Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing.

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

Positive Selection of Squalene Synthase in Cucurbitaceae Plants.

Triterpenoid saponins are secondary metabolites synthesized through isoprenoid pathways in plants. Cucurbitaceae represent an important plant family in which many species contain cucurbitacins as secondary metabolites synthesized through isoprenoid and triterpenoid pathways. Squalene synthase (SQS) is required for the biosynthesis of isoprenoids, but the forces driving the evolution of SQS remain undetermined. In this study, 10 SQS cDNA sequences cloned from 10 species of Cucurbitaceae and 49 sequences of SQS downloaded from GenBank and UniProt databases were analyzed in a phylogenetic framework to identify the evolutionary forces for functional divergence. Through phylogenetic construction and positive selection analysis, we found that SQS sequences are under positive selection. The sites of positive selection map to functional and transmembrane domains. 180L, 189S, 194S, 196S, 265I, 289P, 389P, 390T, 407S, 408A, 410R, and 414N were identified as sites of positive selection that are important during terpenoid synthesis and map to transmembrane domains. 196S and 407S are phosphorylated and influence SQS catalysis and triterpenoid accumulation. These results reveal that positive selection is an important evolutionary force for SQS in plants. This provides new information into the molecular evolution of SQS within the Cucurbitaceae family.

Molecular and biochemical characterization of squalene synthase from Siraitia grosvenorii.

OBJECTIVES: To clone and characterize the squalene synthase from Siraitia grosvenorii (SgSQS). RESULTS: The gene encoding SgSQS was cloned. SgSQS has 417 amino acid residues with an pI of 7.3. There are 32 phosphorylation sites in its sequence: S48 as well as S196 play important roles in regulation of enzyme activity. The enzyme is a monomeric protein with a cave-like active center formed by α helixes and has two transmembrane domains at its C-terminus. SgSQS mRNA expression in stem and root were about twice as much as that in leaf and peel. Full-length SgSQS with measurable catalytic activity was expressed in Escherichia coli. SgSQS activity was optimal at 37 °C and pH 7.5 respectively. CONCLUSION: SgSQS gene was cloned, and the molecular structure and biochemical function of SgSQS were characterized.

Positive selection and functional divergence of farnesyl pyrophosphate synthase genes in plants.

BACKGROUND: Farnesyl pyrophosphate synthase (FPS) belongs to the short-chain prenyltransferase family, and it performs a conserved and essential role in the terpenoid biosynthesis pathway. However, its classification, evolutionary history, and the forces driving the evolution of FPS genes in plants remain poorly understood. RESULTS: Phylogeny and positive selection analysis was used to identify the evolutionary forces that led to the functional divergence of FPS in plants, and recombinant detection was undertaken using the Genetic Algorithm for Recombination Detection (GARD) method. The dataset included 68 FPS variation pattern sequences (2 gymnosperms, 10 monocotyledons, 54 dicotyledons, and 2 outgroups). This study revealed that the FPS gene was under positive selection in plants. No recombinant within the FPS gene was found. Therefore, it was inferred that the positive selection of FPS had not been influenced by a recombinant episode. The positively selected sites were mainly located in the catalytic center and functional areas, which indicated that the 98S and 234D were important positively selected sites for plant FPS in the terpenoid biosynthesis pathway. They were located in the FPS conserved domain of the catalytic site. We inferred that the diversification of FPS genes was associated with functional divergence and could be driven by positive selection. CONCLUSIONS: It was clear that protein sequence evolution via positive selection was able to drive adaptive diversification in plant FPS proteins. This study provides information on the classification and positive selection of plant FPS genes, and the results could be useful for further research on the regulation of triterpenoid biosynthesis.

Molecular identification of Uncaria (Gouteng) through DNA barcoding.

BACKGROUND: While DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng). METHODS: Genomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke's Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbA-trnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0. RESULTS: The accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbA-trnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbA-trnH was higher than the corresponding intraspecific distance. Additionally, psbA-trnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method. CONCLUSION: While psbA-trnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbA-trnH was a more suitable barcode for authentication of Uncaria macrophylla.

Transcriptome Sequencing of Gynostemma pentaphyllum to Identify Genes and Enzymes Involved in Triterpenoid Biosynthesis.

