Content Determination Of Volatile Oil, Molecular Cloning And Expression Analysis Of The Gene Encoding HMG-CoA Reductase From Eucalyptus

Eucalyptus essential oil, which is one of the most volatile oil output in the world, is used for medicinal, perfumery and industrial purposes. Now, the eucalyptus essential oil is mainly supplied by China in international market. On one hand the people's requirement is increasing, on the other hand the eucalyptus essential oil yield is about 1%lowly. It's an effective method to improve the yield by genetic engineering. So, the volatile oil from different tissue were isolated and the composition contents,1,8-cineol andβ-eudesmol, were determined; Simultaneously the relative HMGR gene was cloned in the essention oil metabolic pathway in this study. It can provide some information to transformation of Eucalyptus and determination of essential oil composition.Eucalyptus essential oil from root, stem and leaf of Eucalyptus urophylla×E. grandis were extracted by Clevenger apparatus. The result indicated the total oil from leaf was more than any other tissue and there wasn't essential oil in root. The oil quantity from leaf and stem was 252.6 mg·100g-1 and 18.2 mg·100g-1 respectively, corresponding the oil yield was 0.25%and 0.018%; The proportion of 1,8-cineol in total oil from leaf and stem was 0.06% and 0.04%, and the proportion ofβ-eudesmol in total oil from leaf and stem was 6.69% and 11.5% respectively by HPLC. The result indicatedβ-eudesmol was the mainly composition in Eucalyptus urophylla×E. grandis volatile oil while 1,8-cineol was low relatively.Monoterpenes and sesquiterpenes constitute of the eucalyptus essential oil. GPP and FPP, whose precursors of monoterpenes and sesquiterpenes, are synthesized in both mevalonate pathway and methylerythritol 4-phosphate pathway. The precursors generate the different composition by abscising phosphorylation, isomerization, cyclization and atom rearrangement. The gene encoding 3-Hydroxy-3-methylglutaryl-CoA reductase regarded as the first key enzyme in MVA pathway was cloning through Rapid Amplification of cDNA Ends technology from Eucalyptus urophylla×E. grandis. The full-length HMGR cDNA was 1955 bp. containing 66 bp 5'untranslated region,329 bp 3'untranslated region, poly A tail and a 1560 bp open reading frame encoding a peptide of 519 amino acids with a calculated molecular mass of 55.1202 kDa and an isoelectric point of 5.37. HMGR protein predicted from Eucalyptus urophyla×E.grandis showed 100% identities with Cyclocarya paliurus, Hevea brasiliensis, Camptotheca acuminate and Pyrus pyrifolia by Blast P on-line NCBI. By bioinformatics analysis, HMGR belonged to hydrophobic protein; The 23 amino acid residues, including 15 Serines localized in A60, A62, A63, A83, A111, A121, A147, A149, A191, A282, A344, A363, A437, A510, A511,5 Threonines localized in A144, A197, A237, A350, A362 and 3 Tyrosines localized in A120, A158, A172, were the phosphorylation sites after being translated; There were a signal peptide composed of the A1 to A25 amino acid residues at N-terminus which max possible cleavage site was between the A25 (S) and A26(L) and a transmembrane localized in A5(V) to A27(I); The subcellular prediction found that HMGR hadn't transit peptide at N-terminus and localized in secondary metabolism pathway with a signal peptide; There were two HMG-CoA binding motifs: EMPVGYVQLP and TTEGCLVA, localized in A167 to A176 and A196 to A203 amino acid residues respectively, the G(Glu) amino acid residue had an important promotion; Also there were two NADP(H) binding motifs:RCDGHEH and GTVGGGT localized in A292 to A298 and A441 to A447 amino acid residues respectively; HMGR from Eucalyptus urophylla×E.grandis belonged to plants group by the phylogenetic tree of HMGRs from other plants, the result was accurate and reliable; The secondary structure analysis revealed that HMGR was composed of 47.40% alpha helix,15.41% extended strand,7.13% beta turn and 30.06% random coil. HMGR had a three dimensional structure with "V" homology-based on the HMGR model of Homo sapiens, containing N-domain, S-domain and L-domain.Through the gene construction analysis, there was an intron of 319 bp in genome DNA, the function of the intron was indefinite. Tissue expression profile analysis by Real Time quantitative PCR indicated HMGR expression was the highest in branch, following leaf and the lowest in root. The protein,55.1202 kD of HMGR predicted, was induced expression by IPTG in E. coli M15 successfully.