Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii / 中国中药杂志

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Role of PfGCN5 in nutrient sensing and transcriptional regulation in Plasmodium falciparum

Malaria is a deadly, infectious disease caused by the parasite Plasmodium, leading to millions of deathsworldwide. Plasmodium requires a coordinated pattern of sequential gene expression for surviving in bothinvertebrate and vertebrate host environments. As parasites largely depend on host resources, they also developefficient mechanisms to sense and adapt to variable nutrient conditions in the environment and modulate theirvirulence. Earlier we have shown that PfGCN5, a histone acetyltransferase, binds to the stress-responsive andvirulence-related genes in a poised state and regulates their expression under temperature and artemisinintreatment conditions in P. falciparum. In this study, we show upregulation of PfGCN5 upon nutrient stresscondition. With the help of chromatin immunoprecipitation coupled high-throughput sequencing (ChIP-seq)and transcriptomic (RNA-sequencing) analyses, we show that PfGCN5 is associated with the genes that areimportant for the maintenance of parasite cellular homeostasis upon nutrient stress condition. Furthermore, weidentified various metabolic enzymes as interacting partners of PfGCN5 by immunoprecipitation coupled withmass spectroscopy, possibly acting as a sensor of nutrient conditions in the environment. We also demonstratedthat PfGCN5 interacts and acetylates PfGAPDH in vitro. Collectively, our data provides important insights intotranscriptional deregulation upon nutrient stress condition and elucidate the role of PfGCN5 during nutrientstress condition.

Establishment of Thioltransferase knockout mouse model and the function of thioltransferase in cataractogenesis / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To examine the morphological and biochemical alterations in the eyes of Thioltransferase knockout(TTase KO)mouse model as a function of age, and to explore the important function in redox homeostasis in the lens and in the age-related cataractogenesis.<p>METHODS: TTase KO model was established in this laboratory. TTase KO and WT mice were examined and the lens opacity was classified by using a slit lamp. Each lens was homogenized in lysis buffer and processed for measurement of glutathione(GSH)level. Examination of Protein-GSH mixed disulfides(PSSG)formation in the lens by Western blot analysis. Immunoprecipitation was used to identify the proteins formed PSSG. Dethiolation of lens proteins was carried out using purified recombinant human lens TTase(RHLT). <p>RESULTS: The slit lamp examination showed an age-dependent nuclear cataract development in both eyes of the WT and TTase KO mice. The onset of cataract was 4mo in the KO mice and 9mo in the WT mice. The GSH loss showed in both groups during aging and was prominent in the TTase KO mice after 9mo old. PSSG in the lenses of both groups showed progressive elevation, whereas the lenses of the KO group had a higher level of PSSG after 9mo. These GSH-conjugated proteins were confirmed as actin and glyceraldehyes 3-phosphate dehydrogenase(GAPDH)by immunoprecipitation and they could be eliminated when the homogenates were treated with RHLT. <p>CONCLUSION: The results showed that deletion of TTase gene in the mouse could lead to an early age-dependent cataract formation and the PSSG formation in these lenses appeared to link directly to lens opacity. The PSSG could be dethiolated by TTase. This data strengthens that TTase plays an essential role in maintaining lens clarity.

Selection of reference genes for quantitative real-time PCR in Morinda officinalis / 中草药

Objective: To select the appropriate reference genes for calibrating the quantitative real-time PCR detection of gene expression in different tissues and leaves with different treatments of Morinda officinalis. Methods: With different groups and different processing leaves of M. officinalis as materials, 10 internal genes, including GAPDH, CYP, TUA, Actin and so on, were selected as candidate genes according to the M. officinalis transcriptome data. The expression stability of internal reference genes was analyzed by using real-time fluorescence quantification technique combined with software such as geNorm, NormFinder and BestKeeper, so as to select stable reference genes in different tissues and leaves of M. officinalis with different treatments. Finally, appropriate internal reference genes were selected to analyze the relative expression levels of DXS and DXR genes in different tissues and leaves with different treatments. Results: Internal reference genes GAPDH and UBQ were the most stable in different tissues of M. officinalis, the double internal reference combination of GAPDH + UBQ can more accurately analyze the relative expression levels of target genes in different tissues of M. officinalis, while the most stable reference genes in leaves with different treatments were GAPDH and Actin; The selection of the double reference combination of GAPDH + Actin can ensure the reliability of the target gene expression results. In different tissues of M. officinalis, the relative expression of DXS target gene was in sequence of root < stem < leaf, while the relative expression of DXR was stem < root < leaf. The relative expression levels of DXS and DXR genes in leaves with different treatments were increased compared with those untreated leaves (CK). Conclusion: The selected stable internal reference genes lay a foundation for the subsequent study on the expression of related genes of M. officinalis. Using the combination of two stable internal references to homogenize the target genes is conducive to improving the accuracy of the analysis of the expression of target genes.

Screening and validation of reference genes for quantitative RT-PCR analysis in Ampelopsis grossedentata / 中草药

Objective: To screen reference genes for real time quantitative PCR (qRT-PCR) research in Ampelopsis grossedentata. Methods: On the basis of the conserved sequences among plant species, six candidate reference genes (including Actin, 18 S-rRNA, GAPDH, α-Tubulin, β-Tubulin, and UBQ) were cloned from A. grossedentata by RT-PCR in this study. The expression stability of each reference gene in different tissues (shoot tip, young leaf, mature leaf, old leaf, stem, and root) were analyzed by three softwares (GeNorm, NormFinder, and BestKeeper), followed by validation of the expression pattern of AgPAL by qRT-PCR. Results: Actin, 18 S-rRNA, and GAPDH expressed most stably in all samples and were suitable for reference genes, which were further confirmed by the transcript level analysis result of AgPAL in different tissues. Conclusion: This is the first report on the screening and validation of reference genes for qRT-PCR in A. grossedentata, which benefits future studies on gene expression in this species.

