丹参环阿屯醇合酶基因克隆及表达分析

以丹参cDNA为模板,克隆了丹参环阿屯醇合酶(cycloartenol synthase,CAS)基因的cDNA序列(SmCAS),对其序列进行生物信息学分析,并采用实时荧光定量PCR方法研究了该基因在丹参不同器官及不同胁迫处理下的表达模式。结果显示:该基因全长2 346bp,包含2 271bp开放阅读框,编码756个氨基酸。预测其编码蛋白分子量为86.16kD,具有氧化鲨烯环化酶超家族典型的DCTAE结构域和QW结构域。该基因推测的氨基酸序列与人参、田七、积雪草、甘草、拟南芥的相似性分别为83%、84%、83%、81%和80%。SmCAS基因在丹参根、茎、叶、花中均有表达,在花中表达量最高;而且SmCAS基因能够响应ABA、低温和干旱的诱导。     

SmGGPPS2对丹参酮合成的影响

香叶基香叶基焦磷酸合酶(GGPPS)是植物二萜类次生代谢物合成过程中的重要调控位点。在药用模式植物丹参(Salvia miltiorrhiza)中, GGPPS基因家族成员SmGGPPS2的生物学功能及其在丹参酮有效成分合成过程中的作用尚不明确。分别在丹参植株中过表达和RNA干涉SmGGPPS2基因, 并对转基因丹参中丹参酮含量和丹参酮合成相关基因表达量 以及转基因植物生理指标进行检测, 结果表明, 过表达SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量高于野生型; RNA干涉SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量均低于野生型。调节表达SmGGPPS2后, 丹参株系中SmHMGR1和SmCPS1等多个关键酶基因的表达都呈现明显的变化。此外, 调节表达SmGGPPS2还影响丹参植株抗性。以上结果表明, SmGGPPS2在丹参酮等萜类物质的合成中起重要的调控作用。     

Overexpression of SmLEA enhances salt and drought tolerance in Escherichia coli and Salvia miltiorrhiza

Salinity and drought are important abiotic stresses limiting plant growth and development. Late embryogenesis abundant (LEA) proteins are a group of proteins associated with tolerance to water-related stress. We previously cloned an LEA gene, SmLEA, from Salvia miltiorrhiza Bunge. Phylogenetic analysis indicated that SmLEA belongs to Group LEA14, which is involved in the dehydration response. To determine its function in detail, we have now overexpressed SmLEA in Escherichia coli and S. miltiorrhiza. The logarithmic increase in accumulations of SmLEA proteins in E. coli occurred earlier under salinity than under standard conditions. SmLEA-transformed S. miltiorrhiza plants also showed faster root elongation and a lower malondialdehyde concentration than the empty vector control plants did when cultured on MS media supplemented with 60 mM NaCl or 150 mM mannitol. Moreover, SmLEA-overexpressing transgenics experienced a less rapid rate of water loss. Under either salinity or drought, overexpressing plants had greater superoxide dismutase activity and a higher glutathione concentration. These results suggest that SmLEA may be useful in efforts to improve drought and salinity tolerance in S. miltiorrhiza. Our data also provide a good foundation for further studies into the stress resistance mechanism and molecular breeding of this valuable medicinal plant.     

Regulation of gap junctional communication by astrocytic mitochondrial K(ATP) channels following neurotoxin administration in in vitro and in vivo models

