银杏植物各部位及银杏组织培养细胞中银杏内酯B和白果内酯含量的初步研究

用高压液相色谱法对五年生扦插银杏各部位及银杏组织培养细胞中银杏内酯B和白果内酯的含量进行了测定.结果表明银杏内酯B和白果内酯在银杏植物各部位的含量差异很大.银杏内酯B在银杏叶中含量最高,白果内酯在银杏侧根中含量最高.在6,7-v培养基下银杏组织培养细胞中同时测出银杏内酯B和白果内酯,提示用植物组织培养方法有可能同时产生银杏内酯B和白果内酯.     

The small subunit ADP-glucose pyrophosphorylase (<Emphasis Type="Italic">ApS</Emphasis>) promoter mediates okadaic acid-sensitive<Emphasis Type="Italic"> uidA</Emphasis> expression in starch-synthesizing tissues and cells in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Transgenic plants of Arabidopsis thaliana Heynh., transformed with a bacterial beta-glucuronidase (GUS) gene under the control of the promoter of the small subunit (ApS) of ADP-glucose pyrophosphorylase (AGPase), exhibited GUS staining in leaves (including stomata), stems, roots and flowers. Cross-sections of stems revealed GUS staining in protoxylem parenchyma, primary phloem and cortex. In young roots, the staining was found in the root tips, including the root cap, and in vascular tissue, while the older root-hypocotyl axis showed prominent staining in the secondary phloem and paratracheary parenchyma of secondary xylem. The GUS staining co-localized with ApS protein, as found by tissue printing using antibodies against ApS. Starch was found only in cell and tissue types exhibiting GUS staining and ApS labelling, but not in all of them. For example, starch was lacking in the xylem parenchyma and secondary phloem of the root-hypocotyl axis. Sucrose potently activated ApS gene expression in leaves of wild-type (wt) plants, and in transgenic seedlings grown on sucrose medium where GUS activity was quantified with 4-methylumbelliferyl-beta-glucuronide as substrate. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, completely blocked expression of ApS in mature leaves of wt plants and prevented GUS staining in root tips and flowers of the transgenic plants, suggesting a similar signal transduction mechanism for ApS expression in various tissues. The data support the key role of AGPase in starch synthesis, but they also underlie the ubiquitous importance of the ApS gene for AGPase function in all organs/tissues of Arabidopsis.     

Evidence for a plasma membrane proton pump in phloem cells of higher plants

Metabolic energy is required for the loading of sucrose into the phloem and translocation of sugars throughout the plant. The proton electrochemical gradient generated by a plasma membrane proton pump (H(+)-ATPase) is thought to provide energy for these processes. The plasma membrane H(+)-ATPase is encoded by a multigene family in Arabidopsis thaliana. Here we characterize the expression of isoform AHA3 (Arabidopsis H(+)-ATPase isoform 3). The AHA3 mRNA start site was mapped and 464 bp of the putative upstream regulatory region sequenced. A translational fusion of AHA3 to the beta-glucuronidase (GUS) reporter gene was constructed and used to generate transgenic Nicotiana and Arabidopsis plants. Using a histochemical stain, expression of the AHA3/GUS fusion was found predominantly in phloem cells of leaves, stems, roots, and flowers. Biochemical measurements of GUS activity in pith and vascular explants confirmed the histochemical localization. Our results support the hypothesis that a proton pump is present in phloem cells, possibly providing energy to drive plasma membrane cotransport systems required for phloem loading and translocation of photosynthates. In addition to AHA3/GUS expression in phloem, expression was observed in pollen and regions of the ovule, tissues whose physiological functions correlate with a requirement for high levels of solute transport.     

Molecular cloning, characterization and functional analysis of a 2C-methyl- D-erythritol 2, 4-cyclodiphosphate synthase gene from ginkgo biloba

2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS, EC: 4.6.1.12) is the fifth enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate (MEP) pathway for ginkgolide biosynthesis. The full-length mecps cDNA sequence (designated as Gbmecps) was cloned and characterized for the first time from gymnosperm plant species, Ginkgo biloba, using RACE (rapid amplification of cDNA ends) technique. The full-length cDNA of Gbmecps was 874 bp containing a 720 bp open reading frame (ORF) encoding a peptide of 239 amino acids with a calculated molecular mass of 26.03 kDa and an isoelectric point of 8.83. Comparative and bioinformatic analyses revealed that GbMECPS showed extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family.Phylogenetic analysis indicated that GbMECPS was more ancient than other plant MECPSs. Tissue expression pattern analysis indicated that GbMECPS expressed the highest in roots, followed by in leaves, and the lowest in seeds. The color complementation assay indicated that GbMECPS could accelerate the accumulation of beta-carotene. The cloning, characterization and functional analysis of GbMECPS will be helpful to understand more about the role of MECPS involved in the ginkgolides biosynthesis at the molecular level.     

Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis

Levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis, was cloned and functionally characterized. A Ginkgo biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl diphosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme.     

Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate

The rate-limiting step of cytokinin biosynthesis in Arabidopsis thaliana Heynh. is catalyzed by ATP/ADP isopentenyltransferases, A. thaliana IsoPentenyl Transferase (AtIPT)1, and AtIPT4, and by their homologs AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8. To understand the dynamics of cytokinins in plant development, we comprehensively analyzed the expression of isopentenyltransferase genes of Arabidopsis. Examination of their mRNA levels and the expression patterns of the beta-glucuronidase (GUS) gene fused to the regulatory sequence of each AtIPT gene revealed a specific expression pattern of each gene. The predominant expression patterns were as follows: AtIPT1::GUS, xylem precursor cell files in the root tip, leaf axils, ovules, and immature seeds; AtIPT3::GUS, phloem tissues; AtIPT4::GUS and AtIPT8::GUS, immature seeds with highest expression in the chalazal endosperm (CZE); AtIPT5::GUS, root primordia, columella root caps, upper part of young inflorescences, and fruit abscission zones; AtIPT7::GUS, endodermis of the root elongation zone, trichomes on young leaves, and some pollen tubes. AtIPT1, AtIPT3, AtIPT5, and AtIPT7 were downregulated by cytokinins within 4 h. AtIPT5 and AtIPT7 was upregulated by auxin within 4 h in roots. AtIPT3 was upregulated within 1 h after an application of nitrate to mineral-starved Arabidopsis plants. The upregulation by nitrate did not require de novo protein synthesis. We also examined the expression of two genes for tRNA isopentenyltransferases, AtIPT2 and AtIPT9, which can also be involved in cytokinin biosynthesis. They were expressed ubiquitously, with highest expression in proliferating tissues. These findings are discussed in relation to the role of cytokinins in plant development.     

LE-ACS6启动子在LE-ACS6::GUS转基因拟南芥中的特异性

通过对不同发育时期LE-ACS6::GUS转基因拟南芥中GUS表达特异性的研究,证明LE-ACS6基因的启动子在拟南芥中也表现启动参与第一系统乙烯合成的关键酶基因的活性.在转基因拟南芥中,LE-ACS6启动子还表现响应外源生长素处理、伤害处理等多种刺激因子的特点.     

pib基因启动子及其诱导启动性初探

将pib基因上游5.7 kb区段取代pCAMBIA1301中gus基因上游的35S启动子构建了pib拟启动区-GUS+ 35S-hpt 基因表达载体pNAR604。经农杆菌介导转化水稻成熟胚愈伤,获得了转基因抗潮霉素愈伤和36株转基因水稻植株。 转基因抗性愈伤和转基因植株根的组织化学GUS活性检测表明,光照培养下的抗性愈伤和转基因植株根不能使X-gluc显色,而暗处理24 h后的抗性愈伤和定植后转基因植株的根能使X-gluc显色。转基因植株GUS荧光定量分析结果表明,GUS表达具有器官特异性,黑暗处理前根的GUS活性最高、茎次之,分别是是叶片的7倍和3倍,叶片中仅有痕量本底。24 h黑暗处理后根、茎、叶中GUS活性都有增加,且叶片中的增加比例最大,其活性仅次于根。5 mmol/L水杨酸和0.3 mol/L NaCl叶面喷施转基因植株24 h后叶片中GUS活性分别为处理前的2.7和3.6倍。初步确定pib拟启动区是一个诱导型启动子。黑暗、水杨酸和NaCl能诱导该启动子启动活性。     

A new geranylgeranyl diphosphate synthase gene from Ginkgo biloba, which intermediates the biosynthesis of the key precursor for ginkgolides.

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for ginkgolide biosynthesis. Here we reported for the first time the cloning of a new full-length cDNA encoding GGPPS from the living fossil plant Ginkgo biloba. The full-length cDNA encoding G. biloba GGPPS (designated as GbGGPPS) was 1657bp long and contained a 1176bp open reading frame encoding a 391 amino acid protein. Comparative analysis showed that GbGGPPS possessed a 79 amino acid transit peptide at its N-terminal, which directed GbGGPPS to target to the plastids. Bioinformatic analysis revealed that GbGGPPS was a member of polyprenyltransferases with two highly conserved aspartate-rich motifs like other plant GGPPSs. Phylogenetic tree analysis indicated that plant GGPPSs could be classified into two groups, angiosperm and gymnosperm GGPPSs, while GbGGPPS had closer relationship with gymnosperm plant GGPPSs.     

Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.     

Functional characterization of a putative nitrate transporter gene promoter from rice

Drought is one of the most significant abiotic stresses that influence plant growth anddevelopment.Expression analysis revealed that OsNRT1.3,a putative nitrate transporter gene in rice,wasinduced by drought.To confirm if the OsNRT1.3 promoter can respond to drought stress,a 2019 bpupstream sequence of OsNRT1.3 was cloned.Three OsNRT1.3 promoter fragments were generated by5′-deletion,and fused to the β-glucuronidase (GUS) gene.The chimeric genes were introduced into riceplants.NRT2019::GUS,NRT1196::GUS and NRT719::GUS showed similar expression patterns in seeds,roots,leaves and flowers in all transgenic rice,and GUS activity conferred by different OsNRT1.3 promoterfragments was significantly upregulated by drought stress,indicating that OsNRT1.3 promoter responds todrought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements.