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.

In recent years， food safety incidents such as adulteration of mutton and its meat products have been occurring， and in order to improve the legal basis for market enforcement and inspection， a standard substance for sheep genomic DNA has been developed by using digital PCR to quantify the sheep HELZ gene. Since the standard substance can be used to measure the accuracy of detection methods， it can determine the adulteration of mutton in food and related products. In this research， the standard material of sheep genome DNA was developed as the positive control for the detection of mutton in food and related products. The uniformity and stability of the reference materials were evaluated by digital PCR. The results showed that the reference materials had good uniformity and could be stably stored at 4 ℃ and 25 ℃ for 14 days， and at -20 ℃ for 6 months. The standard values of sheep-derived genomic DNA reference material （high concentration） and sheep-derived DNA reference material （low concentration） and their extended uncertainties were （5.44±0.45） ×10³ copies·μL^-1 and （5.68±0.54） ×10² copies·μL^-1， respectively， which were jointly determined by nine different laboratories. The development of sheep-derived genomic DNA reference materials provides a technical basis for the development of animal-derived reference materials， and will establish a technical platform for the preparation， quantitative detection， quality control and quantitative value traceability of sheep-derived genomic DNA reference materials.

The diversification of meat adulteration in the market has made it increasingly difficult for regulatory agencies to identify ingredients of animal origin with ease， accuracy and sensitivity. In particular， beef products with high economic value on the market have become the hardest hit areas for counterfeiting. Therefore， it is urgent to establish a fast and efficient molecular detection method for bovine components. Based on the requirement， this study designed and screened several groups of bovine specific amplified primers and probe combinations with β-actin， a reference gene expressed constitutively in cattle. Through sensitivity detection and specificity verification， recombinase polymerase amplification （RPA） method for rapid detection of bovine-derived components was established. At the same time， the efficiency of one-step DNA extraction from beef products was improved by adjusting and optimizing the reagent ratio. PRA technology was combined with colloid gold immunotest strip color development technology to realize the convenience of detection and visualization of results. Application results showed that the proposed method could detect bovine RPA specifically， with a minimum detection sensitivity of 14.8 copies， and could visualize the visualized results obtained by colloidal gold immunotest strips， and the accuracy and sensitivity of the results were the same as that of real-time fluorescence RPA. The method has strong specificity and high sensitivity， and the whole process can be completed in 25 min， greatly shortening the detection time.

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.

Genetically modified （GM） maize MON87411 is an event developed by Monsanto， and it has obtained the biological safety certificate imported as raw materials for processing. In this paper， the primers and probes were designed with the specific sequences as targets. Through specificity test， system optimization， sensitivity test and limit of detection （LOD） test， a real-time PCR method for the qualitative detection of GM maize MON87411 was established. The results showed that the method could detect the components of GM maize MON87411， and had the characteristics of good stability， strong specificity and high sensitivity and the LOD could reach 0.05%. The specific test， LOD test and reproducibility test of the method were carried out by 8 domestic GMO safety testing institutions. The cyclic verification report showed that the method met the requirements of the national standard method and could be promoted and applied in the testing industry. The establishment of this method could provide effective technical support for the safety supervision of GM maize MON87411 strain in China.

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.

