Abstract:

Abstract:The xanthine dehydrogenase (XDH), a molybdenum-containing oxidoreductase belonging to the molybdenum hydroxylase flavoprotein family, has been identified in a variety of eukaryotes, bacteria, and archaea. XDH catalyzes the conversion of xanthine and hypoxanthine to uric acid, which then undergoes further reactions to form allantoin and allantoic acid. Studies have shown that XDH plays a role in various metabolic processes, including purine metabolism, nitrogen metabolism, hormone metabolism, reactive oxygen species metabolism, and responses to biotic and abiotic stresses. Here, we introduced the structural features, metabolic pathways, and biological functions of XDH. In addition, we summarized the research progress in XDH to give insights into the molecular mechanism of purine metabolism in plants and prospected the application of XDH, with the aim to facilitate future research on the growth, development, and stress resistance of crops.

Abstract:Calcium-dependent protein kinases (CDPKs/CPKs) are members of the Ca²⁺-sensitive Ser/Thr protein kinase family and play a crucial role in plant growth and development and responses to abiotic stress. CDPKs are capable of rapidly sensing changes in intracellular Ca²⁺ signals and recognizing and phosphorylating specific substrates, thereby transmitting and amplifying Ca²⁺ signal cascades downstream. They are involved in plant responses to stress conditions such as drought, saline-alkali stress, and injuries and regulate plant growth and development, gene expression, ion channel activity, and stomatal movement. The autophosphorylation of CDPKs can affect their activities and substrate specificity. CDPKs have the ability to bind to and phosphorylate multiple substrates. In addition to participating in respiratory burst oxidase homolog (RBOH), mitogen-activated protein kinase (MAPK), and plant hormone signaling pathways, CDPKs can also bind to 14-3-3 proteins, which enables the regulation of plant responses to stress and promotes plant growth and development. This paper summarized the research findings on the discovery, structure, classification, and roles of CDPKs in plant responses to stress and proposed the future research directions, aiming to provide the genetic resources and a theoretical basis for improving the stress tolerance of crops.

Abstract:Camellia oleifera is an important woody oil crop in China, and its seed oil has a high economic value. Anthracnose, one of the main diseases in C. oleifera, occurs in a wide range in the production areas, limiting the growth and development of plants and causing serious losses of oil production. With the rapid development of the C. oleifera industry in recent years, great progress has been achieved in the research on anthracnose in C. oleifera. This paper summarized the resistance mechanisms, the mining of resistance genes, and the evaluation of resistant germplasm resources, aiming to provide a theoretical basis for the prevention and control of anthracnose and the breeding of C. oleifera germplasm with resistance to anthracnose.

Abstract:Sorghum (Sorghum bicolor) is a significant crop serving food, energy, feed, and industrial raw materials, featuring extensive growth adaptability and diverse utility values. Despite the achievements in sorghum breeding in the last decades, conventional breeding methods still confront challenges such as lengthy breeding cycles, low efficiency, and complex genetic backgrounds. With the rapid advancement of molecular biology, genetics, and bioinformatics, molecular breeding has carved new pathways for enhancing sorghum yield and quality. This article reviews the molecular basic research progress in the key agronomic and adaptive traits of sorghum, including grain yield, grain quality, flowering time, plant height, tillering, stress resistance, and male sterility, and discusses future research priorities, offering novel insights and approaches for sorghum breeding.

Abstract:Plant endophytes spend at least part of their life cycle in plants without causing diseases in the hosts, being the microbial resources with rich species and diverse functions. With the advancement in sequencing technology, the microbiological study of endophytes has become increasingly intensive. Being praised for the targeted validation and low cost, Sanger sequencing has been preferred by researchers. However, Sanger sequencing is no longer suitable for deeper genomic study of endophytes due to the low throughput. In this paper, we briefly summarize the research history of endophytes, review the applications of next-generation sequencing characterized by high throughput and third-generation sequencing (single-molecule real-time sequencing) in the research on endophytes, and summarize the research results of different sequencing technologies. Furthermore, we summarize the advantages and limitations of different sequencing technologies and discuss how to choose the appropriate sequencing technology according to the research needs. This review provides a reference for researchers to further explore the potential value of plant endophytes.

Abstract:Microbial resources are diverse and abundant, serving as a crucial source for the discovery of bioactive substances. However, as the research on microbial secondary metabolites deepens, discovering new microorganisms and novel bioactive secondary metabolites from conventional environments is becoming increasingly challenging. The microorganisms inhabiting extreme environments have unique physiological characteristics and can develop distinctive metabolic pathways, holding immense potential for producing chemically diverse and novel bioactive secondary metabolites. This article comprehensively overviews the recent advancements in the isolation strategies of microorganisms from extreme environments and the research progress in their bioactive substances, including antimicrobial, anticancer, and antioxidant compounds. This review aims to serve as a reference for the development and utilization and the related studies of the microbial resources in extreme environments.

Abstract:Heavy metal pollution in water has become a global environmental problem, threatening aquatic ecosystems and human health. Physical and chemical methods can effectively remove heavy metal pollutants, while their applications are limited due to the high costs, complex operation, and susceptibility to secondary pollution. Bioremediation is the most promising method for eliminating toxic pollutants. Microorganisms including bacteria, fungi, and algae can convert toxic heavy metals into less toxic forms, which has become an effective and environmentally friendly solution for the remediation of heavy metal pollution in water environments. This paper expounds the toxicity and mechanism of heavy metal pollution, microbial remediation mechanisms, and primary microbial remediation strategies, providing a reference for the removal or reduction of metal pollutants in water environments as well as the development of related technologies.

