Target Gene Fragment Research Articles

Unravelling the Molecular Identity of Bulgarian Jumping Plant Lice of the Family Aphalaridae (Hemiptera: Psylloidea).

Psyllids (Hemiptera: Psylloidea) are plant sap-sucking insects whose identification is often difficult for non-experts. Despite the rapid development of DNA barcoding techniques and their widespread use, only a limited number of sequences of psyllids are available in the public databases, and those that are available are often misidentified. Here, we provide 80 sequences of two mitochondrial genes, cytochrome c oxidase I (COI) and cytochrome b (Cytb), for 25 species of Aphalaridae, mainly from Bulgaria. The DNA barcodes for 15 of these species are published for the first time. In cases where standard primers failed to amplify the target gene fragment, we designed new primers that can be used in future studies. The distance-based thresholds for the analysed species were between 0.0015 and 0.3415 for COI and 0.0771 and 0.4721 for Cytb, indicating that the Cytb gene has a higher interspecific divergence, compared to COI, and therefore allows for more accurate species identification. The species delimitation based on DNA barcodes is largely consistent with the differences resulting from morphological and host plant data, demonstrating that the use of DNA barcodes is suitable for successful identification of most aphalarid species studied. The phylogenetic reconstruction based on maximum likelihood and Bayesian inference analyses, while showing similar results at high taxonomic levels to previously published phylogenies, provides additional information on the placement of aphalarids at the species level. The following five species represent new records for Bulgaria: Agonoscena targionii, Aphalara affinis, Colposcenia aliena, Co. bidentata, and Craspedolepta malachitica. Craspedolepta conspersa is reported for the first time from the Czech Republic, while Agonoscena cisti is reported for the first time from Albania.

Molecular dissection of the parental contribution in Paeonia Itoh hybrids.

Hybrid breeding between herbaceous peonies (the maternal parent) and tree peonies (the paternal parent) results in Paeonia Itoh hybrids (Itoh peonies), a triploid species that combines advantageous traits from both parental species, thus offering great economic value. However, the exact genetic contribution of the two parents is unclear. In this study, we introduce a straightforward approach utilizing heterozygous single-nucleotide polymorphisms (SNPs) and Sanger sequencing of targeted gene fragments to trace the original bases back to their parents in Itoh peonies. Our results indicate that in triploid Itoh peonies, only one set of genes is derived from herbaceous peonies, and two sets of genes are derived from the tree peonies. Notably, the presence of three distinct bases of heterozygous SNPs across multiple Itoh cultivars suggests that the gametes from the paternal parents carry two sets of heterozygous homologous chromosomes, which could be due to Meiosis I failure during gamete formation. To validate our method's effectiveness in parentage determination, we analyze two Itoh hybrids and their parents, confirming its practical utility. This research presents a method to reveal the parental genetic contribution in Itoh peonies, which could enhance the efficiency and precision of hybrid breeding programs of triploids in Paeonia and other plant species.

Using a DNA mini-barcode within the ITS region to identify toxic Amanita in mushroom poisoning cases

Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning.Key points• Development of a DNA mini-barcoding method for Amanita species identification without fresh samples.• The ITS-a primer set was optimized for robust universality in Amanita samples.• The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.Graphical

Determination of molecular types and resistance to macrolides in <i>Treponema pallidum</i> isolates isolated in the Russian Federation

Background. The number of syphilis cases in the Russian Federation increased significantly in 2022. Control of heterogeneity of Treponema pallidum subtypes is important to monitor the emergence and spread of antibiotic-resistant strains Aims. To determine molecular subtypes and resistance to macrolides in T. pallidum isolates isolated in the Russian Federation in 2022. Methods. We analyzed DNA isolated from 49 samples of clinical material obtained from patients from dermatovenerological treatment and prevention facilities in three federal districts (CFD, SFD, SCFD) of the Russian Federation in 2022 with diagnoses of primary syphilis and secondary syphilis. T. pallidum DNA isolation and confirmation of the presence of genetic material were performed according to the existing algorithms. To search for genetic determinants of resistance to macrolides, a fragment of the 23S rRNA gene was analyzed. Primary decoding of nucleotide sequences was performed in Sequencing Analysis 5.3.1. Mega 11 program was used to align the analyzed fragments of target genes to T. pallidum reference sequences. Results. In 2022, three subtypes of T. pallidum were identified in the territory of the represented federal districts of the Russian Federation: 14d/f, 14d/g, 14d/d with continued dominance of subtype 14d/f. The macrolide-resistant subtype 14d/d was identified in two federal districts, which is new for the Russian Federation. Conclusions. The population of T. pallidum continues to expand in the Russian Federation, including the emergence of azithromycin-resistant strains. The data obtained confirm the need for continuous monitoring of circulating strains and may facilitate understanding of their geographic distribution.

