The research aimed to study the influence of different enzyme preparations on fermentation of tobacco. In this experiment， tobacco produced in Yunnan Province were used as materials. Different enzyme preparations were applied to enzymatically treat tobacco， followed by fermentation using wine yeast. The aim was to prepare tobacco products with richer and fuller flavor. The effects of different enzyme treatments on the conventional chemical composition， volatile flavor substances， and sensory quality of tobacco were analyzed. The results showed that， there was no significant change in moisture content and nitrogen content of tobacco after enzymatic treatment， with nitrogen content ranging from approximately 0.98% to 1.18%. The group treated with 100 U·g^-1 flavor protease exhibited the highest content of soluble total sugars （12.11%） and reducing sugars （5.93%）. The total amount of volatile flavor substances in all groups of tobacco increased， with the highest being 180.029 μg·g^-1. Moreover， the content of characteristic flavor substances in each group of tobacco， such as neophytadiene， phenylethanol， solanone， and megastigmatrienone， also increased. The sensory quality of tobacco improved after enzymatic treatment. Comprehensive analysis revealed that enzymatic treatment using 70 U·g^-1 flavor protease and 50 U·g^-1 α-amylase resulted in a more balanced chemical composition， abundant aroma components， and better sensory indicators of tobacco. Therefore， enzymatic fermentation can improve the quality of tobacco leaves， providing a basis for further commercial development of tobacco leaves.

Soil salinity is a major constraint on global agricultural production， posing a severe threat to agriculture sustainable development and food security. Maize （Zea mays L.） is one of the three major crops in China， while saline-alkali land represents a crucial reserve of arable land resources. Lignin， as a principal structural component of plant cell walls， makes it significant to study the accumulation of lignin in maize and the thickening of cell walls in response to high salinity. This study selected salt-tolerant maize inbred lines （Zhongke4M， Zheng58） and salt-sensitive maize inbred lines （PH4CV， Chang7-2） as subjects. Using a water control and a treatment of 200 mmol·L^-1 NaCl， the morphological changes and cytological characteristics of maize roots under different salt concentrations were analyzed. The differences in enzyme activity， lignin content， and gene expression were also examined. Toluidine blue staining results indicated that the reduction in the area of the root cortex and endodermis under salt stress was significantly lower in the salt-tolerant inbred lines Zhongke4M and Zheng58 compared to the salt-sensitive inbred lines PH4CV and Chang7-2. Furthermore， fluorescence microscopy observations showed that the degree of lignification in the salt-tolerant inbred lines remained stable or increased under salt stress， whereas the salt-sensitive inbred lines exhibited a decrease in lignification. The results demonstrated that the lignin content in the salt-tolerant inbred lines Zhongke4M and Zheng58 remained stable under salt stress， while it significantly decreased in the salt-sensitive inbred lines. Enzyme activity analysis revealed that under salt stress， the activity of phenylalanine ammonia-lyase （PAL） and cinnamate-4-hydroxylase （CAD） decreased in the salt-sensitive inbred lines， while the activity of cinnamate-4-hydroxylase （C4H） increased in the salt-tolerant inbred lines. RNA-seq analysis identified three genes related to lignin biosynthesis， whose expression levels varied among different maize varieties. This study provided new insights into the mechanisms by which maize responds to salt stress through the regulation of lignin accumulation and cell wall structure， contributing to the understanding of maize salt tolerance mechanisms.

Hydraulic fracturing technology has unique advantages in the efficient extraction of shale oil， but the chemicals contained therein pose a high risk of polluting groundwater. By using the fermentation broth produced by shale oil endogenous functional bacteria as biological fracturing fluid， participating in the green exploitation of shale oil can significantly improve the recovery rate of shale oil and reduce the negative impact on the environment. The functional strains were screened by blood plates， and the yield of surfactant was used as the evaluation criterion， and the three functional strains were determined and the species information was identified by 16S rDNA sequencing. According to the different inoculation ratios of 1， 2 and 3 μL， the three strains were compounded into different strains of the same volume to obtain the optimal strain ratio， and the most suitable factors were screened out by single factor experiments， and then the orthogonal and response surfaces were further optimized to obtain high-yield culture conditions. The results showed that the three strains of high-efficiency functional bacteria were Pseudomonas， Bacillus and Taureella， with a combination ratio of 2∶2∶1. The optimal cultivation formula was lactose concentration 13.87 g·L^-1， ammonium persulfate 2.13 g·L^-1， iron sulfite 1.75 g·L^-1 and pH 6. Under these conditions， the surfactant yield of the strain was 315.51 mg·L^-1， an increase of 59.37% compared to the initial yield of 187.30 mg·L^-1. The results of this study can provide a reference for the development of biological fracturing fluid for shale oil.

