Selection and Validation of Reference Genes for Gene Expression in Bactericera gobica Loginova under Different Insecticide Stresses.

Insecticide resistance has long been a problem in crop pest control. Bactericera gobica is a major pest on the well-known medicinal plants Lycium barbarum L. Investigating insecticide resistance mechanisms of B. gobica will help to identify pesticide reduction strategies to control the pest. Gene expression normalization by RT-qPCR requires the selection and validation of appropriate reference genes (RGs). Here, 15 candidate RGs were selected from transcriptome data of B. gobica. Their expression stability was evaluated with five algorithms (Delta Ct, GeNorm, Normfinder, BestKeeper and RefFinder) for sample types differing in response to five insecticide stresses and in four other experimental conditions. Our results indicated that the RGs RPL10 + RPS15 for Imidacloprid and Abamectin; RPL10 + AK for Thiamethoxam; RPL32 + RPL10 for λ-cyhalothrin; RPL10 + RPL8 for Matrine; and EF2 + RPL32 under different insecticide stresses were the most suitable RGs for RT-qPCR normalization. EF1α + RPL8, EF1α + ß-actin, ß-actin + EF2 and ß-actin + RPS15 were the optimal combination of RGs under odor stimulation, temperature, developmental stages and both sexes, respectively. Overall, EF2 and RPL8 were the two most stable RGs in all conditions, while α-TUB and RPL32 were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target cytochrome P450 CYP6a1 gene between adult and nymph stages and under imidacloprid stress. The results of CYP6a1 expression were consistent with transcriptome data. This study is the first research on the most stable RG selection in B. gobica nymphs exposed to different insecticides, which will contribute to further research on insecticide resistance mechanisms in B. gobica.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Identification of sex-specific markers based on gene expression in adult Colorado potato beetles.

The Colorado potato beetle, Leptinotarsa decemlineata, (Coleoptera: Chrysomelidae) is an economically important pest insect of potatoes. Understanding how the mechanisms driving its invasiveness vary between sexes will be critical for developing modern control methods. However, the currently available methods for sexing adult Colorado potato beetles are either inefficient or unsuitable for projects that require RNA as an input, like those measuring gene expression. Therefore, the development of simple molecular tools that are tailored to these studies is important. In this study, we used publicly available RNA-seq data to select 5 candidate genes for sex-specific markers in adult Colorado potato beetles. We confirmed that our 5 marker candidates exhibit a sex-specific expression pattern and can be used as PCR markers for sex determination. This method of sex detection will allow researchers to distinguish the sex of the individual with a simple PCR reaction using cDNA as the template and assign sex to RNA-seq samples post hoc.

Bacterial Inhibition Mechanism of Rhamnolipid-Modified ß-Carotene/Rutinoside Complex Liposomes.

In this study, a new cholesterol-free delivery system named RL-ßC-Rts was developed using rhamnolipid (RL) as the surfactant and encapsulating both ß-carotene (ßC) and rutinoside (Rts). The purpose was to examine its antibacterial properties against four food-borne pathogenic microorganisms including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. m), and Salmonella typhimurium (S. typhimurium) and to investigate the mechanism behind the inhibition. Results from bacterial viability tests and minimum inhibitory concentration (MIC) showed RL-ßC-Rts possessed antibacterial activity. Upon further examination of the cell membrane potential, it was observed that E. coli, S. aureus, L. m, and S. typhimurium exhibited a reduction in mean fluorescence intensity by 50.17%, 34.07%, 34.12%, and 47.05%, respectively. These decreases suggested damage to the structure of the cell membrane, which subsequently resulted in the discharge of proteins from the bacteria and the consequential impairment of crucial functions. This was supported by alterations in protein concentration. The results of the RT-qPCR showcased that the expression of genes associated with energy metabolism, tricarboxylic acid cycle, DNA metabolism, virulence factor formation and cell membrane formation could be suppressed by RL-ßC-Rts. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01077-6.

Genome-wide identification and expression analysis of terpene synthases in Gossypium species in response to gossypol biosynthesis.

