Genetic Diversity Assessment of a Chinese Medicinal Endemic Species, Aspidopterys obcordata var. obcordata, by Combined Molecular Marker Methods (ISSR & SRAP).

Aspidopterys obcordata var. obcordata, a medicinal plant endemic to China, is a narrowly distributed species and wild resources are extremely limited. To evaluate the genetic variability and degree of genetic divergence of A. obcordata var. obcordata, and to make rational scientific decisions on its harvest and germplasm conservation, we collected 122 samples from across nearly all of its distribution area and studied genetic diversity using inter-simple sequence repeats (ISSRs), sequence-related amplified polymorphisms (SRAPs), and a method combining the two techniques. The results revealed the high genetic diversity of A. obcordata var. obcordata, mainly due to its intra-population diversity, and the top two populations with the highest levels of intra-population diversity were ML and DH, individuals of which can serve as excellent germplasm candidates during the processing of germplasm screening and conservation. In general, the combining method was prior to the ISSR analyses and SRAP analyses results, except for a slight difference in the genetic structure of individual populations. Therefore, we suggest that a combination analysis of the two marker methods is ideal for evaluating the genetic diversity and genetic relationships of A. obcordata var. obcordata.

[DNA barcoding identification of original plants of a rare medicinal material Resina Draconis and related Dracaena species].

Resina Draconis, a rare and precious traditional medicine in China, is known as the "holy medicine for promoting blood circulation". According to the national drug standard, it's derived from the resin extracted from the wood of Dracaena cochinchinensis, a Liliaceae plant. In addition, a variety of Dracaena species all over the world can form red resins, and there is currently no molecular identification method that can efficiently identify the origin of Dracaena medicinal materials. In this study, seven species of Dracaena distributed in China were selected as the research objects. Four commonly used DNA barcodes(ITS2, matK, rbcL and psbA-trnH), and four highly variable regions(trnP-psaJ, psbK-psbI, trnT-trnL, clpP) in chloroplast genome were used to evaluate the identification efficiency of Dracaena species. The results showed that clpP sequence fragment could accurately identify seven species of Dracaena plants. However, due to the long sequence of clpP fragment, there were potential problems in the practical application process. We found that the combined fragment "psbK-psbI+ trnP-psaJ" can also be used for accurate molecular identification of the Resina Draconis origin plants and relative species of Dracaena, which were both relatively short sequences in the combined fragment, showing high success rates of amplification and sequencing. Therefore, the "psbK-psbI+ trnP-psaJ" combined fragment can be used as the DNA barcode fragments for molecular identification of Resina Dracon's origin plants and relative species of Dracaena. Research on the identification of Dracaena species, the results of this study can be used to accurately identify the original material of Resina Draconis, and providing effective means for identification, rational development and application of Resina Draconis base source.

Ectopic expression of a phytochrome B gene from Chinese cabbage (Brassica rapa L. ssp. pekinensis) in Arabidopsis thaliana promotes seedling de-etiolation, dwarfing in mature plants, and delayed flowering.

Phytochrome B (phyB) is an essential red light receptor that predominantly mediates seedling de-etiolation, shade-avoidance response, and flowering time. In this study, we isolate a full-length cDNA of PHYB, designated BrPHYB, from Chinese cabbage (Brassica rapa L. ssp. pekinensis), and we find that BrphyB protein has high amino acid sequence similarity and the closest evolutionary relationship to Arabidopsis thaliana phyB (i.e., AtphyB). Quantitative reverse transcription (RT)-PCR results indicate that the BrPHYB gene is ubiquitously expressed in different tissues under all light conditions. Constitutive expression of the BrPHYB gene in A. thaliana significantly enhances seedling de-etiolation under red- and white-light conditions, and causes dwarf stature in mature plants. Unexpectedly, overexpression of BrPHYB in transgenic A. thaliana resulted in reduced expression of gibberellins biosynthesis genes and delayed flowering under short-day conditions, whereas AtPHYB overexpression caused enhanced expression of FLOWERING LOCUS T and earlier flowering. Our results suggest that BrphyB might play an important role in regulating the development of Chinese cabbage. BrphyB and AtphyB have conserved functions during de-etiolation and vegetative plant growth and divergent functions in the regulation of flowering time.

[Fungal composition in massa medicata fermentata based on culture dependent method and independent PCR-SSCP technique].

