| Terpenoid substances, including sterols, carotenoid, dolichol, plant hormones and quinines, are widespread in the nature. So far more than65,000varieties of terpenoid substances have been discovered, in which most of them play an important role in the organism. Terpenoid substances are synthesized from two pathways: mevalonate pathway and pyruvic acid/phosphoric acid glyceraldehydes pathway. In two ways, a critical step to produce metabolic intermediate named isopentenyl diphosphate which has high bioactivity. Under the catalysis of isopentenyl diphosphate isomerase, isopentenyl diphosphate reacts with its double-bond isomer dimethylallyl diphosphate to form precursors of torpedoed substances, and after a series of chemical reactions various substances are gained. Isopentenyl diphosphate isomerase (IPI), a member of the Nudix hydrolase super-family, is a key enzyme in the mevalonic acid biosynthetic pathway. Based on the initial analysis of EST (Expressed Sequence Tag.EST) of cDNA library of Ch.praecox, a gene named CpIPI encoding isopentenyl diphosphate isomerase (IPI) was isolated from Chimonanthus praecox. Bioinformatics analysis, real time quantitative PCR and over-expression in tobaccoes was used to study the function of CpIPI gene. The main results in this paper are as follows:1. The analysis of the CpIPI sequence and its bioinformatics.The sequnce analysis showed that the full-length cDNA of CpIPI is1207bp, having879bp ORF, encoding a predicated protein of293amino acid. BLAST analysis on NCBI showed that the amino acid sequence of the protein encoded by CpIPI gene shares a high similarity with other reported IPI proteins, such as Amblyomma maculatum, Ipomoea sp, Kenyan, Glycine max and Pinus taeda. The structure characteristics of CpIPI protein were analyzed with bioinformatic methods. The results showed that there were no signal peptide and transmembrane domain in the deduced amino acid sequence. Hydrophilic domain and hydrophobic domain interlaces with each other, this suggested that the protein consisted of a mass of hydrophobic domain. Subcellular localization prediction showed that the protein was most likely to play a role in matrix, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial outer membrane.12phosphorylation sites were found in the protein, including4serine phosphorylation sites,4thremnine phosphorylation sites, and4tyrosine phosphorylation sites.2. Transcriptional level analysis of CpIPI geneReal-time quantitative PCR analysis showed that the CpIPI gene exhibited different transcription levels in different tissues and had a higher expression in flowers than other tissues. In the bloomed flower, the expression of CpIPI gene gradually increased from the interior to the exterior and reached the maximum at the external petal, while stamen had minimum expression among all the flower organs detected. Along with the flowers gradually open to wilt, the gene expression gradually increased and presented a peak at wilting stage.3. The overexpression of CpIPI gene in tobaccoesThe plant over-expression vector of CpIPI named pCAMBIA2301g-CpIPI was constructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101(including pCAMBIA2301g-CpIPI recombinant plasmid). Eight transgenical tobacco plantlets were obtained, in which the transgenic ones were identified by GUS (β-glucuronidase) histochemical staining and PCR analysis. Further, in order to validate the fuction of the CpIPI gene in terpenoid synthesis pathway, we measured the terpenoid content of transgenic tobaccoes and control plant (wildtype tobacco).In subsequent molecular detection by qPCR, the high expression level of CpIPI gene in transgenic tobaccoes proved that the content change of terpenoid substances was related to CpIPI gene. |
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