Cottonseed is an invaluable resource, providing protein, oil, and abundant minerals that significantly contribute to the well-being and nutritional needs of both humans and livestock. However, cottonseed also contains a toxic substance called gossypol, a secondary metabolite in Gossypium species that plays an important role in cotton plant development and self-protection. Herein, genome-wide analysis and characterization of the terpene synthase (TPS) gene family identified 304 TPS genes in Gossypium. Bioinformatics analysis revealed that the gene family was grouped into six subgroups TPS-a, TPS-b, TPS-c, TPS-e, TPS-f, and TPS-g. Whole-genome, segmental, and tandem duplication contributed to the evolution of TPS genes. According to the analysis of selection pressure, it was predicted that TPS genes experience predominantly negative selection, with positive selection occurring subsequently. RT-qPCR analysis in TM-1 and CRI-12 lines revealed GhTPS48 gene as the candidate gene for silencing experiments. To summarize, comprehensive genome-wide studies, RT-qPCR, and gene silencing experiments have collectively demonstrated the involvement of the TPS gene family in the biosynthesis of gossypol in cotton.

Selection and Validation of Optimal RT-qPCR Reference Genes for the Normalization of Gene Expression under Different Experimental Conditions in Lindera megaphylla.

Lindera megaphylla, a broad-leaved evergreen that is used as a landscape ornamental plant and medicinal plant, is an ecologically important and dominant tree species. However, little is known about the molecular mechanisms of its growth, development, and metabolism. The selection of suitable reference genes is critical for molecular biological analyses. To date, no research on reference genes as a foundation for gene expression analysis has been undertaken in L. megaphylla. In this study, 14 candidate genes were selected from the transcriptome database of L. megaphylla for RT-qPCR assay under different conditions. Results showed that helicase-15 and UBC28 were most stable in different tissues of seedlings and adult trees. For different leaf developmental stages, the best combination of reference genes was ACT7 and UBC36. UBC36 and TCTP were the best under cold treatment, while PAB2 and CYP20-2 were the best under heat treatment. Finally, a RT-qPCR assay of LmNAC83 and LmERF60 genes were used to further verify the reliability of selected reference genes above. This work is the first to select and evaluate the stability of reference genes for the normalization of gene expression analysis in L. megaphylla and will provide an important foundation for future genetic studies of this species.

Phosphorus and Serendipita indica synergism augments arsenic stress tolerance in rice by regulating secondary metabolism related enzymatic activity and root metabolic patterns.

The multifarious problems created by arsenic (As), for collective environment and human health, serve a cogent case for searching integrative agricultural approaches to attain food security. Rice (Oryza sativa L.) acts as a sponge for heavy metal(loid)s accretion, specifically As, due to anaerobic flooded growth conditions facilitating its uptake. Acclaimed for their positive impact on plant growth, development and phosphorus (P) nutrition, 'mycorrhizas' are able to promote stress tolerance. Albeit, the metabolic alterations underlying Serendipita indica (S. indica; S.i) symbiosis-mediated amelioration of As stress along with nutritional management of P are still understudied. By using biochemical, RT-qPCR and LC-MS/MS based untargeted metabolomics approach, rice roots of ZZY-1 and GD-6 colonized by S. indica, which were later treated with As (10 µM) and P (50 µM), were compared with non-colonized roots under the same treatments with a set of control plants. The responses of secondary metabolism related enzymes, especially polyphenol oxidase (PPO) activities in the foliage of ZZY-1 and GD-6 were enhanced 8.5 and 12-fold, respectively, compared to their respective control counterparts. The current study identified 360 cationic and 287 anionic metabolites in rice roots, and the commonly enriched pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was biosynthesis of phenylalanine, tyrosine and tryptophan, which validated the results of biochemical and gene expression analyses associated with secondary metabolic enzymes. Particularly under As+S.i+P comparison, both genotypes exhibited an upregulation of key detoxification and defense related metabolites, including fumaric acid, L-malic acid, choline, 3,4-dihydroxybenzoic acid, to name a few. The results of this study provided the novel insights into the promising role of exogenous P and S. indica in alleviating As stress.