OBJECTIVE: To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique. METHOD: Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on ß-tubulin gene were used to identify the mycobiota. RESULT: According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples. CONCLUSION: PCR-SSCP based on ß-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

Transcriptome profiling reveals candidate flavonoid-related genes during formation of dragon's blood from Dracaena cochinchinensis (Lour.) S.C.Chen under conditions of wounding stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Dragon's Blood (Resina Draconis) is a red resin that has been used in traditional medicine to promote blood circulation, regenerate muscles, reduce swelling and pain, stop bleeding, etc., and its main chemical constituents are flavonoids. Dracaena cochinchinensis (Lour.) S.C.Chen is the only plant defined by the Pharmacopoeia of the People's Republic of China as a source of dragon's blood. AIM OF THE STUDY: We aimed to reveal genes involved in the biosynthesis and accumulation of flavonoids of D. cochinchinensis which is under wounding stress by performing a de novo transcriptome analysis. MATERIALS AND METHODS: D. cochinchinensis samples were collected for transcriptome sequencing and bioinformatics analysis at 0 days (0 d), 3 days (3 d), 6 days (6 d), and 10 days (10 d) after induction wounding stress, and tissues were microscopically observed after wounding stress. RESULTS: A total of 63,244 unigenes were obtained through bioinformatics analysis, and genes associated with the biosynthesis of flavonoids were identified. Through the analysis of DEGs after wounding stress in D. cochinchinensis, based on gene expression consistent with flavonoid accumulation levels, 20 genes in connection with the flavonoid synthesis pathway and 56 genes that may be responsible for flavonoid modification and transport, and also revealed TFs (MYB, bHLH) that may be responsible for flavonoid biosynthesis. Analysis of DEGs between the four periods revealed that after wounding stress, the greatest number of significant DEGs were enriched during the first 3 days, while fewer DEGs were enriched after day 3, which corresponding to only about 1/10 (353/3883) the number of DEGs during the first 3 days. In addition, putative unigenes involved in lignin biosynthesis, such as CSE, HCT, CCR, F5H, and CAD, were significantly down-regulation after D. cochinchinensis wounding stress, but the putative unigenes responsible for flavonoid biosynthesis, such as CHS, CHI, DFR, F3'5'H, F3H, ANR, FLS, and ANS were significantly up-regulation. CONCLUSION: We performed de novo transcriptome analysis of D.cochinchinensis under wounding stress, candidate genes and TFs involved in the biosynthesis and accumulation of flavonoids were identified, which is the first report on the transcript variants in flavonoid form accumulation in D. cochinchinensis under wounding stress. According to the results of DEGs analysis, wounding stress attenuated lignin biosynthesis meanwhile promoted flavonoid biosynthesis. In addition, we also compared the transcriptomics of the two different original plants (D.cochinchinensis and D.cambodiana) that form dragon's blood in order to provide further understanding of the formation of dragon's blood.

Complete Chloroplast Genome Analysis of Two Important Medicinal Alpinia Species: Alpinia galanga and Alpinia kwangsiensis.

Most Alpinia species are valued as foods, ornamental plants, or plants with medicinal properties. However, morphological characteristics and commonly used DNA barcode fragments are not sufficient for accurately identifying Alpinia species. Difficulties in species identification have led to confusion in the sale and use of Alpinia for medicinal use. To mine resources and improve the molecular methods for distinguishing among Alpinia species, we report the complete chloroplast (CP) genomes of Alpinia galanga and Alpinia kwangsiensis species, obtained via high-throughput Illumina sequencing. The CP genomes of A. galanga and A. kwangsiensis exhibited a typical circular tetramerous structure, including a large single-copy region (87,565 and 87,732 bp, respectively), a small single-copy region (17,909 and 15,181 bp, respectively), and a pair of inverted repeats (27,313 and 29,705 bp, respectively). The guanine-cytosine content of the CP genomes is 36.26 and 36.15%, respectively. Furthermore, each CP genome contained 133 genes, including 87 protein-coding genes, 38 distinct tRNA genes, and 8 distinct rRNA genes. We identified 110 and 125 simple sequence repeats in the CP genomes of A. galanga and A. kwangsiensis, respectively. We then combined these data with publicly available CP genome data from four other Alpinia species (A. hainanensis, A. oxyphylla, A. pumila, and A. zerumbet) and analyzed their sequence characteristics. Nucleotide diversity was analyzed based on the alignment of the complete CP genome sequences, and five candidate highly variable site markers (trnS-trnG, trnC-petN, rpl32-trnL, psaC-ndhE, and ndhC-trnV) were found. Twenty-eight complete CP genome sequences belonging to Alpinieae species were used to construct phylogenetic trees. The results fully demonstrated the phylogenetic relationship among the genera of the Alpinieae, and further proved that Alpinia is a non-monophyletic group. The complete CP genomes of the two medicinal Alpinia species provides lays the foundation for the use of CP genomes in species identification and phylogenetic analyses of Alpinia species.

Effects of genetic variation and environmental factors on bergenin in Rodgersia sambucifolia Hemsl.

