[Identification and functional characterization of candidate genes involved in biosynthesis of ginsenoside Rg_1].

Panax ginseng is a perennial herb with the main active compounds of ginsenosides. Among the reported ginsenosides, ginsenoside Rg_1 not only has a wide range of medicinal functions and abundant content but also is one of the major ginsenoside for the quality evaluation of this herb in the Chinese Pharmacopoeia. The main biosynthesis pathway of ginsenoside Rg_1 in P. ginseng has been clarified, which lays a foundation for the comprehensive and in-depth analysis of the biosynthesis and regulatory mechanism of ginseno-side Rg_1. However, the biosynthesis of ginsenoside Rg_1 is associated with other complex processes involving a variety of regulatory genes and catalyzing enzyme genes, which remain to be studied comprehensively. With the transcriptome data of 344 root samples from 4-year-old P. ginseng plants and their corresponding ginsenoside Rg_1 content obtained in the previous study, this study screened out 217 differentially expressed genes(DEGs) with Rg_1 content changes by DEseq2 analysis in R language. Furthermore, the weighted gene co-expression network analysis(WGCNA) revealed 40 hub genes among the DEGs.Pearsoncorrelation analysis was further perforned to yield 20 candidate genes significantly correlated with ginsenoside Rg_1 content, and these genes were annotated to multiple metabolic processes including primary metabolism and secondary metabolism. Finally, the treatment of P. ginseng adventitious roots with methyl jasmonate indicated that 16 of these genes promoted the biosynthesis of ginsenoside Rg_1 in response to methyl jasmonate induction. Finally, one of the 16 genes was randomly selected to verify the function of the gene by genetic transformation and qRT-PCR and to confirm the rationality of the methodology of this study. The above results lay a foundation for studying the mechanism for regulation on the synthesis of ginsenoside Rg_1 and provide genetic resources for the industrial production of ginsenoside Rg_1.

[Transcriptome-wide identification and expression pattern analysis for Dof gene family in Panax ginseng from Jilin].

Dof(DNA binding with one finger), a unique class of transcription factors in plants, play an important role in seed development, tissue differentiation, and metabolic regulation. To identify the number and function of Dof gene family members in Panax ginseng, this study identified the members of Dof gene family in P. ginseng and systematically analyzed their structures, evolution, functional differentiation, expression patterns, and interactions using bioinformatics methods at the transcriptome level. At the same time, the association analysis of Dof genes from P. ginseng with key enzyme genes for ginsenoside synthesis was carried out to screen the candidate PgDof genes involved in the regulation of ginsenoside biosynthesis. The results showed that there were 54 genes belonging to the Dof gene family in P. ginseng from Jilin. All PgDof genes had Zf-Dof conserved motifs, implying that they were evolutionarily conserved and could be divided into five groups. Expression pattern analysis confirmed that the expression of PgDof gene family members in different tissues, different year-old P. ginseng, and different farm varieties varied significantly. Simultaneously, as revealed by "gene-saponin content" and "gene-gene" linkage analysis, an important candidate PgDof14-1 gene involved in the regulation of ginsenoside biosynthesis was obtained. From the established genetic transformation system of this gene in the hairy roots of P. ginseng, a positive hairy root clone was determined. This study has laid a theoretical foundation for the study of Dof gene family in P. ginseng.

[Expression of saponin biosynthesis related genes in different tissues of Panax quinquefolius].