Abstract:Nitrate (NO₃^–-N) is a common inorganic nitrogen pollutant in water. Excessive NO₃^–-N can lead to water eutrophication and threaten human health. Nanoscale zero-valent iron (nZVI) has attracted much attention in NO₃^–-N removal due to its high specific surface and excellent electron donor properties. The combination of nZVI and denitrifying bacteria (DNB) demonstrates high efficiency in converting NO₃^–-N into N₂. This approach not only substantially enhances the removal rate of NO₃^–-N but also exhibits superior environmental sustainability compared with conventional chemical denitrification methods. Accordingly, it holds substantial promise for mitigating NO₃^–-N pollution and warrants further exploration in the pollution control. Therefore, it is necessary to understand the interaction mechanism between nZVI and DNB for NO₃^–-N removal. This paper details the factors affecting the removal of NO₃^–-N by nZVI combined with DNB, reviews the latest research progress in this field, elaborates on the interaction mechanism between nZVI and DNB for NO₃^–-N removal, and discusses the challenges and future research directions of NO₃^–-N removal by nZVI combined with DNB. This review aims to provide a theoretical basis for the development of efficient approaches for the remediation of NO₃^–-N pollution.

Abstract:Microbial electrochemical technology (MET) represents a novel approach demonstrating promising application prospects in emerging strategic industries such as environment protection, energy saving, and sustainable energy production. Among different METs, microbial electrochemical snorkels (MES) are praised for the simple design, high flexibility, and low costs. Several pilot MESs have been employed to mitigate environmental issues in European and American countries. Despite the rapid development, only one review article on MES has been published so far. Here we review the latest achievements in this field and introduce the principles, structures, functions, and applications of MESs. Moreover, we summarize the key challenges and the future research areas in this field, aiming to give insights into the research on MESs and other METs and improve the applications of such technologies.

Abstract:Rice (Oryza sativa L.) is a major food crop and increasing rice yield is the primary objective of rice research. Photosynthesis and nitrogen utilization efficiency directly affect the tiller number of rice, which affects the yield of rice. In this study, a stable yellow leaf and less tillering rice mutant yllt10 (yellow leaf and less tillering 10) was obtained by heavy-ion beam mutagenesis of rice variety ‘Ke-fu-geng 7’. Compared with the wild type, yllt10 showed reduced chlorophyll content, decreased photosynthesis rate, and abnormal chloroplast structure. The genetic analysis indicated that the phenotype of yllt10 was controlled by a single recessive nuclear gene. Map-based cloning localized YLLT10 between two molecular markers J4 and J5 on chromosome 10. The sequencing of candidate genes within this interval revealed that YLLT10 was an allelic mutation of CAO1/PGL with a single base deletion in the first exon resulting in the frame shift mutation of CAO1/PGL, and YLLT10 was a new allelic variation of CAO1/PGL. The mutant yllt10 was insensitive to changes in nitrogen concentration when being incubated with different nitrogen concentrations. YLLT10 controls leaf color and tiller number and affects photosynthesis and yield of rice. The study of this gene provides a theoretical basis for molecular breeding of rice.

Abstract:Vitamin B6 (VB6), as an essential component involved in numerous biological activities of animals and plants, reflects the nutritional quality of cereal crops such as rice. Few studies have been conducted to mine the genes controlling the VB6 content in rice grains, and the available studies remain to be deepened. In this study, the recombinant inbred lines created from parents ‘HZ’ and ‘Nekken2’ served as the experimental materials. Based on QTL mapping, the initial screening identified ten candidate genes. The expression levels of LOC_Os01g52450, LOC_Os01g52500, LOC_Os05g09500, LOC_Os05g09440, LOC_Os05g20570, and LOC_Os05g36270 showed significant differences between the parents. According to the gene expression and parental VB6 content, we hypothesized LOC_Os05g09500 as the key gene affecting the VB6 content in rice grains, and the high expression of this gene significantly influenced the VB6 content. The results of this study fill a gap in the QTL mapping on the VB6 content of rice grains and provide theoretical support for elucidating the molecular genetic mechanisms and cloning the related genes of VB6 synthesis in rice. In addition, the findings have significant implications for identifying, screening, and breeding new rice cultivars with high VB6 content.

Abstract:Improving the nitrogen use efficiency is an effective way to increase the yield of rice, and maintaining carbon-nitrogen balance is essential for the normal growth and development of rice. To investigate the impact of the sucrose transporter protein OsSTP1 on nitrogen absorption in rice, in this study, we constructed transgenic lines overexpressing the sucrose transporter gene OsSTP1 (OsSTP1-OE1, OsSTP1-OE2) and mutant transgenic lines (osstp1-1, osstp1-2) from the wild type TB309. Further, we conducted a hydroponic experiment with four nitrogen supply levels of free nitrogen (FN, 0 mg/L), low nitrogen (LN, 10 mg/L), normal nitrogen (NN, 40 mg/L), and high nitrogen (HN, 80 mg/L) to study the responses of each line to different nitrogen supply levels during the seedling stage. The results showed that compared with the wild type and mutant lines in the LN group, the OsSTP1-overexpressing lines exhibited significantly increased biomass, root length, and plant height, decreased soluble sugar content in the leaves, and increased soluble sugar content in the roots. The results indicate that the soluble sugars produced by leaf photosynthesis are transported to the roots through the phloem to promote root growth and nitrogen uptake, thus increasing the aboveground biomass. This study has identified that OsSTP1 can affect the long-distance transport of carbohydrates from source to sink to promote root growth, ultimately influencing rice’s absorption and accumulation of nitrogen, improving nitrogen use efficiency and providing reference for reducing nitrogen fertilizer application.