Currently， the clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein 9 （Cas9） system （CRISPR/Cas9） stands out as a primary technology for enhancing genome editing efficiency in eukaryotes. However， for species with longer reproductive cycles， such as the Nile tilapia， the application of CRISPR/Cas9 technology faces challenges due to its low homozygous efficiency， especially in large-scale genetic screening studies. To solve this problem， a highly efficient CRISPR/Cas9 method was developed using SLC24A5 gene as an example in tilapia， which can directly achieve F₀ generation biallelic knockout with a relatively stable probability in injected embryos. Specifically， two highly effective guide RNAs （gRNAs） were used for mixing， the concentration of Cas9 protein was 800 ng·μL^-1， the mass ratio of Cas9 protein to gRNA was 4∶1， and the injection dose was controlled at 1 nL， that is， 800 pg Cas9 protein and 200 pg gRNA. This knockout technique enabled the direct production of individuals with a significant phenotype expressivity （Lv.1， Lv.2， Lv.3， and Lv.4） of 71% in F₀ generation embryos of the new GIFT Nile tilapia， with a significantly phenotypic penetrance （Lv.1 and Lv.2） of 17%. This breakthrough technology provided a convenient and efficient means for genetic screening in Nile tilapia.

Auxin （IAA） signaling pathway plays an important role in plant growth and response to biological and abiotic stress. Powdery mildew （PM） is a common and serious disease in pumpkin. In order to explore the molecular mechanism of IAA signaling pathway in response to PM stress， transcriptomic sequencing and whole genome DNA methylation sequencing were performed on pumpkin leaves treated with PM. As a result， we found 25 differentially expressed genes in the IAA signaling pathway. Fifty-three genes were differentially methylated， among which 16 austerin-upregulated small RNA （SAUR） genes were methylated to different degrees， suggesting that these genes may be involved in the response to powdery mildew stress. The methylation level of SAUR50 （CmoCh19G007170） gene was reduced， and the methylation region was located in the promoter region of the gene. The expression level of SAUR50 was significantly up-regulated under PM stress and significantly down-regulated under IAA induction. Therefore， this gene may regulate its expression level through DNA methylation and participate in the regulation of PM stress in pumpkin through IAA signaling pathway. The results provided a theoretical basis for IAA signaling pathway in response to powdery mildew stress molecular breeding of pumpkin resistant to PM.

Given the time-consuming and technically demanding nature of commonly used methods such as specific PCR and strip tests for detecting transgenic plants， we hoped to explore a simplified and efficient method for identifying transgenic wheat throughout its entire growth cycle in field conditions. We optimized a leaf painting method using BASTA （glufosinate herbicide）， which is low-cost， straightforward， and suitable for large-scale screening of transgenic plants in wheat fields. Selecting transgenic wheat with BASTA resistance， we determined that a 200 mg·L^-1 BASTA solution effectively identified transgenic positive plants during both seedling and flowering stages in field environments. Additionally， we compared this method with Bar strip tests and specific PCR on 20 T₀ generation transgenic wheat plants. Results indicated that the BASTA leaf painting method correlated with the Bar strip test and covered the findings of specific PCR. Compared to traditional methods， the BASTA leaf painting method is cost-effective， efficient， operationally simple， and applicable throughout the entire growth cycle， making it particularly suitable for large-scale field screening of transgenic plants.