The relationship between saponin content of Panax quinquefolius in different parts of the organization and expression of ginsenoside biosynthesis related gene was obtained by the correlation analysis between saponin content and gene expression. The 14 tissue parts of P. quinquefolius were studied, six saponins in P. quinquefolius. Samples (ginsenoside Rg1, Re, Rb1, Rc, Rb2 and Rd), group saponins and total saponins were determined by high performance liquid chromatography and vanillin-sulfuric acid colorimetric method. Simultaneously, the expression levels of 7 ginsenoside biosynthesis related genes (SQS, OSC, DS, ß-AS, SQE, P450 and FPS) in different tissues of P. quinquefolius were determined by Real-time fluorescence quantitative PCR. Although 7 kinds of ginsenoside biosynthesis related enzyme gene in the P. quinquefolius involved in ginsenoside synthesis, the expression of ß-AS and P450 genes had no significant effect on the content of monosodium saponins, grouping saponins and total saponins, FPS, SQS, OSC, DS and SQE had significant or extremely significant on the contents of single saponins Re, Rg1, Rb1, Rd, group saponin PPD and PPT, total saponin TMS and total saponin TS (P<0.05 or P<0.01). The biosynthesis of partial saponins, grouping saponins and total saponins in P. quinquefolius was affected by the interaction of multiple enzyme genes in the saponin synthesis pathway, the content of saponins in different tissues of P. quinquefolius was determined by the differences in the expression of key enzymes in the biosynthetic pathway. Therefore, this study further clarified that FPS, SQS, OSC, DS and SQE was the key enzyme to control the synthesis of saponins in P. quinquefolius by correlation analysis, the biosynthesis of ginsenosides in P. quinquefolius was regulated by these five kind of enzymes in cluster co-expression of interaction mode.

[Research of genes expression related to ginsenosides biosynthesis by methyl jasmonic acid in ginseng hair roots].

In order to obtain the expression of ginsenoside biosynthetic pathway related enzyme gene in ginseng hairy root under the control of elicitors, methyl jasmonate (MeJA) was added exogenously as elicitors. Ginseng hairy root clones induced by 4-year-old ginseng root was used as material, total saponin content in ginseng hairy root before and after MeJA treatment was determined by vanillin-sulfuric acid colorimetry, Meanwhile, relative expression of squalene synthase genes, squalene epoxidase genes, oxidized squalene cyclase genes, dammarenediol synthase genes, ß-amyrin synthase genes, cycloartenol synthase genes before and after MeJA treatment were determined by Real-time PCR. The optimum conditions of MeJA which added to ginseng hairy root were obtained, the optimum additional concentration was 6×10⁻4 µmol•L⁻¹, the optimum additional time was 22 d, and the optimum action time was 5 d. The addition of MeJA could improve the enzymatic activity of peroxidase (PPD), catalase (CAT) and peroxidase (PPD) in ginseng hairy root. The expression of SQS，SQE，OSC，DS and ß-AS genes of ginsenoside biosynthetic pathway increased significantly after MeJA treatment, while the change of CAS gene expression were not significant. The expression of key enzyme SQS，SQE，OSC，DS and ß-AS genes in ginsenoside biosynthetic pathway was consistent with the changes of PPD，CAT，PPO enzymatic activity.

[Research achievements on ginsenosides biosynthesis from Panax ginseng].

Panax ginseng is one of the famous rare medicinal herbs, and ginsenosides are the main active ingredient of ginseng is ginsenoside.They can be divided into three chemotypes: oleanane type, protopanaxadiol (PPD) type and the protopanaxatriol (PPT)type. Ginsenosides possess anti-thrombotic, anti-fatigue, anti-aging, cancer control, strengthening the immune system and many other effects. Rrogress has remarkably been made in pharmacology, efficacy and blosynthesis of ginsenosides.This review covers the recent research achievements of ginsenasides, which would be helpful for the relevant researchers to get useful information.

[Correlation of gene expression related to amount of ginseng saponin in 15 tissues and 6 kinds of ginseng saponin biosynthesis].

