,2013,44(8):942-946. Luo C T,Mao S S,Chen H R,et al. Chemical constituents from Swertia mussotii[J]. Chinese Traditional and Herbal Drugs,2013,44(8):942-946.
5. 张建胜,王雪梅,董秀华,等. 川西獐牙菜化学成分研究[J]. 中药材,2009,32(4):511-514. Zhang J S,Wang X M,Dong X H,et al. Studies on chemical constituents of Swertia mussotii[J]. Journal of Chinese Medicinal Materials,2009,32(4):511-514.
6. 孟宪华,陈德道,张樱山,等. 川西獐牙菜的化学成分、药理作用和临床应用研究进展[J]. 现代药物与临床,2012,27(2):176-179. Meng X H,Chen D D,Zhang Y S,et al. Research progress on chemical constituents,pharmacological actions,and clinical applications of Swertia mussotii[J]. Drugs & Clinic,2012,27(2):176-179.
7. 朱钰叶,张喜德,张洪泉. 藏药獐牙菜属植物的药理研究进展[J]. 中国野生植物资源,2008,27(1):5-7. Zhu Y Y,Zhang X D,Zhang H Q. Research progress on pharmacological actions of Swertia mussotii[J]. Chinese Wild Plant Resources,2008,27(1):5-7.
8. Wang J F,Zhao C Z,Liu C,et al. Introgression of Swertia mussotii gene into Bupleurum scorzonerifolium via somatic hybridization[J]. BMC Plant Biology,2011,11:71.
9. Vaidya H,Goyal R K,Cheema S K. Anti-diabetic activity of swertiamarin is due to an active metabolite,gentianine,that upregulates PPAR-γ gene expression in 3T3-L1 cells[J]. Phytotherapy Research,2013,27(4):624-627.
10. 朴金花,张杨,杨郁,等. 胡黄连属植物环烯醚萜类化学成分和药理活性研究进展[J]. 中国实用医药,2007,2(34):142-144. Piao J H,Zhang Y,Yang Y,et al. Research progress on chemical constituents and pharmacological actions of Picrorhiza Royel[J]. China Practical Medicine,2007,2(34):142-144.
11. 贾诩,李茂星,陶锐,等. 藏药独一味中的主要化学成分环烯醚萜苷化合物在动物的镇痛抗炎作用研究[J]. 中国临床药理学杂志,2015,31(8):635-639. Jia X,Li M X,Tao R,et al. Analgesic and anti-inflammatory activities of iridoid glycosides from Tibetan medicine Lamiophlomis rotata in Balb/C mice[J]. The Chinese Journal of Clinical Pharmacology,2015,31(8):635-639.
12. 匡雪君,王彩霞,邹立秋,等. 长春花萜类吲哚生物碱生物合成与调控研究[J]. 中国中药杂志,2016,41(22):4129-4137. Kuang X J,Wang C X,Zou L Q,et al. Advance in biosynthesis of terpenoid indole alkaloids and its regulation in Catharanthus roseus[J]. China Journal of Chinese Materia Medica,2016,41(22):4129-4137.
13. 向蓓蓓,李晓雪,王勇,等. 川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J]. 中草药,2017,48(5):962-970. Xiang B B,Li X X,Wang Y,et al. Cloning and expression of geranyl pyrophosphate synthase gene in Swertia mussotii[J]. Chinese Traditional and Herbal Drugs,2017,48(5):962-970.
14. Xiang B B,Li X X,Wang Y,et al. Cloning and characterization of two iridoid synthase homologs from Swertia mussotii[J]. Molecules,2017,22(8):E1387.
15. 李晓雪,王勇,孙继奇,等. 川西獐牙菜SmDL7H基因原核表达及组织表达[J]. 西北植物学报,2018,38(8):1375-1381. Li X X,Wang Y,Sun J Q,et al. Prokaryotic expression and tissue specific expression of SmDL7H in Swertia mussotii Franch[J]. Acta Botanica Boreali-Occidentalia Sinica,2018,38(8):1375-1381.
