Selection and validation of reference genes for quantitative real-time polymerase chain reaction analyses of <i>Serratia ureilytica</i> DW2

Background: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula; nonetheless, until now, no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction (RT–qPCR) data. Methods: To screen stable reference genes of S. ureilytica DW2, the expression of its eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT–qPCR. The expression stability of these genes was analyzed using different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels was quantified using the most stably expressed reference gene. Results: The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable genes were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium. Conclusion: Therefore, this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies.     

Targeting the “undruggable” cancer driver genes: <i>Ras</i>, <i>myc</i>, and <i>tp53</i>

The term “undruggable” is to describe molecules that are not targetable or at least hard to target pharmacologically. Unfortunately, some targets with potent oncogenic activity fall into this category, and currently little is known about how to solve this problem, which largely hampered drug research on human cancers. Ras, as one of the most common oncogenes, was previously considered “undruggable”, but in recent years, a few small molecules like Sotorasib (AMG-510) have emerged and proved their targeted anti-cancer effects. Further, myc, as one of the most studied oncogenes, and tp53, being the most common tumor suppressor genes, are both considered “undruggable”. Many attempts have been made to target these “undruggable” targets, but little progress has been made yet. This article summarizes the current progress of direct and indirect targeting approaches for ras, myc, two oncogenes, and tp53, a tumor suppressor gene. These are potential therapeutic targets but are considered “undruggable”. We conclude with some emerging research approaches like proteolysis targeting chimeras (PROTACs), cancer vaccines, and artificial intelligence (AI)-based drug discovery, which might provide new cues for cancer intervention. Therefore, this review sets out to clarify the current status of targeted anti-cancer drug research, and the insights gained from this review may be of assistance to learn from experience and find new ideas in developing new chemicals that directly target such “undruggable” molecules.     

Cloning and characterization of 5-enopyruvylshikimate-3-phosphate synthase from <i>Phragmites australis</i>

Glyphosate is a non-selective broad-spectrum herbicide that blocks plant growth by inhibiting 5- Enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme of the shikimate pathway in microorganisms and plants. The full-length epsps cDNA sequence (paepsps, Genebank: KY860582.1) was cloned and characterized for the first time from Phragmites australis. The full-length cDNA of paepsps was 1308 bp encoding a polypeptide of 435 amino acids. The bioinformatic analyses showed that PaEPSPS has highly homologous with EPSPS from other plants. RT-PCR analysis of paepsps expression indicated that the gene expressed in leaves, stems, and roots, with higher expression in leaves. The expression of the paepsps gene increased with glyphosate application. In addition, the transgenic tobacco containing the paepsps gene showed glyphosate resistance in comparison with control. The novel paepsps is a good candidate gene in transgenic crops with glyphosate tolerance in the future.     

The chloroplast genome comparative characteristic of artificial breeding tree,a case about <i>Broussonetia kazinoki</i> × <i>Broussonetia papyrifera</i>

Broussonetia kazinoki × Broussonetia papyrifera (ZJGS) is a hybrid species in Moraceae family, which has a very complicated hybrid origin. The excellent characteristics of fast growth, strong soil and water conservation ability, high leaf protein content and stem fiber content in ZJGS make it both ecological benefits in the mining area and economically valuable. This study aims to further understand ZJGS and other Moraceae taxa through the ZJGS chloroplast (cp) genome structure and the comparison with 12 closely related Moraceae species. Among the 13 Moraceae species, the cp genome length of seven Broussonetia species (ranges from 160,239 bp to 162,594 bp) is larger than that of six Morus species (ranges from 158,459 bp to 159,265 bp). Among the 77 shared protein-coding genes (PCGs) in Moraceae species, the obvious positive selection of Ka/Ks ratios acted on petD and rpl16 genes of B. kazinoki and B. papyrifera, respectively. Phylogenetic analysis based on shared PCGs from 28 species shows that ZJGS is closely related to maternal B. kazinoki. These findings provide data support for the origin of ZJGS hybridization and provide genomic resources for future ZJGS resource development and molecular breeding.     