Fifteen tissues of 4-year-old fruit repining stage Jilin ginseng were chosen as materials, six kinds of monomer saponins (ginsenosides Rg1, Re, Rb1, Rc, Rb2 and Rd) content in 15 tissues was measured by HPLC and vanillin-sulfuric acid method. The relative expression of FPS, SQS, SQE, OSC, ß-AS and P450 genes in 15 tissues was analyzed by real-time PCR. The correlations between ginseng saponin content in 15 tissues of Jilin ginseng and biosynthetic pathway -related genes were obtained. The results showed that was a synergistic increase and decrease trend of positive linear correlation among six kinds of monomer saponin content, and there was a significantly (P < 0.01) positive correlation between monomer saponin content and total saponins content. Monomer saponin content and 6 kinds of enzyme gene correlation were different. Biosynthesis of ginseng total saponins and monomer saponin were regulated by six kinds of participation ginsenoside biosynthesis enzyme genes, the expression of these six kinds of genes in different tissues of ginseng showed collaborative increase and decrease trend, and regulated biosynthesis of ginseng ginsenoside by group coordinative manner.

Regulatory control of carotenoid accumulation in winter squash during storage.

MAIN CONCLUSION: Storage promotes carotenoid accumulation and converts amylochromoplasts into chromoplasts in winter squash. Such carotenoid enhancement is likely due to continuous biosynthesis along with reduced turnover and/or enhanced sequestration. Postharvest storage of fruits and vegetables is often required and frequently results in nutritional quality change. In this study, we investigated carotenoid storage plastids, carotenoid content, and its regulation during 3-month storage of winter squash butternut fruits. We showed that storage improved visual appearance of fruit flesh color from light to dark orange, and promoted continuous accumulation of carotenoids during the first 2-month storage. Such an increased carotenoid accumulation was found to be concomitant with starch breakdown, resulting in the conversion of amylochromoplasts into chromoplasts. The butternut fruits contained predominantly ß-carotene, lutein, and violaxanthin. Increased ratios of ß-carotene and violaxanthin to total carotenoids were noticed during the storage. Analysis of carotenoid metabolic gene expression and PSY protein level revealed a decreased expression of carotenogenic genes and PSY protein following the storage, indicating that the increased carotenoid level might not be due to increased biosynthesis. Instead, the increase likely resulted from a continuous biosynthesis with a possibly reduced turnover and/or enhanced sequestration, suggesting a complex regulation of carotenoid accumulation during fruit storage. This study provides important information to our understanding of carotenogenesis and its regulation during postharvest storage of fruits.

[Fermentation transformed ginsenoside by Lactobacillus plantarum].

OBJECTIVE: To explore ginseng fermentation process by Lactobacillus plantarum, and to make part of total saponins transformed into more reactive ginsenoside Rd. METHOD: Microbial fermentation was carried out by still dark culture. Total saponins were extracted by Soxhlet extraction, and determined by UV visible spectrophotometry with colours reaction by vanillin-sulfuric acid. Ginsenoside Rd was determined by HPLC method. RESULT: The fermentation process was: MRS medium, 35 degrees C, pH 5.0, cultured for 2 days. The content of total saponins was inhance 32%, and the content of ginsenoside Rd was increased 4.864 mg x g(-1). CONCLUSION: The fermentation system's process was reasonable, and it's suitable for mass production, important significance for ginsenoside microbial transformation.

[Characteristics and Risk Evaluation of Heavy Metal Contamination in Paddy Soils in the Three Gorges Reservoir Area].

Soil Cd, Hg, Pb, As, Cr, Cu, Zn, and Ni of 12 districts in the Three Gorges Reservoir area (Chongqing section) were analyzed, and different evaluation methods were used to assess the degree of contamination, potential ecological risk, and human health risk of soil heavy metals in paddy soils. The results showed that the average values of all heavy metals except Cr in paddy soils in the Three Gorges Reservoir area exceeded the background values of soils in the Three Gorges Reservoir area, and the contents of Cd, Cu, and Ni in 12.32%, 4.35%, and 2.54% of the soil samples exceeded the screening values, respectively. The variation coefficients of the eight heavy metals were 29.08%-56.43%, which belonged to the medium and above-intensity variation levels and were influenced by anthropogenic activities. The eight heavy metals were contaminated in the soil, and 16.30%, 6.52%, and 2.90% of the soil Cd, Hg, and Pb were heavily contaminated. At the same time, the potential ecological risk of soil Hg and Cd were in the medium risk level on the whole. Wuxi County and Wushan County had relatively high pollution levels among the 12 districts, the Nemerow pollution index showed a moderate pollution level, and the comprehensive potential ecological risks were also at a moderate ecological hazard level. The results of the health risk evaluation showed that hand-mouth intake was the main exposure path of non-carcinogenic risk and carcinogenic risk. Soil heavy metals presented no non-carcinogenic risk for adults (HI<1), but 12.68% of the sites had non-carcinogenic risk for children (HI>1). As and Cr were the main influencing factors for non-carcinogenic and carcinogenic risks in the study area, and their total contributions to non-carcinogenic and carcinogenic risks were more than 75% and 95%, respectively, which was cause for concern.

