Combined transcriptome sequencing and prokaryotic expression to investigate the key enzyme in the 2-C-methylerythritol-4-phosphate pathway of Osmanthus fragrans

Terpenoids are one of the main components of plant aromas. In the present study we investigated these compounds in Osmanthus fragrans Lour., which is a fragrant plant widely used for the production of essential oils. Quantitative real-time PCR (qRT-PCR) results of enzymes associated with the 2-C-methylerythritol-4-phosphate pathway confirmed that the TPS is a key enzyme for terpenoid synthesis in O. fragrans. In a series of experiments, we identified the TPS candidate genes in O. fragrans and revealed the underlying catalytic activity and subcellular localisation of the encoded proteins. Because there is no available O. fragrans reference genome, we sequenced and analysed its transcriptome and identified two putative TPS genes, OfTPS1 and OfTPS2. According to qRT-PCR analysis, both genes were most highly expressed at the full-bloom stage, suggesting that OfTPS1 and OfTPS2 contribute to O. fragrans terpenoid synthesis. To verify this hypothesis, we constructed prokaryotic expression vectors to obtain protein. In order to study the function of OfTPS1 and OfTPS2 in the synthesis of monoterpenes, the obtained proteins were reacted with geranyl pyrophosphate. As a result, two kinds of monoterpenes, (E)-β-ocimene and linalool, were detected from reaction products, respectively. In conclusion, OfTPS1 and OfTPS2 are both monoterpene synthases.

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ITRAQ-based quantitative proteomic analysis of Fusarium moniliforme (Fusarium verticillioides) in response to Phloridzin inducers

Proteome Science ◽

10.1186/s12953-021-00170-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Rong Zhang ◽

Weitao Jiang ◽

Xin Liu ◽

Yanan Duan ◽

Li Xiang ◽

...

Keyword(s):

Fusarium Moniliforme ◽

Abiotic Factors ◽

Fusarium Verticillioides ◽

Protein Profile ◽

Sugar Metabolism ◽

Differentially Expressed ◽

Pcr Analysis ◽

Differentially Expressed Proteins ◽

Apple Replant Disease ◽

Qrt Pcr

Abstract Background Apple replant disease (ARD) has been reported from all major fruit-growing regions of the world, and is often caused by biotic factors (pathogen fungi) and abiotic factors (phenolic compounds). In order to clarify the proteomic differences of Fusarium moniliforme under the action of phloridzin, and to explore the potential mechanism of F. moniliforme as the pathogen of ARD, the role of Fusarium spp. in ARD was further clarified. Methods In this paper, the quantitative proteomics method iTRAQ analysis technology was used to analyze the proteomic differences of F. moniliforme before and after phloridzin treatment. The differentially expressed protein was validated by qRT-PCR analysis. Results A total of 4535 proteins were detected, and 293 proteins were found with more than 1.2 times (P< 0.05) differences. In-depth data analysis revealed that 59 proteins were found with more than 1.5 times (P< 0.05) differences, and most proteins were consistent with the result of qRT-PCR. Differentially expressed proteins were influenced a variety of cellular processes, particularly metabolic processes. Among these metabolic pathways, a total of 8 significantly enriched KEGG pathways were identified with at least 2 affiliated proteins with different abundance in conidia and mycelium. Functional pathway analysis indicated that up-regulated proteins were mainly distributed in amino sugar, nucleotide sugar metabolism, glycolysis/ gluconeogenesis and phagosome pathways. Conclusions This study is the first to perform quantitative proteomic investigation by iTRAQ labeling and LC-MS/MS to identify differentially expressed proteins in F. moniliforme under phloridzin conditions. The results confirmed that F. moniliforme presented a unique protein profile that indicated the adaptive mechanisms of this species to phloridzin environments. The results deepened our understanding of the proteome in F. moniliforme in response to phloridzin inducers and provide a basis for further exploration for improving the efficiency of the fungi as biocontrol agents to control ARD.