Microbiome of periodontitis and peri-implantitis before and after therapy: Long-read 16S rRNA gene amplicon sequencing.

The microbial profiles of peri-implantitis and periodontitis (PT) are inconclusive. The controversies mainly arise from the differences in sampling sites, targeted gene fragment, and microbiome analysis techniques. The objective of this study was to explore the microbiomes of peri-implantitis (PI), control implants (CI), PT and control teeth (CT), and the microbial change of PI after nonsurgical treatment (PIAT). Twenty-two patients diagnosed with both PT and peri-implantitis were recruited. Clinical periodontal parameters and radiographic bone levels were recorded. In each patient, the subgingival and submucosal plaque samples were collected from sites with PI, CI, PT, CT, and PIAT. Microbiome diversity was analyzed by high-throughput amplicon sequencing using full-length of 16S rRNA gene by next generation sequencing. The 16S rRNA gene sequencing analysis revealed 512 OTUs in oral microbiome and 377 OTUs reached strain levels. The PI and PT groups possessed their own unique core microbiome. Treponema denticola was predominant in PI with probing depth of 8-10 mm. Interestingly, Thermovirga lienii DSM 17291 and Dialister invisus DSM 15470 were found to associate with PI. Nonsurgical treatment for peri-implantitis did not significantly alter the microbiome, except Rothia aeria. Our study suggests Treponemas species may play a pivotal role in peri-implantitis. Nonsurgical treatment did not exert a major influence on the peri-implantitis microbiome in short-term follow-up. PT and peri-implantitis possess the unique microbiome profiles, and different therapeutic strategies may be suggested in the future.

Systematic investigation of the Borrelia miyamotoi spirochetes in ticks, wildlife and domestic animal hosts in Yunnan province, Southwest China

BackgroundBorrelia miyamotoi is a spirochete species transmitted via hard ticks. Following its discovery in Japan, this pathogen has been detected around the world, and is increasingly confirmed as a human pathogen causing febrile disease, namely relapsing fever. Its presence has been confirmed in the Northeast China. However, there is little information regarding the presence of B. miyamotoi and other hard-tick-borne relapsing fever spirochetes in southern China including Yunnan province, where tick and animal species are abundant and many people both inhabit and visit for recreation. MethodsFor the present study, we collected samples of ticks, wildlife, and domestic animal hosts from different counties in Yunnan province. Nucleic acids from samples were extracted, and the presence of B. miyamotoi and other relapsing fever spirochetes was confirmed using polymerase chain reaction (PCR) for the 16S rRNA specific target gene fragment. The positive samples were then amplified for partial genome of the flaB and glpQ genes. Statistical differences in its distribution were analyzed by SPSS 20 software. Sequence of partial 16S rRNA, flaB and glpQ genome were analyzed and phylogenetic trees were constructed. ResultsA total of 8260 samples including 2304 ticks, 4120 small mammals and 1836 blood of domestic animal hosts were collected for screening for infection of B. miyamotoi and other relapsing fever spirochetes. Cattle and sheep act as the main hosts and Rhipicephalus microplus, Haemaphysalis nepalensis, H. kolonini and Ixodes ovatus were identified as the important vector host with high prevalence or wide distribution. Only one Mus caroli (mouse) and one Sorex alpinus (shrew) were confirmed positive for relapsing fever spirochetes. Evidence of vertical transmission in ticks was also confirmed. Two known strains of B. miyamotoi and one novel relapsing fever spirochetes, B. theileri-like agent, were confirmed and described with their host adaptation, mutation, and potential risk of spreading and spillover for human beings. ConclusionsOur results provide new evidence of relapsing fever spirochetes in vector and animal hosts in Yunnan province based on large sample sizes, and offer guidance on further investigation, surveillance and monitoring of this pathogen.