[The imprinting status of genetic imprinted gene PEG10 in human hepatocellular carcinomas].

OBJECTIVE: To study the imprinting status of genetic imprinted gene PEG10 (perternally expressed gene 10) in human hepatocellular carcinoma (HCC) tissues and liver cancer cell lines. METHODS: Genomic DNA was extracted from 20 HCC tissues and its adjacent tissues, 15 normal liver tissues, 5 liver cancer cell lines (PLC/PRF/5, smmc-7721, HepG2, Hep3B, SK-HEP-1) and 2 normal human liver cell lines (changliver, HL7702). The DNA fragments containing single nucleotide polymorphism (SNP) site of PEG10 were amplified by polymerase chain reaction (PCR) and the genotype of samples was detected by DNA sequencing. Total RNA was extracted from heterozygous samples, the imprinting status and expression level of PEG10 were evaluated by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) and DNA sequencing. RESULTS: It was found that 16/40 of HCC and its adjacent tissues were heterozygous, 3/15 of normal liver tissue were heterozygous. A site of heterozygous mutation was found in HepG2 cells by DNA sequencing. The other liver tissues and cell lines were all homozygous. PEG10 was biallelically expressed and showed loss of imprinting (LOI) in 82.4% (14/17) liver cancer samples (16 HCC tissues and HepG2), however it was a monoallelic expression and showed genomic imprinting in17.6% (3/17) liver cancer samples. In HCC, the expression levels of PEG10 were increased apparently, but it was negative expressed in cancer adjacent tissues and normal liver tissues. Expression levels of PEG10 were not significantly different between imprinted HCC tissues and HCC tissues with LOI (t = 1.311, P value is more than 0.05). CONCLUSION: PEG10 imprinting is lost in a majority of HCC and no correlation exists between the imprinting status of PEG10 and its expressions in HCC tissues.

The relationship between cyclooxygenase-2, CD44v6, and nm23H1 in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal cancer is the fourth most prevalent malignancy in China. The relationship between COX-2, CD44v6, and nm23H1 in esophageal squamous cell carcinoma (ESCC) remains unclear. MATERIAL AND METHODS: Expression of COX-2, CD44v6, and nm23H1 was examined, using the streptavidin-peroxidase method, in 82 ESCC and 30 normal esophageal mucosa (NEM) samples from the Shaanxi Province in China. RESULTS: The positive rates of COX-2, CD44v6, and nm23H1 were 73.2% (60/82), 64.6% (53/82), and 24.4% (31/82), respectively in ESCC, but 6.7% (2/30), 3.3% (1/30), and 90% (27/30), respectively in NEMs. There was a statistically significant difference between NEMs and ESCCs (p < 0.05). Expression of COX-2 showed a positive statistical correlation with expression of CD44v6 (r = 0.4732, p < 0.0001), and an inverse correlation with nm23H1 (r = -0.3226, p = 0.0035). Expression of COX-2, CD44v6, and nm23H1 had no significant correlation with gender or age (p > 0.05), but increased expression of COX-2 and CD44v6 showed statistical correlation with invasion and lymph node metastasis, respectively (p < 0.05). Decreased expression of nm23H1 was statistically correlated with lymph node metastasis (p = 0.0007) but had no correlation with invasion (p = 0.8221). CONCLUSIONS: This is the first report of a significant correlation between COX-2, CD44v6, and nm23H1 in ESCC. This knowledge might help us to further understand the molecular mechanisms of carcinogenesis and progression of ESCC.

