N-glycanase is a class of deglycosylation enzymes, widely used in the study of N-glycosylation modification of glycoprotein. In this study, an N-glycanase gene (OsPNGase A, XM_015775832) with high GC content (69.48%) was cloned from rice and then the yeast secretory expression vector pPICZ(α)A-OsPNGase A was constructed for the purpose of transformation to Pichia pastoris. After induction in Pichia pastoris SMD1168H, the target protein was purified by DEAE Sepharose and HisTrap HP chromatography, with a yield of 12.3 mg OsPNGase A from 1 L fermentation medium, showing a specific activity of 258 U/mg. SDS-PAGE revealed that the purified OsPNGase A was a single band and showed consistentcy with the expected molecular weight. OsPNGase A could act on transferrin recombinantly expressed in rice, avidin recombinantly expressed in corn and horseradish peroxidase. Furthermore, OsPNGase A showed higher activity than commercial PNGase F towards avidin. OsPNGase A displayed the highest digestion activity at pH 6.0 and 40 °C, and was also active in the neutral and alkaline environment. Despite the fact that OsPNGase A was inhibited by reducing agents and surfactants, it still maintained partial enzymatic activity in 100 mmol/L β-ME or DTT. Therefore, the successful expression of rice OsPNGase A provides a new tool for the study of plant glycoproteins and the establishment of yeast secretion expression system lays the foundation for the preparation of PNGase A.

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.
Animals ; Codon ; Electrophoresis, Polyacrylamide Gel ; Endonucleases ; biosynthesis ; Glycoproteins ; Hot Temperature ; Penaeidae ; enzymology ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis

Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²⁺, Zn²⁺, Mn²⁺ and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.

The study is aimed to construct high quality Salvia miltiorrhiza cDNA library and obtain the SmJAZ8 gene of S. miltiorrhiza by yeast two-hybrid system. In this study， full-length cDNA was synthesized from roots， stems， leaves， flowers and hairy roots of S. miltiorrhiza. The full-length cDNA library was synthesized by SMART method and constructed with DSN homogenization technique. The results showed that the library capacity was 1.45×10⁶， the recombination rate was 100%， and the average size of the insert was 500-2 000 bp. The recombinant vector of pDEST-pGADT7-SmJAZ8 was constructed and transformed into Y2HGold strain. The interaction protein was screened by yeast two-hybrid system. The DnaJ protein and UBQ protein were screened by yeast two-hybrid system. This study has successfully constructed a full-length cDNA library of S. miltiorrhiza， and laid the foundation for the follow-up study on functional gene screening and gene function of S. miltiorrhiza.
Co-Repressor Proteins ; genetics ; DNA, Complementary ; Gene Library ; Plant Proteins ; genetics ; Salvia miltiorrhiza ; genetics ; Two-Hybrid System Techniques

Exonuclease Ⅷ (Exo Ⅷ), an ATP-independent dsDNA 5'-3' exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ (tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×10⁵ U/mg. In a 10 μL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×10⁶ cfu/μg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×10⁴ recombinant clones per μg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.
Cloning, Molecular ; Escherichia coli ; genetics ; Exonucleases ; genetics ; Recombinant Proteins ; metabolism ; Recombination, Genetic

The aim of this paper was to investigate the flavonoids of callus of transgenic and non-transgenic Saussurea involucrate and its antitumor activity on the esophageal cancer CaEs-17 cells. The species and content of mono-phenols were detected by high performance liquid chromatography. The growth of human esophageal cancer CaEs-17 cells was detected using CCK-8 assay, apoptosis morphology observation and flow cytometry. Expression of related apoptotic genes Bax and Bcl-2 were determined by qPCR. The results showed that the content of total flavonoids in the transgenic callus was 2.72 times that of the non-transgenic callus. The cyanidin-galactoside was detected in the transgenic callus, but not in the non-transgenic callus. The inhibitory effect of the extracts from the transgenic callus on CaEs-17 cells was more significant than that of the non-transgenic callus, and the IC₅₀ value had a decreased of 26.4%. Flow cytometry analysis results showed that the apoptosis induction effect of the extracts from the transgenic callus on CaEs-17 cells was significantly better than that of the non-transgenic callus. Fluorescence quantitative PCR analysis results showed that the extracts from the transgenic callus could up-regulate the expression of proapoptotic gene Bax and down-regulate the expression of apoptotic gene Bcl-2, and the regulation effect of the transgenic callus was more significant. Therefore, compared with the non-transgenic callus, the antitumor activity of the extracts from the callus of transgenic S. involucrate on the esophageal cancer CaEs-17 cells was significantly increased, which was closely related to the accumulation of cyanidin-galactoside and its metabolism-related flavonoid compounds in the transgenic callus.
Apoptosis ; Chromatography, High Pressure Liquid ; Flavonoids ; Humans ; Phenols ; Plant Extracts ; Saussurea

To research the expression of key enzymes in saikosaponin biosynthesis and the content of saikosaponin under the drought stress, the study focused on the gene-level and the end product responses to environmental change. Taking the five months of Bupleurum chinense as research materials, the contents of saikosaponin A and saikosaponin D under different stress levels were measured by HPLC. The drought was simulated by poly ethylene glycol. The real-time fluorescence quantitative PCR was used to analyze the expression of four key enzymes genes HMGR, IPPI, FPS, β-AS and the expression of β-tubulin was set as a reference gene. The results showed that drought stress significantly improved the content of saikosaponin. The contents of SSa and SSd were highest researching 0.648% and 0.781%, respectively when the concentration of PEG was 10%. Meanwhile, the results reflected that the expression of four key enzymes had risen differently and FPS, β-AS raised significantly(P<0.01). In addition, the results of correlation analysis showed that there was a significant positive correlation between the expression of the four key enzymes genes and the content of saikosaponin. In a word, the contents of secondary metabolites were regulated by the expression of key enzymes genes under the drought stress in B. chinense.