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Lactobacillus reuteri BM53-1 Produces a Compound That Inhibits Sticky Glucan Synthesis by Streptococcus mutans

Microorganisms ◽

10.3390/microorganisms9071390 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1390

Author(s):

Masafumi Noda ◽

Naho Sugihara ◽

Yoshimi Sugimoto ◽

Ikue Hayashi ◽

Sachiko Sugimoto ◽

...

Keyword(s):

Antibacterial Activity ◽

Lactic Acid ◽

Dental Caries ◽

Lactobacillus Reuteri ◽

Early Stage ◽

Pcr Analysis ◽

Cariogenic Bacteria ◽

Qrt Pcr ◽

Actinidia Polygama ◽

Glucan Synthesis

Cariogenic bacteria, such as Streptococcus (S.) mutans and S. sobrinus, produce insoluble and sticky glucans as a biofilm material. The present study demonstrates that a lactic acid bacterium (LAB) named BM53-1 produces a substance that inhibits the sticky glucan synthesis. The BM53-1 strain was isolated from a flower of Actinidia polygama and identified as Lactobacillus reuteri. The substance that inhibits sticky glucan synthesis does not exhibit antibacterial activity against S. mutans. The cariogenic S. mutans produces glucans under the control of three glucosyltransferase (GTF) enzymes, named GtfB, GtfC, and GtfD. Although GtfB and GtfC produce insoluble glucans, GtfD forms soluble glucans. Through quantitative reverse-transcriptional (qRT)-PCR analysis, it was revealed that the BM53-1-derived glucan-production inhibitor (GI) enhances the transcriptions of gtfB and gtfC genes 2- to 7-fold at the early stage of cultivation. However, that of gtfD was not enhanced in the presence of the GI, indicating that the glucan stickiness produced by S. mutans was significantly weaker in the presence of the GI. Our result demonstrates that Lb. reuteri BM53-1 is useful to prevent dental caries.

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Identification of miRNAs and Their Response to Cold Stress in Astragalus Membranaceus

Biomolecules ◽

10.3390/biom9050182 ◽

2019 ◽

Vol 9 (5) ◽

pp. 182 ◽

Cited By ~ 6

Author(s):

Merhaba Abla ◽

Huigai Sun ◽

Zhuyun Li ◽

Chunxiang Wei ◽

Fei Gao ◽

...

Keyword(s):

Cold Stress ◽

Redox Homeostasis ◽

Astragalus Membranaceus ◽

Pcr Analysis ◽

Small Rna Sequencing ◽

Factors Affecting ◽

Gene Regulation Network ◽

Qrt Pcr ◽

Yield And Quality ◽

Regulation Network

Astragalus membranaceus is an important medicinal plant widely cultivated in East Asia. MicroRNAs (miRNAs) are endogenous regulatory molecules that play essential roles in plant growth, development, and the response to environmental stresses. Cold is one of the key environmental factors affecting the yield and quality of A. membranaceus, and miRNAs may mediate the gene regulation network under cold stress in A. membranaceus. To identify miRNAs and reveal their functions in cold stress response in A. membranaceus, small RNA sequencing was conducted followed by bioinformatics analysis, and quantitative real time PCR (qRT-PCR) analysis was performed to profile the expression of miRNAs under cold stress. A total of 168 conserved miRNAs belonging to 34 families and 14 putative non-conserved miRNAs were identified. Many miRNA targets were predicted and these targets were involved in diversified regulatory and metabolic pathways. By using qRT-PCR, 27 miRNAs were found to be responsive to cold stress, including 4 cold stress-induced and 17 cold-repressed conserved miRNAs, and 6 cold-induced non-conserved miRNAs. These cold-responsive miRNAs probably mediate the response to cold stress by regulating development, hormone signaling, defense, redox homeostasis, and secondary metabolism in A. membranaceus. These cold-corresponsive miRNAs may be used as the candidate genes in further molecular breeding for improving cold tolerance of A. membranaceus.

