|
|
|
| Cloning and Expression Analysis of FhSMT1 Gene in Fritillaria hupehensis |
| DUAN Yuan-Yuan1,2, XIONG Jing-Wen1, WANG Xiao-Yue1, WANG Fan-Fan1, TANG Tao1, ZHOU Wu-Xian1, YOU Jing-Mao1,2,* |
1 Key Laboratory of Biology and Cultivation of Chinese Medicinal Materials, Ministry of Agriculture and Rural Affairs/Hubei Standardized Production of Traditional Chinese Medicine (GAP) Engineering Research Center/Hubei Enshi Selenium-rich Resource Observation and Research Station, Institute of Chinese Herbel Medicine, Hubei Academy of Agricultural Science, Enshi 445000, China;
2 Hubei Provincial Engineering Research Center of Under-forest Economy, Hubei Academy of Agricultural Science, Wuhan 430064, China |
|
|
|
|
Abstract Isosteroidal alkaloids are the main active phytochemicals in Fritillaria hupehensis. Sterol C-methyltransferase 1 (SMT1) is an important rate limiting enzyme, playing a significant role in regulating phytosterol biosynthesis. In this study, FhSMT1 gene (GenBank No. PV826664) was successfully cloned from the bulbs of F. hupehensis, based on the gene sequence identified from the prior transcriptomic analysis. Bioinformatics analysis, protein expression profiling, tissue-specific expression analysis, and isosteroidal alkaloid content detection were conducted to study the function of FhSMT1 protein and its expression characteristics. The results showed that the CDS sequence of FhSMT1 gene was 483 bp, which encoded a protein characterized by instability, consisting of 160 amino acids with molecular weight of 17.9 kD and pI of 8.85. There were no signal peptide and transmembrane domains in this protein. The protein had a classical SMT domain, which was a typical SMT family member. The subcellular localization results indicated that FhSMT1 protein was located on the nucleus and the cytoplasmic compartment. On this basis, an expression vector was constructed for prokaryotic expression, and the recombinant expression vector transformed into Escherichia coli. The recombinant protein was obtained through inducing expression with 0.5 mmol/L isopropyl-β-D-thiogalactoside (IPTG) at 16 ℃. His-FhSMT1 and pET28a-SUMO-FhSMT1-His proteins with higher expression were purified using imidazole concentrations with 100 and 250 mmol/L, respectively. FhSMT1 exhibited a tissue-specific expression gradientwithleaf>stem>bulb, consistenting the distribution patterns of peiminine and peimisine, which indicated that FhSMT1 may be involved in regulating the peiminine and peimisine biosynthesis in F. hupehensis. This study provides a theoretical basis for further analysis of the FhSMT1 function in the isosteroidal alkaloid biosynthesis.
|
|
Received: 21 April 2025
|
|
|
|
Corresponding Authors:
*jingmaoyou@126.com
|
|
|
|
[1] 蔡芷辰, 赵君谊, 陈海杰, 等. 2025. 金银花糖基转移酶基因的原核表达及其编码蛋白纯化[J]. 中草药, 56(04): 1338-1345.
(Cai Z C, Zhao J Y, Chen H J, et al.2025. Prokaryotic expressions and encode protein purification of glycosyltransferase in Lonicerae Japonicae Flos[J]. Chinese Traditional and Herbal Drugs, 56(04): 1338-1345.)
[2] 陈晓仪, 张甜甜, 赵琦. 2018. 卷叶贝母环阿屯醇合成酶基因的克隆及表达分析[J]. 中草药, 49(18): 4380-4385.
(Chen X Y, Zhang T T, Zhao Q.2018. Cloning and expression analysis of cycloartenol synthase gene in Fritillaria cirrhosa[J]. Chinese Traditional and Herbal Drugs, 49(18): 4380-4385.)
[3] 冯亚斌, 李沫宇, 吴秋丽, 等. 2017. 浙贝母FPS基因的克隆及其在生物碱代谢中的调控作用研究[J]. 中草药, 48(05): 971-978.
(Feng Y B, Zhang T T, Wu Q L, et al.2017. Cloning of FPS gene and its role in regulation of alkaloid synthesis in Fritillaria thunberfii[J]. Chinese Traditional and Herbal Drugs, 48(05): 971-978. )
[4] 冯亚斌, 俞信光, 庄欣晨, 等. 2016. 浙贝母HMGR基因保守区序列的克隆及生物信息学分析[J]. 核农学报, 30(12): 2289-2294.
(Feng Y B, Yu X G, Zhuang X C, et al.2016. Cloning and bioinformatics analysis of conserved fragment of HMGR gene in Fritillaria thunbergii[J]. Journal of Nuclear Agricultural Sciences, 30(12): 2289-2294.)
[5] 林江波, 王伟英, 邹晖, 等. 2019. 基于转录组测序的铁皮石斛植物甾醇生物合成相关基因分析[J]. 热带亚热带植物学报, 27(06): 693-701.
(Lin J B, Wang W Y, Zou H, et al.2019. Analysis of related genes in phytosterol biosynthesis in Dendrobium officinale based on transcriptome sequencing technology[J]. Journal of Tropical and Subtropical Botany, 27(06): 693-701.)
[6] 苗朝悦, 杜乐, 王佳琦, 等. 2023. 重组蛋白质在大肠杆菌体系中的可溶性表达策略[J]. 中国生物工程杂志, 43(09): 33-45.
