According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.
Calmodulin ; genetics ; Cloning, Molecular ; Genes, Plant ; Pinellia ; genetics ; Plant Tubers ; genetics

To research the correlation between accumulation of triterpenoids and expression of key enzymes genes in triterpenoid biosynthesis of Alisma orientale,the study utilized UPLC-MS/MS method to detect eight triterpenoids content in the tuber of A. orientale from different growth stages,including alisol A,alisol A 24 acetate,alisol B,alisol B 23 acetate,alisol C 23 acetate,alisol F,alisol F 24 acetate and alisol G,and then the Real time quantitative PCR was used to analyze the expression of key enzymes genes HMGR and FPPS in triterpenoid biosynthesis. Correlation analysis showed that there was a significant positive relation between the total growth of these eight triterpenoids and the average relative expression of HMGR and FPPS(HMGR: r = 0. 998,P<0. 01; FPPS: r = 0. 957,P<0. 05),respectively. Therefore,the study preliminarily determined that HMGR and FPPS genes could regulate the biosynthesis of triterpenoids in A. orientale,which laid a foundation for further research on the biosynthesis and regulation mechanism of triterpenoids in A. orientale.
Alisma ; chemistry ; genetics ; Chromatography, Liquid ; Geranyltranstransferase ; genetics ; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent ; genetics ; Phytochemicals ; analysis ; Plant Extracts ; Plant Proteins ; genetics ; Plant Tubers ; chemistry ; Tandem Mass Spectrometry ; Triterpenes ; analysis

Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; genetics ; secretion ; Helianthus ; metabolism ; Kluyveromyces ; genetics ; Metabolic Engineering ; methods ; Plant Tubers ; metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae ; enzymology ; genetics

OBJECTIVETo study mRNA differential expression from microtubes of Pinellia ternata in vitro, and give more information of the molecular mechanism in the formation of microtubers.

METHODDDRT-PCR was used to identify the expressed gene fragments related to the microtubers development of P. ternata in vitro.

RESULTS AND CONCLUSIONFifteen cDNA fragments differentially expressed in the induction of microtubers of P. ternata in vitro were identified and characterized by using mRNA differential display DDRT-PCR. The deduced amino-acid sequences of six fragments of cDNA showed no significant homology with ESTs and genes in the Genbank databases, and they could be new cDNA fragments. However, the remaining three showed significant homologies with sequences encoding components of eukaryotic translation initiation factor 3 subunit H, MADS-box protein and ethylene signal transcription factor, respectively. Their differential expression patterns were confirmed by semi-quantitative RT-PCR analysis. And the expression level of the induction of microtubers of P. ternata in vitro was different.

Base Sequence ; Cloning, Molecular ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Pinellia ; genetics ; metabolism ; Plant Tubers ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment ; Sequence Homology, Amino Acid

OBJECTIVEIn this study, orthogonal design was used to optinize DDRT-PCR amplification system on Pinellia ternata microtubers in vitro in five factors four levels respectively.

METHODP. ternata stems and microtubers in vitro were selected as explants. The effects of five kinds of factors were studied by orthogonal design method including emplate cDNA, Mg2+, dNTPs, primers and Taq DNA polymerase, and in order to establish the optimum DDRT-PCR system of P. ternata microtubers in vitro.

RESULT AND CONCLUSIONA satisfactory DDRT-PCR technique system for P. ternata microtubers in vitro with desirable repeatability and polymorphic bands was established. In a total volume of 20 microL DDRT-PCR system, it contained 10 x buffer, 150 micromol L(-1) dNTPs, 2 micromol L(-1) anchor primer, 1 micromol L(-1) arbitrary primer, 2.5 mmol L(-1) Mg2+, 0.6 U Taq DNA polymerase and 2.5 microg template cDNA. The effect of the five factors was in sequence of Taq DNA polymerase > template cDNA > dNTPs > Mg2+ > Primers. The optimum DDRT-PCR system will provide scientific reference basis for studying effecting character of P. ternata microtubers associated with genes expression.

DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Electrophoresis, Polyacrylamide Gel ; Pinellia ; genetics ; Plant Tubers ; genetics ; Polymerase Chain Reaction ; methods ; Silver Staining ; Taq Polymerase ; genetics

OBJECTIVETo extract RNA from Pinellia ternata and lay a foundation for studying the formation mechanism of P. ternata.

METHODBy modifying the method recommended by Guanidinium for extracting total RNA from plant tissues rich in phenolic and polysaccharidic compounds, a simple and convenient method for extraction of total RNA from the tubers, stems and leaves of P. ternate containing abundant polyphenols and polysaccharides was established. High concentrated p-mercaptoethanol was added in the RNA extracted buffer to remove polyphenols, phenol and chloroform were used to eliminate proteins, and isopropanol and sodium acetate were used to precipitate polysaccharides.

