Construction of a directional T vector for cloning PCR products and expression in Escherichia coli.

In order to clone PCR products and express them effectively in Escherichia coli, a directional cloning system was constructed by generating a T vector based on pQE-30Xa. The vector was prepared by inserting an XcmI cassette containing an endonuclease XcmI site, a kanamycin selective marker, a multiple-cloning-site (MCS) region and an opposite endonuclease XcmI site into the vector pQE-30Xa. The T vector pQE-T with single overhanging dT residues at both 3' ends was obtained by digesting with the restriction enzyme XcmI. For directional cloning, a BamHI site was introduced to the ends of the PCR products. A BamHI site was also located on the multiple cloning site of pQE-T. The PCR products were ligated with pQE-T. The directionally inserted recombinants were distinguished by using BamHI to digest the recombinants because there are two BamHI sites located on the both sides of PCR fragment. In order to identify the T-vector functions, the 14-3-3-ZsGreen and hRBP genes were amplified and a BamHI site was added to the ends of the genes to confirm this vector by ligation with pQE-T. Results showed that the 14-3-3-ZsGreen and hRBP were cloned to the vector pQE-T directly and corresponding proteins were successfully produced. It was here demonstrated that this directional vector is capable of gene cloning and is used to manipulate gene expression very easily. The methodology proposed here involves easy incorporation of the construct into other vectors in various hosts.

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques.

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three-dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waal's forces are the main forces between pepsin and acteoside. According to the theory of Förster's non-radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside.

Real-time video surveillance on highways using combination of extended Kalman Filter and deep reinforcement learning.

Highways, as one of the main arteries of transit and transportation in today's world, play a fundamental role in accelerating transportation, and for this reason, continuous monitoring of them is of great importance. Among these, monitoring compliance with transportation laws by vehicles is of utmost importance; for automation, efficient and vehicle-specific models can be used. In this article, a new method for video surveillance of highways is presented using an extended Kalman filter (EKF) and reinforcement learning models. There are three primary stages to the suggested approach. During the first stage, the extended Kalman filter (EKF) is used to identify and track multiple targets. Next, in the second stage, a convolutional neural network (CNN) processes each detected moving item to determine the kind of vehicle. During this stage, the CNN model's ideal configuration is ascertained using a new optimization approach that combines Particle Swarm Optimization (PSO) and reinforcement learning. After identifying the type of vehicle, in the third phase, the proposed method uses a separate CNN model for each target vehicle to assess its compliance with transportation safety principles. It should be mentioned that each vehicle's associated CNN model is configured during this phase using the suggested optimization methodology. Investigations have been conducted into the effectiveness of the suggested method in identifying violations of road safety laws as well as how well it performed in the two phases of vehicle type identification. According to the findings, the suggested approach can identify the kind of vehicle with 98.72% accuracy, which is at least 3.41% better than the approaches that were compared. On the other hand, this model can detect the violation of road safety laws for each vehicle with an average accuracy of 91.5%, which shows at least a 3.49% improvement compared to the other methods.

New species of Gyrodactylus von Nordmann, 1832 (Monogenoidea: Gyrodactylidae) from Gymnodiptychus dybowskii (Kessler, 1874) (Schizothoracinae) in the Kunes River (Yili River basin), China.

Yili River system hosts a diverse fauna of fishes and parasites. Gymnodiptychus dybowskii is a rare and endangered aboriginal cold-water fish inhabit in the Yili river system. Our research identified a new species Gyrodactylus gymnodiptychi n. sp. isolated from G. dybowskii in the Kunes River (Yili River, China). Morphological comparison revealed identifiable differences between the new species and other parasites, including Gyrodactylus aksuensis, and Gyrodactylus tokobaevi, which are two known parasites living in G. dybowskii inhabit in the Aksu River west of Frunze (Kyrgyzstan), as well as Gyrodactylus montanus living in Shizothorax intermedius inhabited in the Tadzhikistan or Uzbekistan. Especially, the dorsal bar of G. gymnodiptychi n. sp. was raised at both ends with a hollow, and its hamulus roots were curved inward. The BLASTN search of GenBank did not detect any other ITS1-5.8S-ITS2 rDNA sequences same as G. gymnodiptychi's. Using the Bayesian Information and Maximum Likelihood methods to analyze the ITS1-5.8S-ITS2 rDNA gene sequences, we constructed phylogenetic trees for G. gymnodiptychi n. sp. Accordingly, our morphological and molecular research indicated that G. gymnodiptychi n. sp. was not only a new species of parasites but also the first Gyrodactylus member identified in the Yili River in China.

