[Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum].

Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced ß-sandwich fold. A large ß-sheet( ß4-ß11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning ß4,ß5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

A merged molecular docking, ADME-T and dynamics approaches towards the genus of Arisaema as herpes simplex virus type 1 and type 2 inhibitors.

An attempt toward screening of phytoconstituents (Arisaema genus) against herpes viruses (HSV-1 and HSV-2) was carried out using in silico approaches. Human HSV-1 and HSV-2 are accountable for cold sores genital herpes, respectively. Two drug targets, namely thymidine kinase (TK; PDB: 2ki5) serine protease (PDB: 1at3) were selected for HSV-1 and HSV-2. Initially, molecular docking tool was employed to screened apex hits phytoconstituents against herpes infections. ADME-T studies of top ranked were also further highlighted to achieve their effectiveness. Following, molecular dynamics studies were also examined to further optimize the stability of ligands. Glide scores and binding interactions of phytoconstituents were compared with Acyclovir, the main drug used in treatment of HSV, the screened top hits exhibited more glide scores and better binding for both HSV-1 and HSV-2 receptors. Additionally, ADME-T showed an ideal range for top hits while molecular dynamics results also illustrated stability of models. Ultimately, the whole efforts reveal to top three most promising hits for HSV-1 (39, 21, 19) and HSV-2 (20, 51, 19) receptors which can be explored further in wet lab experiments as promising agents against HSV infections.

De novo sequencing and transcriptome assembly of Arisaema heterophyllum Blume and identification of genes involved in isoflavonoid biosynthesis.

Arisaema heterophyllum Blume (AhBl) is one of the valued medicinal plants. However, its genetic information is limited, which impedes further studies of this valuable resource. To investigate the genes involved in the isoflavonoid biosynthesis, we deeply performed transcriptome sequencing for AhBl. An average of 10.98 Gb clean reads were obtained based on root, tuber and leaf tissues, and 109,937 unigenes were yielded after de novo assembly. In total, 72,287 of those unigenes were annotated in at least one public database. The numbers of expressed unigenes in each tissue were 35,686, 43,363 and 47,783, respectively. The overall expression levels of transcripts in leaf were higher than those in root and tuber. Differentially expressed genes analysis indicated that a total of 12,448 shared unigenes were detected in all three tissues, 10,215 of which were higher expressed in tuber than that in root and leaf. Besides, 87 candidate unigenes that encode for enzymes involved in biosynthesis of isoflavonoid were identified and analyzed, and some key enzyme genes were experimentally validated by quantitative Real-Time PCR (qRT-PCR). This study provides a unique dataset for the systematic analysis of AhBl functional genes and expression characteristics, and facilitates the future study of the pharmacological mechanism of AhBl.

Molecular identification of the traditional herbal medicines, Arisaematis Rhizoma and Pinelliae Tuber, and common adulterants via universal DNA barcode sequences.

Methods to identify Pinelliae Tuber and Arisaematis Rhizoma are required because of frequent reciprocal substitution between these two herbal medicines and the existence of several closely related plant materials. As a result of the morphological similarity of dried tubers, correct discrimination of authentic herbal medicines is difficult by conventional methods. Therefore, we analyzed DNA barcode sequences to identify each herbal medicine and the common adulterants at a species level. To verify the identity of these herbal medicines, we collected five authentic species (Pinellia ternata for Pinelliae Tuber, and Arisaema amurense, A. amurense var. serratum, A. erubescens, and A. heterophyllum for Arisaematis Rhizoma) and six common adulterant plant species. Maturase K (matK) and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes were then amplified using universal primers. In comparative analyses of two DNA barcode sequences, we obtained 45 species-specific nucleotides sufficient to identify each species (except A. erubescens with matK) and 28 marker nucleotides for each species (except P. pedatisecta with rbcL). Sequence differences at corresponding positions of the two combined DNA barcodes provided genetic marker nucleotides that could be used to identify specimens of the correct species among the analyzed medicinal plants. Furthermore, we generated a phylogenetic tree showing nine distinct groups depending on the species. These results can be used to authenticate Pinelliae Tuber and Arisaematis Rhizoma from their adulterants and to identify each species. Thus, comparative analyses of plant DNA barcode sequences identified useful genetic markers for the authentication of Pinelliae Tuber and Arisaematis Rhizoma from several adulterant herbal materials.