Abstract:The Rho GDP dissociation inhibitor gene OsRhoGDI1 was isolated as the putative partner of OsRac5 from rice young panicles by the yeast two-hybrid screening, but the characterization and functions of the gene remains unknown. In this study, on the basis of the bioinformatics analysis, expression profile chip mining and RT-qPCR were employed to analyze the expression pattern of OsRhoGDI1, and it was found that the gene was widely expressed in various tissues of rice, especially with high abundance during the development of young panicles, and its expression was regulated by abscisic acid (ABA), brassinolide (BL), salicylic acid (SA), indole-3-acetic acid (IAA), gibberellin (GA) and high salinity. Subcellular localization identification showed that OsRhoGDI1 was distributed in the cytomembrane, cytoplasm, and nucleus. Bimolecular fluorescence completion (BiFC) was further performed to identify the in vivo interaction between OsRhoGDI1 and OsRac5, the results suggested that the interaction occurred in the nucleus, cytomembrane, and cytoplasm. In addition, OsRhoGDI1 interacted with OsRac1 in the cytomembrane. Our results indicated that OsRhoGDI1 may be involved in rice panicle development, and participate in regulating various biological processes such as rice growth and development, hormone response, and abiotic stress by regulating the activities of different Rho/Rop proteins via interactions.

Abstract:DNA fingerprinting can reveal the genetic diversity of Elaeagnus angustifolia germplasm resources and clarify the source and genetic background of E. angustifolia germplasm, which are the preconditions for the breeding of new varieties, the identification and protection of germplasm resources, and the comprehensive development of the E. angustifolia industry considering both ecological and economic benefits. We employed 11 pairs of primers with high polymorphism, clear bands, and high reproducibility to analyze the genetic diversity of 150 E. angustifolia germplasm accessions from Gansu and Beijing by the simple sequence repeat (SSR) molecular markers. We then employed the unweighted pair-group method with arithmetic means (UPGMA) to perform the cluster analysis based on genetic distance and analyzed the genetic structure of the 150 germplasm accessions based on a Bayesian model in Structure v2.3.3. The genetic diversity analysis revealed the mean number of alleles (Na) of 7.636 4, the mean number of effective alleles (Ne) of 2.832 6, the mean Shannon genetic diversity index (I) of 1.178 1, the mean Nei’s gene diversity index (H) of 0.582 1, the mean observed heterozygosity (Ho) of 0.489 9, the mean expected heterozygosity (He) of 0.584 0, the mean polymorphism information content (PIC) of 0.535 4, and the mean genetic similarity (GS) of 0.831 5. These results suggested that the E. angustifolia germplasm resources we studied exhibited significant genetic differences and rich genetic diversity. The cluster analysis revealed that the tested materials can be classified into 3 groups, with the main genetic distance (GD) of 0.422 9. The clustering results were not completely consistent with the geographic origin. The population structure analysis classified the germplasm accessions into 2 populations. We used 8 pairs of primers with high PIC to construct the fingerprints of 150 E. angustifolia germplasm accessions. The present study successfully constructs the DNA fingerprints and clarified the genetic relationship of the E. angustifolia germplasm resources in Gansu and Beijing, providing a theoretical basis for germplasm resource identification, breeding of elite varieties, application in gardening, and molecular-assisted breeding of E. angustifolia.

Abstract:With unique advantages, gibberellin GA₄ has broad application prospects. To explore the regulatory mechanism for the biosynthesis of GA₄, we combined liquid chromatography- mass spectrometry (LC-MS)-based metabolomics with principal component analysis (principal component analysis, PCA) and partial least squares-discriminant analysis (PLS-DA) to screen and identify the differential metabolites between the GA₄-producing strains S (industrial high-yield strain CGMCC 17793) and wild-type strain Y (NRRL 13620) of Gibberella fujikuroi fermented for the same time and the differential metabolites of strain S fermented for different time periods. KEGG and MBROLE 2.0 were used to analyze the metabolic pathways involving the differential metabolites. The results showed that compared with strain Y, strain S significantly upregulated and downregulated 107 and 66, 136 and 47, and 94 and 65 metabolites on days 3, 6, and 9, respectively. Compared with that on day 3 of fermentation, strain S upregulated 29 metabolites and downregulated 40 metabolites on day 6 and upregulated 52 metabolites and downregulated 67 metabolites on day 9. The differential metabolites between strain S and strain Y after fermentation for the same time were mainly enriched in amino acid metabolism, tricarboxylic acid (TCA) cycle, and terpenoid biosynthesis. The differential metabolites of strain S after fermentation for different time periods were mainly enriched in amino acid and sugar metabolism pathways. Pathway annotation results indicated that strain S increased the production of acetyl-CoA by promoting amino acid and sugar metabolism and TCA cycle, thereby enhancing the mevalonic acid pathway and increasing the content of isopentenyl pyrophosphate (IPP), a precursor for the synthesis of terpenoids, which ultimately led to increased GA₄ production. This study explored the metabolic rules of Gibberella fujikuroi GA₄, providing a theoretical basis for regulating Gibberella fujikuroi to improve GA₄ production.