G. pentaphyllum (Gynostemma pentaphyllum), a creeping herbaceous perennial with many important medicinal properties, is widely distributed in Asia. Gypenosides (triterpenoid saponins), the main effective components of G. pentaphyllum, are well studied. FPS (farnesyl pyrophosphate synthase), SS (squalene synthase), and SE (squalene epoxidase) are the main enzymes involved in the synthesis of triterpenoid saponins. Considering the important medicinal functions of G. pentaphyllum, it is necessary to investigate the transcriptomic information of G. pentaphyllum to facilitate future studies of transcriptional regulation. After sequencing G. pentaphyllum, we obtained 50,654,708 unigenes. Next, we used RPKM (reads per kilobases per million reads) to calculate expression of the unigenes and we performed comparison of our data to that contained in five common databases to annotate different aspects of the unigenes. Finally, we noticed that FPS, SS, and SE showed differential expression of enzymes in DESeq. Leaves showed the highest expression of FPS, SS, and SE relative to the other two tissues. Our research provides transcriptomic information of G. pentaphyllum in its natural environment and we found consistency in unigene expression, enzymes expression (FPS, SS, and SE), and the distribution of gypenosides content in G. pentaphyllum. Our results will enable future related studies of G. pentaphyllum.

Natural selection and functional diversification of the epidermal growth factor receptor EGFR family in vertebrates.

BACKGROUND: Genes that have been subject to adaptive evolution can produce varying degrees of pathology or differing symptomatology. ErbB family receptor activation will initiate a number of downstream signaling pathways, such as mitogen-activated protein kinase (MAPK), activator of transcription (STAT), the modulation of calcium channels, and so on, all of which lead to aggressive tumor behavior. However, the evolutionary mechanisms operating in the retention of ErbB family genes and the changes in selection pressures are not clear. RESULTS: Sixty-two full-length cDNA sequences from 27 vertebrate species were extracted from the UniProt protein database, NCBI's GenBank and the Ensembl database. The result of phylogenetic analysis showed that the four ErbB family members in vertebrates might be formed by gene duplication. In order to determine the mode of evolution in vertebrates, selection analysis and functional divergence analysis were combined to explain the relationship of the site-specific evolution and functional divergence in the vertebrate ErbB family. Our results indicate that the acceleration of asymmetric evolutionary rates and purifying selection together were the main force for the production of ErbBs, and positive selections were detected in the ErbB family. CONCLUSION: An evolutional phylogeny of 27 vertebrates was presented in our study; the tree showed that the genes have evolved through duplications followed by purifying selection, except for seven sites, which evolved by positive selection. There was one common site with positive selection and functional divergence. In the process of functional differentiation evolving through gene duplication, relaxed selection may play an important part.

[Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

OBJECTIVE: To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. METHODS: Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. RESULTS: There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. CONCLUSION: RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

[Molecular characters of Centella asiatica found with RAPD technology].

OBJECTIVE: To analyze the DNA molecular characters of Centella asiatica with RAPD technology. METHODS: With the genomic DNA as templates extracted from various source of Centella asiatica samples, optimized RAPD PCR reaction systems had been used. The random promers had been screened to amplify the specific molecular fragments of Centella asiatica. RESULTS: The specific genetic bands of Centella asiatica species from various habitats were established which were highly stable and repeatable and obviously different from those of other families, genuses of plants such as Gynostemma pentaphylum, Tobacco, Cayratia japonica. CONCLUSION: The developed method of RAPD analysis for the genetic character bands of Centella asiatica could be applied to identify real Centella asiatica from its spurious breed plants. The genetic character bands of Centella asiatica amplified with the RAPD method show high homogeneous in several samples from different habitats.

Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP.

Embryo dormancy in flowering plants is an important dispersal mechanism that promotes survival of the seed through time. The subsequent transition to germination is a critical control point regulating initiation of vegetative growth. Here we show that the Arabidopsis COMATOSE (CTS) locus is required for this transition, and acts, at least in part, by profoundly affecting the metabolism of stored lipids. CTS encodes a peroxisomal protein of the ATP binding cassette (ABC) transporter class with significant identity to the human X-linked adrenoleukodystrophy protein (ALDP). Like X-ALD patients, cts mutant embryos and seedlings exhibit pleiotropic phenotypes associated with perturbation in fatty acid metabolism. CTS expression transiently increases shortly after imbibition during germination, but not in imbibed dormant seeds, and genetic analyses show that CTS is negatively regulated by loci that promote embryo dormancy through multiple independent pathways. Our results demonstrate that CTS regulates transport of acyl CoAs into the peroxisome, and indicate that regulation of CTS function is a major control point for the switch between the opposing developmental programmes of dormancy and germination.