In silico prediction and expression analysis of vaccine candidate genes of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common food-borne pathogen that causes human gastroenteritis in the United States. Consumption of contaminated poultry products is considered as the major source of human Campylobacter infection. An effective vaccine would be a promising alternative to antibiotic supplements to curb C. jejuni colonization in poultry gastrointestinal (GI) tract. However, the genetic diversity among the C. jejuni isolates makes vaccine production more challenging. Despite many attempts, an effective Campylobacter vaccine is not yet available. This study aimed to identify suitable candidates to develop a subunit vaccine against C. jejuni, which could reduce colonization in the GI tract of the poultry. In the current study, 4 C. jejuni strains were isolated from retail chicken meat and poultry litter samples and their genomes were sequenced utilizing next-generation sequencing technology. The genomic sequences of C. jejuni strains were screened to identify potential antigens utilizing the reverse vaccinology approach. In silico genome analysis predicted 3 conserved potential vaccine candidates (phospholipase A [PldA], TonB dependent vitamin B12 transporter [BtuB], and cytolethal distending toxin subunit B [CdtB]) suitable for the development of a vaccine. Furthermore, the expression of predicted genes during host-pathogen interaction was analyzed by an infection study using an avian macrophage-like immortalized cell line (HD11). The HD11 was infected with C. jejuni strains, and the RT-qPCR assay was performed to determine the expression of the predicted genes. The expression difference was analyzed using ΔΔCt methods. The results indicate that all 3 predicted genes, PldA, BtuB, and CdtB, were upregulated in 4 tested C. jejuni strains irrespective of their sources of isolation. In conclusion, in silico prediction and gene expression analysis during host-pathogen interactions identified 3 potential vaccine candidates for C. jejuni.

Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the gold-standard technique for detecting and quantifying messenger RNA. However, without proper validation, the method may produce artefactual and non-reproducible cycle threshold values generating poor-quality data. The newer droplet digital PCR (ddPCR) method allows for the absolute quantification of targeted nucleic acids providing more sensitive and accurate measurements without requiring external standards. This study compared these two PCR-based methods to measure the expression of well-documented genes used in ecotoxicology studies. We exposed Mediterranean mussels (Mytilus galloprovincialis) to copper and analyzed gene expression in gills and digestive glands using RT-qPCR and ddPCR assays. A step-by-step methodology to optimize and compare the two technologies is described. After ten-fold serial complementary DNA dilution, both RT-qPCR and ddPCR exhibited comparable linearity and efficiency and produced statistically similar results. We conclude that ddPCR is a suitable method to assess gene expression in an ecotoxicological context. However, RT-qPCR has a shorter processing time and remains more cost-effective.

Effects of fatty acid-ethanol amine (FA-EA) derivatives on lipid accumulation and inflammation.

This study aimed to investigate the effect of fatty acid-ethanol amine (FA-EA) derivatives (L1-L10) on the mitigation of intracellular lipid accumulation and downregulation of pro-inflammatory cytokines in vitro. First, the series of FA-EA derivatives were synthesized and characterized. Then, their cytotoxic, intracellular lipid accumulation and inhibition of pro-inflammatory cytokines were evaluated. The oil red O staining experiment showed that the tested compounds L4, L6, L8, L9, and L10 could reduce intracellular lipid accumulation induced by palmitic acid (PA). Moreover, ω-3/ω-6 PUFA-EA derivatives showed inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. ω-3/ω-6 PUFA-EA derivatives at a concentrations of 10 µM could significantly decrease mRNA levels of IL-6, IL-1ß, and TNF-α, inhibit NO production, and alleviate the protein expression of IL-1ß in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These data suggest that ω-3 PUFA-EA derivatives can be beneficial for further pharmaceutical development to treat chronic low-grade inflammation diseases such as obesity.

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata.

Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai-Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

Molecular Mechanism of the Role of Apigenin in the Treatment of Hyperlipidemia: A Network Pharmacology Approach.