[The effect of physical properties of chitosan on cell activity and on its mechanics property].

Chitosan is a natural biopolymer and is made up of D-glucosamine subunits linked by beta-(1,4) glycosidic bond. In recent years, the application of chitosan has attracted more and more attention because of its good biological function in cell biology. The properties of chitosan-based biomaterial are attributed to the physical properties and chemical composition of chitosan. The author of this paper summarized recent related studies and progresses of the influence of physical properties of chitosan on cell activity and cell mechanics property at home and abroad. The findings show that most studies mainly focused on the influence of chitosan and cell activity, while few were on cell mechanics property. The related studies of the influence of chitosan on cell will contribute to the explanation for the mechanism of the interaction between chitosan and cell, and provide the theoretical support for the further study.

A New Species of Paradiplozoon (Monogenea: Diplozoidae), A Gill Parasite of the Schizothorax Fish (Cyprinidae: Schizothoracinae) from the Yarkand River, Xinjiang, China.

INTRODUCTION: Monogeneans of the genus Paradiplozoon were found on the gills of specimens of five species of schizothoracid caught using fyke nets in the upper stream of the Yarkand River, Xinjiang, China in May-August 2019. METHODS: The preserved parasite were stained with boric acid magenta and hematoxylin, respectively. Morphological observations, line drawings, photomicrographs and measurements were made in Nikon ECLIPSE E200 imaging optical microscope and digitally edited. The molecular analysis included the study of the sequence of the second internal transcribed spacer (ITS 2) of the ribosomal DNA region, calculation and analysis of genetic distance, with phylogenetic reconstructions based on the Bayesian inference and Maximum Likelihood analysis. RESULTS: The natural infection rate of host fish was 10-88%. Morphological analysis indicated that the average length of the new species was 2.125 mm while the width was 0.69 mm. The anterior part was 1.387 mm in length and the average length of the posterior part was 0.545 mm. The vitellaria was well-developed and located in the front of the body. A single ovary (oval shaped) was located at the back end of the reproductive binding area. A testis (irregular mass) was located behind or parallelled to the ovary. The new species can be distinguished from all the recorded Paradiplozoon species in terms of morphological characteristics such as haptor, clamp and central hook morphology, intestine shape and body size. In addition, the second internal transcribed spacer (ITS 2) of the ribosomal DNA region of the diplozoid was compared with that of known diplozoids previously published. It indicated that there were significant differences between the new species and the published diplozoids. CONCLUSION: Both morphological and molecular analysis support that the diplozoid is a new species. Based on the sampling location, the new species was named Paradiplozoon yarkandense n. sp.

Interacting domains of P14-3-3 and actin involved in protein-protein interactions of living cells.

14-3-3 proteins are conserved regulatory proteins present in all eukaryotic cells that control numerous cellular activities via targeted protein interactions. To elucidate the interaction between P14-3-3 from Physarum polycephalum and actin in living cells, PCR and DNA recombination were used to generate various P14-3-3 and actin constructs. Yeast two-hybrid assay and FRET were employed to characterize the interaction between P14-3-3 and actin. The two-hybrid assay indicated that P14-3-3 N-terminal 76-108 amino acids and the C-terminal 207-216 amino acids played an important role in mediating interactions with actin, and the actin N-terminal 1-54 amino acids and the C-terminal 326-376 amino acids are also crucial in the interactions with the mPa, a P14-3-3 with mutations at Ser62 (Ser62 â†’ Gly62). Mutations to potential phosphorylation sites did not affect interactions between P14-3-3 and actin. FRET results demonstrated that P14-3-3 co-localized with actin with a FRET efficiency of 22.2% and a distance of 7.4 nm and that P14-3-3 N-terminal 76-108 and C-terminal 207-216 amino acids were important in mediating this interaction, the truncated actin peptides without either the N-terminal 1-54 or C-terminal 326-376 amino acids interacted with P14-3-3, consistent with the results obtained from the yeast two-hybrid assay. Based on data obtained, we identified critical actin and P14-3-3 contact regions.

Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites.

The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3's transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures.

Developing multidrug-resistant cells and exploring correlation between BCRP/ABCG2 over-expression and DNA methyltransferase.

Expression of breast cancer resistance protein/ATP-binding cassette sub-family G member 2 (BCRP/ABCG2) is the major cause of chemotherapy failure. It is important to establish and characterize the multidrug resistance cells and to investigate the mechanism of multidrug resistance. Multidrug-resistant cells expressing BCRP/ABCG2 based on human breast cancer MCF-7/wt cells were developed by gradually increasing application of low concentration of mitoxantrone. Real-time quantitative PCR, western blot, and immunofluorescence assay were employed to analyze BCRP mRNA and protein expression. Drug accumulation in the cells was measured by flow cytometry and DNA methyltransferases were analyzed by western blot. The results indicated that the inhibitory ratio of cell proliferative growth exhibited an exponential relation with the concentration of mitoxantrone. The IC50 of MCF-7/wt cells to mitoxantrone was found to be 0.42 µM. 3-(4,5-Dimethylthlthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide assay indicated that the mitoxantrone-resistant cells at different stages exhibited cross-resistance to adriamycin and taxol. BCRP/ABCG2 mRNA and protein levels in the mitoxantrone-resistant cells at different stages increased with increasing concentration of mitoxantrone. Intracellular accumulation of mitoxantrone in the cells decreased with the increase of the BCRP/ABCG2 expression levels. DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3a (DNMT3a) expressions in the cells at different stages decreased slightly, whereas DNA methyltransferase 3b (DNMT3b) expression decreases significantly. BCRP/ABCG2 overexpression and its drug-efflux function in the drug-resistant cells are the main factors to produce multidrug resistance. Our results suggest that multidrug resistance is related to overexpression of BCRP/ABCG2 and the decrease of DNA methyltransferases, especially DNMT3b.

Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum.

BACKGROUND: Serine/arginine (SR) protein-specific kinases (SRPKs) are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS) of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379) from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. RESULTS: We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS). Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328) was localized in the alkaline Omega-loop of a helix-loop-helix motif (HLHM) of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. CONCLUSION: This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Omega-loop structure could play a crucial role in the NLS function of PSRPK.

A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF/SF2.

A 1591-bp cDNA of a serine-rich protein kinase (SRPK)-like protein has been identified in Physarum polycephalum (GenBank accession No. DQ140379). The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK (PSRPK). Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains (CDs). However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coli cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.

[Effects of Yisui Jiedu Recipe on JAK2-STAT5 signal transduction pathway in bone marrow hematopoietic cells from patients with myelodysplastic syndrome-refractory anemia].