Abstract:Terpene synthases (TPSs) play a crucial role in the synthesis of terpenoids that contribute to the scent profiles of flowers. However, few studies report the genome-wide analysis of TPSs gene in Jasminum sambac var. Fuzhou bifoliatum and their expression pattern in response to methyl jasmonate (MeJA). In this study, we employed bioinformatics tools for genome-wide analysis of the J. sambac TPS (DJTPS) gene family and determined the physical and chemical properties, subcellular location, protein-protein interactions, phylogenetic relationship, subfamily classification, chromosomal location and collinearity, gene structure, conserved motifs, and promoter cis-acting elements. The expression patterns of DJTPSs in different tissues and in response to MeJA treatment were analyzed based on the transcriptome data combined with quantitative real-time PCR (qRT-PCR). We identified 32 intact DJTPS genes in the genome of J. sambac, which presented uneven distribution across nine chromosomes. All the deduced proteins were hydrophilic, predominantly localized in the cytoplasm. The phylogenetic analysis classified the DJTPS genes into five subfamilies: TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g. The results of the collinearity analysis showed a total of 10 sets of replication events in DJTPSs, most of which underwent purifying selection. A comparative analysis of TPS homologous gene pairs was performed among J. sambac var. Fuzhou bifoliatum and other six species, which revealed different number of homologous gene pairs. The number of exons and motifs was conserved within the same subfamily. DJTPS genes carried multiple elements that may be involved in the response to MeJA. In addition, the transcriptome and qRT-PCR data unveiled that several TPS genes exhibited tissue-specific expression patterns, and the genes with specific expression in flowers were the most. Upon exposure to MeJA, 14 TPS genes showcased upregulated expression 5 h or 6 h post-treatment, and DJTPS03, DJTPS04 and DJTPS21 showed significantly increased expression levels after MeJA treatment. This study provides preliminary evidence that MeJA possesses the ability to enhance the expression of DJTPS genes during the critical flowering stage, which will facilitate the synthesis of terpenoids and improve the quality of floral fragrance.

Abstract:Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean (Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a-Gs5PTase8 and pGEX4T1-Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP₃ [inositol(1,4,5)trisphosphate], IP₄ [inositol(1,3,4,5)tetrakisphosphate], PI(4,5)P₂ [phosphatidylinositol(4,5) bisphosphate] and PI(3,4,5)P₃ [phosphatidylinositol(3,4,5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.

Abstract:The zinc uptake regulator (Zur) has highly conserved sequences in the plant pathogen Xanthomonas, while its functions are diverse in different strains or races. To elucidate the functions of Zur in Xanthomonas axonopodis pv. glycines (Xag), we constructed a zur-deleted mutant (Δzur) by homologous recombination. Compared with the wild type, Δzur demonstrated reduced pathogenicity in the host soybean and reduced ability to trigger hypersensitive responses (HR) in nonhosts such as tobacco, tomato, chili pepper, and eggplant. Additionally, the deletion of zur significantly enhanced Xag’s sensitivity to Zn²⁺, Fe³⁺, and Cu²⁺, induced an imbalance in intracellular zinc homeostasis, decreased extracellular polysaccharide (EPS) production, and down-regulated the expression of extracellular hydrolases (cellulase, endo-glucanase, amylase, and protease). Functional complementation restored the defective properties of Δzur to the wild-type levels. The qRT-PCR results showed that zur expression was remarkably induced by Zn²⁺. Moreover, the deletion of zur evidently reduced the expression levels of hrp representative genes (hrpB1, hrpD6, hrpE, hrcV, and hrcC), extracellular hydrolase encoding genes (engXCA, egl2, pro1, pro2, pro8, pro11, and alpha1), and EPS synthesis genes (gumB, gumD, gumK, gumM, gumG, and gumH) relative to the wild type. In summary, the results suggested that Zur may be involved in pathogenicity in the host soybean and in triggering HR in nonhosts of Xag by regulating the synthesis of virulence factors and the expression of hrp genes. This laid a foundation for further analysis of the mechanism of Zur in Xanthomonas-plant interaction.

Abstract:Abscisic acid insensitive 5 (ABI5) is a basic leucine zipper transcription factor regulating ABA-mediated seed germination, and TaABI5 is closely related to the pre-harvest sprouting in wheat. Studies have shown that TaABI5-D3, one of multiple copies of TaABI5 gene, encodes the intact TaABI5 protein. In this study, we constructed the prokaryotic expression vector pET-28a-TaABI5-D3 for expression of TaABI5-D3 in Escherichia coli and obtained the purified recombinant protein His-TaABI5-D3. This protein existed in the form of inclusion bodies, with the best expression induced by incubation with 0.6 mmol/L IPTG at 16 ℃, 150 r/min overnight (for 12 h). Subsequently, the purified His-TaABI5-D3 was injected into Balb/C mice for the preparation of polyclonal antibodies. Western blotting analysis indicated that the polyclonal antibody had relatively high specificity, laying foundations for clarification of the function of TaABI5 protein in wheat.

