Objective: Aspergillus cristatus is a homothallic fungus, whose sporulation is regulated by osmotic pressure, which is quite different from the light-regulated sporulation mechanism of Aspergillus nidulans. The sexual reproduction of A. cristatus is mainly regulated by MAT1-1-1 and MAT1-2-1, but the regulation mechanism of the MAT gene on the sexual reproduction is still unclear. This study aims to screen the interaction proteins of A. cristatum MAT, and lay the foundation for the further study of the sexual sporulation mechanism of A. cristatum.Methods: This study screened the interaction protein with MAT1-1-1 and MAT1-2-1 by GST pull-down combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We analyzed the biological information of the interaction protein using ProteinPilot, Gene Ontology and the genome databank of A. cristatus. The study also detected the expression level of SI65_00917 and SI65_03348 in sexual development by RT-qPCR, and used yeast two-hybrid technology to verify their interaction with MAT protein.Results: The GST-MAT1-1-1 and GST-MAT1-2-1 vectors were successfully constructed, and the target bait proteins were induced to express and purify. The bait proteins were used to capture the interacting proteins from the total protein of A. cristatus. The results showed that 56 proteins interacted with MAT1-1-1, and 413 proteins interacted with MAT1-2-1, respectively. GO analysis shows that these interaction proteins are involved in translation regulation, metabolic processes, protein transport, protein binding and other biological processes, and share nucleotide binding activity, catalytic activity, and protein binding activity. The results from RT-qPCR indicated that the interaction proteins SI65_00917 would participate in the sexual development in A.cristatus. Yeast two-hybrid results show that SI65_00917 protein has auto-activation and may be a transcription factor, and SI65_03348 protein interacts with MAT1-1-1 and MAT1-2-1 in yeast.Conclusion: These results indicate that MAT regulates the sexual development of A.cristatus through direct or indirect interaction with the other proteins.
Objective: To identify the Folprp4 gene in Fusarium oxysporum f. sp. lini and reveal its function and pathogenic correlation in Fusarium oxysporum f. sp. lini. Methods: Based on the principle of homologous recombination, the Split-Marker strategy was applied to construct the gene deletion cassette containing hygromycin resistance gene (hph) according to the genomic sequence of Folprp4 gene determined. The constructs were transformed into wild-type protoplasts mediated by PEG and the transformants were screened on TCC medium containing hygromycin B. The Folprp4 gene deletion mutants were confirmed by PCR with positive and negative primers. To do a complement test, the vector pZDH1 containing Folprp4 gene was constructed, and transformed into the deletion mutants. Results: Compared with wild type (hm) and ectopic insertion mutant (ecFolprp4), the growth of the mycelia of the deletion mutants was seriously obstructed and its colony looked like a tiny dot when the wild type and ectopic insertion mutant extended full of the plate. Another striking change of the deletion mutant was that the conidial production of ΔFolprp4 were significantly decreased. The infection assay showed that the virulence of ΔFolprp4 on flax seedlings was significantly reduced. The complement test indicated that the transformants of the complementary vector (Folprp4-C) recovered in colony morphology, growth rate, conidial yield and virulence of wild-type strain. Conclusion: The results revealed that Folprp4 gene was tightly associated with mycelial growth, conidial formation and pathogenicity in Fusarium oxysporum f. sp. lini.
