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.
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