Abstract:To explore the role and metabolic differences of plant hormones in regulating the flower bud size of daylily, we collected the flower buds from two daylily varieties ‘Datong Huanghua’ and ‘Dongbei Huanghua’ at the young bud, green, yellowing, and yellow stages for transcriptome sequencing. The differentially expressed genes (DEGs) were screened, and the exogenous plant hormone spraying experiments were conducted. A total of 199 DEGs related to the biosynthesis and metabolism of plant hormones was screened out at different flower development stages of ‘Datong Huanghua’ and ‘Dongbei Huanghua’. These genes regulated the biosynthesis and metabolism of six plant hormones: abscisic acid, gibberellin, auxin, jasmonate, cytokinin, and ethylene. The DEGs associated with auxin were the most, which suggested that auxin played a role in regulating flower bud development. The auxin response factor (ARF) presented up-regulated expression at all the four stages of flower bud development, indicating that ARF played a positive regulatory role throughout the flower bud development of daylily. The experiments with exogenous spraying of six hormones further verified that indole-3-acetic acid (IAA) significantly promoted the growth and increased the nutrient content in the flower buds of ‘Datong Huanghua’, suggesting that IAA played a role in regulating flower bud development. Our results laid a theoretical foundation for probing into the regulatory mechanism of flower bud development of ‘Datong Huanghua’ and ‘Dongbei Huanghua’.

Abstract:The Toll and immune deficiency (IMD) signaling pathways in insects are highly conserved in evolution and regulate the expression of antimicrobial peptides (AMPs) and other immune-related genes mainly through nuclear factor-kappa B (NF-κB) transcription factors. However, the differences of NF-κB transcription factors Rels and Relish in the expression regulation of AMPs and other immune-related genes in silkworm (Bombyx mori) have not been systematically reported. In this study, the BmRelA, BmRelB and BmRelish1 genes were cloned and their eukaryotic cell overexpression vectors were constructed. After the recombinant vectors were transfected into BmE and BmN cells, the expression of AMPs and immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that the expression of AMP genes Defensin2 and Gloverin2 was mainly regulated by Relish, the expression of Moricin was mainly regulated by RelA and RelB, and the expression of other AMP genes was jointly regulated by both. In addition, the expression levels of peptidoglycan recognition proteins (PGRPs), β-1,3-glucan recognition proteins (βGRPs), lysozymes (Lys) and lysozyme-like proteins (LLPs), and nitric oxide synthase (NOS) were up-regulated to varying degrees in different cell lines in response to RelA, RelB and Relish1, suggesting that the expression of these immune-molecules was also regulated by Toll or IMD pathways in silkworm. Compared with the regulatory specificity of transcription factors in Drosophila Toll and IMD signaling pathways on the expression of AMPs, this study found that the regulatory patterns of Rels and Relish1 on the expression of AMPs in silkworm are more complex, which provides an experimental basis for further analysis of the effect mechanism and feedback mechanism of Toll and IMD pathways in insects.

Abstract:In order to mitigate the adverse effects of madrassa poisoning disease on our livestock industry and to fully utilize the potential pasture resources, biodegradation of locoweed can remove swainsonine, the major toxic component of locoweed, so that the locoweed can be used as high-quality forage. Arthrobacter nitroquajacolicus HW08 can stably and efficiently degrade swainsonine. In this study, Lactococcus lactis, as a food-grade microorganism, was used as a vector to express four key degradation genes from A. nitroquajacolicus HW08. Subsequently, liquid chromatography was employed to evaluate the swainsonine-degrading performance. The crude enzyme solution extracted from the L. lactis strain transformed with the ethanol dehydrogenase gene A1R6C3 degraded 323.4 μg of swainsonine in 24 h at 30 ℃. The crude enzyme solutions from the L. lactis strains transformed with the genes encoding glutathione synthase, esterase/acyl hydrolase, and glycosyltransferase did not show any degradation ability for swainsonine when being used alone but degraded about 140.5 μg of swainsonine when being used in mixture. The findings will help the clinical promotion of swainsonine-degrading engineering strains and provide new research ideas for the prevention and treatment of swainsonine poisoning in animals and the detoxification and utilization of locoweed.

Abstract:To clarify the roles of the heat shock protein gene Hsp70 in the sclerotial formation and pathogenicity of Sclerotinia sclerotiorum, we employed reverse transcription PCR (RT-PCR) to clone Hsp70 from S. sclerotiorum and performed sequence analysis. Quantitative real-time PCR (qRT-PCR) was employed to determine the relative expression levels of Hsp70 at different growth stages and under the stress induced by cyclic adenosine monophosphate (cAMP) and low and high temperatures. The thermal stability of Hsp70 was measured. The Agrobacterium-mediated method was employed to construct the Hsp70-silenced strain. The pathogenicity and fungicide resistance of strains were tested by inoculation in detached rapeseed leaves and cultivation in the media containing procymidone and thiophanate-methyl, respectively. The results showed that the cloned Hsp70 had a total length of 1 890 bp and close relationship with the Hsp70 gene of Ciborinia. Hsp70 showcased the highest expression level in sclerotia, which was more than 30 times higher than that in hyphae. The cAMP stress significantly induced the expression of Hsp70. The expression level of Hsp70 showed an increasing-decreasing-increasing trend at 40 ℃ and no significant change at 4 ℃. Recombinant strain with high expression of Hsp70 showed good thermal stability. The Hsp70-silenced transformant did not form sclerotia, with decreased pathogenicity and fungicide resistance. This study reveals that Hsp70 plays an important role in the sclerotial formation and stress resistance of S. sclerotium, providing reference for further in-depth research on the biological roles of Hsp70 in S. sclerotium.