It is known that neuronal ATP-sensitive potassium (K(ATP)) channels and astrocytic gap junctions (GJs) are involved in the mechanism underlying neurodisorders. The K(ATP) channels exist also in glial cells, and the objective of this study was to determine whether the astrocytic K(ATP) channels exert their effect on neurotoxin-induced neurodysfunction through regulating the astrocytic GJ function. The results showed that diazoxide, a selective mitochondrial K(ATP) (mitoK(ATP)) channel opener, enhanced the GJ coupling, but 5-hydroxydecanoate, a selective mitoK(ATP) channel blocker that significantly inhibits GJ coupling in vitro did not. Activation of astrocytic mitoK(ATP) channels alleviated kainic acid-induced dysfunction of GJ intercellular communication. Finally, activation of mitoK(ATP) channels improved the astrocytic GJ coupling in the hippocampus after seizures due to the colabeling of GJ subunit connexin 43 and connexin 45 with glial marker and was increased substantially by the administration of diazoxide. Western blot demonstrated that the mitoK(ATP) channels regulated the expression of connexin 43 (P2; active form) and connexin 45 in the epileptic hippocampus. These findings demonstrate that activation of astrocytic mitoK(ATP) channels improves the GJ function in astrocytes, indicating that the effect of the astrocytic mitoK(ATP) channels on neurotoxin-induced neurodysfunction might be, in part, through the regulation of the GJ-coupled spatial buffering in the hippocampus.     

Molecular cloning and characterization of the promoter of <Emphasis Type="Italic">SmGGPPs</Emphasis> and its expression pattern in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. It regulates the formation of diterpenoid, such as tanshinones. We cloned a gene for GGPP synthase SmGGPPs involved in diterpenoid biosynthesis from Salvia miltiorrhiza. At 2,767 bp long, this gene comprises an intron and two exons that encode a polypeptide of 364 amino acid residues. Then the 5′ flanking sequence of SmGGPPs was characterized by bioinformatics method. Deletion analysis of the promoter of SmGGPPs using tobacco plant displayed that the promoter was induced by heat and cold. To further search these cis-elements involved in induction regulation in the 5′ flanking sequence of SmGGPPs, many putative cis-elements were predicted with the PlantCARE and PLACE databases. A group of putative cis-acting elements are involved in induction regulation, including G-Box, WRKY, MYC and ATCT motifs. Real-time PCR analysis revealed that SmGGPPs is mainly expressed in the leaves and can also be induced by various factors, such as NaCl, wounding, high temperature, darkness, pathogen, methyl jasmonate, abscisic acid, salicylic acid, and gibberellins. This study provides useful information for further study of SmGGPPs and its regulator effect on the biosynthetic process of tanshinones so that researchers can improve the tanshinone contents in S. miltiorrhiza.     

Downregulation of Cinnamoyl CoA Reductase Affects Lignin and Phenolic Acids Biosynthesis in Salvia miltiorrhiza Bunge

The biosynthesis of salvianolic acid B shares the phenylpropanoid pathway with lignin, and cinnamoyl CoA reductase (CCR; EC 1.2.1.44) is a specific enzyme in the lignin pathway. In this study, a CCR gene (SmCCR1) from Salvia miltiorrhiza Bunge was cloned using DNA walking technology (GenBank ID: JF798634). The full-length SmCCR1 is 2,489?bp long and consists of four introns and five exons encoding a polypeptide of 324 amino acid residues. Sequence alignment revealed that SmCCR1 shares 83?% identity with CCR sequences reported in Camellia oleifera and other plant species. Expression pattern analysis indicated that expression of SmCCR1 can be induced by exposure to Xanthomonas campestris pv. Campestris or methyl jasmonate. To demonstrate its functioning, we selected a 296-bp fragment and established an RNA interference construct that was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants exhibited dwarfing phenotypes, and both syringyl and guaiacyl lignin monomers were decreased more than 60?%. In contrast, biosynthesis of phenolic acids??danshensu, rosmarinic acid, and salvianolic acid B??was strongly induced by 2.03-, 1.41-, and 1.45-fold, respectively, in the roots of transgenic plants from line CCR-10. Consistent with these phytochemical changes, downregulation of SmCCR1 also affected the expression of related genes in the phenolics and lignin biosynthetic pathways. Our results also provide potential opportunities for engineering danshensu and salvianolic acid B production in S. miltiorrhiza.