Objective: Halophilic bacteria are widely distributed and have strong adaptability, but the research on their mechanism is not deep enough. In order to strengthen the exploration and research on the mechanism of salt tolerance of halophilic bacteria, potential genes related to salt tolerance were screened from H. alkaliphila DSM 16354 T, informatics analysis was carried out, and the physiological functions of the related proteins were verified. Methods: Using the combination of gene library screening and functional complementarity, through the salt tolerance functional complementarity experiment with E.coli salt sensitive defective strain KNabc (ΔnhaA、ΔnhaB、ΔchaA), the protein coding gene with salt tolerance function was screened, and then the reverse transport activity and substrate affinity of the protein were determined by fluorescence quenching recovery experiment. Results: Two protein coding genes with salt tolerance function were screened. Bioinformatics analysis showed that the gene encoded membrane proteins with unknown function from DUF1538(domain of unknown function with No.1538 family), named DUF1 and DUF2, respectively. Phylogenetic tree analysis showed that DUF1 and DUF2 from H. alkaliphila DSM 16354 T belonged to an independent branch. It was predicted that these two proteins may be new members of DUF1538 family transporters. The physiological functions of DUF1 and DUF2 were analyzed which did not have salt tolerance when expressed alone, but showed significant salt tolerance when expressed together, indicating that duf1 and duf2 subunits jointly supported the salt tolerance function of the protein. The activity of protein DUF1-2 reverse transport assay showed that the two-component protein DUF1-2 had Na + (Li+、K+)/H+ antiporter activity. Conclusion: The selected duf1 and duf2 transporters had salt-alkali tolerance function and antiporter activity when they were co-expressed. This laid a foundation for screening new DUF1538 family transporter genes and further exploring the function of DUF1538 family transporters.
Objective: A functional α1β2γ2L-GABAAR-CHO human cell line is constructed in which α1 subunit is inducd expression and β2 and γ2L subunits are stable expression. Methods: The coding genes of human subunit α1, β2 and γ2L were amplified from human cDNA library, and the subunit vectors were respectively constructed. The three subunit vectors were cotransfected into CHO-K1 cells, and the stable expression clones were screened by resistance screening and membrane potential detection. The expression of subunits were identified by qPCR and western blot;the pharmacological function of α1β2γ2L-GABAAR-CHO line were identified by whole-cell patch clamp detection and membrane potential detection method. Results: The α1β2γ2L-GABAAR-CHO with high expression level was obtained by screening the clones. The cells stably expressed α1, β2 and γ2L subunits. The constructed α1β2γ2L-GABAAR-CHO cells expressed α1 subunit in the presence of tetracycline, and assembled with β2 and γ2L subunits to form α1β2γ2L-GABAAR with functional activity. Whole-cell patch clamp detection of α1β2γ2L-GABAAR-CHO showed that GABA could stimulate it and cause the characteristic current change of chloride channel in α1β2γ2L-GABAAR-CHO, and diazepam could enhance the activation effect of GABA on α1β2γ2L-GABAAR. Membrane potential detection showed that EC50 of agonist GABA was (177.72±15.92) nmol/L, EC50 of allosteric agent diazepam was (3.63±0.52) μmol/L, and IC50 of antagonist bicuculine was (538.83±29.55) nmol/L, respectively. Conclusion: α1β2γ2L-GABAAR-CHO cell line is successfully constructed by the induced expression strategy, which has the pharmacological function of specific detection of agonists, positive allosteric agents and antagonists.
Objective: Base on spore products of Bacillus licheniformis can be produced comparatively easily and incorporated into everyday foods/feeds with high stability retaining their viability. The spore production with a good sporulation efficiency is a key step in bio-products development. It is important to study a production method that can reduce manufacturing costs and realize automatic control to enhance the productivity of Bacillus licheniformis and provide a way for other bacilli to heighten the productivity of spores. Methods: The optimal culture temperature, carbon sources and nitrogen sources of Bacillus licheniformis BF-002 were determined by the single factor experiment in shake flasks. On this basis, the experiment of the 5 L fermentor was carried out. It explored the relationship between spore formation and nitrogen sources that different concentrations of nitrogen sources were added initially. It controlled relative amino nitrogen level by fed-batch, constant speed of fed-batch and fed-batch on tail gas CO2 concentration feedback. The computer control program was written in Python language to realize automatic control. Results: The optimal culture temperature, carbon source and nitrogen source determined by the single factor experiment were: 37℃, glucose and peptone from fish: soybean meal=1∶1. The results showed that the lower the relative amino nitrogen was, the higher the spore rate was. The relative amino nitrogen was controlled at the level of 8.42 mg / OD600 by fed-batch on tail gas CO2 concentration feedback, and the number of spores can reach 4.25×10 9 cfu/ mL. The number of spores can get 1.87×1010 cfu / mL by using an automatic fed-batch of ammonium chloride, 4.4 times the optimal batch in the early stage. Conclusion: There was a correlation between spore rate and relative amino nitrogen. The strategy of using an automatic fed-batch of ammonium chloride can reduce the production cost and realize automatic control, which provides an idea for the study of Bacillus.