The therapeutic effect of apigenin (APG) on hyperlipidemia was investigated using network pharmacology combined with molecular docking strategy, and the potential targets of APG in the treatment of hyperlipidemia were explored. Genetic Ontology Biological Process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment analysis of common targets were performed. Then, molecular docking was used to predict the binding mode of APG to the target. Finally, Sprague Dawley rats were used to establish a hyperlipidemia model. The expression levels of insulin (INS) and vascular endothelial growth factor A (VEGFA) mRNA in each group were detected by quantitative reverse transcription-polymerase chain reaction. Network pharmacological studies revealed that the role of APG in the treatment of hyperlipidemia was through the regulation of INS, VEGFA, tumor necrosis factor, epidermal growth factor receptor, matrix metalloprotein 9, and other targets, as well as through the regulation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway, fluid shear stress, and atherosclerosis signaling pathways, vascular permeability; APG also participated in the regulation of glucose metabolism and lipid metabolism, and acted on vascular endothelial cells, and regulated vascular tone. Molecular docking showed that APG binds to the target with good efficiency. Experiments showed that after APG treatment, the expression levels of INS and VEGFA mRNA in the model group were significantly decreased (p<0.01). In conclusion, APG has multiple targets and affects pathways involved in the treatment of hyperlipidemia by regulating the HIF-1 signaling pathway, fluid shear stress, and the atherosclerosis pathway.

Systematic Identification and Validation of Suitable Reference Genes for the Normalization of Gene Expression in Prunella vulgaris under Different Organs and Spike Development Stages.

The quantitative real-time PCR (qRT-PCR) is an efficient and sensitive method for determining gene expression levels, but the accuracy of the results substantially depends on the stability of the reference gene (RG). Therefore, choosing an appropriate reference gene is a critical step in normalizing qRT-PCR data. Prunella vulgaris L. is a traditional Chinese medicine herb widely used in China. Its main medicinal part is the fruiting spike which is termed Spica Prunellae. However, thus far, few studies have been conducted on the mechanism of Spica Prunellae development. Meanwhile, no reliable RGs have been reported in P. vulgaris. The expression levels of 14 candidate RGs were analyzed in this study in various organs and at different stages of Spica Prunellae development. Four statistical algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) were utilized to identify the RGs' stability, and an integrated stability rating was generated via the RefFinder website online. The final ranking results revealed that eIF-2 was the most stable RG, whereas VAB2 was the least suitable as an RG. Furthermore, eIF-2 + Histon3.3 was identified as the best RG combination in different periods and the total samples. Finally, the expressions of the PvTAT and Pv4CL2 genes related to the regulation of rosmarinic acid synthesis in different organs were used to verify the stable and unstable RGs. The stable RGs in P. vulgaris were originally identified and verified in this work. This achievement provides strong support for obtaining a reliable qPCR analysis and lays the foundation for in-depth research on the developmental mechanism of Spica Prunellae.

Genome-wide identification and analysis of bZIP gene family reveal their roles during development and drought stress in Wheel Wingnut (Cyclocarya paliurus).

BACKGROUND: The bZIP gene family has important roles in various biological processes, including development and stress responses. However, little information about this gene family is available for Wheel Wingnut (Cyclocarya paliurus).  RESULTS: In this study, we identified 58 bZIP genes in the C. paliurus genome and analyzed phylogenetic relationships, chromosomal locations, gene structure, collinearity, and gene expression profiles. The 58 bZIP genes could be divided into 11 groups and were unevenly distributed among 16 C. paliurus chromosomes. An analysis of cis-regulatory elements indicated that bZIP promoters were associated with phytohormones and stress responses. The expression patterns of bZIP genes in leaves differed among developmental stages. In addition, several bZIP members were differentially expressed under drought stress. These expression patterns were verified by RT-qPCR. CONCLUSIONS: Our results provide insights into the evolutionary history of the bZIP gene family in C. paliurus and the function of these genes during leaf development and in the response to drought stress. In addition to basic genomic information, our results provide a theoretical basis for further studies aimed at improving growth and stress resistance in C. paliurus, an important medicinal plant.

Molecular Cloning and Functional Analysis of IrUGT86A1-like Gene in Medicinal Plant Isodon rubescens (Hemsl.) Hara.