Maize， an important food and feed crop， faces severe growth inhibition， yield reduction， and quality deterioration under salt stress. Jasmonic acid and its derivatives （JAs）， crucial phytohormones involved in plant defense mechanisms， have been shown through studies in model plants to play essential roles in salt stress responses. To investigate JAs-mediated salt stress adaptation mechanisms in maize， we subjected seedlings to combined treatments of 200 mmol·L^-1 NaCl and 100 μmol·L^-1 methyl jasmonate （MeJA） for six hours. Transcriptomic analysis of shoots and roots identified differentially expressed genes （DEGs） associated with both JA signaling and salt stress response. Eight overlapping DEGs from shoot-root comparisons were subsequently validated using RT-qPCR. The study revealed 362 and 803 stress-responsive DEGs in overground and subterranean tissues， respectively. Functional enrichment analyses （GO and KEGG） demonstrated these genes participate in carbohydrate metabolism/transport， defensive secondary metabolite biosynthesis， antioxidant enzyme production， along with abscisic acid and ethylene signaling pathways. These findings indicated that JA signaling activates specific genetic and metabolic networks underlying salt stress adaptation of maize， providing critical insights for further elucidating the molecular mechanisms of JA-mediated salt tolerance regulation.

To investigate the effect of cold plasma treatment on salt tolerance of oats under salt stress， the oat seeds of Bayou 14 were used as experimental materials， and different durations （30 s， 15 s×2） of 5 and 6 kV were applied to the oat seeds at different times cold plasma treatment was used to determine the germination of oat seeds， seedling growth， and physiological indicators under different concentrations of NaCl solution （0.5， 1.0， 1.5 g·L^-1） stress. The results showed that cold plasma can significantly improve the hydrophilicity of oat seed coat and the water absorption rate of oat seeds， and could alleviate the effects of salt stress on oat plant height and root length. Based on the comprehensive evaluation and analysis of oat salt resistance using membership functions， it was found that the cold plasma treatment parameters were 5 kV， 30 s， and 6 kV， 15 s×2， respectively， the salt resistance of oats under stress concentration of 1.5 g·L^-1 was increased. Moreover， as the stress intensity increases， the alleviating effect of cold plasma treatment on salt stress gradually increases under 5 kV and 30 s parameters.

Quantitative GMO detection is essential for ensuring food safety and protecting consumer rights to information. Although digital PCR is currently considered the gold standard for accurate nucleic acid quantification， the lack of validated new quantification technologies limits its applications. However， the emergence of high-throughput sequencing has opened up new possibilities for solving this challenge. While high-throughput sequencing is primarily used for qualitative nucleic acid sequence determination， its potential for quantitative analysis has yet to be fully explored. In this study， plasmid DNA standard materials containing transgenic T-NOS， P-35S， CP4-EPSPS， and soybean housekeeping gene Lectin were used as the detection objects. The method of library construction without amplification was adopted to compare the differences between NGS， third-generation sequencing， and digital PCR quantification. The results showed significant differences between NGS sequencing results and digital PCR results， highlighting the challenges and demands in current nucleic acid quantitative analysis technologies. However， it is worth noting that the results of third-generation sequencing were consistent with the digital PCR detection results， demonstrating its potential as a precise method for quantifying transgenic nucleic acids.

Plasmid DNA serves as the most commonly used gene delivery vehicle， playing a crucial role in gene synthesis technology. Achieving accurate and rapid detection of synthesized plasmid DNA is the key to ensuring the integrity of the genome and improving the efficiency of gene synthesis. DNA detection methods based on first-generation sequencing have established their accuracy as an industry standard， but they have limitations in terms of detection throughput， speed， and cost， which has prompted scientists to continuously seek new solutions. Based on the biological enzyme library， the DNA library construction enzyme TN5 was developed， and a high-throughput plasmid DNA detection solution， named Fast NGS， was established. The feasibility of Fast NGS was evaluated using plasmid DNA samples of different lengths and qualities and high-throughput sequencing of plasmid DNA samples was performed. Finally， the efficiency of Fast NGS and Sanger sequencing was compared. The results showed that the purity and quality of the DNA library construction enzyme TN5 protein met the requirements of next-generation sequencing. Fast NGS is suitable for sequencing of 3~8 kb gene synthesis plasmids， and its detection throughput is up to 2 500 per 12 h， with a sequencing success rate of over 95%. The sequencing accuracy is comparable to that of first-generation sequencing， and there is no significant sequence preference. Fast NGS achieves high-throughput， rapid， and low-cost detection of plasmid DNA， providing a new direction for the development of gene synthesis technology.