Detection of human papillomavirus type 16 DNA in formalin-fixed, paraffin-embedded tissue specimens of gastric carcinoma.

OBJECTIVES: Human papillomavirus type 16 (HPV16) is regarded as one of the important tumor-related viruses, which are known to have a role in cervical carcinoma; however, there are few reports on HPV16 in gastric carcinoma (GC). Our study aimed to investigate the relationship between HPV16 and the occurrence of GC. METHODS: Liquid PCR (LPCR) and in-situ PCR (ISPCR) methods were carried out to detect the HPV16 oncogene E6 cell-type-specific enhancer in the long control region of HPV16 in 40 GCs and corresponding gastric adjacent normal mucosa (GANM). The patients were from Shaanxi Province in China; Helicobacter pylori (Hp) was detected by immunohistochemistry and by hematoxylin and eosin staining in their gastric tissues. RESULTS: The HPV16 E6 gene was detected in 37.5% (15/40) of the GCs and 5% (2/40) of the GANMs with LPCR, as was the cell-type-specific enhancer; however, the positive rate of E6 was 27.5% (11/40) in GCs and 0% (0/40) in GANMs, respectively, with ISPCR. HPV16 DNA was mainly located in the nucleus of gastric glandular epithelium cells. The infection rate of HPV16 DNA in GCs was higher than that in GANMs (P=0.0004), and the HPV16 had no statistical correlations with sex, age, invasion, grading or lymph node metastasis (P>0.05). The infection rate of HPV16 in cardiac GCs was significantly higher than that in noncardiac ones (P=0.0136), and HPV16 had no correlation with Hp in GCs (P=0.0829). Receiver operator characteristic curve analysis indicated that there was no statistical difference between the LPCR and ISPCR methods in our study through optimizing parameters in ISPCR procedures (P=0.768). CONCLUSIONS: These findings suggested that HPV16 can infect gastric glandular epithelium cells and that viral infection might play a role in the occurrence of GCs independent of or without the cooperation of an Hp infection.

Inflammatory monocyte/macrophage modulation by liposome-entrapped spironolactone ameliorates acute lung injury in mice.

AIM: To examine the therapeutic/preventive potential of liposome-encapsulated spironolactone (SP; Lipo-SP) for acute lung injury (ALI) and fibrosis. MATERIALS & METHODS: Lipo-SP was prepared by the film-ultrasonic method, and physicochemical and pharmacokinetic characterized for oral administration (10 and 20 mg/kg for SP-loaded liposome; 20 mg/kg for free SP) in a mouse model bleomycin-induced ALI. RESULTS: Lipo-SP enhanced bioavailability of SP with significant amelioration in lung pathology. Mechanistically, SP-mediated mineralocorticoid receptor antagonism contributes to inflammatory monocyte/macrophage modulation via an inhibitory effect on Ly6C(hi) monocytosis-directed M2 polarization of alveolar macrophages. Moreover, Lipo-SP at lower dose (10 mg/kg) exhibited more improvement in body weight gain. CONCLUSION: Our data highlight Lipo-SP as a promising approach with therapeutic/preventive potential for ALI and fibrosis.

The relationship between HPV16 and expression of cyclooxygenase-2, P53 and their prognostic roles in esophageal squamous cell carcinoma.