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Small Non-Coding RNAome of Ageing Chondrocytes

International Journal of Molecular Sciences ◽

10.3390/ijms21165675 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5675

Author(s):

Panagiotis Balaskas ◽

Jonathan A. Green ◽

Tariq M. Haqqi ◽

Philip Dyer ◽

Yalda A. Kharaz ◽

...

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Differential Expression Analysis ◽

Cartilage Tissue ◽

Pcr Analysis ◽

Small Rna Sequencing ◽

High Grade ◽

Human Cartilage ◽

Fragment Analysis ◽

Qrt Pcr ◽

Age Related

Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.

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The Reactions of Substituted Germyl- and Silylhydrazines with BX3 and CY2 Acceptor Species

Canadian Journal of Chemistry ◽

10.1139/v72-086 ◽

1972 ◽

Vol 50 (4) ◽

pp. 562-566 ◽

Cited By ~ 6

Author(s):

L. K. Peterson ◽

K. I. Thé

Keyword(s):

Molecular Weight ◽

Nitrogen Atom ◽

Low Temperatures ◽

Adduct Formation ◽

Steric Effects ◽

Reaction Products ◽

Hydrazine Derivatives ◽

Basic Catalysts ◽

Series Of Experiments ◽

Electronic And Steric Effects

Boron trimethyl reacts with each of the organometalloid-hydrazine derivatives Me2NN(R)GeMe3 (R = H, Me, SiMe3) except Me2NN(SiMe3)GeMe3, and with Me2NNHSiMe3, forming 1:1 adducts. With Me2NN(SiMe3)GeMe3 and BMe3, no adduct was formed even at low temperatures. On the basis of p.m.r. spectra, the metalloid-substituted nitrogen atom rather than the terminal nitrogen, is indicated as the basic center at which adduct formation occurs. With BF3, the Si—N and Ge—N bonds of the hydrazine derivatives were cleaved, again indicating attack at the central nitrogen atom. In a series of experiments involving CY2 (Y = O, S) and Me2NN(R)MMe3 (R = H, Me, SiMe3; M = Si, Ge), reaction occurred readily for R = H, but less readily for R = Me and for the compound Me2NN(GeMe3)2, while no reaction was observed for the compounds Me2NN(SiMe3)2 and Me2NN(SiMe3)GeMe3, even when heat or basic catalysts were employed. Reaction products, carbamate and thiocarbamate derivatives, Me2NN(R)C(Y)YMMe3, were characterized on the basis molecular weight, i.r., p.m.r., and mass spectroscopy, and m.p. data. Ease of formation is discussed in terms of electronic and steric effects.

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MiR-107 inhibits proliferation of lung cancer cells through regulating TP53 regulated inhibitor of apoptosis 1 (TRIAP1)

Open Life Sciences ◽

10.1515/biol-2017-0023 ◽

2017 ◽

Vol 12 (1) ◽

pp. 200-205 ◽

Cited By ~ 2

Author(s):

Bing Wang ◽

Zhanjie Zuo ◽

Fang Lv ◽

Liang Zhao ◽

Minjun Du ◽

...

Keyword(s):

