For a long time, the production of common wheat (Triticum aestivum) in the Huang-Huai and northern regions of China has been severely threatened by drought stress. Traits related to the root system architecture (RSA) of wheat are closely associated with the absorption and translocation of nutrients and water, which are crucial for high-yield and stable-yield of wheat. Thus, identifying loci for RSA traits and developing available markers are crucial for high and stable yield wheat breeding. In this study, RSA-related traits, including average root diameter (ARD), total root volume (TRV), and root dry weight (RDW), were evaluated in the 'Doumai'/'Shi4185' recombinant inbred line (RIL) population under hydroponics. In addition, both the RILs and parents were genotyped using the wheat 90K SNP array. Using whole genome linkage analysis, 2 loci related to ARD (QARD.caas-2B and QARD.caas-6A), 2 loci related to RDW (QRDW.caas-2A and QRDW.caas-5B), and 1 locus related to TRV (QTRV.caas-3A) were identified and each explaining 6.25%~7.59%, 7.21%~10.30% and 7.59% of the phenotypic variances, respectively. Among these, QARD.caas-6A, QRDW.caas-2A and QTRV.caas-3A were novel loci. The favorable allele of QRDW.caas-5B was contributed by the 'Doumai', whereas the favorable allele of QARD.caas-6A, QRDW.caas-2A and QTRV.caas-3A were originated from 'Shi4185'. Five candidate genes related to plant hormone regulation, stress tolerance, and signal transduction were identified, respectively. Additionally, one competitive allele-specific PCR (KASP) marker, Kasp_2A_RDW (QRDW.caas-2A) was developed and validated in 115 wheat accessions, this marker was significantly associated with wheat RDW. The study provides new genes and available KASP markers for optimizing wheat root RSA, improving wheat resistance to abiotic stress and high and stable yield breeding.

NAC (NAM, ATAF1/2, CUC2) transcription factors are a superfamily of transcription factors that are unique to plants and play an important role in plant development and stress response. In this study, BnaNAC069 was cloned from Brassica napus. Sequence analysis and phylogenetic analysis revealed that BnaNAC069 had a classical NAC domain in its N-terminal and a transmembrane domain in its C-terminal, which was a typical membrane-bound NAC transcription factor. BnaNAC069 had the closest genetic relationship with Arabidopsis thaliana AtNTL13/ANAC069. Subcellular localization showed that BnaNAC069 was located on the endoplasmic reticulum membrane. The expression pattern analysis showed that high salinity (NaCl), salicylic acid (SA) and hydrogen peroxide (H₂O₂) could significantly induce the expression of BnaNAC069. Phenotypic analysis showed that the A. thaliana anac069 mutants were tolerant to high salinity, while plants overexpressing BnaNAC069 were sensitive to drought and high salinity. The results of qRT-PCR showed that BnaNAC069 negatively regulated the expression of BnaRD29B (responsive to dehydration) and BnaAREB1 (ABA responsive element binding protein 1), suggesting that BnaNAC069 negatively modulates plant response to drought and high salinity in an ABA-dependent pathway. This study provides basic data for further investigation of the function of the BnaNAC069 gene in regulating plant response to drought and salt stress in B. napus.

Diacylglycerol kinase (DGK) is a key enzyme in lipid signalling pathways and plays an important role in phytohormone signalling, biotic and abiotic stresses. This study analysed the sequence characteristics of the StDGKs genes in potato (Solanum tuberosum) with reference to the protein sequence of the model plant Arabidopsis thaliana AtDGKs as well as information on the conserved structural domains of DGK, and sequence cloning and analysis of this multigene family were performed, followed by analysis of its expression pattern in response to drought, salt stress and potato tuber development. The results showed that 11 StDGKs were screened from the potato genome database (S. tuberosum group Phureja DM1-3 v6.1) and 6 of them (StDGK1, StDGK2, StDGK5, StDGK6, StDGK7, StDGK8) were successfully cloned. Sequence analysis showed that the full length of the 6 StDGKs were 1 460~2 220 bp, and the similarity of sequence comparison was more than 95%. Real-time fluorescence quantification PCR showed that the expression of 6 StDGKs were induced by drought and salt stress. Compared with the control, the expression of StDGK2 and StDGK8 were significantly up-regulated and peaked at 12 h of drought treatment and StDGK6 at 4 h. The expression of StDGK1, StDGK5, StDGK7 were significantly higher than that of the control under salt stress treatment and peaked at 4 h, and that of StDGK2 and StDGK6 peaked at 15 min of salt treatment. In addition, this study found that StDGKs played an important role in the early stage of potato tuber development. For example, the expression of StDGK1 and StDGK8 gradually increased from the early to the middle stage of tuber development, and then slowly decreased at the late stage of tuber expansion. This study provides basic materials for a deeper understanding of StDGKs gene function.