OBJECTIVE: To investigate the effect of Yisui Jiedu Recipe (YSJDR), a compound traditional Chinese herbal medicine, on cytokines and their corresponding just another kinase 2-signal transducers and activators of transcription 5 (JAK2-STAT5) signal transduction pathway in bone marrow hematopoietic cells from patients with myelodysplastic syndrome-refractory anemia (MDS-RA). METHODS: Fluorogenic quantitative polymerase chain reaction (FQ-PCR) method was established to detect the levels of JAK2, STAT5 and Bcl-xL mRNA expressions, and JAK2-STAT5 signal transduction pathway was activated by granulocyte-macrophage-colony stimulating factor (GM-CSF) in cultured bone marrow hematopoietic cells from 10 patients with MDS-RA. The levels of interleukin-2 (IL-2), interleukin-3 (IL-3), gamma-interferon (gamma-INF) and tumor necrosis factor-alpha (TNF-alpha) in the cultural supernatant of untreated control, AG490-treated and YSJDF-treated cells were measured by enzyme-linked immunosorbent assay. RESULTS: The levels of IL-2 and TNF-alpha in YSJDR-treated group were significantly lower than those in untreated control group and AG490-treated group (P<0.01, P<0.05), and IL-3 level in YSJDP-treated group was remarkably higher than that in the other two groups (P<0.01). There were no significant differences in the levels of IL-2 and IL-3 between AG490-treated group and untreated control group (P>0.05), while the TNF-alpha level in AG490-treated group was decreased obviously as compared with the untreated control group (P<0.01). There was no significant difference in gamma-INF level between YSJDR-treated group and AG490-treated group (P>0.05), while TNF-alpha level in the two groups were significantly lower than that in the untreated control group (P<0.01). The expressions of JAK2, STAT5 and Bcl-xL mRNAs were significantly down-regulated in the YSJDR-treated and the AG490-treated groups as compared with those in the untreated control group (P<0.05, P<0.01), while there were no differences in the expressions of JAK2, STAT5 and Bcl-xL mRNAs between YSJDR-treated group and AG490-treated group. CONCLUSION: YSJDR can modulate cytokine level in bone marrow hematopoietic cells of MDS-RA, suppress JAK2-STAT5 signal transduction, and inhibit the Bcl-xL mRNA expression.

Sulcisporasupratumida sp. nov. (Phaeosphaeriaceae, Pleosporales) on Anthoxanthumodoratum from Italy.

Sulcispora is typified by S.pleurospora. We collected a sulcispora-like taxon on leaves of Anthoxanthumodoratum L. in Italy and obtained single ascospore isolates. Combined ITS, LSU, SSU and tef1 sequence analyses suggested that Sulcispora is placed in the family Phaeosphaeriaceae and a newly collected Sulcispora species is introduced here as S.supratumida sp. nov. Detailed descriptions and illustrations are provided for Sulcisporasupratumida and it is compared with the type species, S.pleurospora.

A new design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency.

BACKGROUND: RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied. RESULTS: To explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system. CONCLUSIONS: We reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.

Study on interaction between curcumin and pepsin by spectroscopic and docking methods.

The interaction between curcumin and pepsin was investigated by fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD), and molecular docking. Under physiological pH value in stomach, the fluorescence of pepsin can be quenched effectively by curcumin via a combined quenching process. Binding constant (Ka) and binding site number (n) of curcumin to pepsin were obtained. According to the theory of Förster's non-radiation energy transfer, the distance r between pepsin and curcumin was found to be 2.45 nm within the curcumin-pepsin complex, which implies that the energy transfer occurs between curcumin and pepsin, leading to the quenching of pepsin fluorescence. Fluorescence experiments also suggest that curcumin is located more closely to tryptophan residues than tyrosine residues. CD spectra together with UV-vis absorbance studies show that binding of curcumin to pepsin results in the extension of peptide strands of pepsin with loss of some ß-sheet structures. Thermodynamic parameters calculated from the binding constants at different temperatures reveal that hydrophobic force plays a major role in stabilizing the curcumin-pepsin complex. In addition, docking results support the above experimental findings and suggest the possible hydrogen bonds of curcumin with Thr-77, Thr-218, and Glu-287 of pepsin, which help further stabilize the curcumin-pepsin complex.

Uptake of silica nanoparticles: neurotoxicity and Alzheimer-like pathology in human SK-N-SH and mouse neuro2a neuroblastoma cells.

Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 µg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular ß-amyloid 1-42 (Aß(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer's disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-ß-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3ß at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3ß. This is the first study with direct evidence indicating that in addition to neurotoxicity induced by SiNPs, the application of SiNPs might increase the risk of developing AD.

RNA interference with carbon catabolite repression in Trichoderma koningii for enhancing cellulase production.