Evaluation of candidate reference genes for quantitative expression studies in Asian seabass (Lates calcarifer) during ontogenesis and in tissues of healthy and infected fishes.

Quantitative real-time PCR (qRT-PCR), used to determine the gene expression profile, is an important tool in functional genomic research, including fishes. To obtain more robust and meaningful result, the best possible normalization of the data is of utmost significance. In the present study, we have evaluated the potential of five commonly used housekeeping genes i.e., elongation factor 1-α (EF1A), β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-2-Microglobulin (B2M) in normal physiological conditions, developmental stages and in response to bacterial infection in Asian seabass, Lates calcarifer (Bloch), an important food fish cultured in the Asia-Pacific region. The expression levels of these five genes were estimated in 11 tissues of normal seabass juveniles, 14 embryonic and larval developmental stages and six tissues of Vibrio alginolyticus-challenged animals. Further, the expression stability of these genes was calculated based on three algorithms i.e. geNorm, NormFinder and BestKeeper. The results showed that although there are tissue-specific variations for each gene, ACTB and EF1A are the most stable genes across the tissues of normal animals. However, in bacteria-challenged animals, EF1A and 18S were found to be the best reference genes for data normalization. The expression of all the genes tested showed an increasing trend in developmental stages and the increase was significant at blastula stage. Among the five genes tested, EF1A and ACTB were found to be the genes with least variation and highest stability across the developmental stages. This forms the first report on validation of housekeeping genes in L. calcarifer, in the context of ontogenic development and in response to infection.

Cloning and analysis on GAPDH genomic DNA from Eleutherococcus senticosus / 中草药

Objective: To clone and analyze the full length DNA and promoter sequence of GAPDH gene from Eleutherococcus senticosus. Methods: PCR and TAIL-PCR techniques were used to clone the full length DNA sequence and promoter sequence of GAPDH gene of E. senticosus, then these two sequences were analyzed by bioinformatics methods. Results: Length of 4 103 bp of E. senticosus GAPDH gene DNA and promoter sequence was cloned. Gene structure analysis showed that it contained 12 exons and 11 introns, and the splicing principles of its exon and intron were consistent with GT-AG; The promoter sequence length was 1 304 bp, and the transcription start site located 61 bp upstream of the initiation codon ATG; The promoter elements such as TATA-box, CAAT-box, as well as many cis-regulatory elements were related to hormone signal response, light response and stress signals. Conclusion: The full length DNA and promoter sequence of GAPDH gene in E. senticosus is successfully cloned and reported for the first time, and it provides a stable foundation for further study of GAPDH gene structure and function of E. senticosus.

Cloning and sequence analyses of Eleutherococcus senticosus GAPDH / 中草药

Objective In order to make the gene a valuable internal control gene, GAPDH of Eleutherococcus senticosus was cloned and analyzed in sequence. Methods Total RNA of E. senticosus was extracted using improved isothiocyanate method. Part sequence of GAPDH was coined by RT-PCR, and the sequence was acted as internal control gene for semiquantitative PCR. Results Length 627 bp of E. senticosus GAPDH was cloned, speculating coding 209 amino acids. To compare the amino acid sequence of £. senticosus GAPDH with those of Panax notoginseng, P. ginseng, and Arabidopsis thaliana, the amino acid homology was 97%, 93%, and 93%, respectively, and the nucleotide homology was 94%, 86%, and 84%, respectively. When the sequence acted as internal control gene, the semiquantative PCR has benign amplification effect and good reproducibility. Conclusion The cDNA clone of E. senticosus GAPDH is first reported, the results prove that the sequence is able to be internal control gene for analysis on gene expression. This study could make a foundation for the key enzyme expression and regulate mechanism analysis in eleutheroside biosynthesis pathway.

Purification, Enzyme Activity and Immunology Study of Recombinant Protein Glyceraldehyde-3-phosphate Dehydrogenase of Clonorchis sinensis / 中国寄生虫学与寄生虫病杂志

Objective To produce prokaryotic recombinant protein glyceraldehyde-3-phosphate dehydrogenase of Clonorchis sinensis (CsGAPDH), analyze its enzyme activity and immunological function. Methods The recombinant CsGAPDH was purified according to the protocol of GST?Bind~TM kit and was digested with thrombin proteinase and eluted with wash buffer. The BALB/c mice were inoculated with the purified protein. The antisera collected from the mice were used to detect the titres of IgG antibodies by ELISA, and Western blotting was used to identify the specificity of the antisera with the purified CsGAPDH. S-P immunohistochemistry method was used to confirm the expression and distribution of CsGAPDH in adult Clonorchis sinensis with the polyclonal antibodies from immunized BALB/c mice. The CsGAPDH catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate (3-GAP). Results SDS-PAGE showed a single purified protein band. Gel scanning analysis revealed that the protein purity of CsGAPDH was 90%. ELISA analysis showed an increased IgG value. S-P immunohistochemistry analysis demonstrated that the recombinant plasmid pGEX-4T-1-GAPDH expressed and distributed in muscle cell membrane of immune mice. Western blotting result suggested that CsGAPDH protein contained essential epitopes with high antigenic activities. This protein CsGAPDH could catalyzed 3-GAP with enzymatic active unit of 2 872 U min~-1ml~-1. Conclusion The recombinant protein CsGAPDH shows a proper enzymatic activity and immunogenicity.