C3H transcription factors belong to the zinc finger protein (ZFP) family and play a crucial role in plant responses to both biotic and abiotic stresses. To investigate the potential function of the MtC3H gene in Medicago truncatula, the full-length sequence of the MtC3H gene was cloned from Medicago truncatula R108. the full length of CDS of MtC3H was 672 bp and encoded 223 amino acids. Phylogenetic tree analysis showed that MtC3H protein was closely related to Cicer arietinum and Pisum sativum. Expression pattern analysis demonstrated that MtC3H exhibited the highest expression level in the stems of Medicago truncatula, followed by the roots. Furthermore, the MtC3H gene was found to respond to exogenous hormones such as gibberellin (GA), auxin (IAA), and methyl jasmonate (MeJA), with a particularly heightened sensitivity to MeJA induction. Subcellular localization analysis indicated that the MtC3H protein predominantly localized in the nucleus and cytoplasm. Notably, yeast expression analysis revealed that the MtC3H protein exhibited non-toxic properties and possessed transcriptional self-activation activity. This study provides a reference for further exploring the role of MtC3H gene.

Low temperature is the major stress factor limiting the high-quality development of the grape (Vitis vinifera) industry. The cuticular wax on leaves is closely related to the plant's stress resistance, but the biological functions and synthesis pathways of cuticular wax during cold stress response is still unclear. The wax ester synthase 1 (WSD1) catalyzes the production of wax esters, an important component of cuticular wax, which play a crucial role in the synthesis of cuticular wax. The present study initially examined the correlation between leaf cuticular wax and cold resistance of grape by utilizing 24 diverse grape germplasm with varying levels of cold resistance. The results showed that the total wax content of leaf epidermis of cold resistant grape varieties was significantly higher than that of sensitive grape varieties, low temperature could promote the wax accumulation in grape leaves, and the wax content in the epidermis of cold resistant grape varieties increased significantly, with the most obvious increase in esters and alkanes. Meanwhile, the wax ester synthase gene VvWSD1 (GenBank No. XM_002263373.5) induced by low temperature stress was cloned from the cold-resistant grape variety 'Zuoyouhong', bioinformatics analysis and expression characteristic analysis of the VvWSD1 were conducted. Using transgenic Arabidopsis plants, the function of VvWSD1 in cold tolerance was studied. The results showed that VvWSD1 cDNA was 1 524 bp in length, encoding 514 amino acids. VvWSD1 was expressed in the stems, leaves, buds and berries of grapes, with the highest expression level observed in the mature leaves. VvWSD1 was induced by cold stress, and the expression levels in resistant grape varieties were significantly higher than those in sensitive ones. Under cold stress, the content of cuticular wax increased significantly after VvWSD1 transiently transformed, the growth of the VvWSD1 overexpressing lines was better than wild-type. Compared with the wild type, the VvWSD1 transgenic lines accumulated wax in the leaf epidermis at low temperatures, decreased leaf water loss rate and chlorophyll leaching, decreased malondialdehyde (MDA) and reactive oxygen species (ROS) content, and decreased relative permeability of cell membranes, indicating that VvWSD1 mediated wax accumulation and participated in grape response to low temperature stress. This study establishes a theoretical framework for elucidating the molecular mechanisms through which leaf cuticular waxes and their associated synthesis enzyme genes contribute to plant responses to cold stress, thereby holding significant implications for the advancement of cold-resistant grape cultivation and germplasm selection.

Huanglongbing is a major impediment to the growth of the citrus industry. It is caused by Candidatus Liberibacter asiaticus which causes the production of reactive oxygen species in the phloem of citrus trees, resulting in cell death. The catalase (CAT) is essential for the elimination of hydrogen peroxide. In order to gain insights into the expression pattern of the catalase gene CsCAT2 in Citrus sinensis, the promoter of CsCAT2 was cloned, the cis-acting elements on the promoter were investigated, and truncations of the CsCAT2 promoter were constructed. Moreover, the response of the CsCAT2 promoter to various phytohormone treatments was evaluated. Analysis of the CsCAT2 promoter sequence revealed that it contained 15 cis-acting elements associated with biotic and abiotic stress and phytohormone signal response, predominantly for hormone signal response. The CsCAT2 promoter with its full-length had the highest promoter activity, and the activity of the truncated mutants decrease as their length decreasing. The activity of CsCAT2 promoter was significantly increased by abscisic acid, jasmonic acid, auxin, gibberellin and salicylic acid treatment, but treatment with ethephon significantly inhibited the activity of the CsCAT2 promoter. This study furnish data for further exploration of the regulatory process of the CsCAT2 gene from C. sinensis, and has broadened the theories concerning active oxygen scavenging metabolism during the interaction between Citrus spp. and Candidatus Liberibacter asiaticus.