In order to reveal the molecular mechanism of blue labeled genic male sterility (BM-type GMS) and utilize the heterosis of BM-type GMS, we used the anthers of white-seed plants WS (sterile) and light blue seed plants WF (normal fertility) as experimental materials to analyze the differences in gene expression between them by transcriptome technology. And we also verified the genes expressed in anthocyanin synthesis in this study. Compared with WF, a total of 2352 differentially expressed genes were detected in WS. According to GO functional annotation, these genes could be divided into 3 categories and 43 subgroups. They are mainly involved in biosynthesis, phenylpropane metabolism, L-phenylalanine catabolism, membrane components, plasma membrane, cytoplasm, ATP binding, protein serine/threonine kinase activity, etc. KEGG pathway analysis showed that there were 159 genes enriched in the phenylpropanoid biosynthesis pathway, followed by the phenylalanine pathway, including 136 differentially expressed genes. Other genes are also involved a variety of amino acid metabolism, purine metabolism, pyrimidine metabolism and sugar metabolism pathway. Related to anthocyanin metabolism, several structural genes of key enzymes were differentially expressed, and most of them were up-regulated in WF, while only Flavanone 3-hydroxylase (F3H) and colorless anthocyanin dioxygenase (ANS) were down-regulated. Quantitative real-time PCR showed that the expression of 10 genes related to anthocyanin metabolism had the same trend as that in transcriptome sequencing data. Sequence homology analysis showed that the two selected transcription factors (DN48762c2g1 and DN25944c0g1) are clustered into the same cluster as the transcription factors regulating anthocyanin biosynthesis in maize, rice and Arabidopsis thaliana, which might be candidate genes for the blue aleurone layer of light blue seed plants in wheat. And fluorescence quantitative analysis showed that the expression level of DN48762c2g1 and DN25944c0g1 in WF was significantly higher than that in WS. In conclusion, the genes related to the anthocyanin biosynthesis pathway are not only related to the blue grain trait, but also may be involved in the anther abortion of BM-type GMS.

To efficiently produce non-specific nuclease (NU) of Serratia marcescens through recombinant overexpression approach and to characterize the purified NU. The nuclease gene was amplified from the genomic DNA of Serratia marcescens by PCR and fused into vector pMAL-c4X with maltose binding protein (MBP) tag. The recombinant vector verified by DNA sequencing was transformed into Escherichia coli BL21. The expressed MBP-NU was purified through the amylose resin and its catalytic characters were analyzed. The results showed the NU gene had 97% identities with the reported S. marcescens nuclease gene and intracellularly expressed in E. coli BL21. The optimal expression conditions were 37 degrees C, 0.75 mmol/L IPTG with 1.5 h induction. The purified MBP-NU exhibited non-specific nuclease activity, able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Its optimal temperature was 37 degrees C and optimal pH 8.0. From 1 L culture broth 10.8 mg NU could be purified with a specific activity of 1.11x10(6) U/mg. The catalytic activity of NU was not inhibited by reagents such as EDTA (0.5 mmol/L), PMSF (1 mmol/L) and KCl (150 mmol/L) commonly used in protein purification.
Base Sequence ; Endodeoxyribonucleases ; biosynthesis ; genetics ; Endoribonucleases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Maltose-Binding Proteins ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serratia marcescens ; enzymology

The aim of this research is to find an effective cardiomyocyte-induced method derived from porcine amniotic fluid stem cells (pAFS). For cardiac differentiation, the cells were formed embryoid bodies (EBs) firstly, then cultured in induced-medium including 5-azacytidine (5-aza) and vitamin C (Vc). We detected the specific markers of cardiomyocyte by immunocytochemistry, RT-PCR and transmission electron microscope. The results showed that some embryoid bodies beat rhythmically after 10 days of induction. Furthermore, analysis of t test revealed that the percentage of beating cardiomyocyte-like cell clusters was highest (33%) when induction using 0.1 mmol/L Vc and 5 micromol/L 5-aza. Immunocytochemistry analysis demonstrated that cardiomyocyte-like cell clusters expressed alpha-actin, Tnni3. RT-PCR analysis also illustrated that TbX5, Gata4, alpha-MHC and Tnni3 were expressed positive in cardiomyocyte-like cell clusters. Especially, we observed basic structures of myocardium, such as myofilament, glycogen granule and so on by transmission electron microscope. In conclusion, 5-azacytidine and vitamin C could promote differentiation of pAFS into myocardium.
Amniotic Fluid ; cytology ; Animals ; Ascorbic Acid ; pharmacology ; Azacitidine ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Embryoid Bodies ; Embryonic Stem Cells ; cytology ; Female ; Myocytes, Cardiac ; cytology ; Swine