Abstract:Algae has been proven to have the potential to be efficient biosorbents in the detection and remediation of heavy metal pollution such as cadmium in the environment. This study aims to enhance the cadmium adsorption capacity of Chlamydomonas reinhardtii by expressing the cadmium-binding protein CADR on the cell wall by the surface display technology. Firstly, the golden gate technique was employed to construct the transformation vector PET-X-CADR, which anchored CADR to the cell wall with the cell wall protein GP1. The high-throughput screening with the fluorescence signal of the fusion tag YFP resulted in three engineered algal strains with high expression of CADR on the cell wall. Physiological experiments demonstrated that the CADR displayed on the cell wall did not affect the growth of the engineered algal strains exposed to cadmium with the concentration below 200 μmol/L. In the presence of 200 μmol/L cadmium, the growth rates of CADR-engineered algal strains were three times as that of the wild-type, indicating stronger tolerance of the CADR-engineered algae to cadmium. The protein lyase GLE released during the mating of Chlamydomonas was used to isolate the cell walls of wild-type and engineered strains, the cadmium content of which was compared. The results showed that the cell wall of the engineered strain exhibited an increase of 33% in cadmium adsorption capacity. This study gives insights into the application of algae in the management of cadmium pollution in the environment, especially in the recycling of heavy metals from the environment.

Abstract:Polyethylene terephthalate (PET) is a largely-produced polymer worldwide. However, its extensive waste generation and resistance to degradation pose significant environmental concerns. Consequently, there is considerable interest in researching enzymatic degradation of PET. Relevant studies have shown that the addition of a carbohydrate binding module (CBM) can increase the affinity between the enzyme and the substrate, enhancing the enzyme’s degradation ability. In order to develop more efficient PET hydrolytic enzymes, this study introduced carbohydrate binding domains (CBMs) from different families with different substrate affinities into the PET-degrading enzyme LCC-ICCG. High crystallinity PET powder and amorphous PET film were used as substrates to characterize the degradation efficiency of the modified enzymes, aiming to explore the enzyme with the optimal degradation ability. The results showed that the fusion of type B CBM reduced the degradation rate and T_m value of the enzyme towards PET, while the introduction of type A and type C CBMs significantly improved the degradation rate of the enzyme towards the film-like substrate. The degradation rate and T_m value of PET were also enhanced, especially with the fusion enzyme LCC-ICCG-CBM9-2, which showed an over 10-fold increase in the degradation rate compared to the original enzyme LCC-ICCG. Therefore, this study demonstrates that by introducing type A and type C CBMs, the degradation rate and thermal stability of LCC-ICCG towards film-like PET can be improved, addressing the issue of its low activity and enabling more effective PET degradation. This research provides support for plastic degradation technology and contributes to environmental conservation efforts.

Abstract:To address the potential pollution caused by the carcinogen 1,4-dioxane in aquatic environments, we isolated a highly efficient 1,4-dioxane-degrading bacterial strain, designated as DXTK-010, from the groundwater contaminated by 1,4-dioxane. According to the morphological characteristics, the phylogenetic tree established based on the 16S rRNA gene sequence, and the whole genome sequence, we identified DXTK-010 as Aminobacter aminovorans. This strain demonstrated robust degradation capacity within a temperature range of 20 ℃ to 37 ℃ and a pH range of 5.0 to 8.0. Furthermore, single-factor experiments indicated the optimal degradation conditions at 30 ℃ and pH 7.5. Under the optimal conditions, the strain completely degraded 200 mg/L of 1,4-dioxane within 24 h, achieving a maximum degradation rate of 9.367 mg/(L·h). The Monod equation was adopted to fit the degradation kinetics of 1,4-dioxane at different initial concentrations, which revealed a maximum specific degradation rate of 0.224 mg 1,4-dioxane/(mg protein·h), a half-saturation constant (K_s) of 41.350 mg/L, and a cell yield of 0.130 mg protein/(mg 1,4-dioxane). Whole genome sequencing revealed a circular chromosome and three plasmids within DXTK-010. Functional gene annotation and analysis underscored the significance of the propane monooxygenase gene cluster and alcohol dehydrogenase gene in facilitating the efficient degradation of 1,4-dioxane by this strain. DXTK-010 outperformed the existing degraders for 1,4-dioxane, expanding the strain resources for the bioremediation of 1,4-dioxane pollution. This study provides a theoretical basis for the practical application of DXTK-010 in the remediation of 1,4-dioxane pollution.