Adipocyte determination and differentiation factor 1 (ADD1) is an important eukaryotic transcription factor that plays an important role in the regulation of lipid metabolism. The mechanism of its action in the process of tail fat deposition in sheep (Ovis aries) remains unclear. In this study, ADD1 gene was selected as a candidate gene to search for genetic polymorphic loci associated with fat deposition in sheep tail and to be used as genetic molecular markers. A total of 98 Tibetan sheep and 264 Altay sheep were selected as research objects, and 30 blood samples from each of the 2 breeds were mixed, and DNA mixing pools were constructed, respectively. All exon regions of ADD1 gene were amplified, and the gene types of the selected mutation sites were analyzed by Haploview 4.2 software. The results showed that there were 3 SNPs on the second exon of sheep ADD1 gene, which were g.73 C>T , g.154 C>T and g.270 C>T , and they were in Hardy-Weinberg equilibrium. Genetic polymorphism analysis showed that g.154 C>T locus was moderately polymorphic. At the g.270 C>T locus, the CC and TT genotypes were significantly different between Altay sheep and Tibetan sheep (P<0.05), and CC genotype was the dominant genotype of Altay sheep and TT genotype was the dominant genotype of Tibetan sheep. Linkage disequilibrium analysis showed that there was a strong linkage between g.154 C>T and g.270 C>T, which constituted 3 haplotypes, among which haplotype CC had the highest frequency and was the dominant haplotype of Altay sheep, while the dominant haplotype of Tibetan sheep was TC. Therefore, TT genotype at the g.270 C>T locus of ADD1 gene can be used as an important candidate molecular marker for lean tail breeding of sheep, which also provides a reference basis for the breeding work of sheep.

Mesenchymal stem cells (MSCs) have the characteristics of promoting tissue repair, activating cytokine secretion, regulating organism immunity, and low moral and ethical constraints, which can provide effective treatment for various complex diseases in veterinary clinic. At present, MSCs are often cultured in a medium containing 10% fetal bovine serum (FBS), and the high cost of serum limits the mass production and application of MSCs. In this study, MSCs with a density of 70% were treated with α-MEM medium with gradient FBS of 0.5%~10% respectively. Cells were collected at 6, 12, 18, 24 and 48 h for Giemsa staining, EDU (5 ethynyl-2' deoxyuridine) staining and cell migration experiments, respectively. The results showed that Serum Starvation (SS) of 2% FBS for 6 h could promote the migration ability of MSCs in vitro. Twelve diabetic dogs (Canine lupus familiaris) were given 1×10⁷ MSCs and SS-MSCs by intravenous injection, and the changes of blood sugar and weight of dogs were monitored within 2 weeks. Serological examination and histological examination were carried out 2 weeks after stopping treatment. The results showed that SS-MSCs group achieved similar therapeutic effects to normal culture environment, including restoring blood sugar and weight of diabetic dogs, remodeling islets, and improving liver glucose metabolism. This study provides a feasible strategy for clinical large-scale preparation of medical-grade MSCs, and improves the application value of MSCs in clinical disease treatment.

Angiotensin converting enzyme 2 (ACE2) can serve as a protective and regulatory agent for various pathophysiological processes, as well as a functional receptor for human coronavirus. However, there have been no reports on research related to chickens (Gallus gallus domesticus). In this study, broiler chickens were used as research subjects. Firstly, the coding region of the ACE2 gene was amplified by PCR and connected to the prokaryotic expression vector pET32a to construct a chicken ACE2 prokaryotic expression system. The expression of the ACE2 protein was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the induction conditions were optimized to determine the expression pattern. The purified recombinant chicken ACE2 protein obtained through KCl staining gel purification was identified for specificity and molecular weight using SDS-PAGE and Western blot techniques. The isoelectric point of the ACE2 protein was determined using isoelectric focusing polyacrylamide gel electrophoresis. The purified recombinant ACE2 protein was used as an immunogen to produce chicken ACE2 polyclonal antibodies in rats (Rattus norvegicus). The efficacy of the antibodies was measured, and their specificity was validated to determine the tissue distribution and cellular localization of ACE2 in chickens. The study successfully constructed a prokaryotic expression system for ACE2 in chickens using the pET32a vector, prepared chicken ACE2 polyclonal antibodies with good efficacy and specificity, and confirmed the widespread distribution of ACE2 in chickens for the first time. This study provides an experimental basis and data for further research on ACE2 in chickens.

