Molecular Cloning, Expression Of FPS And Analysis Of Salt Stress In Matricaria Chamomilla L.

German Chamomile(Mauritania chamomile L.), an annual herbs from Asteraceae family, is one of the medicinal and aromatic plants most used worldwide. German Chamomile essential oil has been proved to having anti-inflammatory, antimicrobial, antispasmodic, sedative, analgesic and anti-carcinogenic effects. The essential oil mainly consists of sesquterpenoids which is the main active component in German Chamomile. The biosynthetic pathway of sesquterpenoids is related to isoprenoid biosynthesis. And the Farnesyl pyrophosphate synthase (FPS) is one of the important rate-limiting enzymes in the the pathway of isoprenoid biosynthesis. In the present research, FPS gene was cloned from M. chamomile by RT-PCR, then sub-cloned into prokaryotic expression vector pET-30a(+) and induced in E. coli BL21(DE3). Moreover, effects of salt stress on physiological and biochemical characteristics of Mauritania chamomile L. and its microstructure were analyzed. These studies facilitated the application of medicinal plants through gene engineering in future.1. A pair of primer was designed with the software Primer Premier5.0according to the mRNA sequence of German Chamomile FPS gene in GenBank databases(Accession No: EF675758.1). After cloning of FPS gene from Mauritania chamomile L. by RT-PCR with the total RNA of German Chamomile, sequence analysis showed that the cloned cDNA consisted of1032bp encoded a protein of343amino acid (aa) residues. The German Chamomile FPS was a non-transmembrane peptide with the signal peptide and Hydrophobicity plot analysis.2. The FPS gene was sub-cloned into the multiple clone sites of plasmid pET-30a(+) to get the prokaryotic expression vector of pET-30a(+)-FPS, which was transformed into E.coli BL21(DE3). The results indicated that the deduced amino acid sequence showed a high identity to other plant FPS. As temperature, isopropylthiogalactoside (IPTG) concentration and induction time were optimized; the FPS protein was expressed highly under lmmol/L IPTG concentrations at37℃. The results showed that the prokaryotic expression vector pET-30a(+)-FPS was constructed successfully. Sequence analysis shows that FPS protein consisting of343amino acids, was expressed with molecular weight of38kDa. Western blotting analysis was carried out to test the product which laying a foundation for further purification and functional study of the FPS protein.3. The variation of germination characteristics of German Chamomile, such as relative germination percentage, germination energy, root length, stem length, biomass under different salt stress concentrations were tested. We also researched the variability of the chlorophyll content, soluble sugar, peroxidase(POD) and proline, and observed the changes of microstructure through the paraffin slicing. The results showed that the seed germination, seed germination energy root length, grass height, fresh weight and dry weight were decreased with the increase of NaCl concentration. The tendency of chlorophyll content was ascend in first and descend at last while the soluble sugar, POD and proline elevated with the increase of concentration of NaCl. The layers of mesophyll cells obviously increased. There were some changes of microstructure which adapt to environment after treatment of salt stress.