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International Journal of Medicinal Mushrooms
IF: 1.423 5-Year IF: 1.525 SJR: 0.431 SNIP: 0.661 CiteScore™: 1.38

ISSN Print: 1521-9437
ISSN Online: 1940-4344

International Journal of Medicinal Mushrooms

DOI: 10.1615/IntJMedMushrooms.v18.i5.80
pages 445-455

Cloning and Characterization of a Squalene Synthase Gene from the Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes)

Panpan Zhang
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, China
Xiaoying Cao
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, China
Changgen Li
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, China
Zhujun Zheng
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, China
Sun Yong
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, China
Ji-Hong Jiang
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, China

ABSTRACT

Squalene synthase catalyzes the condensation of 2 molecules of farnesyl diphosphate to produce squalene, the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. A squalene synthase gene, designated IoSQS, was isolated from Inonotus obliquus, a medicinal mushroom that produces a plethora of bioactive triterpenes. IoSQS complementary DNA was found to contain an open reading frame of 1476 bp, encoding a protein of 491 amino acids with a calculated molecular mass of 55.85 kDa. The IoSQS genomic DNA sequence consisted of 1813 bp and contained 4 exons and 3 introns. The restriction fragment polymorphisms revealed by Southern blot analysis suggested that IoSQS was a single-copy gene. Promoter analysis indicated that the 5' upstream region of IoSQS possessed various potential elements associated with physiological and environmental factors. The expression pattern of IoSQS in different stages and under methyl jasmonate treatment correlated with the accumulation of total triterpenoids and was consistent with the predicted results of the IoSQS promoter region. The N-terminal 466 residues of the hydrophilic sequence were expressed as a His-tagged protein in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. Squalene was detected in vitro in reaction mixture by high-performance liquid chromatography analysis. These results suggest that the IoSQS enzyme is involved in squalene production in I. obliquus.


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