Feed mold is prevalent, with zearalenone (ZEN) and deoxynivalenol (DON) being the most common mycotoxins found in cereal feed contamination. This study aimed to investigate potential health risks to broilers from the long-term consumption of low doses of ZEN and DON-contaminated diet, as well as the protective effect of licorice (Glycyrrhiza uralensis) extract. A total of 315 one-day-old male Liangfeng broilers were randomly assigned to 3 groups with 7 replicates per group and 15 broilers per replicate. They were fed a basal diet (CON group), a diet contaminated with mycotoxins ZEN and DON (MYC group; with 5% natural moldy corn (Zea mays) replacing an equal amount of normal corn in the basal diet), and the contaminated diet supplemented with 0.1% Licorice extract (LIC group), respectively. The experiment lasted for 84 d. The results of slaughter experiments and HE staining of liver paraffin sections showed that the liver index of broilers at 28 and 56 d of age in the MYC group was significantly increased (P<0.05), with hepatocytes exhibiting edema, ballooning deformity and some apoptosis. The real-time fluorescence PCR detection revealed that the expression of inflammatory-related genes in liver of MYC group broiler, such as myeloid differentiation factor 88 (MYD88), interleukin 12α (IL-12α),nuclear factor kappa-B1 (NF-kB1), and FOS protooncogene (c-FOS), as well as apoptosis-related genes such as tumor necrosis factor- α (TNF-α), NADPH oxidase 2 (NOX2), were significantly increased (P<0.05), In contrast, the expression of heme oxygenase-1 (HO-1) was significantly reduced (P<0.05). After adding licorice extract to the diet, the liver index decreased (P<0.05), the liver tissue morphology was normal, the expression of inflammation and apoptosis related genes significantly decreased (P<0.05), and the expression of HO-1 increased (P<0.05). The content of serum pro-inflammatory cytokines IL-2, interferon (IFN-γ), and TNF-α in the MYC group significantly increased, while the content of immune factors IgA and IgG significantly decreased (P<0.05). After adding licorice extract, the level of pro-inflammatory cytokines significantly decreased, while the level of immune factors increased (P<0.05). The serum and liver antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were significantly reduced, malondialdehyde (MDA) content was significantly increased (P<0.05), and the expression of GSH-Px and SOD1 genes in the liver was significantly reduced (P<0.05) in broiler chickens of MYC group. After adding licorice extract, the liver antioxidant enzyme activity and its coding gene expression, and serum GSH-Px activity were significantly increased (P<0.05), and the MDA content in the serum of 28 d old broilers and in the liver of 56 and 84 d old broilers significantly decreased (P<0.05). In summary, long-term intake of low-dose ZEN and DON-contaminated diet could impair the antioxidant and immune functions of broilers, induce inflammatory responses and hepatocyte apoptosis, and the addition of licorice extract to the diet could effectively alleviate these effects. The results provide a basis for utilizing medicinal plants and their extracts to prevent and manage the damage caused by long-term low-dose mycotoxin contamination in broiler production.

Gymnocypris przewalskii is a kind of Schizothoracinae with low temperature and salt and alkali resistance. Heavy metal stress is one of the main abiotic stresses that limit the anadromous migration and breeding of G. przewalskii. To investigate the effects of Cd²⁺ and Zn²⁺ stress on the acute toxicity, antioxidant ability and non-specific immune function of G. przewalskii. G. przewalskii was exposed to different concentrations of Cd²⁺ and Zn²⁺ at 24, 48, 72, and 96 h.The semi-lethal concentration for 50% (LC₅₀) and the safe concentration (SC) were determined by static bioassay. The activities of superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), and alkaline phosphatase (AKP) in gills, kidney and liver tissues of G. przewalskii were detected by the automatic microplate reader, and the relative expression of NF-κB related immune genes, such as transforming growth factor β-activatrd kinase 1 (TAK1), inhibitory nuclear factor kinase-κB α (IKKα), inhibitor nuclear factor-κB α (IκBα), nuclear transcription factor-κB (NF-κB), interleukin 8 (IL-8), zinc finger protein A20 (A20), and tumor necrosis factor α (TNF-α) were detected by qPCR. The results showed that the LC₅₀ of G. przewalskii under Cd²⁺ stress for 24, 48, 72, and 96 h were 1.455, 1.022, 0.579, and 0.428 mg/L, respectively. The LC₅₀ of G. przewalskii under Zn²⁺ stress for 24, 48, 72, and 96 h were 4.634, 2.797, 1.843, and 1.157 mg/L, respectively. The SC of Cd²⁺ and Zn²⁺ were 0.004 3 and 0.115 7 mg/L, respectively, and the toxicity magnitude was Cd²⁺>Zn²⁺. After Cd²⁺ and Zn²⁺ stress, the activities of SOD and CAT in the kidney and liver of G. przewalskii shown varying degrees of response to Cd²⁺ and Zn²⁺ stress, but in high concentration, the activities were higher at 24 h in the gills. The activities of ACP were significantly reduced (P<0.05) in the gills and kidney,and the activities of AKP were significantly decreased (P<0.05) in the gills and liver. The expression of TAK1, IκBα, NF-κB, and IL-8 were highly up regulated (P<0.05) in gills, kidney, and liver, while the expressions of IKKα, A20, and TNF-α were relatively lower, especially in the liver. In conclusion, both Cd²⁺ and Zn²⁺ stress could induce oxidative stress and inflammatory response in G. przewalskii, activate the system of antioxidant defense and non-specific immune, regulate immune mechanism to cope with the adverse external environment. This study provides basis for further exploring the molecular mechanism of physiological response and artificial proliferation and release of G. przewalskii under heavy metal stress.

