Cloning And Expression The Gene Of Squalene Synthase From Dryopteris Fragrans (L.)Schott

Dryopteris fragrans（L.） Schott is a deciduous fern in Dryopteris family of Dryopteris genus, which mainly grows on the volcanics of north of Heilongjiang Province and it is used as an effective perennial herb against various skin diseases and rheumatoid arthritis. Rresearches in recent years have discovered that D. fragrans owns significant effects of antibiosis, antiinflammatory and anti-tumor. Through chemistry extraction and chemical analysis and investigation of pharmacology and pharmacodynamic action, researchers found and identified a variety of active components in D. fragrans, indicating the fern has the development potential and application foreground. Terpenoids synthesized in D. fragrans have many biological activities, including the volatile aroma compounds, secretion of antimicrobial, antitumor. And in previous studies, the β-sitosterol derived from tetracyclic triterpenoids isolated by Ito H. is validated is one of the main active ingredient in the anti-tumor. Therefore, researches on triterpenoids in D. fragrans have significan development value fort medicine.The squalene synthase gene （SQS） is the first key enzyme gene in triterpenoid biosynthesis pathway and is the starting point for all triterpenoids ramifications. It is significantly important for the triterpenoids synthesi of downstreams and accumulation in plant. Meanwhile, the position of squalene synthase is in the midway of terpene biosynthesis pathway, changes of its expression level and activity would affect the whole pathway of terpene synthesis and change the metabolic flux flow in plants, which plays a pivotal role. Therefore, the SQS regulation studies in D. fragrans would not only provide a theoretical basis in molecular biology, but also found the foundation for understanding terpene metabolic pathways in D. fragrans deeply.In this work, germfree D. fragrans sporophyte was used as materials. The transcriptome database sequenced by Illumina which was completed in previous work was employed to select candidate gene fragments of squalene synthase. The full-length cDNA of selected gene fragment was cloned and analyzed by bioinformatics tools and softwares. The squalene synthase gene of D. fragrans was expressed by prokaryotic expression. The Real-time PCR was employed to analyse the level of gene expression in different tissues. The results would provide evidences for triterpenoids synthesis researches in D, fragrans.The mainly results obtained in this work are as follows:1. According to the transcriptome database of D. fragrans, there were4Unigenes which were likely to squalene synthase gene by annotation and function forecast. Those sequences were aligned by BLAST and we picked the Unigene₃1852All out. 2. Total RNA was extracted from the leaves of D. fragrans sporophyte and converted to cDNA by reverse transcription. The primers of conservative sequence of Unigene₃1852All were used to amplify the sequence of Unigene₃1852All. The amplifatation products was linked to the pMD18-T vector for sequencing. The5’&3’-RACE primers were designed by sequence and we obtained the full length cDNA of squalene synthase gene in D. fragrans. The new gene was the first squalene synthase gene cloned from fern.3. We predicted the ORF （open reading frame） of squalene synthase gene in D. fragrans and its amino acid sequence by bioinformatics software. After BLAST alignment and conservation domain forecast, the obtained gene was affirmed as squalene synthase gene. We submitted the sequence of gene to GenBank, the accession number was KJ728647. Subsequently, we predicted basic chemical property, secondary and tertiary structure, conservation domain, hydrophilicity/hydrophobicity, sites of phosphorylation and glycosylation, disulfide bond, membrane-spanning domain, its cellular localization. A phylogeny of various plant species was drawn, which reflected the evolution position of squalene synthase gene of D. fragrans.4. The primer pair of ORF was disigned according to the sequences of5’&3’-UTR. The ORF was amplified by PCR, which was used to construct prokaryotic expression vector and the vecter was transformed into expression strain BL21（DE3）. The IPTG was employed to induce BL21（DE3） and the protein was expressed.We employed the Real-time PCR to identify the levels of squalene synthase gene expression in different tissues of D. fragrans. The results showed that the roots had the maximal expressed value, the leaves and petioles of aerial part was the second and the rhizome was the lest, implicating that the roots were the most vigorous tissue for the triterpenoids biosynthesis in D. fragrans.