The 60 nm gold nanoparticles improve qPCR amplification efficiency through specific palindromic sequences (GGATCC or ACCGGT) in primers

BackgroundPolymerase chain reaction (PCR) technology and quantitative real-time PCR (qPCR) technology are widely used in clinical diagnosis and research, but amplification efficiency and sensitivity are still key problems for researchers. An increasing number of reports show that gold nanoparticles (AuNPs) can be used to improve the sensitivity and amplification efficiency of PCR. Here, we found that 60 nm gold nanoparticles with a positive charge (60 nm- Au+) can enhance the amplification efficiency of qPCR. MethodsMouse DNA was extracted by the alkaline lysis method. Primer 5.0 software was used to design primers and mutation primers, and the DNA fragments were obtained by the method of synthesizing plasmids. The qPCR was applied to amplify target gene fragments. ResultsThe amplification efficiency of qPCR was improved by about 1.828 times in the experimental group with 60 nm- Au+ compared with the control group without 60 nm- Au+. The primer pair contains a specific palindromic sequence (GGATCC or ACCGGT). And 60 nm Au+ did not enhance the amplification efficiency of qPCR when the above primer was mutated. ConclusionsThe primers contain special palindrome sequences (GGATCC or ACCGGT) with 60 nm- Au+ can enhance the amplification efficiency of qPCR. Therefore, it suggests a more in-depth understanding of the mechanism and function of gold nanoparticles and primer sequences. This study has presented some implications for gold nanoparticles application in the development of qPCR technology.

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses.

To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Cas13a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Cas13a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Cas13a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 100 copies/μL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1 h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection.

Gene Silencing via RNA Interference in Cryptococcus.

RNA interference (RNAi) is a molecular biology technique for silencing specific eukaryotic genes without altering the DNA sequence in the genome. The silencing effect occurs because of decreased levels of mRNA that then result in decreased protein levels for the gene. The specificity of the silencing is dependent upon the presence of sequence-specific double-stranded RNA (dsRNA) that activates the cellular RNAi machinery. This chapter describes the process of silencing a specific target gene in Cryptococcus using a dual promoter vector. The plasmid, pIBB103, was designed with two convergent GAL7 promoters flanking a ura5 fragment that acts as a reporter for efficient RNAi. The target gene fragment is inserted between the promoters to be transcribed from both directions leading to the production of dsRNA in cells that activate the RNAi pathway.

Association between TRAF1/C5 Gene Polymorphisms and IgA Vasculitis in Chinese Children

ABSTRACT Objective To investigate the association between loci rs3761847 and rs10818488 of tumor necrosis factor receptor-associated factor 1/complement C5 (TRAF1/C5) gene and the susceptibility to IgAV. Methods 100 blood samples of children with IgAV and 100 blood samples of healthy children were collected from the Third Xiangya Hospital of Central South University from June 2017 to June 2019. The target gene fragment was amplified by polymerase chain reaction (PCR), and the single nucleic acid gene polymorphism of the gene loci was detected by PCR sequencing based typing technique. The association between gene polymorphism of each locus and susceptibility to IgAV was analyzed. Results There were significant differences in both genotype (P < .05) and allele frequencies （P < .05) of rs3761847 of TRAF1/C5 gene between the IgAV group and the control group.Besides, the risks of developing IgAV in children with the TT genotype was 0.495 times and in children with the C allele was 1.627 times of that in children with other genotypes and alleles, respectively (P < .05). For IgAV patients, renal involvement risk in children with CC genotype was 5.859 times of that in children with other genotypes (P < .05). There were no significant differences in genotype (P > .05) and allele frequencies (P > .05) of rs10818488 of TRAF1/C5 gene between the IgAV group and the control group. IgAV patients with TT genotype had a 3.2 times higher risk of renal involvement than those with other genotypes (P < .05). Conclusions There is an association between locus rs3761847 of TRAF1/C5 gene single nucleotide polymorphisms and susceptibility to IgAV. The T allele at locus rs3761847 of TRAF1/C5 gene may be a protective factor for IgAV. The C allele at locus rs3761847 and the T allele at locus rs10818488 of TRAF1/C5 gene may be associated with kidney injury in IgAV.