Gene editing technology currently has become an important molecular tool， which can selectively modify genome sequences and has wide applications in gene function analysis and crop genetic breeding. Due to the rapid development of gene editing technology， gene editing techniques can perform almost seamless editing on receptor organisms， which increases the difficulty of detecting the gene editing product. Therefore， the establishment of detection methods for gene editing products is conducive to strengthening market supervision of the gene editing product. This study was based on TaqMan real-time fluorescence quantitative PCR （TaqMan-qPCR） technology for detection to design specific primers and probes for the editing site of OsWx gene by developed with CRISPR/Cas9. In this study， the detection system had good specificity and sensitivity， and could effectively distinguish OsWx single base mutants from wild-type rice.

Fusarium verticillioides is a major pathogen responsible for maize ear and stalk rot， posing a serious threat to maize yield and quality. To investigate the gene function and improve frequency of gene knock-out in F. verticillioides， two key genes in the non-homologous end joining （NHEJ） pathway， FvKu70 and FvKu80， were individually knocked out. A comparative analysis was conducted on various aspects， including vegetative growth rate， colony morphology， conidiation， pathogenicity on maize， and knockout efficiency. The results showed that ΔFvKu70 and ΔFvKu80 had no significant differences in their morphological characteristics （mycelium morphology， vegetative growth rate， conidiation， and colony diameter） on the PDA plate. Moreover， they displayed similar pathogenicity in maize stalks compared to the wild-type strain FvLNF15-11. Notably， the frequency of homologous recombination was significantly higher in the deletion mutant strains of ΔFvKu70 and ΔFvKu80 compared to the wild type， and ΔFvKu70 exhibited the highest efficiency of homologous recombination. By successfully constructing ΔFvKu70 and ΔFvKu80 deletion mutants， it becomes feasible to rapidly and effectively achieve gene knockout mutants in F. verticillioides， thereby facilitating the study of key gene functions in this pathogen.

To investigate the microbial contamination and antibiotic resistance characteristics of pathogenic bacteria isolated from raw milk in pastures of northern Fujian Province. From December 2023 to June 2024， 46 bulk tank raw milk samples were collected from 14 pastures in northern Fujian. The samples were analyzed for aerobic plate count， coliform count， and screened for contamination by five pathogenic bacteria： Staphylococcus aureus， Salmonella spp.， Listeria monocytogenes， Cronobacter spp.， and Klebsiella spp. Additionally， antibiotic susceptibility tests were conducted on the isolated strains of Staphylococcus aureus， Klebsiella spp.， and Escherichia coli. The average total bacterial count in the 46 raw milk samples was 5 500 CFU·mL^-1， and the average coliform count was 39 CFU·mL^-1. The overall antibiotic resistance rate of the isolated pathogenic bacteria was 63.5%， with resistance rates of 38.3% for Staphylococcus aureus， 96.6% for Klebsiella spp.， and 84.6% for Escherichia coli. The highest resistance was observed to β-lactam antibiotics， with 93.2% of the pathogenic bacteria exhibiting multidrug resistance. The microbial contamination risk in the raw milk from northern Fujian pastures is low， meeting the “premium milk” standard. However， there is a significant issue with antibiotic resistance among the pathogenic bacteria， particularly to β-lactam antibiotics， with a high risk of multidrug resistance.

Phytophthora nicotianae is a soil-borne oomycete that can cause black shank disease of tobacco， which is very harmful to tobacco production. A strain XC-29 with strong biocontrol function against P.nicotianae was screened， and its antagonistic metabolites and genes were mined by analyzing its genomic information. The antibacterial activity and control effect of biocontrol bacterium XC-29 were verified by the plate confrontation method and pot test. The strain was identified by morphological observation and 16S rRNA amplicon sequencing. The antagonistic mechanism of strains was verified the disease prevention effect of the strain. The strains were identified by exploration by whole genome and transcriptomic sequencing. The results showed that the antagonistic strain XC-29 isolated from rhizosphere soil was identified as Bacillus safensis. Its whole genome sequencing predicted 136 carbohydrate-active enzymes and 11 gene clusters encoding the synthesis of secondary metabolites， of which six were identified as the synthesis clusters of antimicrobial substances， encoding lichenysin， zwittermicin， schizokinen， fengycin， bacilysin and bacilibactin. The results of transcriptome sequencing further confirmed that the strain genes could encode corresponding antibacterial substances. In conclusion， Bacillus safensis XC-29 has a strong antagonistic effect on Phytophthora nicotianae. The whole genome and transcriptome analysis preliminarily revealed the disease inhibition mechanism of Bacillus safensis XC-29， which provides a theoretical basis for further exploration of the antibacterial mechanism and biological prevention and control of antagonistic bacteria.