16. Wang J F,Liu Y L,Cai Y F,et al. Cloning and functional analysis of geraniol 10-hydroxylase,a cytochrome P450 from Swertia mussotii Franch[J]. Bioscience,Biotechnology,and Biochemistry,2010,74(8):1583-1590.
17. Pandey S S,Singh S,Babu C S V,et al. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis[J]. Scientific Reports,2016,6:26583.
18. Liu Y,Wang H,Ye H C,et al. Advances in the plant isoprenoid biosynthesis pathway and its metabolic engineering[J]. Journal of Integrative Plant Biology,2005,47(7):769-782.
19. Dugé De Bernonville T,Foureau E,Parage C,et al. Characterization of a second secologanin synthase isoform producing both secologanin and secoxyloganin allows enhanced de novo assembly of a Catharanthus roseus transcriptome[J]. BMC Genomics,2015,16(1):619.
20. Liu Y,Wang Y,Guo F X,et al. Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii[J]. Scientific Reports,2017,7:43108.
21. Irmler S,Schröder G,St-Pierre B,et al. Indole alkaloid biosynthesis in Catharanthus roseus:new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[J]. The Plant Journal,2000,24(6):797-804.
22. 吴昕怡,刘小莉. 环烯醚萜类成分生物合成途径及关键酶基因研究进展[J]. 中国民族民间医药,2017,26(8):44-48. Wu X Y,Liu X L. Progress of biosynthetic pathway and the key enzyme genes of iridoids[J]. Chinese Journal of Ethnomedicine and Ethnopharmacy,2017,26(8):44-48.
23. 韩梅,赵博,安志刚,等. 长春花萜类吲哚生物碱生物合成途径中重要酶(DXR、SLS、G10H、STR)基因的克隆与表达[J]. 植物研究,2007,27(5):564-568. Han M,Zhao B,An Z G,et al. Cloning and expression of cDNA encoding key enzymes (DXR,SLS,G10H and STR) in terpene indole alkaloids biosynthesis pathway from Catharanthus roseus[J]. Bulletin of Botanical Research,2007,27(5):564-568.
24. 常贺. 喜树SLS候选基因的分离及功能预测[D]. 哈尔滨:黑龙江大学,2009. Chang H. Cloning and function prediction of Camptothecia acuminate SLS gene[D]. Harbin:Heilongjiang University,2009.
25. 张晓东,李彩霞,王连春,等. 滇龙胆GrSLS1基因的克隆与原核表达[J]. 西北植物学报,2014,(7):1311-1317. Zhang X D,Li C X,Wang L C,et al. Cloning and prokaryotic expression of GrSLS1 gene in Gentiana rigescens[J]. Acta Botanica Boreali-Occidentalia Sinica,2014,34(7):1311-1317.
26. Hall T A. BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J]. Nucleic Acids Symposium Series,1999,41:95-98.
27. Tamura K,Peterson D,Peterson N,et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Molecular Biology and Evolution,2011,28(10):2731-2739.
28. 卢晟晔,王丽颖. 大肠杆菌中外源蛋白高效表达的影响因素及策略研究的新进展[J]. 中国实验诊断学,2006,10(9):1100-1103. Lu S Y,Wang L Y. New progress in the study of influencing factors and strategies for high-level expression of foreign protein in Escherichia coli[J]. Chinese Journal of Laboratory Diagnosis,2006,10(9):1100-1103.

[1]	Haonan ZHANG, Shanshan CHEN, Jianmin XU, Ping LUO, Xiaoping WANG, Zhiru XU, Chunjie FAN. Cloning and Functional Analysis of EgrWAT1 Gene in Eucalyptus grandis [J]. Bulletin of Botanical Research, 2023, 43(4): 601-611.
[2]	Haiyun SONG, Tao ZHANG, Peng HE, Shufang ZHENG, Lifeng WANG, Wenlin WANG. Cloning and Expression Analysis of MibZIP1 from Macadamia integrifolia [J]. Bulletin of Botanical Research, 2023, 43(1): 131-139.
[3]	Yuning Yang, Hao Dong, Shiwei Dong, Nairui Wang, Yue Song, Hanguo Zhang, Shujuan Li. Cloning and Expression Analysis of Transcription Factor LobHLH34 from Larix olgensis [J]. Bulletin of Botanical Research, 2022, 42(1): 112-120.
