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Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.
Subject(s)
Escherichia coli/enzymology , Escherichia coli/genetics , Glycoside Hydrolases/metabolism , Transformation, Genetic , Gene Expression , Reverse Transcriptase Polymerase Chain Reaction , RNA Stability , Fermentation , Genetic Vectors , Glycoside Hydrolases/geneticsABSTRACT
Objective: To clone the geranyl pyrophosphate synthase gene from Swertia mussotii (SmGPPS), analyze the bioinformation of SmGPPS, and perform the gene expression. Methods: According to the SmGPPS gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector pET-28a-SmGPPS was constructed and transformed into Escherichia coli BL-21 (DE3) for expression under 37℃ and induced by 1 mmol/L IPTG. The relative expression of gene SmGPPS in the leaf, stem, and flower of S. mussotii was also studied. Results: The results showed that SmGPPS cDNA complete sequences had a length of 1 119 bp encoding 372 amino acid residues. And the protein secondary and tertiary structures were analyzed and forecasted. The SmGPPS protein shared high identity with other GPPS proteins of plants. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Relative RT-PCR analysis indicated that SmGPPS showed the highest transcript abundance in the leaf. Conclusion: This work will provide a foundation for further functional research of SmGPPS protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.
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Abstract The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level.
Subject(s)
Enzymes/metabolism , Freeze Drying , Lactobacillus/drug effects , Lactobacillus/radiation effects , Sodium Chloride/metabolism , Gene Expression Profiling , Glycolysis/drug effects , Glycolysis/radiation effects , Lactobacillus/enzymology , Lactobacillus/physiology , Microbial Viability/drug effects , Microbial Viability/radiation effectsABSTRACT
Objective To analyze the differences between the semi-quantitative RT-PCR and real time quantitative fluorescence RT-PCR assays for detecting XDH/XO mRNA expression in various organ tissues of rhesus monkey, and provide useful reference in methodology of experimental studies.Total RNA was extracted from the myocardium, kidney, testis, skin, and liver tissues, respectively, for detecting XDH/XO mRNA expression in rhesus monkey by semi-quantitative RT-PCR and real time quantitative fluorescence RT-PCR assays.The sensitivity and specificity of the two assays were compared with each other using the same primer sequences and reference genes.Results The expression of XDH/XO mRNA in different organ tissues were detected by both the two PCR assays.The sensitivity of quantitative fluorescence real-time RT-PCR for the XDH/XO mRNA expression in the liver tissue was 39 times higher than that by semi-quantitative RT-PCR.Conclusions Both the quantitative and semi-quantitative fluorescence RT-PCR assays can be used to detect the expression of XDH/XO mRNA in different organ tissues of rhesus monkey.The sensitivity of quantitative fluorescence real-time PCR assay is more sensitive than that of the semi-quantitative RT-PCR assay.
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The mRNA of OsGSTL1 was detected in the roots and leaves of rice plants at seedling and tillering stages, and their roots, leaves and panicles at the heading stage. The full-length open reading frame of OsGSTL1 cDNA was 732 bp and encoded a putative polypeptide of 243 amino acids with a calculated molecular mass of 27.30 kDa and a theoretical pI of 5.50. The protein sequences of OsGSTL1 exhibited typical feature of the lambda class GST, which contained the conserved domain "GST_C_Lambda" in C-terminal alpha helical domain and a highly conserved Cys42 in active center. In silico predictions showed that the OsGSTL1 protein was strongly hydrophilic. The phylogenetic analysis revealed OsGSTL1 belonged to monocots subgroup and was closer to IN2-1 of Z. may. The OsGSTL1 gene was cloned into pYTV vector and was introduced into yeast strain PEP4. Western blot analysis showed that the exogenous OsGSTL1 was expressed in the transformed yeast. The GST activity of the crude extracts of yeast showed that the OsGSTL1 transgenic yeast had higher levels of GST activities than the control yeasts. These findings suggested that the OsGSTL1 was a glutathione S-transferase and could play an important role during the growth and development processes in rice.
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Background & objectives: The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer. Methods: Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibroblasts and the cultured cells studied for the expression of ABCG2 and p63 by immunohistochemistry and Western blot. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done on the cultured cells at varying intervals of time for expression of ABCG2, p63, connexin43 (Cnx43), and keratin 3 (K3) and keratin 12 (K12). Results: The growth rates were similar in both denuded AM and denuded AM + 3T3. The cells cultured over AM + 3T3 showed the expression of p63 and ABCG2 till 21 days of incubation by immunohistochemistry and Western blot. The expression of p63 and ABCG2 were retained till 21 days of incubation on the cells cultured over denuded AM + 3T3, whereas it was expressed only till day 8 on the cells cultured over the denuded membrane by semi quantitative RT-PCR. Cnx43 and K3/K12 were observed in both the conditions. Interpretation & conclusions: The limbal epithelial cells cultured in the presence of mitomycin C treated 3T3 feeder layer were able to maintain the expression of putative stem cell markers. Further in vitro studies using feeder layer will enable us to understand the factors, which play a role in maintaining the limbal stem cell niche.