ETHNOPHARMACOLOGICAL RELEVANCE: Bergenin is a well-known active compound that exhibits antioxidant, antiarrhythmic, hepatoprotective, and anti-inflammatory activities. However, the resource reserve of Rodgersia sambucifolia, one of the main raw materials for extracting bergenin, have sharply declined, and the bergenin content in different germplasms differs vastly, resulting in a serious shortage of the market supply of bergenin. AIM OF THE STUDY: To investigate the influence of genetic diversity and environmental factors on bergenin content in Rodgersia sambucifolia. MATERIALS AND METHODS: Fifty Rodgersia sambucifolia samples with a growth period of 2-3 years were collected from different areas across China and the bergenin content was determined via HPLC. Meanwhile the total genomic DNA was extracted and ISSR was performed. The bergenin content as measured using HPLC and the environmental data gathered from the meteorological stations and field work were combined and analyzed using correlation tests in XLSTAT 2018 to detect the key factors affecting bergenin content. The genetic UPGMA tree constructed based on genetic distances of the 50 samples and the chemical dendrogram constructed according to the distance between the bergenin content were compared to determine the correlation between genetic and chemical differentiation. RESULTS: Among the 50 individuals, bergenin content varied from 2.83 to 12.54%, with the highest content being 4.43-fold that of the lowest content. The survey of the 50 individuals produced a total of 193 amplified bands, 187 of which were polymorphic (96.89%). In the study, bergenin content was positively correlated with annual mean temperature (AMT) (r = 0.583, P < 0.0001) and 1-12 month monthly mean temperature (MMT) (P < 0.0001). A comparison of the genetic dendrogram with the AHC dendrogram found no corresponding relationship between them. Mantel correlation analyses also showed that there was no significant correlation between them (r = 0.144). CONCLUSIONS: There were large differences in bergenin content among different germplasms that were not correlated with the high genetic variation in Rodgersia sambucifolia but were significantly correlated with environmental factors, such as temperature. This study lays the foundation for subsequent superior germplasm selection and artificial breeding of Rodgersia sambucifolia to improve the bergenin content and meet market demands.

Sema 3A as a biomarker of the activated mTOR pathway during hexavalent chromium-induced acute kidney injury.

Semaphorin 3A (sema 3A) is one of a class of secretory proteins belonging to a family of axon-directed factors found in podocytes, distal tubules, and collecting tubes of the kidney. It is considered to be a potential target molecule involved in the mammalian target of the rapamycin (mTOR) pathway in renal injury or renal diseases, but it has an unknown role in the course of hexavalent chromium-Cr(VI) induced nephrotoxicity. In the present study, an acute kidney injury (AKI) model in rats or cultured tubular epithelial HK-2 cells was employed for Cr(VI) exposure alone or in combination with rapamycin (Rap) or N-acetyl-l-cysteine (NAC) or recombinant sema 3A. The methods of histopathology, biochemics, and western blotting were applied to evaluate tubular injury and the role of sema 3A. The results showed that a significant increase of urinary sema 3A indicates an early occurrence of AKI exposed to Cr(VI), accompanied with a significant increase of tubular injury score and phosphorylated mTOR proteins. Further, Cr(VI) treatment, in combination with pretreatment of the mTOR pathway inhibitor, Rap, showed a considerably stronger protective effect of Rap in protecting against Cr(VI)-induced nephrotoxicity than that seen with the free radical scavenger NAC, highlighting the dominant renal protective role of the mTOR pathway in inhibiting toxicity by downregulating the expressed levels of sema 3A in renal tissue. This study has demonstrated that an increased expression of sema 3A occurs in Cr(VI)-induced AKI resulting from activation of the mTOR pathway, and that inhibition of this pathway has been shown to decrease the severity of the toxicity. In conclusion, this study has shown that increased urinary sema 3A is indicative of an activated mTOR pathway and is a valuable biomarker of the early AKI induced by Cr(VI) exposure.

The effects of genetic variation and environmental factors on rhynchophylline and isorhynchophylline in Uncaria macrophylla Wall. from different populations in China.

Uncaria macrophylla Wall. is an important Chinese medicinal herb. Rhynchophylline (RIN) and isorhynchophylline (IRN) are its major active compounds. We investigated the influence of genetic differentiation and environmental factors on the RIN and IRN to find the main influencing factors of their contents and lay the foundation for the following cultivation and breeding. We used inter-simple sequence repeat (ISSR) markers to investigate the genetic diversity, and high-performance liquid chromatography (HPLC) to measure the contents of RIN and IRN in 200 samples of U. macrophylla obtained from nine natural populations, and then to analyze the correlation between genetic differentiation, environmental factors of sampling sites and the contents of RIN and IRN. We found that High intra-population (80.05%) and low inter-population (19.95%) genetic diversity existed in the samples of U. macrophylla. To some extent, genetic differentiation and the contents of RIN and IRN had correlation in individual populations (such as JH, MH, XM, and ML). The RIN and IRN contents were significant negatively correlated with the precipitation in May (RIRN = -0.771, p = 0.015) and June (RRIN = -0.814, p = 0.008; RIRN = -0.921, p = 0.000), indicating that precipitation was the main affecting factor of their contents. Interestingly, the analysis results showed that the RIN content had a significant positive correlation (r = 0.585, p = 0.000) with the IRN content (they are isomers); the proportion of RIN had a significant negative correlation with the sum of the two (r = -0.390, p<0.0001), while the proportion of IRN had a significant positive correlation (r = 0.390, p<0.0001). It meant that, with the total quantity of the two compounds increased, the proportion of RIN decreased and the proportion of IRN increased, illustrating that their conversion exist some regularity. Moreover, the content ratio of RIN and IRN was significant positively correlated with the January precipitation (r = 0.716, p = 0.030), implying that January may be the key period for the mutual transformation of RIN and IRN.