长春花萜类吲哚生物碱含量测定及相关基因的表达分析

采用反相高效液相色谱法(RP-HPLC)对23个长春花品种中的重要萜类吲哚生物碱文多灵、长春质碱和长春碱含量进行了测定,发现这3种生物碱的含量在不同品种中存在较大差异。相关性分析表明,文多灵、长春质碱和长春碱这3种生物碱中,文多灵含量和长春碱含量有极显著的相关性(P0.01)。利用RT-PCR分析了香叶醇10-脱羧酶基因(g10h)和异胡豆苷合成酶基因(str)在萜类吲哚生物碱含量有显著差异的品种之间的表达水平差异,并结合生物碱含量数据结果,发现g10h和str的表达水平和文多灵和长春质碱的总含量有显著的正相关性(P0.05),说明g10h和str基因可以作为长春花中文多灵和长春质碱含量的参考基因标记。该研究对为选育高萜类吲哚生物碱含量长春花品种及长春花萜类吲哚生物碱代谢工程具有重要意义。     

Tissue Compartment Analysis for Biomarker Discovery by Gene Expression Profiling

Background

Although high throughput technologies for gene profiling are reliable tools, sample/tissue heterogeneity limits their outcomes when applied to identify molecular markers. Indeed, inter-sample differences in cell composition contribute to scatter the data, preventing detection of small but relevant changes in gene expression level. To date, attempts to circumvent this difficulty were based on isolation of the different cell structures constituting biological samples. As an alternate approach, we developed a tissue compartment analysis (TCA) method to assess the cell composition of tissue samples, and applied it to standardize data and to identify biomarkers.

Methodology/Principal Findings

TCA is based on the comparison of mRNA expression levels of specific markers of the different constitutive structures in pure isolated structures, on the one hand, and in the whole sample on the other. TCA method was here developed with human kidney samples, as an example of highly heterogeneous organ. It was validated by comparison of the data with those obtained by histo-morphometry. TCA demonstrated the extreme variety of composition of kidney samples, with abundance of specific structures varying from 5 to 95% of the whole sample. TCA permitted to accurately standardize gene expression level amongst >100 kidney biopsies, and to identify otherwise imperceptible molecular disease markers.

Conclusions/Significance

Because TCA does not require specific preparation of sample, it can be applied to all existing tissue or cDNA libraries or to published data sets, inasmuch specific operational compartments markers are available. In human, where the small size of tissue samples collected in clinical practice accounts for high structural diversity, TCA is well suited for the identification of molecular markers of diseases, and the follow up of identified markers in single patients for diagnosis/prognosis and evaluation of therapy efficiency. In laboratory animals, TCA will interestingly be applied to central nervous system where tissue heterogeneity is a limiting factor.     

Analysis of Catharanthus roseus alkaloids by HPLC

Catharanthus roseus is a medicinal plant from which secondary metabolites used in chemotherapy to treat diverse cancers are extracted. The well known high value metabolites vincristine and vinblastine are just 2 of 130 alkaloids that can be found in C. roseus. However, only few (∼11) of this high number of chemical entities are frequently analyzed and even fewer (∼8) are available commercially. For more than 30 years, different analytical techniques have been developed to isolate and identify C. roseus metabolites, and then allowing revealing the therapeutic potential of C. roseus metabolites. Among few approaches, high performance liquid chromatography (HPLC) technique is still widely used for the separation and analysis of secondary metabolites such as those from C. roseus. This article thus reviews the most recent developments in HPLC analysis of alkaloids from C. roseus. Diverse considerations that are crucial to the efficiency of secondary metabolites separation and identification steps, such as biomass manipulation, extraction phase and protocols, HPLC separation and analysis protocols are reviewed in details. Examples of spectra obtained using the most common detectors are also shown and suggestions are made on how to proceed in developing efficient separation and identification methods at the analytical and semi-preparative scales.     

Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots

Madagascar periwinkle [Catharanthus roseus (L.) G Don] is a pantropical plant of horticultural value that produces the powerful anticancer drugs vinblastine and vincristine that are derived from the dimerization of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine. The present study describes the genetic engineering and expression of the terminal step of vindoline biosynthesis, deacetylvindoline-4-O-acetyltransferase (DAT) in Catharanthus roseus hairy root cultures. Biochemical analyses showed that several hairy root lines expressed high levels of DAT enzyme activity compared to control hairy root cultures expressing β-gulucuronidase activity (GUS) activity. Metabolite analysis using high performance liquid chromotagraphy established that hairy root extracts had an altered alkaloid profile with respect to hörhammericine accumulation in DAT expressing lines in comparison to control lines. Further analyses of one hairy root culture expressing high DAT activity suggested that DAT expression and accumulation of hörhammericine (9) were related. It is concluded that expression of DAT in hairy roots altered their MIA profile and suggests that further expression of vindoline pathway genes could lead to significant changes in alkaloid profiles. Evidence is provided that hörhammericine (9) accumulates via a DAT interaction with the root specific minovincinine-19-O-acetyltransferase (MAT) that inhibits the MAT mediated conversion of hörhammericine (9) into 19-O-acetyl-hörhammericine (12).     