Conjugation Vector Delivery System for Molecular Cloning into Cells of Bacteria of The Genus &lt;i&gt;Bacillus&lt;/i&gt;

A delivery system for vectors for molecular cloning into bacterial cells of the genus Bacillus has been developed. A specific feature of the developed system is the use of the pBS72 plasmid, which provides the conjugative transfer of the conditionally lethal vector pKS1mob obtained into the cells of the studied bacteria. The ability of vector pKS1mob to replicate in Escherichia coli and B. subtilis cells at a low temperature (30°C), the presence of two polylinkers around the kanamycin resistance gene makes it possible to clone target gene fragments into its composition using traditional genetic engineering approaches. Inactivation of the rok gene in the pBS72 plasmid made it possible to transform the strain containing it with the constructed vector pKS1mob with high efficiency. Crossing the donor strain B. subtilis 168, containing conjugative pBS72 and pKS1mob mobilizable plasmids, with a recipient strain of the genus Bacillus made it possible to introduce the pKS1mob plasmid into it. The possibility of using the created system for inactivation of gene codY of bacteria Bacillus licheniformis was shown.

Research Progress of Virus-Induced Gene Silencing and its Application in Gramineae

With the proliferation of genome sequence information, biological science research has entered the era of big data, but how to annotate the function of genome information has become an important research goal. Virus-induced gene silencing (VIGS) is a powerful tool that has been used to study key genes in multiple plant growth and development processes. This technology uses the innate antiviral defense system of plants. And generates small interfering RNA (siRNA) in the plant by inserting the target gene fragment into the viral vector, so that the transcript of the endogenous gene of the plant becomes a target for degradation, resulting in the expression of the target gene being down-regulated. This paper systematically reviews the development, mechanism, and advantages of VIGS technology, focuses on the vectors that can be applied in grasses, and finally discusses the limitations and development trend of VIGS technology in the application of Gramineae.

Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis

ObjectiveThis study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. MethodsThe standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. Resultsobtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997–0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%−72.73; kappa 0.37–0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2–100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). ConclusionsThe uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.

Bacterial Communities Associated with Houseflies (Musca domestica L.) Inhabiting Hospices in South Africa.

Houseflies are alleged reservoirs as well as vectors of human and animal pathogens, including bacteria, because they frequently have contact with animal excreta and decaying organic substances. The rapid adaptation process of ingested microbes in the insect gut may involve gene transfer, including antibiotic resistance determinants among different bacterial strains. Six hundred and fifty-seven (n = 657) houseflies were collected from hospices and were identified morphologically and genetically using the 16S rRNA, CO1, and ITS2 barcoding genes. This study also characterized the bacterial communities harboured by the captured houseflies using 16S rRNA metabarcoding on the next-generation sequencing (NGS) platform and further sought to detect antibiotic resistance traits by using gene-specific PCR assays. Generated sequences for the targeted gene fragments matched with Musca domestica and all the sequences were deposited to the GenBank database. The 16S rRNA metabarcoding analysis revealed that the most abundant phyla detected with variable abundance observed among all the houseflies were Proteobacteria, followed by Firmicutes, and Bacteroidetes. Furthermore, the NGS data revealed the presence of multiple bacterial genera, including Providencia, Enterobacter, Dysgonomonas, Escherichia-Shigella, Klebsiella, Pseudomonas, and Streptococcus, which are known to harbour potentially pathogenic species of animals and humans. Antibiotic resistance genes detected from the housefly DNA in this study included ermB, tetA, blaSHV, and blaTEM. Moreover, these genes are associated with resistance to erythromycin, tetracycline, and beta-lactams antibiotics, respectively. The presence of bacterial pathogens and the detection of antibiotic resistance genes from the houseflies collected from the hospices indicates the possible health risk to patients in hospices and the surrounding community. Therefore, it is imperative to keep high standards of hygiene, food preparation, safety, and control of houseflies in hospices.