Mesenchymal stem cells (MSCs) have attracted great attention in regenerative medicine due to their capacities for multilineage differentiation, immunomodulation and migration. However, increased donor age and prolonged in vitro culture inevitably trigger senescence. MSC senescence and cellular dysfunction are considered one of the main causes of aging in an individual and the development of degenerative diseases, while they hinder the application of MSCs in regenerative medicine. As a major lysosome-dependent degradation and recycling pathway, autophagy is the mechanism through which the cytoplasmic components can be renewed, contributing to maintaining intracellular homeostasis and resisting environmental stress, and may become a potential therapeutic target for regulating MSC secescence. This review focuses on the phenotypic characterizations, functional alterations and molecular mechanisms in senescent MSCs, and the relationship between autophagy and senescence, which develop a theoretical foundation for the research and clinical application of MSCs.
Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease caused by insufficient insulin secretion or insulin resistance. The rapid increase in the number of patients with T2DM makes the treatment and prevention of T2DM an urgent problem in the world. With the advancement of microbiome technology, the research on the intestinal flora, its metabolites and the T2DM has gradually deepened. Perhaps the intestinal flora can be used as a target for the treatment and prevention of T2DM. The potential mechanism of intestinal flora acting on T2DM is reviewed. It mainly participates in the inflammatory response in the body, increasing the production of intestinal short-chain fatty acids, regulating the metabolism of intestinal bile acids and their product distribution, and ajusting the metabolism of branched-chain amino acids. Currently, drugs for T2DM may have some side effects. Measures based on the intestinal flora to intervene in T2DM are relatively safe and harmless. Long-term intake of a strictly controlled diet with a specific structure can be used to control blood sugar or increase the long-term intake of probiotics. It can also affect the ecological structure of the intestinal flora through oral administration. Sugar drugs (metformin and acarbose) effectively regulate blood sugar levels. The potential mechanism of T2DM induced by the intestinal flora and its metabolites is reviewed, and measures for the intervention of T2DM based on the intestinal flora are also discussed. In addition, new methods for the treatment of T2DM from a new perspective of the intestinal flora are explored, which may provide a thorough treatment of T2DM in the future.
In recent years, therapeutic recombinant protein drugs have become a research hot topic in the field of biopharmaceuticals research. Chinese hamster ovary cell lines are commonly used for recombinant protein expression in industrial production. The traditional expression of CHO cell lines is mostly based on random integration. However, random integration may result in target genes targeted into heterochromatin regions or unstable chromatin regions, causing unstable expression in CHO cell lines, and in order to obtain the ideal expression cell line researchers have to do multiple rounds of screening. The latest research proves that GOI integrates specifically in the prediction or specific genomic sites of CHO cells, which can maintain long-term consistency and stability in the expression of recombinant CHO cells. The integration sites for efficient and stable transcription in the genome of CHO cells are called hot spots. This review describes the latest research progress of stable hot spot sites in the CHO cell genome, summarizes the research methods of these sites, and concludes how to locate the GOI at predicted gene locus efficiently, to achieve high-level and stable expression recombinant protein, and to help researchers discover new or effective hot spot sites and then construct stable expression CHO cell lines.