Crooked head disease is one of the most harmful diseases in Pelophylax nigromaculata breeding. It has high mortality and strong infectivity. This disease has a high mortality rate and strong infectivity, causing significant losses in frog farming. Currently, there are no effective prevention or treatment methods for this disease. In view of the current breeding status of P. nigromaculata, it is necessary to explore the pathogenic mechanism of the crooked head disease in order to reduce the breeding risks of P. nigromaculata. In this study, the aseptic operation of P. nigromaculata suffering from the crooked head disease was inoculated with bacteria, and a dominant bacteria HBG0512 was isolated. The brain and liver tissues of diseased and healthy black spotted frogs were sequenced. The differentially expressed genes were obtained and analyzed by GO and KEGG, and the gene expression level was verified by real-time fluorescence quantitative PCR. The results showed that the HBG0512 strain isolated from the diseased P. nigromaculata was Elizabethkingia miricola. Transcriptome sequencing found 212 and 612 differential genes in the brain and liver tissues of the diseased and healthy P. nigromaculata. GO enrichment analysis showed abnormal expression of liver endoplasmic reticulum related genes and accumulation of pro-inflammatory factors in brain tissue. KEGG pathway analysis revealed that activating transcription factor 6 (ATF6) was abnormally expressed, which caused endoplasmic reticulum stress, and brain inflammatory response might be mediated by the IgE/FcεRI pathway. Inflammation occured in the liver and brain of P. nigromaculata with the crooked head disease, the liver endoplasmic reticulum receptor ATF6 gene was significantly down-regulated, the ability of endoplasmic reticulum folding reaction was decreased, and the accumulation of unfolded proteins led to endoplasmic reticulum stress-induced inflammation; brain inflammation was mainly mediated by IgE/FcεRI, which led to the destruction of blood-brain barrier permeability and the occurrence of bacterial crooked head disease. This study revealed the pathogenic mechanism of head crookedness in P. nigromaculata through transcriptomics, provides reference ideas for disease prevention and control of frogs and other amphibians and reptiles.

Microcystins (MCs) is a class of natural toxins produced by cyanobacteria during water bloom outbreaks and can induce a variety of pathological injuries in aquatic animals and pose a great threat to the healthy aquaculture of aquatic economic animals. The aim of this study was to investigate the effects of astaxanthin (AX) on the structural changes of hepatopancreas, antioxidant indexes and gene expression of antimicrobial peptide in Litopenaeus vannamei under microcystin-LR (MC-LR) stress in this study. A total of 1 800 L. vannamei with an average weight of (0.5±0.1) g were randomly divided into 3 groups with 3 replicates in each group. The control group and MC group were fed with basic feed, while the MC+AX group was fed with feed with an AX content of 100 mg/kg for 30 d. After the 30th day, the control group continued to be fed with basic feed, while the MC group was fed with feed supplemented with MC-LR (100 μg/kg). The MC+AX group was fed with feed supplemented with MC-LR (100 μg/kg)+AX (100 mg/kg),, and continued to be fed for 15 d. At 0, 5, 10 and 15 d after the addition of MC-LR, 10 L. vannamei were randomly selected from each replicate, and the hepatopancreas were collected to observe the changes of tissue structure and to determine antioxidant indexes and gene expression of antimicrobial peptide. The results showed that the structure of hepatopancreas of L. vannamei was seriously damaged in MC group, with atrophy of hepatic tubules, separation of epithelial cells from the basement membrane, dissolution of connective tissues between hepatic tubules, enlarged gaps, and abnormal shape of the star-shaped lumen. While in MC+AX group, the hepatic tubules of L. vannamei were arranged neatly, and cell morphology was more normal, which indicated that AX could attenuate the damage of hepatopancreatic caused by the MC-LR. Compared with the control group, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), alkaline phosphatase enzyme (AKP), acid phosphatase enzyme (ACP), lysozyme (LZM) and lactate dehydrogenase (LDH) showed a trend of increasing first and then decreasing in MC group. The activities of catalase (CAT) and malondialdehyde (MDA) increased gradually. Compared with MC group, the activities of SOD, CAT, GSH-Px, AKP, ACP, LZM and LDH increased significantly (P<0.05) in MC+AX group, and the content of MDA decreased significantly (P<0.05). The genes expression of Penaeidin 3 and anti-lipopolysaccharride factor (ALF) increased first and then decreased in MC group , both reaching the maximum on the 5th day, which were 1.35 and 1.31 times of control group, respectively, which were significantly higher than the control group (P<0.05). And then reached the minimum on the 10th and 15th day. The expression of crustin showed a gradual downward trend, reaching the minimum on 15th day, which was 0.3 times of control group, and significantly lower than the control group (P<0.05). In MC+AX group, the expression of Penaeidin 3, crustin and ALF were significantly increased (P<0.05), and reached the maximum at 5th, 10th and 5th day, respectively, which were 1.93, 1.46 and 2.9 times of control group. In conclusion, the addition of AX to the diet enhances antioxidant capacity of L. vannamei and effectively alleviates the toxic effects of MC-LR. This study provides a theoretical basis for developing the healthy aquaculture and carrying out the research of related functional feeds in pacific white L. vannamei.