With the increasing variety and planting area of transgenic maize， the demand for qualitative and quantitative detection of transgenic maize-related products is increasingly urgent. The selection of stable genetic and specific endogenous genes is of great significance for the accuracy and consistency of transgenic maize identification results. In this study， digital PCR technology was used to screen and compare nine endogenous genes in maize， including hmg， adh1-1， adh1-2， ivrI-1， ivrI-2， zSSIIb-1， zSSIIb-2， zein-1 and zein-2. By optimizing the annealing temperature of the above nine kinds of endogenous genes， the optimal annealing temperature for each gene was found. Taking transgenic maize MON810 as the research object， the above endogenous genes were used to determine the actual samples. The results showed that adh1-1 and adh1-2 could be used for copy number detection of actual samples， which would provide reference for endogenous gene selection and related quantitative detection of transgenic maize in the future， and provid a strong technical support for the management of transgenic crops in China.

In order to explore the common genes and molecular interaction mechanism of Newcastle disease virus and Streptococcus after infection， and to gain a deeper understanding of the synergistic infection relationship between the two， the experiment obtained the relevant gene chip datasets of Newcastle disease virus and Streptococcus clinically infected chickens from the GEO database， and conducted secondary bioinformatics data mining， and carried out GO and KEGG pathway analysis and protein interaction network construction. The results showed that there were 34 differentially expressed genes （DEGs） after Newcastle disease virus infection and Streptococcus enteritidis infection. Through GO functional annotation and KEGG enrichment analysis， the above genes were mainly involved in biological processes such as response to viruses， cell death， programmed cell death， regulation of cytokine production， and NOD-like receptor signaling pathway， cytokine receptor interaction， Toll-like receptor signaling pathway. The results of protein interaction network revealed there were 15 interacting proteins with chicken Newcastle disease virus and Streptococcus infection. Further， 10 pivotal genes， including IL6、TNFSF13B、TNRSF1A、IL1B、TLR4、CD83、IRF7、IRF1、SOCS1、SOCS3， were screened， which were all differentially up-regulated genes. The clinical verification results of core genes showed that some core genes were significantly up-regulated consistently in both Newcastle disease virus infection and Streptococcus infection， which further indicated that there was an interaction between the two. In conclusion， Newcastle disease and Avian Streptococcosis not only behaved similar clinical symptoms， but also had interacting genes in the mechanism of action.

To explore the relationship between EPAS1 gene polymorphisms and hemoglobin indices in hypoxia adaptation among local Ovis aries populations in Xinjiang at varying altitudes， exons 9 and 16 of the EPAS1 gene were PCR-amplified and sequenced in 79 Tashkurgan sheep （4 200 m altitude） and 74 Duolang sheep （1 200 m altitude）. 7 SNP loci were identified， gene frequencies and differences in hemoglobin indices were analyzed. The results revealed that the hemoglobin content in Tashkurgan sheep was significantly higher than in Duolang sheep， suggesting that Tashkurgan sheep adapt to high-altitude hypoxia by increasing hemoglobin levels. Additionally， exons 9 and 16 of the EPAS1 gene were relatively conserved， with amino acid-level variation being significantly lower than nucleotide-level variation. Duolang sheep exhibited greater genetic diversity at the same polymorphic loci， indicating a negative correlation between altitude and polymorphic information content. These findings provide insights into the molecular mechanisms underlying Tashkurgan sheep's adaptation to high-altitude hypoxia and changes in blood physiological indices.