Integrated Analyses of Metabolomic Profiling and Associated Gene Expression of Catharanthus roseus Seedling Reveal the Metabolic Alternations of Primary Metabolites and Flavonoids During the Apical Hook Opening Phase

Journal of Plant Growth Regulation - In plants, apical hook opening is critical for the post-germination stage and subsequent photomorphogenesis, and this process is activated by a luminous...     

长春花核型的研究

对长春花属的长春花(Catharanthus roseus(L.)G.Don)、白长春花(C. roseus(L.)G.Don'Albus')和黄长春花(C.roseus(L.)G.Don'Flavus')的染色体数目和核型进行了研究.结果表明,它们的核型公式均为2n=2x=16=2m 12sm 2T,均属于"3A"核型,染色体数目均为2n=16,但它们的端部和中部着丝点染色体在核型分析中的排列次序不同.     

面向药物发现和精准医疗的基因表达谱分析

作为功能基因组学中重要的组成部分,基因表达谱在生物学、医学和药物研发等多个领域发挥着重要作用.特别是随着精准医疗概念的提出,整合多组学数据用于个性化医疗是未来的发展趋势.本文从基因表达谱的基本概念出发,重点介绍面向药物发现的基因表达谱分析方法,即基于关联图谱的方法、基于基因调控网络的方法和基于多组学数据整合的方法.系统整理了各种方法的研究进展,特别是在抗癌药物研发领域的最新进展,为利用基因表达谱数据进行药物研发提供方法借鉴.     

Deep Sequencing of the Scutellaria baicalensis Georgi Transcriptome Reveals Flavonoid Biosynthetic Profiling and Organ-Specific Gene Expression

Scutellaria baicalensis Georgi has long been used in traditional medicine to treat various such widely varying diseases and has been listed in the Chinese Pharmacopeia, the Japanese Pharmacopeia, the Korean Pharmacopoeia and the European Pharmacopoeia. Flavonoids, especially wogonin, wogonoside, baicalin, and baicalein, are its main functional ingredients with various pharmacological activities. Although pharmaological studies for these flavonoid components have been well conducted, the molecular mechanism of their biosynthesis remains unclear in S. baicalensis. In this study, Illumina/Solexa deep sequencing generated more than 91 million paired-end reads and 49,507 unigenes from S. baicalensis roots, stems, leaves and flowers. More than 70% unigenes were annotated in at least one of the five public databases and 13,627 unigenes were assigned to 3,810 KEGG genes involved in 579 different pathways. 54 unigenes that encode 12 key enzymes involved in the pathway of flavonoid biosynthesis were discovered. One baicalinase and three baicalein 7-O-glucuronosyltransferases genes potentially involved in the transformation between baicalin/wogonoside and baicalein/wogonin were identified. Four candidate 6-hydroxylase genes for the formation of baicalin/baicalein and one candidate 8-O-methyltransferase gene for the biosynthesis of wogonoside/wogonin were also recognized. Our results further support the conclusion that, in S. baicalensis, 3,5,7-trihydroxyflavone was the precursor of the four above compounds. Then, the differential expression models and simple sequence repeats associated with these genes were carefully analyzed. All of these results not only enrich the gene resource but also benefit research into the molecular genetics and functional genomics in S. baicalensis.     

长春花钙调素基因克隆及其假基因的识别

以长春花[Catharanthus roseus(L.)G.Don]叶片cDNA和基因组DNA为模板,利用PCR技术扩增得到了长春花钙调素基因447 bp的全长编码cDNA序列和2个大小不同的DNA片段.序列分析表明,DNA长片段全长1 551 bp,由2个外显子和1个内含子构成,为长春花钙调素基因编码区DNA片段;DNA小片段全长447 bp,与447 bp的长春花钙调素基因cDNA核苷酸一致性高达87%,有56个碱基的差异,其中位于226 bp处的碱基A突变为T,即由AAG突变为终止密码子TAG使翻译提前终止.推测此447 bp的DNA小片段可能为长春花钙调素基因的假基因,命名为CCaMP1.