Potential mechanisms of osthole against bladder cancer cells based on network pharmacology, molecular docking, and experimental validation

BackgroundOsthole was traditionally used in treatment for various diseases. However, few studies had demonstrated that osthole could suppress bladder cancer cells and its mechanism was unclear. Therefore, we performed a research to explore the potential mechanism for osthole against bladder cancer.MethodsInternet web servers SwissTargetPrediction, PharmMapper, SuperPRED, and TargetNet were used to predict the Osthole targets. GeneCards and the OMIM database were used to indicate bladder cancer targets. The intersection of two target gene fragments was used to obtain the key target genes. Protein–protein interaction (PPI) analysis was performed using the Search Tool for the Retrieval of Interacting Genes (STRING) database. Furthermore, we used gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to explore the molecular function of target genes. AutoDock software was then used to perform molecular docking of target genes,osthole and co-crystal ligand. Finally, an in vitro experiment was conducted to validate bladder cancer inhibition by osthole.ResultsOur analysis identified 369 intersection genes for osthole, the top ten target genes included MAPK1, AKT1, SRC, HRAS, HASP90AA1, PIK3R1, PTPN11, MAPK14, CREBBP, and RXRA. The GO and KEGG pathway enrichment results revealed that the PI3K-AKT pathway was closely correlated with osthole against bladder cancer. The osthole had cytotoxic effect on bladder cancer cells according to the cytotoxic assay. Additionally, osthole blocked the bladder cancer epithelial-mesenchymal transition and promoted bladder cancer cell apoptosis by inhibiting the PI3K-AKT and Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathways.ConclusionsWe found that osthole had cytotoxic effect on bladder cancer cells and inhibited invasion, migration, and epithelial-mesenchymal transition by inhibiting PI3K-AKT and JAK/STAT3 pathways in in vitro experiment. Above all, osthole might have potential significance in treatment of bladder cancer.SubjectsBioinformatics, Computational Biology, Molecular Biology.

Virus-Induced Silencing of a Sequence Coding for Loricrin-like Protein in Phytophthora infestans upon Infection of a Recombinant Vector Based on Tobacco Mosaic Virus

Phytophthora infestans is the oomycete responsible for late blight disease of Solanaceae that causes both yield and economic losses. With the aim of reducing plant wilt and high management costs mainly due to wide fungicide applications, alternative eco-sustainable control strategies are needed. RNA interference (RNAi) is a powerful tool for gene function studies that can be accomplished by constitutive transformation or transient expression such as virus-induced gene silencing (VIGS) experiments. VIGS makes use of viruses to deliver sequences homologous to a target gene fragment and trigger RNAi. Indeed, a P. infestans ortholog of plant loricrin-like protein (LLP), named PiLLP, has been silenced using the direct infection of a recombinant vector based on the plant virus tobacco mosaic virus (TMV-PiLLP-1056), aiming to reduce the oomycete sexual reproduction. For this purpose, the gene coding for the green fluorescent protein (GFP) present in the TMV-GFP-1056 vector has been replaced with an antisense construct obtained by fusion PCR of the PiLLP 5′-UTR and 3′-UTR sequences. Here, we show that RNAi can be expressed in the A1 mating type of P. infestans strain 96.9.5.1 by VIGS using the direct infection of TMV-PiLLP-1056. We provide evidence that the recombinant vector can enter, replicate, and persist in mycelia of P. infestans where it induces the partial downregulation of the PiLLP transcript. Compared with the wild-type, the PiLLP-silenced A1 mating type had slower colony growth and a diminished virulence in detached tomato leaflets. This seems to be the first evidence of a constitutive gene downregulation of P. infestans using a recombinant vector based on a plus-sense RNA plant virus.

Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitochondrial antibodies (AMAs) are an important diagnostic basis, especially the M2 subtype (AMA-M2) with almost 100% specificity. We selected the BCOADC-E2 protein, a mitochondrial autoantigen that reacts specifically with AMA-M2 antibodies, and carried out DNA recombination and protein mutation experiments by cloning in vitro the homologous target gene sequence BCKD that expresses the antigenic epitope of BCOADC-E2 protein, to provide experience for later exploring the effect of mutations of amino acids around the lysine in the active center of BCOADC-E2 protein on its specific binding to AMA-M2, and to lay the foundation for determining the key amino acids of BCOADC-E2 for the diagnosis and treatment of PBC. In addition, we apply this scientific research content to graduate course teaching. Experimental technology of microbial molecular ecology is a course with the cross-integration of multidisciplinary knowledge and experimental skills offered at our college since 2018. This article derives from the part of this course on the construction of recombinant plasmids. The students first constructed the recombinant plasmid pGEX-BCKD using the vector plasmid pGEX-4T1 and the target gene fragment BCKD provided by the laboratory and used this as a template to construct the pGEX-BCKD-E4A point mutation plasmid by the overlap extension PCR (SOE PCR) technique to achieve the effect of mutating the fifth amino acid glutamate in front of lysine, the active centre of the BCOADC-E2 lipid acyl binding domain, to alanine for subsequent studies. Through the research experiment, combining theoretical knowledge and experimental operation, we aim to deepen the student's understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students' interest in professional knowledge learning, and cultivate students' scientific thinking and innovation consciousness. We examined the quality of the teaching through the process and summative evaluation of the students. In this study, the students successfully completed the construction of pGEX-BCKD-E4A point mutant plasmid, and the average test score increased from 40.4% before teaching to 91.1%. The teaching effect was remarkable. This kind of research experimental teaching mode has good application prospects, and other education and teachers can refer to and reference it.

Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.

Characterization of BbKlf1 as a novel transcription factor vital for asexual and infection cycles of Beauveria bassiana.

The large family of C2H2-type zinc finger transcription factors (TFs) comprise the Kruppel-like factors (KLFs) that evolved relatively late in eukaryotes but remain unexplored in filamentous fungi. Here, we report that an orthologue (BbKlf1) of yeast Klf1 mediating cell wall integrity (CWI) is a wide-spectrum TF evidently localized in nucleus and cytoplasm in Beauveria bassiana. BbKlf1 features conserved domains and multiple DNA-binding motifs predicted to bind multiple promoter DNA fragments of target genes across asexual developmental and stress-responsive pathways. Despite limited impact on normal colony growth, deletion of Bbklf1 resulted in impaired CWI and hypersensitivity to Congo red-induced cell wall stress. Also, the deletion mutant was severely compromised in tolerance to oxidative and osmotic stresses, hyphal septation and differentiation, conidiation capacity (reduced by 95%), conidial quality (viability and hydrocarbon epitope pattern) and virulence. Importantly, these phenotypes correlated well with sharply repressed or nearly abolished expressions of those genes required for or involved in chitin biosynthesis, antioxidant activity, cell division and differentiation, aerial conidiation and conidial maturation. These findings indicate an essentiality of BbKlf1 for the asexual and insect-pathogenic lifecycles of B. bassiana and a novel scenario much beyond the yeast orthologue-mediated CWI, suggesting important roles of its orthologues in filamentous fungi.

A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus

The difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses can infect the host systemically to achieve expression and replication without transgene integration. Previously, a novel four-component barley stripe mosaic virus (BSMV) was reported to overexpress large fragments of target genes in plants over a long period of time, which greatly simplified the study of gene overexpression. However, whether this system can infect cotton and stably overexpress target genes has not yet been studied. In this study, we verified that this new BSMV system can infect cotton through seed imbibition and systemically overexpress large fragments of genes (up to 2340 bp) in cotton. The target gene that was fused with GFP was expressed at a high level in the roots, stems, and cotyledons of cotton seedlings, and stable fluorescence signals were detected in the cotton roots and leaves even after 4 weeks. Based on the BSMV overexpression system, the subcellular localization marker line of endogenous proteins localized in the nucleus, endoplasmic reticulum, plasma membrane, Golgi body, mitochondria, peroxisomes, tonoplast, and plastids were quickly established. The overexpression of a cotton Bile Acid Sodium Symporter GhBASS5 using the BSMV system indicated that GhBASS5 negatively regulated salt tolerance in cotton by transporting Na+ from underground to the shoots. Furthermore, multiple proteins were co-delivered, enabling co-localization and the study of protein–protein interactions through co-transformation. We also confirmed that the BSMV system can be used to conduct DNA-free gene editing in cotton by delivering split-SpCas9/sgRNA. Ultimately, the present work demonstrated that this BSMV system could be used as an efficient overexpression system for future cotton gene function research.