Genome-wide identification of <i>U-box</i> gene family and expression analysis under abiotic stresses in <i>Salvia miltiorrhiza</i>

Plant U-box (PUB) E3 ubiquitin ligases play important roles in hormone signaling pathways and in response to different abiotic stresses, but little is known about U-box genes in Danshen (root of Salvia miltiorrhiza Bunge). Here, we identified and characterized 70 SmPUB genes based on its genome sequence. Phylogenetic analysis of U-box genes from S. miltiorrhiza and Arabidopsis suggested that they can be clustered into seven subgroups (I–VII). Typical U-box domains were found in all identified SmPUB genes through the analysis of conserved motifs. Moreover, qRT-PCR was applied to analyze the relative expression levels of U-box genes in S. miltiorrhiza roots and leaves under PEG-induced water deficit and salt stresses. Results revealed that the SmPUB genes exhibited stronger response to drought than to salt stress. To the best of our knowledge, this report is the first to perform genome-wide identification and analysis of the U-box gene family in S. miltiorrhiza, and the results provide valuable information for better understanding of the function of U-box in S. miltiorrhiza.     

Application of ferrous sulfate alleviates negative impact of cadmium in rice (<i>Oryza sativa</i> L.)

Soil contamination with toxic heavy metals [such as cadmium (Cd)] is becoming a serious global problem due to rapid development of social economy. Iron (Fe), being an important element, has been found effective in enhancing plant tolerance against biotic and abiotic stresses. The present study investigated the extent to which different levels of Ferrous sulphate (FeSO₄) modulated the Cd tolerance of rice (Oryza sativa L.), when maintained in artificially Cd spiked regimes. A pot experiment was conducted under controlled conditions for 146 days, by using natural soil, mixed with different levels of CdCl₂ [0 (no Cd), 0.5 and 1 mg/kg] together with the exogenous application of FeSO₄ at [0 (no Fe), 1.5 and 3 mg/kg] levels to monitor different growth, gaseous exchange characteristics, oxidative stress, antioxidative responses, minerals accumulation, organic acid exudation patterns of O. sativa. Our results depicted that addition of Cd to the soil significantly (P < 0.05) decreased plant growth and biomass, gaseous exchange parameters, mineral uptake by the plants, sugars (soluble, reducing, and non-reducing sugar) and altered the ultrastructure of chloroplasts, plastoglobuli, mitochondria, and many other cellular organelles in Cd-stressed O. sativa compared to those plants which were grown without the addition of Cd in the soil. However, Cd toxicity boosted the production of reactive oxygen species (ROS) by increasing the contents of malondialdehyde (MDA), which is the indication of oxidative stress in O. sativa and was also manifested by hydrogen peroxide (H₂O₂) contents and electrolyte leakage to the membrane bounded organelles. Although, activities of various antioxidative enzymes like superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) and non-enzymatic antioxidants like phenolics, flavonoid, ascorbic acid, anthocyanin and proline contents increased up to a Cd level of 0.5 mg/kg in the soil but were significantly diminished at the highest Cd level of 1 mg/kg in the soil compared to those plants which were grown without the addition of Cd in the soil. The negative impacts of Cd injury were reduced by the application of FeSO₄ which increased plant growth and biomass, improved photosynthetic apparatus, antioxidant enzymes, minerals uptake together with diminished exudation of organic acids as well as oxidative stress indicators in roots and shoots of O. sativa by decreasing Cd retention in different plant parts. These results shed light on the effectiveness of FeSO₄ in improving the growth and upregulation of antioxidant enzyme activities of O. sativa in response to Cd stress. However, further studies at field levels are required to explore the mechanisms of FeSO₄-mediated reduction of the toxicity of not only Cd, but possibly also other heavy metals in plants.     