Antifreeze proteins (AFPs) are a type of macromolecular proteins that can bind to the surface of ice crystals to inhibit the growth of ice crystals. Pichia pastoris is a system that can express foreign proteins. It can use methanol as a carbon source to express the target protein. In order to obtain high-yield active antifreeze protein, the insect snow flea (Hypogastrura harveyi) antifreeze protein (HhAFP) was selected. XhoⅠ and XbaⅠ restriction sites were added to the 5' end and 3' end of the target gene through PCR. 6×His tag was added to the 3' end of the target gene that could be used for identification and screening. The target gene was added to the expression vector pPICZαA suitable for the P. pastoris expression system through transformation, and the recombinant expression vector pPICZαA-HhAFP was successfully constructed. pPICZαA-HhAFP was transformed into P.pastoris expression strain X-33 by electroporation, and cultured in a 29 ℃ incubator until positive yeast transformants grew. Two positive yeast transformants were then screened out using bleomycin (zeocin) and methanol. The expression of these 2 yeast strains was induced and cultured for 72 h using BMM medium containing 1% methanol at 29 ℃, 250 r/min, and pH 6.0. The yeast supernatant was purified using affinity chromatography, and the obtained purified product was dialyzed and concentrated. It was analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF) to verify that the protein was the recombinant protein HhAFP. The recombinant protein HhAFP was added to the cell cryopreservation solution to test its antifreeze activity. The results proved that HhAFP could significantly improve the survival rate of cells after cryopreservation and reduce cell damage. By further optimizing the expression conditions of HhAFP, the culture temperature was determined to be 28 ℃ and the methanol concentration was 1.5%. The recombinant protein HhAFP obtained after 72 h of expression and culture could reach 527 mg/L. This study provides a technical approach for the high-yield expression of active antifreeze proteins by P. pastoris and the industrialization of antifreeze proteins.

The inoculation and fermentation of tea-origin microorganisms can enhance the quality and flavor of green tea, thus offering novel perspectives for the processing and treatment of green tea. In this study, the effects of liquid fermentation of P. parafulva T1 and D. Hansenii T12 isolated from fresh tea (Camellia sinensis) leaves on green tea were investigated. Non-targeted metabolomics studies were carried out on the green tea samples before and after fermentation was performed by ultra high performance liquid chromatography - quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and a total of 118 characteristic differential metabolites were screened out. After fermentation with T1, the contents of significant differential metabolites such as xanthine, theophylline, and 3-methylxanthine were significantly increased, while the contents of caffeine and theobromine were significantly decreased. After fermentation with T12, the contents of differential metabolites such as gallic acid, catechin, epicatechin, gallocatechin, epigallocatechin, and theaflavin significantly increased, while epigallocatechin gallate and hydrolyzable tannins significantly decreased. Through metabolic pathway enrichment analysis in the KEGG database, the result showed that the metabolic pathways in green tea fermented by T1 bacteria were mainly enriched in glyoxylate and dicarboxylate metabolism, phenylalanine metabolism, biosynthesis of phenylpropanoids, and caffeine metabolism, while the metabolic pathways in green tea fermented by T12 bacteria were mainly enriched in tyrosine metabolism, sphingolipid metabolism, alanine, flavone and flavonol biosynthesis, and anthocyanin synthesis. Consequently, the fermentation with tea-origin microorganisms had brought about remarkable changes in the composition and content of metabolites in sun-dried green tea. Through the screening and analysis of differential metabolites, this study provides an experimental basis for the fermentation and utilization of green tea.