The synthesis of secondary metabolites in plants often includes glycosylation modifications. Often, the final step of constructing plant secondary metabolites is completed by glycosylation transferases, which are also involved in many cell processes. In this study, a UDP-glycosyltransferase gene (UGT) was amplified from Isodon rubescens (Hemsl.) Hara with RT-PCR and named IrUGT86A1-like (GenBank: MZ913258). Here, we found that IrUGT86A1-like gene is 1450 bp in length and encodes for 479 amino acids. Bioinformatics analysis revealed that IrUGT86A1-like is a stable and hydrophilic protein, located in the cytoplasm with a transmembrane domain. Phylogenetic analysis showed that IrUGT86A1-like protein has the closest genetic relationship with the UDP-glycosyltransferase 86A1-like protein (XP_042054241.1) of Salvia splendens. RT-qPCR analysis demonstrated that the expression of IrUGT86A1-like gene varied in different tissues; leaves had the highest expression followed by flowers, stems, and roots had the lowest expression. This expression trend is similar to the distribution of oridonin content in different tissues of I. rubescens. Additionally, IrUGT86A1-like gene was found to be positively enhanced by NaCl and MeJA treatment, and in contrast was down-regulated by ABA treatment. Finally, the prokaryotic expression vector pEASY®-Blunt E1-IrUGT86A1 was successfully used to express about 53 KD of IrUGT86A1-like protein. This research builds a foundation for further investigation on the function of this gene in the synthesis and modification of secondary metabolites.

Moringa seed-supplemented diets modulate ACE activity but not its gene expression in L-NAME-induced hypertensive rats.

INTRODUCTION: We investigated the effect of dietary inclusions of Moringa seed (5% and 10%) on blood pressure, angiotensin-1 converting enzyme (ACE) activity, and gene expression, as well as redox status in hypertensive rats. MATERIAL AND METHODS: Wistar strain albino rats were fed moringa seed-based diets for two weeks prior L-NAME (40 mg/kg/day, p.o.) administration for another ten days. Subsequently, the blood pressure was monitored. Furthermore, the kidney homogenates were assayed for ACE activity and gene expression, as well as oxidative stress markers. RESULTS: The increased (systolic = 297 ± 59.30 mmHg; diastolic= 242 ± 51.96 mmHg) blood pressure, arginase activity, and reduced nitric oxide level were significantly ameliorated in hypertensive rats treated with the seed. However, the elevated ACE activity was significantly reduced but not the upregulated ACE1 gene. Also, the reduced antioxidant enzyme activities were ameliorated with a significant downregulation in their regulator-Nrf2. Rutin (4.07 ± 0.02 mg/g) and quercitrin (4.06 ± 0.01 mg/g), among others, were found in the seed. DISCUSSION: This study suggests that moringa seed offers its antihypertensive properties by acting as an ACE inhibitor but not its gene modulator, and also modulates the antioxidant system through interaction with Nrf2. CONCLUSION: Moringa seed could act as an ACE inhibitor and not its gene modulator.

Identification of novel associations of candidate genes with resistance to Rhizoctonia solani AG-3PT in Solanum tuberosum stem canker.

To develop an understanding mechanism to define responding of potatoes to R. solani, we analyzed the expression of ten novel candidate gene-markers using reverse-transcription-quantitative PCR (RT-qPCR) in resistant 'Savalan' and partially resistant 'Agria' in contrast to susceptible 'Sagita', and partially susceptible 'Pashandi'. In addition, oxidant-enzymatic-activity of catalase and superoxide-dismutase, as well as biomass-growth-parameters; shoot and root length, fresh and dry weight, and root volume were considered as complementary factors to the involving mechanism accordingly. Gene-markers up-regulated maximum up to 3.5-fold with the highest correlation, r = 0.939** following R. solani-inoculation, predominantly in resistant genotypes. Surprisingly, WRKY8-gene, basically resistant to late-blight-Phytophtora infestans was also up-regulated to 2.3-fold in resistant 'Savalan' followed by 'Agria'. Similar results with 3.1-fold were obtained on Osmotin-gene resistant to early-blight-Alternaria alternata. Enzymatic-activity of catalase with 1.6-fold and superoxide-dismutase, 6.8-fold also showed the highest level of activity in resistant genotypes, and had a high significant correlation, r = 773** and r = 0.881** with expression levels of related gene-markers respectively. Similarly, there were significant differences in biomass-growth-parameters, but with reductions in partially susceptible 'Sagita' and susceptible 'Pashandi'. Conclusively, S. tuberosum-R. solani interaction revealed that certain gene-markers can cover resistance to more than one disease simultaneously.