Role of foliar spray of plant growth regulators in improving photosynthetic pigments and metabolites in <i>Plantago ovata</i> (Psyllium) under salt stress–A field appraisal

Salinity is one of the major abiotic factors that limit the growth and productivity of plants. Foliar application of plant growth regulators (PGRs) may help plants ameliorate the negative impacts of salinity. Thus, a field experiment was conducted at the Botanical Garden University of Balochistan, Quetta, to explore the potential role of PGRs, i.e., moringa leaf extract (MLE; 10%), proline (PRO; 1 µM), salicylic acid (SA; 250 µM), and thiourea (TU; 10 mM) in ameliorating the impacts of salinity (120 mM) on Plantago ovata, an important medicinal plant. Salinity hampered plant photosynthetic pigments and metabolites but elevated oxidative parameters. However, foliar application of PGRs enhanced photosynthetic pigments, including Chl b (21.11%), carotenoids (57.87%) except Chl a, activated the defense mechanisms by restoring and enhancing the metabolites, i.e., soluble sugars (49.68%), soluble phenolics (33.34%), and proline (31.47%), significantly under salinity stress. Furthermore, foliar supplementation of PGRs under salt stress led to a decrease of about 43.02% and 43.27% in hydrogen peroxide and malondialdehyde content, respectively. Thus, PGRs can be recommended for improved photosynthetic efficiency and metabolite content that can help to get better yield under salt stress, with the best and most effective treatments being those of PRO and MLE to predominately ameliorate the harsh impacts of salinity.     

Ultrastructural analysis of sperm from the genus <i>Idarnes</i> (Hymenoptera: Chalcidoidea,Sycophaginae)

In this study, the sperm ultrastructure of three species of Idarnes genus was investigated using light and transmission electron microscopy. Spermatozoon morphology of the three species was similar to that of most Chalcidoidea, with helicoidally twisted nucleus and flagellum. The head region consists of an acrosome and a nucleus; the nucleus-flagellum transition region characterized by the presence of mitochondrial derivatives and the centriolar adjunct; a flagellum region, which includes the axoneme with microtubular arrangement 9 + 9 + 2 and two mitochondrial derivatives. However, the sperm of these three species exhibit features that discriminate one species from each other: (1) only one species, Idarnes sp. 2 (carme group) exhibited an extracellular sheath surrounding the anterior portion of the nucleus, which extends to the anterior region of the flagellum, but it did not present filaments; (2) the acrosome in the three species was quite different, Idarnes sp. 1 and Idarnes sp. 2 (carme group) has two compartments (acrosomal and subacrosomal vesicles) while Idarnes sp. 3 (flavicollis group) has a third compartment (perforatorium); (3) the centriolar adjunct elongated and its location among the mitochondrial derivatives is similar for the three species analyzed; (4) mitochondrial derivatives differ between the species, with triangular (Idarnes sp. 1 and sp. 3) and elongated or flat shaped (Idarnes sp. 2) appearance. These data shows that sperm structure may differ within the same genus and confirms the potential of these cells in phylogenetic and taxonomic analyses in the Chalcidoidea superfamily, as well as in Hymenoptera in general.     

Genome-wide identification,characterization, and expression analysis of aluminum-activated malate transporter genes (ALMTs) in <i>Gossypium hirsutum</i> L.