Lung Cancer ◽

Cell Proliferation ◽

Western Blot ◽

Cancer Cells ◽

A549 Cells ◽

Lung Cancer Cells ◽

Pcr Analysis ◽

Aberrant Expression ◽

Small Interference Rna ◽

Qrt Pcr

AbstractAimsAccumulating evidence indicates that aberrant expression of miR-107 plays a crucial role in cancers. This study aims to display the function of miR-107 and its novel target genes in the progression of lung cancer.Methods and MaterialMiR-107 or miR-107 inhibitor was transfected into lung cancer cells A549. The levels of miR-107 and TP53 regulated inhibition of apoptosis 1 (TRIAP1) were examined by quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis and Western Blot. Functionally, MTT and colony formation assays were carried out to test the effect of miR-107 inhibitor and/or small interference RNA (siRNA) targeting TRIAP1 mRNA on proliferation of lung cancer cells. Levels of miR-107 or TRIAP1 were detected in clinical lung cancer samples by using qRT-PCR analysis.ResultsQRT-PCR analysis revealed that miR-107 inhibitor or miR-107 was successfully transfected into A549 cells. Western Blot indicated that miR-107 decreased the expression of TRIAP1 protein in the cells. In contrast, miR-107 inhibitor augmented the levels of TRIAP1 protein. Functionally, miR-107 inhibitor remarkably suppressed A549 cell proliferation, whereas, TRIAP1 siRNAs could abrogate the miR-107 inhibitor-induced proliferation of cells. Then, we validated that TRIAP1 was increased in clinical lung cancer samples. MiR-107 expression was negatively related to TRIAP1 expression in clinical lung cancer samples.ConclusionsMiR-107 suppresses cell proliferation by targeting TRIAP1 in lung cancer. Our finding allows new insights into the mechanisms of lung cancer that is mediated by miR-107.

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Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in Amblyomma maculatum (Acari: Ixodidae)

Journal of Medical Entomology ◽

10.1603/me12123 ◽

2012 ◽

Vol 49 (6) ◽

pp. 1339-1346 ◽

Cited By ~ 15

Author(s):

Rebecca Browning ◽

Steven Adamson ◽

Shahid Karim

Keyword(s):

Reference Genes ◽

Stable Set ◽

Pcr Analysis ◽

Amblyomma Maculatum ◽

Qrt Pcr

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Circular RNA Expression Profiling Identifies Prostate Cancer- Specific circRNAs in Prostate Cancer

Cellular Physiology and Biochemistry ◽

10.1159/000494870 ◽

2018 ◽

Vol 50 (5) ◽

pp. 1903-1915 ◽

Cited By ~ 18

Author(s):

Qianlin Xia ◽

Tao Ding ◽

Guihong Zhang ◽

Zehuan Li ◽

Ling Zeng ◽

...

Keyword(s):

Prostate Cancer ◽

Polymerase Chain Reaction Analysis ◽

Circular Rna ◽

Differentially Expressed ◽

Pcr Analysis ◽

High Morbidity ◽

Qrt Pcr ◽

Diagnosis And Prognosis ◽

Novel Biomarkers ◽

Rna Expression Profiling

Background/Aims: Prostate cancer (PCa) is one of the main cancers that damage males’ health severely with high morbidity and mortality, but there is still no ideal molecular marker for the diagnosis and prognosis of prostate cancer. Methods: To determine whether the differentially expressed circRNAs in prostate cancer can serve as novel biomarkers for prostate cancer diagnosis, we screened differentially expressed circRNAs using SBC-ceRNA array in 4 pairs of prostate tumor and paracancerous tissues. A circRNA-miRNA-mRNA regulatory network for the differential circRNAs and their host genes was constructed by Cytoscape3.5.1 software. Quantitative real-time polymerase chain reaction analysis (qRT-PCR) was performed to confirm the microarray data. Results: We found 1021 differentially expressed circRNAs in PCa tumor using SBC-ceRNA array and confirmed the expression of circ_0057558, circ_0062019 and SLC19A1 in PCa cell lines and tumor tissues through qRT-PCR analysis. We demonstrated that combination of PSA level and two differentially expressed circRNAs showed significantly increased AUC, sensitivity and specificity (0.938, 84.5% and 90.9%, respectively) than PSA alone (AUC of serum PSA was 0.854). Moreover, circ_0057558 was correlated positively with total cholesterol. The functional network of circRNA-miRNA-mRNA analysis showed that circ_0057558 and circ_0034467 regulated miR-6884, and circ_0062019 and circ_0060325 regulated miR-5008. Conclusion: Our results demonstrated that differentially expressed circRNAs (circ_0062019 and circ_0057558) and host gene SLC19A1 of circ_0062019 could be used as potential novel biomarkers for prostate cancer.

