Tissue-specific transcriptome and metabolome analyses reveal candidate genes for lignan biosynthesis in the medicinal plant Schisandra sphenanthera.

Schisandra sphenanthera is an extremely important medicinal plant, and its main medicinal component is bioactive lignans. The S. sphenanthera fruit is preferred by the majority of consumers, and the root, stem, and leaf are not fully used. To better understand the lignan metabolic pathway, transcriptome and metabolome analyses were performed on the four major tissues of S. sphenanthera. A total of 167,972,229 transcripts and 91,215,760 unigenes with an average length of 752 bp were identified. Tissue-specific gene analysis revealed that the root had the highest abundance of unique unigenes (9703), and the leaves had the lowest (189). Transcription factor analysis showed that MYB-, bHLH- and ERF-transcription factors, which played important roles in the regulation of secondary metabolism, showed rich expression patterns and may be involved in the regulation of processes involved in lignan metabolism. In different tissues, lignans were preferentially enriched in fruit and roots by gene expression profiles related to lignan metabolism and relative lignan compound content. Furthermore, schisandrin B is an important compound in S. sphenanthera. According to weighted gene co-expression network analysis, PAL1, C4H-2, CAD1, CYB8, OMT27, OMT57, MYB18, bHLH3, and bHLH5 can be related to the accumulation of lignans in S. sphenanthera fruit, CCR5, SDH4, CYP8, CYP20, and ERF7 can be related to the accumulation of lignans in S. sphenanthera roots. In this study, transcriptome sequencing and targeted metabolic analysis of lignans will lay a foundation for the further study of their biosynthetic genes.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis.

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis / 中国天然药物

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Candidate genes involved in the biosynthesis of lignan in Schisandra chinensis fruit based on transcriptome and metabolomes analysis.

Schisandra chinensis Turcz. (Baill.) is a plant species with fruits that have been well known in Far Eastern medicine for a long time. It has traditionally been used as a stimulating and fortifying agent in cases of physical exhaustion and to inhibit fatigue. The major bioactive compounds found in S. chinensis are lignans with a dibenzocyclooctadiene skeleton, but little is known about their biosynthesis in plants. S. chinensis is the ideal medicinal plant for studying the biosynthesis of lignans, especially the dibenzocyclooctadiene skeleton. Genomic information for this important herbal plant is unavailable. To better understand the lignan biosynthesis pathway, we generated transcriptome sequences from the fruit during ripening and performed de novo sequence assembly, yielding 136 843 unique transcripts with N50 of 1778 bp. Putative functions could be assigned to 41 824 transcripts (51.57%) based on BLAST searches against annotation databases including GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, 22 candidate cytochrome P450 genes and 15 candidate dirigent proteins genes that were most likely involved in the lignan biosynthesis pathway were discovered based on transcriptome sequencing of S. chinensis. The genomic data obtained from S. chinensis, especially the identification of putative genes involved in the lignan biosynthesis pathway, will facilitate our understanding of lignan biosynthesis at the molecular level. The lignan metabolite profiles were analyzed by metabolomes, the accumulation patterns of 30 metabolites involved in the lignan pathway were studied. Co-expression network of lignan contents and transcriptional changes showed 355 strong correlations (correlation coefficient, R2 > 0.9) between 21 compounds and 153 transcripts. Furthermore, the comprehensive analysis and characterization of the genes involved in lignan pathways and the metabolite profiles of lignans are expected to provide better insight regarding the diversity of the chemical composition, synthetic characteristics, and regulatory mechanisms of this medical herb.

Detecting Schisandrae Chinensis Fructus and Its Chinese Patent Medicines with a Nucleotide Signature.

Schisandrae Chinensis Fructus (Wuweizi) is often adulterated with Schisandrae Sphenantherae Fructus (Nanwuweizi) in the herbal market. This adulteration is a threat to clinical treatment and safety. In this study, we aimed to develop a nucleotide signature for the identification of Wuweizi and its Chinese patent medicines based on the mini-DNA barcoding technique. We collected 49 samples to obtain internal transcribed spacer 2 (ITS2) sequences and developed a 26-bp nucleotide signature (5'-CGCTTTGCGACGCTCCCCTCCCTCCC-3') on the basis of a single nucleotide polymorphism (SNP) site within the ITS2 region that is unique to Wuweizi. Then, using the nucleotide signature, we investigated 27 batches of commercial crude drug samples labeled as Wuweizi and eight batches of Chinese patent medicines containing Wuweizi. Results showed that eight commercial crude drug samples were adulterants and one of the Chinese patent medicines contained adulterants. The nucleotide signature can serve as an effective tool for identifying Wuweizi and its Chinese patent medicines and can thus be used to ensure clinical drug safety.

[Molecular characters of Schisandra chinensis with white fruit by RAPD and ISSR makers].