Abstract:In order to assess the antibiotic resistance of Escherichia coli and its transmission risk in a rice-frog coculture system in Zhejiang Province, this study collected E. coli from isolated soil, field water, and frog feces from the rice-frog coculture systems in four different areas of Zhejiang Province. The collected isolates were identified by 16S rRNA sequencing, while their antibiotic-resistant phenotypes were determined by Kirby-Bauer (K-B) method. PCR was used to identify the antibiotic-resistant genotypes and integrons, while conjugative transfer experiments were used to assess resistance transmission characteristics. The results showed a high prevalence of antibiotic resistance in the 82 strains of E. coli tested, primarily against tetracycline, sulfisoxazole, amoxicillin, and erythromycin. Most of these strains exhibited multidrug resistance, with the Fuyang area demonstrating the highest resistance rate compared to the other three areas. Further PCR analysis identified the sul1 gene as the most frequently detected resistance gene (63.41%), followed by bla_TEM, tetA, and tetB. Among the 16 antibiotic resistance genes (ARGs) detected, the Fuyang isolates consistencly exhibited higher detection rate of 9 ARGs in comparison to the other regions. Additionally, the integrase gene intI1 displayed the highest detection rate, with 14 strains (34.15%) of integrase-positive bacteria carrying gene cassettes. Four different gene cassette compositions were observed, with dfrA1-aadA1 and dfrA17-aadA5 being the most common combinations. Conjugative transfer experiments demonstrated successful transfer of gene cassettes in 4 out of 14 donor bacteria, with conjugation transfer frequencies ranging from 4.32×10⁻⁵ to 7.13×10⁻⁴. These findings revealed the severity of resistance in the Fuyang area among the four regions. Integrons play a significant role in mediating the resistance to multiple antibiotics in E. coli, facilitating the potential spread of resistance gene cassettes between different bacteria. Overall, this study provides valuable insights into the resistance status and transmission characteristics of E. coli in the rice-frog coculture system in Zhejiang Province, providing a theoretical basis for ensuring the food safety of rice crops.

Abstract:As two efficient broad-spectrum sterilizing agents, triclosan (TCS) and triclocarban (TCC) are widely used, especially during the COVID-19 pandemic. The health risks caused by secondary pollution of TCS and TCC have aroused wide concern. Because of the similar mother nucleus structure and high lipophilicity, it remains unknown about the differences in the effect and mechanism of the toxicity (especially immunotoxicity) between TCS and TCC in organisms in the environment. In this study, we used zebrafish as a model to compare the immunotoxicity and mechanisms between the two pollutants at the same exposure concentration (0.6 µmol/L). The results showed that both TCS and TCC led to a hatching rate below 60% at the time point of 72 hours post fertilization (hpf) and the mortality rates of 40% and 50% at 120 hpf in larval zebrafish, respectively. The zebrafish exposed to TCS and TCC displayed malformations, such as shortened body, swimming sac closure, pericardial edema, yolk cyst deposition, and absorption disorder. Moreover, the developmental abnormalities caused by TCC were significantly severer than those caused by TCS. TCS exposure increased the proliferation rate of innate immune cells to 20% and decreased the number of mature T cells by 35%, while TCC exposure inhibited the differentiation of both innate immune cells and T cells, with the inhibition rates of 25% and 60%, respectively. The results of real-time quantitative PCR (RT-qPCR) and ELISA showed that TCS and TCC exposure up-regulated the expression levels of il-1β, il-6, and tnf-α, while il-10 and IgM exhibited opposite expression patterns. Additionally, both compounds slightly decreased C3 expression. The Pearson correlation analysis showed that the developmental toxicity induced by TCS and TCC had positive and negative correlations with the differentiation of immune cells, respectively. However, the toxicity induced by either TCS or TCC was positively correlated with the expression of pro-inflammatory cytokines. GO function and KEGG pathway enrichment analyses demonstrated that the target molecules of TCS and TCC were enriched in different signaling pathways, and the key network hub genes and the enriched regulatory pathways differed between TCS and TCC. The findings provide compelling evidence that TCS and TCC adopt different mechanisms in triggering immunotoxicity and offer a theoretical reference for the recognition, warning, and management of TCS and TCC-induced health risks.

Abstract:The extraction of rare earth elements (REEs) through in-situ leaching with ammonium sulphate [(NH₄)₂SO₄] had resulted in the production of a large volume of ammonium-rich wastewater, causing severe environmental pollution. This study aimed to assess the ability of an indigenous microalga Chlamydomonas sp. YC, isolated from REEs wastewater, to directly treat real REEs wastewater under outdoor conditions in 50 L airlift photobioreactors (AL-PBRs) and 5.0 m³ open race-way photobioreactors (ORWPs). Additionally, the harvested Chlamydomonas sp. YC biomasses from these two pilot photobioreactors were comprehensively analyzed to evaluate the nutritional values. The results showed that Chlamydomonas sp. YC in AL-PBRs exhibited higher biomass production (1.1 g/L), greater removal efficiencies in NH₄⁺-N (24.9%) and total nitrogen (20.4%), as well as higher CO₂ fixation rate (125.0 mg/(L·d)), compared to those of ORWPs. Moreover, the Chlamydomonas sp. YC biomasses obtained from the two pilot photobioreactors contained 44.5% and 49.4% protein, 9.1% and 14.3% lipids. Moreover, Chlamydomonas sp. YC in the two pilot photobioreactors displayed essential amino acid indexes (EAAI) of 0.900, which was higher than that of soybean protein (0.657), indicating superior nutritional values. In conclusion, the implementation of the process involving Chlamydomonas sp. YC in AL-PBRs under outdoor conditions holds promise as a coupled microalgal biotechnology for the simultaneous removal of NH₄⁺-N from REEs wastewater, and the capture of CO₂ for the production of valuable biomass.