(Miao Z Y, Du L, Wang J Q, et al.2023. Soluble expression strategies for recombinant proteins in Escherichia coli[J]. China Biotechnology, 43(09): 33-45.
[7] 王威威, 李德鑫, 李秋娥, 等. 2021. 暗紫贝母鲨烯合酶基因的克隆及其组织表达分析[J]. 西南农业学报, 34(12): 2596-2603.
(Wang W W, Li D X, Li Q E, et al.2021. Southwest China Journal of Agricultural Sciences, 34(12): 2596-2603.
[8] 许云婷, 徐黎明, 刘妍, 等. 2025. SUMO蛋白和高亲和PDGFβR肽共修饰的Bak BH3可溶性表达条件优化[J]. 牡丹江医科大学学报, 46(01): 9-12.
(Xu Y T, Xu J M, Liu Y, et al.2025. Optimization of expression of Bak BH3 co-modified with SUMO fusion and high-affinity peptide for PDGFβR[J]. Journal of MuDanJiang Medical University, 46(01): 9-12.
[9] 张阳, 李锐, 杨千叶, 等. 2018. 卷叶贝母1-脱氧-D-木酮糖-5-磷酸合成酶基因的克隆与表达分析[J]. 西南农业学报, 31(12): 2492-2497.
(Zhang Y, Li R, Yang Q Y, et al.2018. Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate synthetase gene in Fritillaria cirrhosa D. Don[J]. Southwest China Journal of Agricultural Sciences, 31(12): 2492-2497.
[10] Carland F, Fujioka S, Nelson T.2010. The sterol methyltransferases SMT1, SMT2, and SMT3 influence Arabidopsis development through nonbrassinosteroid products[J]. Plant Physiology, 153: 741-756.
[11] Chen G X, Liu J H, Jiang L Q, et al.2018. Peiminine protects dopaminergic neurons from inflammation-induced cell death by inhibiting the ERK1/2 and NF-kB signalling pathways[J]. International Journal of Molecular Sciences, 19: 821.
[12] Duan Y Y, Liu X H, Wu J Q, et al.2022. Transcriptomic and metabolic analyses reveal the potential mechanism of increasing steroidal alkaloids in Fritillaria hupehensis through intercropping with Magnolia officinalis[J]. Frontiers in Plant Science, 13: 997868.
[13] Guan H Y, Zhao Y J, Su P, et al.2017. Molecular cloning and functional identification of sterol C24-methyltransferase gene from Tripterygium wilfordi[J]. Acta Pharmaceutica Sinica B, 7: 603-609.
[14] Guan H Y, Su P, Zhao Y J, et al.2018. Cloning and functional analysis of two sterol-C24-methyltransferase 1 (SMT1) genes from Paris polyphylla[J]. Journal of Asian Natural Products Research, 20: 595-604.
[15] Kelly L J, Simon R B, Pellicer J, et al.2015. Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size[J]. New Phytologist. 208: 596-607.
[16] Kumar P, Ashrita, Acharya V, et al.2021. Comparative transcriptome analysis infers bulb derived in vitro cultures as a promising source for sipeimine biosynthesis in Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.)-high value Himalayan medicinal herb[J]. Phytochemistry, 183: 112631.
[17] Liu F J, Chen X Y, Yang J, et al.2023. Revealing active components and action mechanism of Fritillariae Bulbus against non-small cell lung cancer through spectrum-effect relationship and proteomics[J]. Phytomedicine, 110: 154635.
[18] Lu X, Zhang L N, Du J F, et al.2020. Comparative analysis and natural evolution of squalene epoxidase in three Fritillaria species[J]. Plant Molecular Biology, 103: 705-718.
[19] Lu Q X, Li R, Liao J Q, et al.2022. Integrative analysis of the steroidal alkaloids distribution and biosynthesis of bulbs Fritillariae Cirrhosae through metabolome and transcriptome analyses[J]. BMC Genomics, 23: 511.
[20] Pal S, Rastogi S, Nagegowda D A, et al.2019. RNAi of sterol methyl transferase 1 reveals its direct role in diverting intermediates towards withanolide/phytosterol biosynthesis in Withania somnifera[J]. Plant and Cell Physiology, 60: 672-686.
[21] Qi P Y, Zhang Y N, Zhao C S, et al.2023. Research progress on biological regulation and biosynthesis of isosteroid alkaloids in Fritillaria[J]. Plant growth regulation, 101: 599-615.
[22] Sawai S, Ohyama K, Yasumoto S, et al.2014. Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato[J]. Plant Cell, 26: 3763-3774.
[23] Sharma B, Seth R S, Thakur S, et al.2021. Genome-wide transcriptional analysis unveils the molecular basis of organ-specific expression of isosteroidal alkaloids biosynthesis in critically endangered Fritillaria roylei Hook[J]. Phytochemistry, 187: 112772.
[24] Sonawane P D, Pollier J, Panda S, et al.2016. Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism[J]. Nature Plants, 2016, 3: 1-13.
[25] Wang Y, Hou H P, Ren Q, et al.2021. Natural drug sources for respiratory diseases from Fritillaria: Chemical and biological analyses[J]. Chinese Medicine, 16(01): 40.
[26] Yin X, Liu J, Kou C, et al.2023. Deciphering the network of cholesterol biosynthesis in Paris polyphylla laid a base for efficient diosgenin production in plant chassis[J]. Metabolic Engineering, 76: 232-246. |
|
|
|