Myxobacteria are a group of higher prokaryotes with abundant and diverse metabolites. Phytophthora infestans is a pathogen of potato (Solanum tuberosum) late blight, which causes 10%~30% loss of potato industry in China every year. Myxobacteria have significant antagonistic activity against P. infestans, so it is of great research value to screen the active substances with antagonistic activity against P. infestans from the metabolites of myxobacteria. In this study, myxobacteria were isolated from soil samples in Ordos area and their antagonistic activity against P. infestans was detected. Then, the strains were identified by morphological and molecular biological methods, and the optimal fermentation conditions for the strains to produce the active substances against P. infestans and the effect of macroporous resin adsorption on the activity of myxobacterial metabolites were investigated by orthogonal experimental design. The results showed that the isolated strain E10 had a strong antagonistic activity against P. infestans, the diameter of the inhibition zone was 26 mm, the colony was circular and expanding, the edge was wavy, the fruiting body was yellow or orange-red, ridge-like protrusions, and coral-like branches.The strain belonged to Corallococcus exiguus. MD1 medium was the optimal fermentation medium for the strain, and 32 ℃, 0 concentration of NaCl and fermentation for 11 d were the optimal fermentation conditions for the strain. The addition of macroporous resin could effectively adsorb the active substances against P. infestans in the fermentation broth and increase the synthesis of these products, thus increasing their antagonistic ability. This study is of great significance for the future research and development of biopesticides against potato late blight.

Seed Zucchini (Cucurbita pepo), a major economic crop in Xinjiang, suffers severe yield and quality losses due to viral infections, and identifying its pathogens is essential for further control measures. In this study, leaf samples displaying mosaic symptoms were collected from Seed Zucchini in Shihezi, Xinjiang. Under an electron microscope, flexuous Potyvirus particles measuring approximately 750 × 11 nm were observed. The double-stranded RNA (dsRNA) of the virus was extracted, RT-PCR was performed on the virus with random primers and diffuse bands XHL2-1, XHL2-2, and XHL56 were obtained. BLAST searches in the NCBI database revealed that XHL2-1 and XHL56 had sequence similarities of 92.22%~98.69% and 96.21%~99.64%, respectively, with the corresponding regions of Zucchini yellow mosaic virus (ZYMV); XHL2-2 showed a sequence similarity of 89.38%~98.45% with the corresponding region of Watermelon mosaic virus (WMV). These findings indicated that the mosaic disease in Seed Zucchini might be caused by infections of both ZYMV and WMV. To further investigate, specific primers for ZYMV and WMV were designed and synthesized. RT-PCR was conducted on 15 samples, and ZYMV-specific amplification fragments from samples SH2, SH12, and SH13 were sequenced. The sequence similarity among these fragments was over 99.29%, with a similarity of 96.09%~99.56% to ZYMV sequences recorded in GenBank. The WMV-specific amplification fragments from samples SH1, SH9, and SH11 had sequence similarities exceeding 94.30% among themselves, and 94.74%~100% similarity with WMV sequences in GenBank. These results further confirmed that the mosaic disease in Seed Zucchini in Xinjiang was caused by co-infection of ZYMV and WMV. Phylogenetic analysis revealed that the ZYMV infecting Seed Zucchini in Xinjiang clusters with isolates from Eastern Europe and India, while the WMV was closely related to isolates from China, France, and other regions. This study identifies the primary pathogens of mosaic virus disease in Xinjiang's Seed Zucchini as a co-infection by ZYMV and WMV, providing a theoretical basis for further control measures against the disease.

Real-time fluorescence quantitative PCR (qPCR) is a common way to study gene regulation at the transcriptional level, but its accuracy depends on the normalization of data by appropriate reference genes. In order to obtain the stable expression of reference genes in quinoa (Chenopodium quinoa) under cold, drought stresses and abscisic acid (ABA), fluridone (FLU)(ABA inhibitor) treatments, five common statistics tools (geNorm, Normfinder, BestKeeper, ΔCt method, RefFinder) were used to evaluate 9 candidate reference genes. The results showed that the optimal reference genes for cold or drought stress were triphosphate guanosinase 3 (GTP638) and 26S proteasome (PRN483), PRN483 and dipeptidyl carboxypeptidase (DCP894) for ABA or fluridone treatments, ubiquitin-conjugating enzyme E2 (UBC822) and PRN483 were the optimal reference genes for cold, drought stresses and ABA, fluridone treatments. Moreover, the stability of the housekeeping gene β-actin (ACT878) was the weakest when subjected to cold, drought stresses, and ABA or fluridone treatments. Additionally, the reliability of the suggested reference gene was confirmed by the expression levels of 9-cis-epoxycarotenoid dioxygenase (NCED185) and oxidoreductase (OXI802). The results showed that when UBC822 or PRN483 were used as reference gene, the expression patterns of NCED185 and OXI802 were comparable, and they could respond to cold, drought stresses and ABA, fluridone treatments. However, with ACT878 as the reference gene, NCED185 and OXI802 did not exhibit a response to the diverse treatments. This research provides appropriate internal reference genes for gene expression analysis of quinoa, as well as technical support to explore related molecular mechanisms.