Recently, tRNA-derived small RNAs (tsRNAs) were gradually recognized as a novel and potential non-coding RNAs (ncRNAs).There are mainly two types of tsRNAs, including tRNA halves (tiRNAs) and tRNA-derived fragments (tRFs), which differ in the cleavage position of the precursor or mature tRNA transcript. Emerging evidence suggests that tsRNAs are implicated in various cellular processes, including translational inhibition, gene silencing, and ribosome biogenesis. They also play an important role in the development of related diseases such as cancer, neurodegeneration, metabolic diseases and viral infections. This review summarizes the functions and mechanisms of action of tsRNAs, the potential application of tsRNAs in related diseases,and the current problems and puts forward future research directions.
The degeneracy in genetic codons further improves the capacity of genetic information. The genetic feature leads to synonymous codon usage bias representing a universal feature across all organisms’ genomes. Although synonymous codon usage variations in genes have no effects on the amino acid compositions, more and more studies point out that synonymous codon usage bias plays an important role in half-life of gene transcriptions, translational efficiency and accuracy and folding structure via fine-tune translation regulation mechanisms. Synonymous codons are used to mediate the translation rates in the process of ribosomal for transferring tRNA following the specific amino acid sequence, and the co-translation mechanism regulates the correct folding structure of the newborn peptide chain to ensure the normal biological function of the protein product. In particular, some synonymous codon usage patterns have the significantly positive correlations with folding structure formation of protein, since changes in synonymous codon usage bias could lead to folding structure of protein. Based on the previous reports in recent years, we review the related context for synonymous codon bias in fine-tune translation regulation.
With the rise of the artificial meat craze, heme, which is the coloring substance of artificial meat, has increasingly attracted the interest of researchers. As a porphyrin compound containing iron, it takes 5-aminolevulinic acid as the only precursor, and is synthesized in organisms through three pathways, namely, coproporphyrin-dependent, protoporphyrin-dependent, and siroheme-dependent, which is considered to be an ideal iron supplement and colorant. Compared with chemical synthesis and biological extraction, microbial synthesis is the promising method to make mass product of heme due to its convenient operation, environmental-friendly and so on. This article introduces the synthetic pathway of heme in detail, and summarizes the latest progress in the production of heme using 5-aminolevulinic acid as the sole precursor by microorganisms. In addition, the challenges and prospects of synthetic microorganisms were briefly analyzed.
Microalgae are sources of many important chemicals, such as lipids, proteins, polysaccharides, pigments and polyunsaturated fatty acids, which have a broad range of applications in biofuels, food, feed, nutraceutical, and pharmaceutical industries. However, the low production of microalgal biomass under photoautotrophic cultivation has limited its applications. In contrast, microalgae can grow fast and achieve ultrahigh cell densities under heterotrophic cultivation. These characteristics make heterotrophic cultivation have a promising potential for industrial production of microalgae-based products. This review aims to provide an overview of the advantages and disadvantages in microalgal heterotrophic cultivation, and the factors and strategies affecting microalgal cell growth as well as the current production of several microalgae under high-cell-density heterotrophic cultivation. The path forward for further economical and efficient production of microalgal target compounds under high-cell density heterotrophic culture with respect to four different opportunities is also discussed, which will in turn meet the huge demand of microalgal feedstocks in the above industries and accelerate the development of microalgae industry.
Xylan is an abundant renewable resource in nature, the content of which is second only to cellulose. Xylanase is a kind of enzyme that can hydrolyze xylan into monosaccharides and oligosaccharides. The products decomposed by xylanase can be widely used in food, papermaking, textile and other industries. Xylanase can be divided into alkaline xylanase, neutral xylanase and acidic xylanase according to its tolerance to acid-base environment. Alkaline xylanase is suitable for paper industry, especially in various processes such as pulping, bleaching and waste paper deinking, which can significantly improve paper quality, effectively reduce chlorine emission, and reduce environmental pollution. With the progress of biotechnology, the molecular modification of alkaline xylanase can be carried out by means of genetic engineering, so as to improve its alkali resistance and heat resistance and expand its condition range in industrial application. This paper introduces the research progress of alkaline xylanase in molecular modification, and briefly introduces its applications in pulping, bleaching and waste deinking.