Aluminum-activated malate transporters (ALMT) are widely involved in plant growth and metabolic processes, including adaptation to acid soils, guard cell regulation, anion homeostasis, and seed development. Although ALMT genes have been identified in Arabidopsis, wheat, barley, and Lotus japonicus, little is known about its presence in Gossypium hirsutum L. In this study, ALMT gene recognition in diploid and tetraploid cotton were done using bioinformatics analysis that examined correlation between homology and evolution. Differentially regulated ALMT genetic profile in G. hirsutum was examined, using RNA sequencing and qRT-PCR, during six fiber developmental time-points, namely 5 d, 7 d, 10 d, 15 d, 20 d, and 25 d. We detected 36 ALMT genes in G. hirsutum, which were subsequently annotated and divided into seven sub-categories. Among these ALMT genes, 34 had uneven distribution across 14/26 chromosomes. Conserved domains and gene structure analysis indicated that ALMT genes were highly conserved and composed of exons and introns. The GhALMT gene expression profile at different DPA (days post anthesis) in different varieties of G. hirsutum is indicative of a crucial role of ALMT genes in fiber development in G. hirsutum. This study provides basis for advancements in the cloning and functional enhancements of ALMT genes in enhancing fiber development and augmenting high quality crop production.     

Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in <i>Escherichia coli</i> to promote L-tryptophan production

In this study, phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas (EMP) pathway and pentose phosphate (PP) pathway in Escherichia coli, thus increasing the L-tryptophan production. Firstly, the effects of disrupting EMP pathway on L-tryptophan production were studied, and the results indicated that the strain with deletion of phosphofructokinase A (i.e., E. coli JW-5 ΔpfkA) produced 23.4 ± 2.1 g/L of L-tryptophan production. However, deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth, especially deletion of glucosephosphate isomerase. Next, the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd) and thus increasing the L-tryptophan production (i.e., 26.5 ± 3.2 g/L vs. 21.7 ± 1.3 g/L) without obviously changing the cell growth (i.e., 0.41 h⁻¹ vs. 0.44 h⁻¹) as compared with the original strain JW-5. Finally, the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated. It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd (i.e., E. coli JW-5 zwf243 gnd361 ΔpfkA) produced 31.9 ± 2.7 g/L of L-tryptophan, which was 47.0% higher than that of strain JW-5. In addition, the glucose consumption rate of strain JW-5 zwf243 gnd361 ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5. The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.     

Characterization of multidrug-resistant <i>Klebsiella pneumoniae</i> isolated from the Chinese cobra <i>Naja atra</i> in a Beijing suburb

The emergence and spread of antibiotic resistance genes among Bacteria are a serious threat to global health. Their occurrence in animals which are in contact with humans is also important. The Chinese cobra (Naja atra, Elapidae), though a highly venomous species, is appreciated as food and as a source of materials used in traditional Chinese medicine. We are here reporting the isolation of multidrug-resistant Klebsiella pneumoniae (Enterobacteriaceae) from the lung of Naja atra, obtained from a snake farm in a Beijing suburb. Our study analyzed, using gene sequencing, the occurrence of antibiotic resistance genes (ARGs) in three K. pneumoniae isolates from two snakes. In addition, bacterial clones were identified by biochemical tests and phylogenetic analysis. Tests of antimicrobial susceptibility showed that all K. pneumoniae isolates were resistant to a host of antibiotics (piperacillin, cefazolin, gentamicin, tetracycline, doxycyclin, ciprofloxacin, levofloxacin, lomefloxacin, ofloxacin, norfloxacin, nalidixic acid, chloramphenicol, nitrofurantoin, sulfamethoxazole, and sulfamethoxazole/trimethoprim) but were susceptible to cefotaxime, cefixime, aztreonam, bramycin, amikacin, kanamycin, netilmicin, and streptomycin. Eighteen ARGs were detected in total DNA extracted from the isolates. Results showed three quinolone resistance genes (oqxA, oqxB, qnrB), the gyrA gene that confers resistance to beta-lactam antibiotics, and the emerging aac(3)-II gene that confers resistance to aminoglycosides. K. pneumoniae is an important opportunistic human pathogen and the emergence of multidrug-resistant K. pneumoniae in N. atra suggests the increasing risk of pathogen transmission between humans, livestock, and wildlife. Given the close association between foodborne pathogenic microorganisms and humans, it is key factor to identify these antibiotic resistance genes profile thereby minimize the risk of K. pneumoniae transmission.     