Transposable element insertions and excisions are prevalent forces in driving phenotypic variations， gene mutations and genome evolution. Mrh/rMrh transposons are the components of the first genetically characterized two-element transposon system from Mutator superfamily in maize， wherein rMrh transposon， the nonautonomous component has been cloned for sequence characterization. Through phenotypic analysis on the segregation ratio of progeny segregants from genetic cross population， we verified the existence of a single copy autonomous Mrh transposon to catalyse the excision of rMrh from the reporter a1 allele in the maize genome. To elucidate the effects of somatic excision and repairing pattern of rMrh on the function of host a1 gene， and the effects of two autonomous Mrh elements from distinct genetic loci on the somatic excision and repairing pattern of rMrh element at the identical a1 locus， PCR amplification and cloning were applied to recover the simple somatic excision products for sequencing. Results suggested two types of major somatic transposition footprints upon the somatic excision of rMrh transposon in maize， one is precise excision repairing， and another is partial retentions of TIRs from rMrh. Meanwhile， under the regulation of Mrh elements from different genetic loci， the repairing patterns of rMrh somatic excision are rather simple at the primary insertion site in maize， and lead to recovery of the function of host gene through precise excision repairing or host gene mutation due to extra base-pair retentions， but the sequences of repairing products are varied between autonomous Mrh elements from different genetic loci regardless. Our results elucidated the excision repairing features of rMrh from the classic Mrh/rMrh two-element transposon system， enriched the knowledge of genetics characteristics of Mutator transposons， and provided theoretical bases for further exploration of Mrh/rMrh transposon system for generating novel maize germplasms and genetic materials for functional genomics.

Utilizing bioinformatics methods and protein property prediction models to mine novel GH45 family cellulases， and achieving their efficient expression in Trichoderma reesei， in order to provide a new enzyme source for animal feed processing. By using the Preoptem protein model prediction tool combined with the public database Uniparc， the GH45 family cellulase TpCel45A from Thielaviopsispunctulate was screened. We synthesized the encoded gene and construct an expression vector， and transformed it into a strain of Trichoderma reesei using protoplasts. The recombinant TpCel45A protein was obtained through expression and purification， and its enzymatic properties were determined. The research showed that the optimal temperature and pH for recombinant TpCel45A were 55 ℃ and 5.5， respectively， and exhibited good thermal stability. After 15 minutes of treatment at 80 ℃， the enzyme activity remained over 84%. In addition， TpCel45A exhibits good stability within the pH range of 4.0~9.0， with residual enzyme activity exceeding 52%. The determination of kinetic parameters showed that the K_m of the cellulase was 8.04 mg·mL^-1， V_max is 19.04 mg·mL^-1·min^-1， the k_cat value is 377.1 s^-1， and the catalytic efficiency k_cat/K_m value is 46.9. The excavation， identification， and heterologous expression of TpCel45A provide a new enzyme source for the industrial application of cellulase. In addition， the excellent thermal stability and pH stability of TpCel45A make it have the potential for application in high-temperature granulation and different pH environments， and have important application value in fields such as feed proessing and biomass conversion utilization.

Tobacco is one of the most important cash crops， and bacterial wilt of tobacco causes a great loss to tobacco production. Therefore， a safe and effective method is needed to prevent the occurrence of tobacco bacterial wilt. In the previous study， it found that the soluble calcium fertilizer could effectively control tobacco bacterial wilt. When tobacco was planted in the field， the best combination was selected by applying different soil conditioner containing calcium and biocontrol bacteria. The quantity of Ralstonia solanacearum was quantified through fluorescence quantitative PCR. The composition and structure of microbial community in tobacco rhizosphere soil under different treatments were studied by high-throughput sequencing. Field experiments showed that the application of soil conditioner and biocontrol agent could effectively reduce the occurrence of tobacco bacterial wilt， but only ammonium calcium nitrate could compound with biocontrol bacteria GT11 better. The combination treatment decreased the quantity of bacterial wilt in soil， improved the composition and structure of microbial community in tobacco rhizosphere soil， and significantly reduced the incidence and disease index of tobacco bacterial wilt. The control effect in the field was up to 60%. The results showed that the combination of calcium ammonium nitrate and Bacillus velezensis GT11 could effectively control tobacco bacterial wilt， which provided a new idea and theoretical basis for tobacco production.