Study On The Mechanism Of Forming Aromatic Heartwood In Santalum Album L. By Ethephon Stimulation

Objective:It took about10years to form the aromatic heartwood (forming the heartwood containing terpenes) under natural condition for Santalum album L.,30years, to be used as medicine. So the author did a experiment of stimulating the3-year-old sandalwoods to form aromatic heartwood with ethephon, focused on the terpenoid synthase genes and its functional expression during the aromatic heartwood forming, in order to clarify the molecular mechanism on the aromatic heartwood forming, and provided a scientific basis and technical support for significantly shorten the sandalwood production cycle, as well as our country’s sandalwood resources sustainable development.Methods:Drilled holes in the sandalwood trees to inject different concentrations of ethephon; G.C-MS and HPLC were introduced to analyze the changes of component and content on sandalwood heartwood before and after the stimulation; Degenerate primers were designed based on the conserved region by comparing the sequenees of Sandalwood terpene synthase gene published in Genbank. CTAB-LiCl treatment was used to extract RNA from the heartwood of Santalum album L. and RT-PCR was used to get TPS1gene, then the TPS1gene was subcloned into the vector PGM-T to get a Cloned plasmid; the TPS1was amplified by PCR, double-digested with EcoR V and Sac I and then inserted into the expression vector pET32a (+) which was digested with the same enzymes, to get recombinant plasmid; E. coli DH5a competent cells were transformed with the recombinant plasmid; Expression of TPS1gene was investigated in the E.coli BL21(DE3) carrying prokaryotic expression plasmid under the induction of IPTG, SDS-PAGE was used to analyze the results of expressed protein;The purified expressed protein was added to the a reaction buffer containing substrates of FPP and GPP, Reaction mixture was extracted with hexane, GC-MS was used to analyze the reaction products.Result:1The test of Stimulating Heartwood by EthephonThe color of the heartwood which was from ethephon stimulated group is significantly deeper than the water Stimulated group, The color of the heartwood which was from the3%concentration group is deeper than the2%concentration group, The color of the upper and lower5centimeter is deeper than10centimeter taken from the heartwood. The powder from the ethephon stimulated group was very fragrant, while the water Stimulated group was not.The microscopic characteristics of the heartwood powder between the three groups were Basically the same, The following structure can be observed under the microscope:Toughness of fiber, Fiber tracheids, Bordered pits catheters and wood rays. Large accumulation of yellow-brown discharge was Observed in the3%concentration group, only a few scattered yellow-brown discharge was Observed in the2%concentration group, while there was no yellow-brown discharge in the water Stimulated group.2Chemical composition analysisAfter1year and a half of ethephon stimulation, the sandalwoods contained volatile oil, that the water stimulated group didn’t contain, Mass spectrometry was used to analyze peaks, The main chemical components of the sandalwood volatile oil are alpha-santalol, α-trans-bengamotol, E cis,Epi-beta-santalol, beta-santalol. Regardless of the total or a single content of the sandalwood volatile oil, the oil from3%ethephon stimulated group were significantly higher than that from2%ethephon stimulated group, The average content of volatile oil was no significant difference between Up and down5centimeter on the stimulation site, but the content of the up5centimeter was higher than the up10centimeter. The content of both alpha-sandalwood alcohol and beta-sandalwood alcohol was less than20%in the2%stimulated group and, was67%in the3%stimulated group.After3year, the sandalwoods of the Water group and the natural group contained the volatile oil, but the content of the oil in these two groups was lower than in the ethephon stimulated groups and reference drug, alpha-santalol, α-trans-bengamotol, E cis, Epi-beta-santalol, beta-santalol were still the main compositions of the oil in all groups except the water group. Percentages of four components in each group are the highest in the natural group, which reaches88.7495%, the second is the reference drug and the3%stimulated group., both of the two groups reach77.000%, the least is2%stimulated group, reaching44.769%. Although the water group contains volatile oil, its main content is dibutyl-hydroxy-toluene(34.715%). The all percentages of alpha-santalol, α-trans-bengamotol, E cis, Epi-beta-santalol, beta-santalol are only24.133%, also the content of N-palmityl acid and9-stearate are very high in water group.3Cloning of the core fragment of TPS1Gel electrophoresis showed that an approximately1.8kb fragment was specifically amplified as expected. At the same time, a terpene synthase gene, which was the key enzyme gene in terpenoids biosynthesis, was isolated by using the method of homology cloning. The gene was consisted of1812bp nucleotides, containes the coding region of1731bp, and encoded576amino acid residues, which was renamed as TPS1. The sequence shares the homology more than95%with the given sequences(Genebank, EU798692) by using the blastn in NCBI.4Construction of prokaryotic expression plasmidThe results of gel electrophoresis and sequence alignment showed that expected expression plasmid containing the TPS1was successfully constructed.5Expression of TPS1as fusion proteinThe results of SDS-PAGE showed that the expected expression plasmid containing the TPS1expressed the target protein which is about7272～95kDa after IPTG induction as expectated. Most of the target protein is still the form of inclusion bodies in the precipitate After sonication, only small amount of the target protein could be able to detect in the supernatant. That was Some of the target protein in soluble expression.6Analysis the function of fusion proteinGC-MS was used to analysis the catalytic reaction product. The results showed that the maps were no difference between positive group and negative control group with the substrate of FPP and GPP. conclusion:1The results showed that two concentrations of ethephon could form heartwood by stimulating. However, the effect of3%was much better than that of2%. The content of essential oil in heartwood had the trend of more farther more smaller to the site that stimulated by ethephon.After3years of the stimulating, The composition of volatile oil had major changes in the ethephon stimulated groups. Regardless of composition or content of the volatile oil reached consensus between the ethephon stimulated groups and the reference drug.2A terpene synthase gene, which was was the key enzyme gene in terpenoids biosynthesis, was isolated by using the method of homology cloning. The gene was consisted of1812bp nucleotides, containes the coding region of1731bp, and encoded576amino acid residues, which was renamed as TPS1. The sequence shares the homology more than95%with the given sequences(Genebank, EU798692) by using the blastn in NCBI.3Constructed the prokaryotic expression plasmid pET32a-TPS1, Mainly expressed as inclusion bodies after IPTG induction, only a small amount of soluble expressed, Increased the expression of soluble protein By changing the express hostel bacteria and optimize the induction conditions, So that subsequent purification experiments to be carried out smoothly.4The results of analysising the activity of purified fusion protein showed that the fusion protease had no catalytic, the reason needs futher research.