Response of <i>MaHMA2</i> gene expression and stress tolerance to zinc stress in mulberry (<i>Morus alba</i> L.)

HMA2 (heavy metal ATPase 2) plays a crucial role in extracellular and intracellular Zn²⁺ transport across biomembranes, maintaining ion homeostasis, and playing an important role in the normal physiological metabolism, growth, and development of plants. In our study, a novel HMA2 gene, named MaHMA2, was isolated and cloned from white mulberry (Morus alba L.). The gene sequence obtained was 1,342 bp long, with an open reading frame of 1,194 bp, encoding a protein of 397 amino acids, with a predicted molecular mass of 42.852 kD and an isoelectric point of 7.53. This protein belonged to the PIB-type ATPase transport protein family. We analyzed the expression of the MaHMA2 gene by quantitative real-time PCR. The results showed that the level of MaHMA2 gene expression decreased to a Zn concentration of 800 mg/kg. Malondialdehyde and proline levels increased and responded to increasing Zn when the MaHMA2 gene was silenced, whereas the activities of peroxidase and superoxide dismutase tended to increase in response to increasing Zn²⁺ ion stress concentrations but were lower in the gene-silenced plants. These findings suggested that the MaHMA2 gene played an active role in the tolerance response of mulberry to Zn stress.     

Overexpression of a sugarcane<i> ScCaM</i> gene negatively regulates salinity and drought stress responses in transgenic <i>Arabidopsis thaliana</i>

Calmodulin (CaM) proteins play a key role in signal transduction under various stresses. In the present study, the effects of a sugarcane ScCaM gene (NCBI accession number: GQ246454) on drought and salt stress tolerance in transgenic Arabidopsis thaliana and Escherichia coli cells were evaluated. The results demonstrated a significant negative role of ScCaM in the drought and salt stress tolerance of transgenic lines of A. thaliana, as indicated by the phenotypes. In addition, the expression of AtP5CS and AtRD29A, two genes tightly related to stress resistance, was significantly lower in the overexpression lines than in the wild type. The growth of E. coli BL21 cells expressing ScCaM showed weaker tolerance under mannitol and NaCl stress. Taken together, this study revealed that the ScCaM gene plays a negative regulatory role in both mannitol and NaCl stresses, and it possibly exerts protective mechanisms common in both prokaryotes and eukaryotes under stress conditions.     

Expression analysis of <i>OsSERK</i>, <i>OsLEC1</i> and <i>OsWOX4</i> genes in rice (<i>Oryza</i> sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.     

Structural characterization of four <i>Rhododendron</i> spp. chloroplast genomes and comparative analyses with other azaleas

Azalea is a general designation of Rhododendron in the Ericaceae family. Rhododendron not only has high ornamental value but also has application value in ecological protection, medicine, and scientific research. In this study, we used Illumina and PacBio sequencing to assemble and annotate the entire chloroplast genomes (cp genomes) of four Rhododendron species. The chloroplast genomes of R. concinnum, R. henanense subsp. lingbaoense, R. micranthum, and R. simsii were assembled into 207,236, 208,015, 207,233, and 206,912 bp, respectively. All chloroplast genomes contain eight rRNA genes, with either 88 or 89 protein-coding genes. The four cp genomes were compared and analyzed by bioinformatics, and the phylogenetic analysis based on chloroplast genomes of 26 species of Ericaceae, Actinidiaceae, and Primulaceae under Ericales was conducted. A comparison of the linear structure of cp genomes of four Rhododendron showed that there were substantial sequence similarities in coding regions, but high differences in non-coding regions. A phylogenetic analysis, based on chloroplast whole genome sequences, showed that all Rhododendron species are in the clade Ericaceae. This study provides valuable genetic information for the study of population genetics and evolutionary relationships in Rhododendron and other azalea species.