Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii
Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the...
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MDPI AG
2018-01-01
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| Series: | Molecules |
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| Online Access: | http://www.mdpi.com/1420-3049/23/2/269 |
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| author | Bin Zhang Yan Liu Mengmeng Chen Juntao Feng Zhiqing Ma Xing Zhang Chuanshu Zhu |
| author_facet | Bin Zhang Yan Liu Mengmeng Chen Juntao Feng Zhiqing Ma Xing Zhang Chuanshu Zhu |
| author_sort | Bin Zhang |
| collection | DOAJ |
| description | Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coli Transetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii. |
| first_indexed | 2024-12-23T05:29:09Z |
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| id | doaj.art-5027754102174d1ca48e00cac8d16848 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-12-23T05:29:09Z |
| publishDate | 2018-01-01 |
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| spelling | doaj.art-5027754102174d1ca48e00cac8d168482022-12-21T17:58:31ZengMDPI AGMolecules1420-30492018-01-0123226910.3390/molecules23020269molecules23020269Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordiiBin Zhang0Yan Liu1Mengmeng Chen2Juntao Feng3Zhiqing Ma4Xing Zhang5Chuanshu Zhu6Research & Development Center of Biorational Pesticides, Northwest A & F University, Yangling 712100, ChinaResearch & Development Center of Biorational Pesticides, Northwest A & F University, Yangling 712100, ChinaResearch & Development Center of Biorational Pesticides, Northwest A & F University, Yangling 712100, ChinaResearch & Development Center of Biorational Pesticides, Northwest A & F University, Yangling 712100, ChinaResearch & Development Center of Biorational Pesticides, Northwest A & F University, Yangling 712100, ChinaResearch & Development Center of Biorational Pesticides, Northwest A & F University, Yangling 712100, ChinaResearch & Development Center of Biorational Pesticides, Northwest A & F University, Yangling 712100, ChinaCelastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coli Transetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii.http://www.mdpi.com/1420-3049/23/2/269Tripterygium wilfordiisqualene synthasecelastrolprokaryotic expressionfunctional characterization |
| spellingShingle | Bin Zhang Yan Liu Mengmeng Chen Juntao Feng Zhiqing Ma Xing Zhang Chuanshu Zhu Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii Molecules Tripterygium wilfordii squalene synthase celastrol prokaryotic expression functional characterization |
| title | Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii |
| title_full | Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii |
| title_fullStr | Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii |
| title_full_unstemmed | Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii |
| title_short | Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii |
| title_sort | cloning expression analysis and functional characterization of squalene synthase sqs from tripterygium wilfordii |
| topic | Tripterygium wilfordii squalene synthase celastrol prokaryotic expression functional characterization |
| url | http://www.mdpi.com/1420-3049/23/2/269 |
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