[Anti-tumor and analgesic activity evaluation and mechanism of Compound Kushen Injection].

This study aims to evaluate the anti-tumor and analgesic activities of Compound Kushen Injection(CKI) based on zebrafish model in vivo and investigate the anti-tumor mechanism. To be specific, zebrafish tumor xenotransplantation model was established by microinjection of murine LPC H12 cells into yolk sac. Then the high-dose CKI(H-CKI), medium-dose CKI(M-CKI), low-dose CKI(L-CKI) groups, and the model group were set. The anti-tumor activity of CKI was evaluated with the tumor area growth fold and integral absorbance(IA) growth fold 72 h after administration. The peripheral pain and central pain in zebrafish were respectively induced with acetic acid(AA) and phorbol myristate acetate(PMA). Zebralab ViewPoint system was employed to monitor behavioral trajectory of zebrafish, and movement times, movement time, movement distance, and movement velocity were used to evaluate the analgesic activity of CKI. Finally, real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression levels of apoptosis-related B lymphocyte tumor-2(Bcl-2) and phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(Akt or PKB) pathway-related genes, for the verification of the anti-tumor mechanism. Compared with the model group, M-CKI and H-CKI significantly reduced the growth folds of tumor area and IA, relief the peripheral pain and central pain. The mechanism was that CKI can up-regulate the expression of cysteine aspartic acid specific protease-3(caspase-3, Casp3) and caspase-9(Casp9), down-regulate the expression of phosphoinositide 3-kinase(PI3 K) and Akt, and significantly reduce the expression of Bcl-2, hypoxia-inducible factor-1α(HIF-1α), and vascular endothelial growth factor(VEGF). In conclusion, CKI has significant inhibitory effect on tumor growth and pain, which is related to the PI3 K/Akt signaling pathway. The pathway mediates cell apoptosis, suppresses tumor growth, and alleviates tumor pain.

Comparative transcriptome and tissue-specific expression analysis of genes reveal tissue-cultured plants as an alternative source for phenylethanoids and phenylpropanoids in Rhodiola imbricata (Edgew.).

Rhodiola imbricata (Crassulaceae) is a traditional trans-Himalayan endangered medicinal herb with immense therapeutic applications. Over the years, over-exploitation, un-managed harvesting, and lack of captive cultivation procedures persuaded threat to its wild habitat. Plant tissue culture and RNA-Seq-based molecular bioprospection of key regulatory genes aid the understanding of molecular dynamics involved in specialized metabolites (phenylethanoids and phenylpropanoids) biosynthesis and its sustainable production. Hence, comparative transcriptomic analysis was performed using leaf and root tissues from the wild and tissue-cultured plants, revealing tissue-specific production of salidroside and rosavin. The transcriptome profiling resulted in 345 million high-quality reads yielding 92,380 unique transcripts with an N50 of 1260 bp. Tissue-specific gene expression analysis revealed that both phenylethanoids and phenylpropanoids biosynthesis are predominantly associated with the shikimate pathway. In addition to RNA-Seq data, the downstream biosynthesis pathways genes viz., phospho-2-dehydro-3-deoxyheptonate aldolase (DAHPS), 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), chorismate mutase (CM), arogenate dehydrogenase (TYRAAT), aromatic-L-amino-acid decarboxylase (TDC), phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4-CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) showed higher expression pattern in wild plant tissues compared to tissue-cultured plants. The transcript fold expression determined by RT-qPCR results followed similar patterns as those observed in RNA-seq and targeted metabolite profiling data. Salidroside and rosavin content in wild plants exhibited 2.40 fold and 1.77 fold increase accumulation compared to the tissue-cultured plant. The present investigation explained the tissue and condition-specific significant differences between the expression of proposed biosynthetic pathway genes and salidroside and rosavin content. Additionally, NAC, bHLH, and ARF were the most abundant transcription factor families found in the transcriptomic analysis of R. imbricata. The generated transcriptome dataset provides a valuable gene(s)/transcription factors hub that can be used for the sustainable production of salidroside and rosavin in R. imbricata under tissue culture conditions.