The cellulase and xylanase genes of filamentous Trichoderma fungi exist under carbon catabolite repression mediated by the regulator carbon catabolite repressor (CREI). Our objective was to find the role of CREI in a cellulase-hyperproducing mutant of Trichoderma koningii, and address whether enzyme production can be further improved by silencing the cre1 gene. cre1 partially silenced strains were constructed to improve enzyme production in T. koningii YC01, a cellulase-hyperproducing mutant. Silencing of cre1 resulted in derepression of cellulase gene expression in glucose-based cultivation. The cre1 interference strain C313 produced 2.1-, 1.4-, 0.8-, and 0.8-fold higher amounts of filter paper activity, ß-1,4-exoglucanase activity (ρ-nitrophenyl-ß-D-cellobioside as substrate), ß-1,4-endoglucanase activity (sodium carboxymethyl cellulose as substrate), and xylanase activity, respectively, than the control strain, suggesting that silencing of cre1 resulted in enhanced enzyme production capability. In addition, downregulation of cre1 resulted in elevated expression of another regulator of xylanase and cellulase expression, xyr1, indicating that CREI also acted as a repressor of xyr1 transcription in T. koningii under inducing conditions. These results show that RNAi is a feasible method for analyzing the regulatory mechanisms of gene expression and improving xylanase and cellulase productivity in T. koningii.

Improving cellulase production in Trichoderma koningii through RNA interference on ace1 gene expression.

Ribonucleic acid interference (RNAi) inhibits the expression of target genes in a sequence-specific manner, and shows potential for gene knockdown in filamentous fungi, in which the locus-specific gene knockout occurs in low frequency. In this study, the function of the repressor of cellulase expression I (ACEI) was verified in Trichoderma koningii (T. koningii) YC01 through RNAi, and ace1- silenced strains with improved cellulase productivity were obtained. An expression cassette that transcribed the interfering double-stranded RNA (dsRNA) of ace1 was constructed and transformed into T. koningii, and the transformants, in which the expression of ace1 was successfully silenced, were selected. As a result of the ace1 gene silencing, the expression levels of the main cellulase and xylanase genes were elevated, and the enhanced production of total proteins, cellulase, and xylanase was observed in the cultivation. In addition, the downregulation of ace1 resulted in an increasing expression of xyr1, but no clear variation in the expression of cre1, which suggested that ACEI acted as a repressor of the xyr1 transcription, but was not involved in the regulation of the cre1 expression. The results of this work indicate that ace1 is a valid target gene for enhancing enzyme production in T. koningii, and RNAi is an appropriate tool for improving the properties of industrial fungi.

Identification of a novel PSR as the substrate of an SR protein kinase in the true slime mold.

Here, a novel cDNA encoding a serine/arginine (SR)-rich protein, designated PSR, was isolated from the true slime mold Physarum polycephalum and expressed in Escherichia coli. The deduced amino acid (aa) sequence reveals that PSR contains RS repeats at its C-terminus, similar to the conventional PSRPK substrate ASF/SF2. To study the novel protein, we generated a variety of mutant constructs by PCR and site-directed mutagenesis. Our analysis indicated that the purified recombinant PSR was phosphorylated by PSRPK in vitro and the SR-rich domain (amino acids 460-469) in the PSR protein was required for phosphorylation. In addition, removal of the docking motif (amino acids 424-450) from PSR significantly reduced the overall catalytic efficiency of the phosphorylation reaction. We also found that the conserved ATP-binding region (62)LGWGHFSTVWLAIDEKNGGREVALK(86) and the serine/threonine protein kinases active-site signature (184)IIHTDLKPENVLL(196) of PSRPK played a crucial role in substrate phosphorylation and Lys(86) and Asp(188) were crucial for PSRPK phosphorylation of PSR. These results suggest that PSR is a novel SR-related protein that is phosphorylated by PSRPK.

[Transient expression in microplasmodia of Physarum polycephalum].

The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRedl-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 micros electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.