| Ginkgo polyprenols, including free polyprenols (PPs) and polyprenyl acetates (PPAs), is another kind of secondary metabolite besides flavonoids and terpene lactones, and has important medicinal and health effects to human being. The current researches of ginkgo polyprenols are focused on the the component analysis and content determination, while there is no research on its releated gene clone and expression.In this paper, leaves of different growth period (May to October) and different organs (leaf, pollens, fruit and root) of Ginkgo biloba was used as material, the contents of PPs were determined, genes related to polyprenols biosynthesis (CPT, PT, FPS) were cloned, and the expression of CPT was also studied. Research results are as follows:1. The PPs contents in male and female leaves of different growth stages of Ginkgo biloba were analyzed, and PPs can be detected in the leaves of different stages. The PPs contents were320.92-1954.38μg.g-1FW and401.93-2291.67μg.g-1FW for male and female leaves of Ginkgo biloba, respectively.2. The PPs contents of the male leaves on young and old plants at different growth stages of Ginkgo biloba were analyzed. It showed that the PPs contents were480.30-1961.89μg.g-1FW and320.92-1004.03μg.g-1FW for male Ginkgo biloba leaves on young and old plants, respectively.3. The PPs contents in different organs of Ginkgo biloba (leaf, pollens, fruit and root) were analyzed, and PPs can be detected in different organs. The PPs contents of root and pollens were35.73μg·g-1FW and58.05μg·g-1FW, respectively, and were significantly lower than those of fruit(545.31μg·g-1FW)and leaf(1389.62μg·g-1FW).4. The female leaves at different growth stages were used as materials, and the effects of CTAB method, glycol monobutyl ether method and modified Trizol method on RNA extraction were compared. The results showed that the higher concentration and production rate were obtained by modified Trizol method, and it can be used in the subsequent molecular biology research.5. PCR primers were designed based on the conserved sequences of cis-prenyltransferase synthase (CPT) genes in other plants, and the1210bp long full-length cDNA of CPT gene was cloned in ginkgo leaf with RNA as template by RT-PCR and RACE techonology. Protein sequence with262amino acid residues was encoded by an789bp open reading frame (ORF) in this gene, and there was high homology between this protein sequence and amino acid sequences encoded by other plant CPT genes. The gene has been named GbCPT, and the accession number in GeneBank was JN108023.6. PCR primers were designed based on the conserved sequences of prenyltransferase synthase (PT) genes in other plants, and the1646bp long full-length cDNA of PT gene was cloned in ginkgo leaf with RNA as template by RT-PCR and RACE techonology. Protein sequence with249amino acid residues was encoded by an750bp open reading frame (ORF) in this gene, and there was high homology between this protein sequence and amino acid sequences encoded by other plant PT genes. The gene has been named GbPT, and the accession number in GenBank was JQ303336.7. PCR primers were designed based on the conserved sequences of farnesyl diphosphate synthase (FPS) genes in other plants, and3’-end sequence was cloned in ginkgo leaf with RNA as template through RT-PCR and3’-RACE method. This fragment was701bp long, encoding104amino acid residues, with an ORF316bp long, UTR385bp long and a complete PolyA tail. There was high homology between between this encoded amino acid sequence and and amino acid sequences encoded by other plant FPS genes. The gene has been named GbFPS, and the accession number in GeneBank was JQ309808.8. The CPT gene relative expression levels of different organs were carried out, and the results demonstrated that CPT gene expression levels of different organs was in accordance with of the biosynthesis and accumulation of PPs, while the CPT gene expression levels of leaves at different developmental stages was inconsistent with PPs content change dynamics. |
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