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COEXPRESSIONFINDER: A NEW ALGORITHM FOR FINDING GROUPS OF COEXPRESSED GENES

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720006002193 ◽

2006 ◽

Vol 04 (04) ◽

pp. 853-864

Author(s):

SERGEY I. ROGOV ◽

KUVAT T. MOMYNALIEV ◽

VADIM M. GOVORUN

Keyword(s):

Gene Expression ◽

Hierarchical Clustering ◽

Parallel Execution ◽

Expression Vectors ◽

Dna Arrays ◽

Java Application ◽

Coordinated Expression ◽

Series Of Experiments ◽

Coexpressed Genes

Results: A new algorithm is developed which is intended to find groups of genes whose expression values change in a concordant manner in a series of experiments with DNA arrays. This algorithm is named as CoexpressionFinder. It can find more complete and internally coordinated groups of gene expression vectors than hierarchical clustering. Also, it finds more genes having coordinated expression. The algorithm's design allows parallel execution. Availability: The algorithm is implemented as a Java application which is freely available at: and .

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Cloning and function analysis of BAG family genes in wheat

Functional Plant Biology ◽

10.1071/fp15317 ◽

2016 ◽

Vol 43 (5) ◽

pp. 393

Author(s):

Shiming Ge ◽

Zhen Kang ◽

Ying Li ◽

Fuzhen Zhang ◽

Yinzhu Shen ◽

...

Keyword(s):

Heat Stress ◽

Salinity Stress ◽

Heat Tolerance ◽

Expressed Sequence Tag ◽

Function Analysis ◽

Pcr Analysis ◽

Subcellular Localisation ◽

Hybrid Technique ◽

Rt Pcr ◽

Conserved Domain

By analysing the cDNA microarray of the salt tolerant mutant of wheat RH8706–49 under salinity stress, our results showed an expressed sequence tag fragment and acquired an unknown gene (designated as TaBAG) with a BAG conserved domain through electronic cloning and RT–PCR technology. The gene was registered into GenBank (No. FJ599765). After homologous alignment analysis, electronic cloning, and amplifying with RT–PCR, the other gene with a BAG conserved domain, TaBAG2, was obtained and registered into GenBank (No. GU471210). Quantitative PCR analysis demonstrated that TaBAG2 expression was induced by saline and heat stress. TaBAG gene expression under salinity stress increased remarkably but showed an insignificant response to heat stress. The adversity stress detection results showed that Arabidopsis overexpressing TaBAG and TaBAG2 exhibited an obvious salt tolerance increase. Under heat stress, Arabidopsis overexpressing TaBAG2 showed increased heat tolerance; however, the heat tolerance of Arabidopsis overexpressing TaBAG did not vary significantly under heat stress. Subcellular localisation results demonstrated that TaBAGs were mainly located in the cytoplasm and the cell nucleus. We applied fluorescence complementation and yeast two-hybrid technique to prove that TaBAG2 can obviously bond with TaHsp70 and TaCaMs. After the respective mutation of aspartic acid (D) and arginine (R) at high conservation in BAG domain of TaBAG2, the bonding interaction between TaBAG2 and TaHsp70 disappeared, indicating that the two amino acids were the key loci for the interaction between TaBAG2 and TaHsp70. Heat tolerance detection results demonstrated that the heat tolerance of Arabidopsis overexpressing and cotransfected with TaBAG2 and TaHsp70 was much higher than that of Arabidopsis overexpressing TaBAG2 and Arabidopsis overexpressing TaHSP70. This finding implies that the synergistic use of TaBAG2 and TaHSP70 can improve heat tolerance of plants.

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