OBJECTIVES: This study aimed to investigate the relationship between human papillomavirus type 16 (HPV16) and expression of cyclooxygenase-2 (COX-2), P53 in esophageal squamous cell carcinoma (ESCC), which has not yet been elucidated. METHODS: HPV16 was detected by amplifying the HPV16 E6 gene by the PCR method, and the expression of COX-2, P53 protein in 69 ESCCs and 32 normal esophageal mucosa (NEM) from Shaanxi Province was examined by the streptavidin-peroxidase method. Estimation of overall survival by HPV16, COX-2, and P53 was calculated with the Kaplan-Meier method and analyzed with the log-rank test. RESULTS: The infection rate of HPV16 in ESCCs (35 of 69, 50.7%) was significantly higher than that in NEMs (two of 32, 6.25%) (P<0.01). The expression rate of COX-2 in ESCCs (44 of 69, 63.8%) was higher than that in NEMs (two of 32, 6.25%) (P<0.01). The expression intensity of COX-2 expression had statistical difference in histological grade (R = 0.4453, P = 0.0019), tumor stage (R = 0.438, P = 0.000), and metastasis (R = 0.417, P = 0.002). P53 expression rate was 49.3% (34 of 69) in ESCC and 18.8% (six of 32) in NEMs. The expression rate of P53 proteins in ESCC was statistically higher than that in N67EMs (P = 0.0037). The infection of HPV16 had inverse correlation with the overexpression of COX-2 in ESCCs (R = -0.321, P = 0.008). The HPV16 DNA in ESCC had no statistical correlation with P53 protein (R = -0.014, P = 0.9055) and the elevated expression of COX-2 had positive correlation with P53 protein in ESCC (R = 0.441, P = 0.000). No statistical correlation was observed between the infection of HPV16 and clinicopathological features in ESCCs including sex, age, tumor stage, and lymph node metastasis, respectively (P>0.05). The COX-2 had no statistical correlation with sex and age (P>0.05), but had association with tumor stage and lymph node metastasis, respectively (P<0.05). The expression of P53 protein had significant association with lymph node metastasis (P = 0.0005), but not with sex, age, and tumor stage, respectively (P>0.05). The overexpression of COX-2, infection of HPV16, and P53 protein in ESCC were not correlated with survival during the 5-year follow-up period (P>0.05). CONCLUSION: We first concluded that the increased expression of COX-2 had inverse correlation with HPV16 in ESCC. COX-2, HPV16, and P53 had no significant effect on the survival of patients with ESCC. These observations might help us to further understand the significant association between HPV16 and other molecules involved in the carcinogenesis and progression of ESCC.

Changes in chloroplast ultrastructure, fatty acid components of thylakoid membrane and chlorophyll a fluorescence transient in flag leaves of a super-high-yield hybrid rice and its parents during the reproductive stage.

In plants, it is well established that chloroplast is one of the early targgeted organelles to breakdown during leaves senescing. Here we applied a newly developed super-high-yield hybrid rice (Oryza sativa) LiangYouPeiJiu (LYPJ) and its parents lines to investigate changes in ultrastructure of chloroplasts, fatty acid composition of thylakoid membrane lipids and chlorophyll (Chl) a fluorescence transient in natural senescing leaves. We found that at full expansion of flag leaves in three lines, chloroplasts often showed oblong shapes with a typical membrane system of stroma and grana thylakoids, whereas their shapes had been changed from oblong to spherical during senescence. Our data showed that the initiation of senescence displayed accumulation of starch and an increase in the number and size of plastoglobuli with the damaged thylakoid membranes; subsequently, swollen thylakoid membranes in stroma and in grana with a significant increase in MDA content, and disorganization of thylakoid membrane system with significant changes in fatty acid composition of thylakoid membrane lipids were developed. Compared with its parents, the newly developed hybrid rice LYPJ had the highest capacity of carbohydrate transport from leaves (sources) to ears (sink), marked with the lowest accumulation of starch grains in the leaf chloroplasts, and the slowest senescing rate of chloroplast in overall leaf senescence process. Chl a fluorescence transients of three kinds of flag leaves were analyzed by so-called JIP-test. The results of analysis suggest that these findings inculding a high inherited activity of antioxidant enzymes and high photosynthetate transport to pretect chloroplast structure in the hybrid rice LYPJ have close relations to its super-high yield.