[4]	Qiu-Jin TAN, Tao ZHANG, Yuan-Rong WEI, Shu-Fang ZHENG, Xiu-Hua TANG, Wen-Lin WANG. Cloning and Expression Vector Construction of a Flowering Related Gene MiMADS-box from Macadamia integrifolia [J]. Bulletin of Botanical Research, 2021, 41(6): 888-895.
[5]	Xin-Tong JI, Lei YU, Ya-Guang ZHAN. Cloning and Expression Analysis of BRASSINAZOLE RESISTANTANT1 (BZR1) Gene from Fraxinus mandshurica [J]. Bulletin of Botanical Research, 2021, 41(5): 744-752.
[6]	Wei-Zhong KE, Wen-Juan ZHONG, Kai-Ying LIU, Xiang-Yuan LI, Ming-Hua YIN. Cloning and Sequence Analysis of Squalene Synthase(SQS) Gene from Dioscorea bulbifera L. Microtuber [J]. Bulletin of Botanical Research, 2021, 41(2): 243-250.
[7]	Wen-Lin WANG, Hai-Sheng CHEN, Shu-Fang ZHENG, Song-Le FAN, Li-Feng WANG, Qiu-Jin TAN, Zhen-Shi QIN, Xi-Yun HUANG, Peng HE, Xiu-hua TANG, Peng XU. Cloning,Structure and Function Analysis of MiMYB2 Gene from Macadamia integrifolia [J]. Bulletin of Botanical Research, 2020, 40(6): 913-922.
[8]	DONG Shi-Wei, YANG Yu-Ning, WANG Nai-Rui, ZHANG Han-Guo, LI Shu-Juan. Gene Cloning and Stress Response Analysis of Natural Disorder Protein in Populus trichocarpa [J]. Bulletin of Botanical Research, 2020, 40(4): 575-582.
[9]	LI Yang, SHI Ya-Kun, GAO Shi-Ke, Li Xiao-Yu, LAN Xing-Guo. Gene Cloning and Expression Analysis of PDIL1-2 of Ornamental Kale(Brassica oleracea var. acephala) [J]. Bulletin of Botanical Research, 2020, 40(2): 293-300.
[10]	LI Xin-Yu, HE Li-Ming, ZHAN Ya-Guang. Cloning and Expression Analysis of FmWRKY44 Gene in Fraxinus mandshurica [J]. Bulletin of Botanical Research, 2019, 39(4): 529-538.
[11]	DING Yi-Wei, ZHAN Ya-Guang, ZHANG Jia-Wei, HE Li-Ming. Cloning and Expression Analysis of Two PLT Transcription Factors Genes in Fraxinus mandshurica [J]. Bulletin of Botanical Research, 2019, 39(1): 139-147.
[12]	ZHANG Ji, ZHANG Li-Li, ZHANG Bei-Bei, ZHANG Fu-Chun. Prokaryotic Expression of Halostachys caspica HcDMC1 and Preparation of Its Antiserum [J]. Bulletin of Botanical Research, 2018, 38(3): 399-405.
[13]	LIAO Xu, HE Ming-Liang, ZHANG Su-Yan, MA Hai-Yan, WANG Xu-Hui, LUO Qiu-Xiang, GUAN Qing-Jie. Prokaryotic Expression,Activity Assay of Chlorella Cyclophilin A and Salt Tolerance Analysis of Arabidopsis Over-expressing CsCyp1A [J]. Bulletin of Botanical Research, 2017, 37(5): 722-729.
[14]	YE Si-Cheng, YAO Xiao-Hua, WANG Kai-Liang, LIN Ping, GONG Hong-En, ZHUO Ren-Ying. Cloning and Expression Analysis of Citrate Synthase(CS) Gene in Camellia oleifera [J]. Bulletin of Botanical Research, 2016, 36(4): 556-564.
[15]	ZHANG Xiao-Dong1；LI Cai-Xia1；WANG Yuan-Zhong2. Cloning and expression analysis of GrCMS* gene in Gentiana rigescens [J]. Bulletin of Botanical Research, 2016, 36(2): 258-265.