Gene editing in poultry is widely used in poultry industry, such as enhancing economic traits of poultry, breeding for disease resistance and developing vector vaccines, improving poultry welfare, improving the safety of poultry egg consumption, and producing pharmaceutical proteins in eggs, and so on. At present, gene editing in poultry relies on the traditional in vitro or in vivo primordial germ cell (PGC) pathways, and gene editing efficiency is much lower. In vitro gene editing of sperm,or sperm transfection assisted gene editing (STAGE), is a promising approach to improve gene editing efficiency, and electroporation is a safe, economical and simple method for sperm transfection. The purpose of this study was to electroporate Cy-3 fluorescein-labeled DNA into Guangxi Partridge chicken (Gallus gallus) sperm cells to screen the optimal electroporation conditions through evaluating sperm dynamics parameters using a computer-assisted sperm analysis system (CASA), investigated sperm cell membrane and acrosome integrity, mitochondrial membrane potential and foreign DNA take-up efficiency by fluorescence microscopy and flow cytometry under different combination of electroporation parameters. The results showed that under the optimized electroporation conditions (100 V, 5 ms, 5 pulses), the DNA take-up efficiency was up to 57.1%, and no significant changes were observed in the fertilization-related parameters such as progressive motility of sperm, sperm acrosome integrity, and sperm mitochondrial membrane potential, significant decrease was seen in total motility of sperm and sperm membrane integrity (P<0.05), showing minimum damage to sperm cells. This study provides a theoretical basis for the application of electroporation mediated STAGE in poultry gene editing.

Choosing an appropriate internal reference gene is an essential requirement for qRT-PCR. However, Lyophyllum decastes has no internal reference genes reported. In this study, mycelia at different after-ripening times, fruiting bodies of different developmental stages and different cap morphologies were selected as the experimental materials. qRT-PCR was used to quantitatively amplify 6 commonly internal reference genes which were cytochrome c oxidase subunit 1 (Cox1), ATPase, glucose-6-phosphate isomerase (PGI), protein phosphatase 2A (PP2A), DNA-directed RNA polymerase subunit 2 (Rpb2), and ubiquitin-conjugating enzyme (UBC) and 4 rarely internal reference genes which were chaperonin containing TCP1, subunit 2 (CCT2), cytochrome b560 subunit (Cyb), 17 beta-hydroxysteroid dehydrogenase type 3 (HSD17B3) and copper/zinc superoxide dismutase (SODC). The stability of 10 internal reference genes were evaluated by ΔCt, geNorm, NormFinder, BestKeeper and RefFinder and validated by β-1,3-glucanases, which was the key enzyme in starch and sucrose metabolism. The results showed that HSD17B3 were the most stable gene in different after-ripening times and different developmental stages; Rpb2 was the most stable gene in different cap morphologies. However, PP2A was the most unstable gene in different after-ripening times and different cap morphologies samples. Moreover, PGI was the most unstable in different developmental stages. The study evaluate the stability of internal reference genes in L. decastes and expect to offer reference for researches about different stages and different cap morphologies in L. decastes.

Fritillaria thunbergii blongs to the genus Fritillaria in the Liliaceae family. The bulb is the main medicinal part, it has the function of clearing heat and dispersing clumps. Leaf spot is one of the main fungal diseases of F. thunbergii, which caused by Diaporthe eres. At present, plant protection personnel and growers mainly rely on classic symptom observation methods to determine the occurrence of leaf spot disease in F. thunbergii, which cannot achieve rapid and accurate diagnosis in its early stages. The purpose of this study was to prepare monoclonal antibodies (McAbs) against D. eres and the immunological detection kits to provide technical support for the rapid and accurate identification of leaf spot in the field. Initially, the protein extract of the hyphae of D. eres was used to immunize Bal b/c mice (Mus musculus). Subsequently, a McAb against D. eres and its hybridoma cell line PoF7 was prepared using hybridoma technology. Indirect ELISA and Western blot analysis showed that the McAb had high specificity against D. eres, with a detection sensitivity of 11 ng/mL. The titer of McAb was ≥ 2.56×10⁴-fold dilution. The antibody type was IgM. It binded to a protein with a molecular weight of 107 kD. The detection sensitivity of the ELISA test kit based on PoF7 was 44 ng/mL, demonstrating good repeatability and specificity. The kit is easy to operate and can be used for large-scale detection of F. thunbergii leaf spot disease in the field after a simple grinding.