Abstract:The nitrate transporter (NRT) and ammonium transporter (AMT) are crucial transmembrane proteins involved in the absorption, transport, and distribution of inorganic nitrogen in wheat. Obtaining NRT/AMT and preparing corresponding antibodies are conducive to probing into their tissue localization and comprehending the nitrogen utilization process in wheat. In this study, four genes (TaNPF4.5, TaNPF8.3, TaNRT3.1, TaAMT1.2) with high expression levels were chosen and cloned from 405 genes of the TaNRT/TaNPF family and 23 genes of the TaAMT family identified previously. The transmembrane domains of the four transporters were predicted by HMMER to determine the putative expression segments, followed by prokaryotic expression and purification. Under the induction with 1 mmol/L IPTG at 37 ℃, the non-transmembrane segments of TaNPF4.5, TaNPF8.3, and TaNRT3.1 reached the highest expression levels (as inclusion bodies) after 4 h, while TaAMT1.2 was expressed at the highest level (as a soluble protein) after 3 h. TaNPF4.5, TaNPF8.3, and TaNRT3.1 were purified by a pH gradient. The purity of TaNPF4.5 and TaNPF8.3 reached about 87% and 85% at pH 2.0 and pH 3.0, respectively, both of which were suitable for antibody preparation. However, the purity of TaNRT3.1 did not reach 85%. TaAMT1.2 was purified by an imidazole gradient, reaching the purity of about 95% at 20 mmol/L imidazole, and the antibody was prepared successfully. The expression, purification, and antibody preparation of TaAMT1.2 not only provides insights into the expression, purification, and antibody preparation of membrane proteins including TaNPF4.5 and TaNPF8.3 but also lays a foundation for studying the expression and localization of membrane proteins in wheat.

Abstract:The WRKY transcription factor gene family is a plant-specific transcription factor that plays important roles defense responses. Studies in model plant Arabidopsis demonstrated that WRKYs function downstream of mitogen activated-protein kinase (MAPK) signaling cascade and participate in defense responses through activating the expression of defense-related genes. However, the roles of WRKYs in defense responses have not been previously investigated in paleopolyploidy soybean. Bioinfomatic analysis revealed that there are three pair of GmWRKY33 genes in the soybean genome. The identity of first two pair of GmWRKY33 genes is greater than 84% (named as GmWRKY33A). The identity of genes within the same pair is greater than 95%. A 300 bp fragment highly homologous to these four GmWRKY33A was chosen to clone into bean pod mosaic virus (BPMV)-based silencing vector (BPMV-VIGS) to achieve the goal of silencing four GmWRKY33A genes simultaneously. In this study, we simultaneously silenced four homologous genes of GmWRKY33A using a bean pod mottle virus (BPMV) vector carrying a single fragment of GmWRKY33A. Comparing the silenced plants with the vector control plants, no evident morphological phenotypes were observed. However, the GmWRKY33A-silenced plants exhibited significantly reduced resistance to Pseudomonas syringae pv. glycinea (Psg), Xanthomonas axonopodis pv. glycine (Xag), as well as to soybean mosaic virus (SMV). Furthermore, we demonstrated that silencing these GmWRKY33A genes significantly inhibited the activation of GmMPK3/GmMPK6 induced by Psg infection. Collectively, our results suggest that GmWRKY33As are involved in soybean immunity through regulating the transcription of GmMPK3/6 genes or activating the kinase activities of GmMPK3/6. Taken together, our results demonstrated that GmWRKY33As are positive regulators of soybean immune responses.

Abstract:In order to obtain more effective cell culture parameters of Taxus anticancer plants, we optimized the callus induction and subculture conditions of the explants (stem segments with buds) of the anticancer medicinal plant Taxus media by using the plant tissue culture technology and orthogonal test. Furthermore, we studied the method to inhibit browning in the culture. The results indicated that the optimal conditions for inducing callus was culture in the medium composed of B₅+0.25 mg/L 2,4-D+1.5 mg/L NAA+0.5 mg/L KT in the dark, which showed short induction time and a high induction rate (86.7%). The formula of the optimal medium for subculture was B₅+0.5 mg/L 2,4-D+2.0 mg/L NAA+1.5 mg/L KT.The proliferation multiple of callus cultured by subculture on the 10th day of callus growth was the highest. Activated carbon inhibited the browning in callus subculture, with the optimal inhibitory concentration of 0.8 g/L. The results of this study lay a foundation for the production of taxol by suspension culture of T. media cells.

Abstract:Uhrf1 is a multi-domain and multifunctional epigenetic regulator playing key roles in DNA methylation, cell metabolism, and cell proliferation. To investigate the role of Uhrf1 in the reproductive physiology of female yaks, we collected three reproductive organs (ovaries, oviducts, and uteri) from healthy yaks during three reproductive phases (follicular, luteal, and gestational phases), with a total of nine groups. Real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry (IHC) were employed to determine the expression levels of Uhrf1 and the subcellular localization of this protein. RT-qPCR and Western blotting results showed that Uhrf1 was expressed highest in the oviduct during the follicular phase, moderate expression in the uterus during the gestational phase, and the lowest expression in the uterus during the luteal phase (P<0.05). IHC results showed that Uhrf1 was mainly expressed in the ovarian germinal epithelium, theca follicular, follicular granulosa, luteal cells, oviduct mucosal epithelial cells, and uterine glands (UG) of yaks. In conclusion, Uhrf1 was differentially expressed in the major reproductive organs during the reproductive cycle of female yaks, indicating its important regulatory role in the reproductive physiology of yaks.