RESULTThe A260/A230 value of RNA extracted with improved method were all over 2.0 and the values of A260/A280 were between 1.7 and 2.0. The electrophoresis bands were cleared on agarosegel and integrity of RNA was good.

CONCLUSIONThe results showed that RNA obtained from the tubers, stems and leaves of P. ternate with this method had high purity and quality and could be used in molecular biological research, as DDRT-PCR and reverse Northern blotting analysis directly. This method is simple, economic, stable performance, and has a good repeatability as well as is suitable for extracting total RNA of medicinal plants with high concentrations of phenolics and polysaccharides.

Blotting, Northern ; Pinellia ; genetics ; Plant Leaves ; genetics ; Plant Stems ; genetics ; Plant Tubers ; genetics ; Polymerase Chain Reaction ; RNA, Plant ; isolation & purification

OBJECTIVETo study lipid-regulating action of 2, 3, 5, 4'-tetrahydroxy stilbene-2-O-beta-D-glucopyranoside (TSG) from Polygonum multiflorum on experimental model hyperlipidemic rats.

METHODTSG 90 and 180 mg x kg(-10 x d(-1), atorvastatin mg kg(-1) x d(-1) and saline 2 mL x d(-1) were administered to hyperlipidemic rats. Groups of rats were determined and compared with those of saline group. The LDLR and HMGR mRNA expression were also detected.

RESULTTSG significantly reduced serum TC and LDL-C level and atherosclerosis index, increased the expression of LDLR in the liver cells.

CONCLUSIONTSG, which shows effects and mechanism in part like atorcastatin, is a major constituent with blood-lipid regulating effect of P. multiflorum and can be explored as a potent medication for hyperlipidemia. Effects on LDL-C and AI, as well as on gene expression of TSG were first reported.

Animals ; Anticholesteremic Agents ; administration & dosage ; pharmacology ; Atorvastatin Calcium ; Cholesterol, LDL ; blood ; Glucosides ; administration & dosage ; isolation & purification ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Heptanoic Acids ; administration & dosage ; pharmacology ; Hydroxymethylglutaryl CoA Reductases ; biosynthesis ; genetics ; Hyperlipidemias ; blood ; prevention & control ; Male ; Plant Tubers ; chemistry ; Plants, Medicinal ; chemistry ; Polygonum ; chemistry ; Pyrroles ; administration & dosage ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, LDL ; biosynthesis ; genetics ; Stilbenes ; administration & dosage ; isolation & purification ; pharmacology ; Triglycerides ; blood

OBJECTIVETo study the ITS sequence variation of Pinellia ternata from different population in China, and it correlation to geographical distribution and morpha of the plant.

METHODThe rDNA ITS regions of various P. ternata were amplified and sequenced. And they were analyzed by means of the software of CLUSTRAL and MEGA.

RESULTComplete sequence of ITS and 5.8S rDNA of 16 different P. ternata population were obtained. The sequences of ITS1, 5.8S and ITS2 are 276,162 and 246 bp, respectively. ITS1 was more conservative than ITS2. Phylogenetic tree based on ITS1 and ITS2 sequences data was conducted by Neighbor-joining method.

CONCLUSIONRibosomal DNA ITS sequence analyses can be applied to the resource research of P. ternata.

Base Sequence ; China ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Ecosystem ; Molecular Sequence Data ; Phylogeny ; Pinellia ; genetics ; Plant Tubers ; genetics ; Plants, Medicinal ; genetics ; RNA, Ribosomal, 5.8S ; genetics ; Sequence Analysis, DNA

Gastrodia elata Bl. is a famous and costful traditional Chinese medicine. Their genomic DNA fingerprints were investigated using a modified Randomly Amplified Polymorphic DNA method. DNA fragments common to all or to fine populations were identified and recovered. Five DNA fragments were proven not to be reported through DNA cloning, PCR identifying, nucleotide sequencing and bioinformatics analyses and were received in and recorded by NCBI GenBank. Gastrodine contents of the Gastrodia tuber samples were determined using high performance liquid chromatography technique. The distribution of the five DNA fragments in 9 Gastrodia elata Blue populations and the correlation with gastromedicine content were studied. The results show the distribution of these DNA sequences varied greatly among the populations whereby DNA Sequence 1 was the common and distinguishing molecular marker for all the populations studied and DNA Sequence 2 may relate to higher gastrodine content. In conclusion, these DNA marker sequences can be employed to identify genuine gastrodia tubers, better varieties and optimize their selection and cultivating.
Base Sequence ; Benzyl Alcohols ; analysis ; Cloning, Molecular ; Computational Biology ; DNA, Plant ; chemistry ; Gastrodia ; genetics ; Glucosides ; analysis ; Plant Tubers ; genetics