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A novel gene, which was a homologue of Arabidopsis COI1 was isolated from rice (Oryza sativa L.) by RT-PCR and designated as OsCOI1. It encoded a protein of 595 amino acids. The similar F-box motif and 16 leucine-rich repeats were found in the deduced protein OsCOI1. OsCOI1 and COI1 showed high homology (74%) at amino acid level. Semi-quantitative RT-PCR and Northern blot analysis demonstrated that the expression of OsCOI1 in rice varied obviously after treatment with MeJA and ABA but was not affected by SA and ET, suggesting that the specific function of OsCOI1 in JA signal pathway and related ABA pathway.
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Objective To study the tissue-specificity of ?-carotene-15,15’-monooxygenase(? CMOOX). Method Semi-quantitative RT-PCR and HPLC were used to study the expression of ?CMOOX mRNA and its activity in different tissues of chicken. Results ?CMOOX mRNA was expressed in tissues including heart, liver, spleen, lung, kidney, duodenum, jejunum, ileum, muscle and testis, not expressed in lung. Furthermore, it was expressed in jejunum with the highest level. ?CMOOX activity was found in every tissue, except lung, and highest in duodenum and jejunum. Conclusion The distribution of ?CMOOX in chicken has tissue specificity.
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Objective:To investigate the gene expression variety of different genotype of F-ATPase subunit uncEBF in Streptococcus mutans (S.mutans) and to evaluate the relationship among uncEBF gene expression levels, genotypes and the acidurance ability of S.mutans. Methods:The relative expression quantity of uncEBF gene against the housekeeping gene recA was determined by the two-step method of semi-quantitative RT-PCR in 18 clinical isolates of S.mutans(7 with genotype A uncEBF and 11 with genotyp B,10 with high acidurance and 8 with low). A gel documentation system and QUANTITY ONES software were used to assay the data. Results:uncEBF mRNA expression level in the isolates with genotype A uncEBF was higher than that in those with genotype B(P
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An effective RT-PCR method was developed to detect exogenous gene xylB transcript levels in Zymomonas mobilis CP4. Total RNAs without genomic DNA contamination were purified from wild-type and gene engineering strains, and were quantified to the same concentration. Then, cDNAs synthesis and PCR analysis of these samples were conducted by reverse transcription PCR. The optimal number of cycles was determined by observing amplification profile of target gene xylB and internal control gene 16s rRNA, and relative expression levels of xylB in various samples were analyzed by RT-PCR. The results indicated that the xylB transcript was not be detected in CP4, however that could be found in recombinant strains, in which xylB transcription abundance was similar. The enzyme assay furthermore confirmed that effective ex-pression of the target gene. The method provided a useful and rapid tool for detecting transcript levels of target genes from various samples of Z. mobilis.
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Objective To establish a Taqman real-time PCR assay for quantitative detection of the expression of metabolic syndrome related gene (MSRG) mRNA, and to study the function of a new gene. Methods Specific primers and probes were designed for real-time PCR according to the MSRG cDNA sequence. The plasmid standard preparations were constructed by T-A clone, and serial 10-fold dilutions of the extracted plasmid standard preparations were prepared for plotting the standard curve which was used for relative quantification of real-time PCR. The sensitivity, specificity and reproducibility of real-time PCR assay were detected. To compare with the semi-quantitative RT-PCR, the expression levels of MSRG were measured by real-time PCR and semi-quantitative RT-PCR, respectively, in HepG2 cells which were incubated with glucose in different concentrations [5.6mmol/L (G5.6), 22mmol/L (G22) and 33.3mmol/L(G33.3)]. Results An effective real-time PCR assay was established for detection of MSRG mRNA expression levels. Significant differences existed in MSRG expression in HepG2 cells between the G33.3, G22 and G5.6 groups detected by real-time PCR assay. The expression levels of MSRG in HepG2 cells increased significantly in G33.3 group, whereas no significant difference on the expression level of MRSG mRNA was found between G22 and G5.6 groups when semi-quantitative RT-PCR was used for detection. It suggested that the real-time PCR assay was more sensitive and even more precise than that of semi-quantitative RT-PCR. Conclusion The real-time PCR assay was a sensitive, specific, quantitative, and reproducible tool for studying the function of MSRG at the mRNA expression levels of gene.
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Objective To investigate the relationship between the expression of endostatin, VEGFR and chronic altiplano cardiopathy,and the mechanism of the curative effect of rhodiola.on treat altiplano cardiopathy. Methods The rat model with chronic altiplano cardiopathy was created.The experimental rats were divided into four groups: control group;smoking and running stimulated group;altiplano cardiopathy group and rhodiola treated group.The expression levels of endostatin and VEGFR were determined by the methods of immunohistochemistry and semi-quantitative RT-PCR. Results The expression levels of VEGFR in hearts and lungs of the stimulated groups were found higher than that of the control group,while endostatin level was contrary.The expression level of VEGFR in hearts and lungs of the rhodiola treated group was lower than that of the altiplano cardiopathy group,while endostatin was higher.Conclusion The pathogenesis of chronic altiplano cardiopathy seems to be closely associated with epithelial lesion.The curative effect of rhodila to this disease may be exerted by reducing the damage of epithelial cells.