[DNA barcoding identification between arisaematis rhizoma and its adulterants based on ITS2 sequences].

Fifty-eight samples belonging to 7 species of Arisaematis Rhizoma and its adulterants were collected. The ITS2 locus was employed as a DNA barcode and amplified, sequenced and assembled for all of the collected samples. Then, ITS2 sequences have been annotated using HMM-based method. The intra- and inter-specific variations were calculated and NJ tree was constructed using MEGA 6.0 software. The results showed that inter-specific K2P distances were significantly larger than intra-specific distances for all of the three origin species of Arisaematis Rhizoma. Furthermore, three origin species, Arisaema amurense, A. erubescens and A. heterophyllum, can be respectively formed to be a single branch with high bootstrap values. It is concluded that ITS2 can be used to correctly identify Arisaematis Rhizoma from its adulterants and the application of ITS2 in the identification of traditional Chinese medicine has an important prospective.

Development and characterization of a novel set of microsatellite markers for Arisaema serratum (Araceae).

PREMISE OF THE STUDY: We developed novel microsatellite markers in Arisaema serratum, a perennial herb that possesses pitfall flowers and exhibits labile sex expression, to facilitate research on parentage and pollination biology in this species. METHODS AND RESULTS: By using procedures for enrichment of desired microsatellite-containing fragments and PCR-based isolation of microsatellite arrays, we detected 18 novel microsatellite loci. Thirteen were highly polymorphic: the number of alleles per locus ranged from six to 46, the observed heterozygosities ranged from 0.320 to 0.940, and the expected heterozygosities ranged from 0.440 to 0.976. Nine of the 13 markers successfully amplified regions in congeneric species. CONCLUSIONS: These highly polymorphic markers will facilitate further studies on the mode of pollination and other aspects of reproductive biology in A. serratum.

Genetic diversity and differentiation of cultivated amochi (Arisaema schimperianum Schott) in Ethiopia as revealed by AFLP markers.

Amochi (Arisaema schimperianum Schott) is an off-season crop plant in southern Ethiopia, grown during the dry season on residual moisture, for its edible tubers. It has gained importance as a "security crop" especially during the years of moisture stress and food shortage. Amochi is irritating in contact to the skin. Removal of this effect is an important question for breeding. As the first step, however we attempt to establish base line information of its breeding system and genetic variability using AFLPs. The extent of genetic differentiation among 11 populations (96 individuals) of amochi sampled along altitudinal gradients that varied from 1700 to 3200 m a.s.l. was investigated. The populations were classified in to three altitudinal groups: lowland (1700 to 2200 m a.s.l.), central-highland (2201 to 2600 m a.s.l.) and highland (2601 to 3200 m a.s.l.). Polymorphic loci (167) scored from four primer pair combinations, were used for principal component analysis (PCA), and analysis of molecular variance (AMOVA). Both PCA and unweighed pair group with arithmetic mean (UPGMA) clearly differentiated populations into their respective altitude groups, with large genetic distances. AMOVA analysis revealed 70.5%, 16.7% and 12.8% variability between altitude groups, between populations and within populations respectively. Average diversity indices within populations were also low. Since the largest proportion of variation is located between altitude groups, rather than within populations, we suggest future studies on the chemical composition, low irritation, and other desirable traits should consider populations from different altitude ranges.

An efficient method for rapid amplification of Arisaema heterophyllum agglutinin gene using a genomic walking technique.

The genomic sequence of Arisaema heterophyllum agglutinin (AHA), a mannose-binding lectin (MBL), was cloned through a novel genomic walking technique. Adaptor ligation reactions and subsequent amplifications with adaptor primer and multiple specific primers were used to generate specificity in this method. The method allowed for the amplification of over 1 kb of genomic DNA sequence immediately upstream and downstream from the 5' and 3' ends of full-length cDNAs. For aha gene, the upstream regions contained a putative transcription initiation start site and other sequences commonly found in eukaryotic promoters. The downstream regions of aha contained two polyadenylation signals. Our study demonstrated that aha had no intron like mannose-binding lectin genes cloned from other plant species so far. This efficient method, based on a genomic walking technique, was useful for the cloning of promoters, insertion sites, and other sequences of interest without constructing and screening genomic libraries.