OBJECTIVE: The characters of Schisandra chinensis with white fruit were represented at molecular levels and the genetic diversity were investigated using RAPD and ISSR. METHODS: 12 primers of RAPD randomized markers and 8 primers of ISSR markers were used to test 21 samples of white fruit Schisandra chinensis, and POPGENE 32 software were used to analyze the results. RESULTS: One or more unique bands were produced to distinguish white fruit Schisandra chinensis from normal Schisandra chinensis using the primers of S83, S180 and S300. RAPD:66 discernible DNA fragments were generated with 52 (78.79%) polymorphic fragments; ISSR: 42 discernible DNA fragments were generated with 25 (59.52%) polymorphic fragments. The genetic variation of white fruit Schisandra chinensis was more unstable than normal Schisandra chinensis, but the genetic distance of them was small at the species level. CONCLUSION: RAPD and ISSR markers can be used to put up the characteristics of Schisandra chinensis with white fruit at molecular levels. Also they can indicate the genetic relationship of the Schisandra chinensis germplasm resource.

[Identification of Schisandra chinensis with white fruits based on ITS2 sequences].

OBJECTIVE: To analyse a special kind of Schisandra chinensis with the white fruit using ITS2 barcode at molecular levels. METHOD: ITS2 regions were sequenced bidirectionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner, MEGA 5.0 software was used to align the sequences. The ITS2 secondary structure was predicted using ITS2 web server, BLAST 1 method was used to identify the S. chinensis with the white fruit. RESULT: The length of the ITS2 sequence was 231 bp. And the sample was identified as S. chinensis using the method of BLAST 1. Their mean interspecific genetic distance (K2P distance) among the populations of the S. chinensis with the white fruit and S. chinensis was far lower than the mean interspecific genetic distance between the S. chinensis and S. sphenanthera. CONCLUSION: By using ITS2 the S. chinensis with the white fruit was identified as S. chinensis, and the ITS2 barcode could be used to identify S. chinensis and S. sphenanthera.

[DNA identification of Zijingpi's adulterant species Schisandra sphenanthera based on NCBI nucleotide database analysis].

OBJECTIVE: To provide basis for quality control of Zijingpi, DNA identification was used based on NCBI nucleotide database analysis. METHOD: Firstly, total DNA of Zijingpi was extracted. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and PCR products was directly sequenced after purification. Finally, ITS sequence similarity and phylogenetic tree were used for identification. RESULT: The ITS sequence information of the mainstream commercial drugs of Zijingpi was obtained. CONCLUSION: It is firstly reported that the mainstream commercial drugs of Zijingpi was the bark of Schisandra sphenanthera.

Genetic diversity and population structure of two important medicinal plant species Schisandra chinensis and Schisandra sphenanthera revealed by nuclear microsatellites.

Seven polymorphic and transferable nuclear microsatellites were used to investigate the population structure of genetic diversity of Schisandra chinensis and Schisandra sphenanthera for facilitating their conservation and sustainable utilization. High levels of gene diversity were revealed in these two medicinal species, the majority of genetic diversity was harbored within populations, and population structure was might due to restricted gene flow among populations. Isolation by distance was close to significance in S. chinensis but not in S. sphenanthera. In S. chinensis, null alleles were identified as a cause for excess of homozygotes at loci G24 and WGA60, but inbreeding might also be partly responsible for the positive F ( IS ) values in this species. In contrast, null allele frequencies were high at all the seven loci in S. sphenanthera and resulted in overestimation of fixation index. The strategy for ex situ conservation of these two medicinal species is discussed based on the genetic results.

[Identification of Schisandra sphenanthera and S. chinensis by random amplified polymorphic DNA sequence characterized applied region].

OBJECTIVE: To establish a new method for the identification of Schisandra sphenanthera and S. chinensis. METHOD: Random amplified polymorphic DNA-Sequence characterized applied region (RAPD-SCAR) method was applied to screen primers. RESULT: Screening from 100 primers, only 2 random primers, which can be used to identify S. sphenanthera and S. chinensis accurately with a good reproducibility. It worked to fit them into sequence characterized applied region. CONCLUSION: RAPD-SCAR can be used to identify S. sphenanthera and S. chinensis accurately.

[Ribosomal DNA ITS sequences analysis of the Chinese crude drug fructus schisandrae sphenantherae and fruts of Schisandra viridis].

OBJECTIVE: To find the patterns of the rDNA ITS sequence variation of Schisandra sphenanthera and S. viridis, and to establish the molecular biological method for the identification of Fructus Schisandrae Sphenantherae and the fruits of S. viridis. METHOD: PCR products were sequenced directly and the sequences were analyzed with PAUP 4.0b10. NJ systematic tree was obtained with neighbor-joining method. RESULT: The Complete ITS sequence of S. sphenanthera was 691-692 bp, of which there were 282 bp of ITS1 and 246-247 bp of ITS2. The complete sequence of S. viridis was 694-695 bp, consisting of 285-286 bp of ITS1 and 246-247 bp of ITS2. There were three informative sites in ITS1 regions for the two species. In the NJ tree with Kadsura anamosma and K. coccinea as outgroups, five different populations of S. viridis were the monophyletic group with the bootstrap value of 68%. These populations included one from Tianmushan, Zhejiang province, three populations from Jigongshan, Henan Province and the other two populations of S. viridis cited the sequences from GeneBank (registration numbers are AF263438 and AF163703 respectively). CONCLUSION: The rDNA internal transcribed spacer is a good marker to distinguish the Fructus Schisandrae Sphenantherae from the fruits of S. viridis.