In order to solve the problems of high cost， narrow detection linear range and poor source controllability of commercial kits for determining the content of p24 protein in lentivirus vector， a pair of mouse monoclonal antibodies against p24 protein were used as capture antibodies and detection antibodies， and the detection antibodies were coupled with biotin to establish a double antibody sandwich ELISA method for quantitative detection of p24 protein. The best working concentration of coated antibody and detection antibody in double antibody sandwich ELISA were determined by square matrix titration. The linearity range， lower limit of quantification， accuracy， precision and specificity of the standard curve were investigated. Six batches of lentivirus vector samples produced by our company were used to detect p24 content and sample stability were investigated for method fitness. The experimental results showed that the optimal working concentrations of coated antibody and biotin labeled antibody in double antibody sandwich ELISA were 0.8 μg·mL^-1 and 0.005 μg·mL^-1， the method had the best linearity in the concentration range of 1.25~80.00 ng·mL^-1， and the correlation coefficient r²>0.95. The recovery of intra and inter assay for high， medium and low quality control sample were 80%~120%， the coefficient of variation was less than 10.0%， and the lower limit of quantitative detection was 1.25 ng·mL^-1. The detection results of p24 protein content of 6 batches of the same lentivirus vector were within 30% deviation， and the detection results of storage stability of 4 batches of samples within 8 hours were within 10% deviation. The double antibody sandwich ELISA method has been optimized， developed and fully validated， which could be used for quantitative detection of lentivirus vector p24 protein content， and provide important data support and theoretical basis for lentivirus vector process development， quality control and quality consistency between batches.

High cellulose content make the tissue of tobacco leaves broken easily， affect the plasticity of tobacco leaves during processing， and make the heavy impurity of tobacco leaves. In order to obtain excellent cellulase producing strains and achieve effective degradation of cellulose in aged tobacco leaves， ten Bacillus subtilis engineering strains of cellulose-degrading were successfully constructed by homologous recombination method. Four recombinant strains C36， CM， KF and GH5 with strong cellulase production capacity were screened by the Congo red plate method， carboxymethyl cellulose sodium enzyme activity assay， filter paper enzyme activity assay and filter paper disintegration rate detection. When aged tobacco leaves were used as substarate， the recombinant strain CM showed the highest cellulase production efficiency， and its carboxymethyl cellulose sodium enzyme activity was 39.55 U·mL^-1 and filter paper enzyme activity was 23.52 U·mL^-1， respectively. The results indicated that the recombinant strains could utilize cellulose in aged tobacco leaves to produce cellulase， which could provide theoretical support for cellulose degradation of aged tobacco leaves in industrial production.

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.

The aim of this study was to investigate the effect of Chinese yam extract （DOE） on CHO cell growth and antibody expression. By adding different concentrations of DOE， the cell density， cell viability， the expression of antibody in cell supernatant， and the content of single antibody were measured respectively. Meanwhile， the antibody charge variants were detected and the glycosyl composition and relative content of antibody and the relative binding activity of protein were analyzed. In addition， the activities of malondialdehyde （MDA） and superoxide dismutase （SOD） in cells were measured during cell growth. The results showed that 5 mg·mL^-1 of DOE could significantly promote the growth of CHO cells， increase the density of living cells， cell viability and SOD activity， and reduce the content of MDA， and also increase the content of intracellular antibodies and monoclonal antibodies. The addition of DOE could change the content of some sugar types of antibody to a certain extent. With the increase of DOE concentration， the relative content of G0F and Man5 showed a trend of first decreasing and then slowly increasing， while the content of G1F sugar type gradually increased， and the change of G0 was relatively stable and small. After the addition of DOE， there was no significant difference in the content of acid charge variant， main peak and basic charge variant in the antibody （P>0.05）， and the relative binding activity of the antibody remained between 102% and 135% of the reference sample. DOE did not affect the binding activity of the antibody. Overall， DOE played an important role in cell growth and antibody expression during cell culture. The addition of 5 mg·mL^-1 of DOE had the best effect on cell growth and antibody expression. The result provided a reference for the future study of the effects of Chinese herbal extracts on CHO cell growth and antibody expression.

Cotton is an important strategic and livelihood material， which faces many unfavorable factors in the cultivation process， among which cotton Verticillium wilt， as a very harmful plant disease， could cause a large reduction in cotton production. Therefore， a safe and efficient method is needed to deal with the occurrence of cotton Verticillium wilt. We extracted and purified the strain BJB01 from a severely diseased cotton field in Xinjiang. Physiological and biochemical identification and Gram staining test were performed. And we also extracted the DNA of the strain for 16S rDNA sequencing， and blasted the sequencing results on NCBI； then we used the plate standoff test to initially test the effectiveness of the prevention and control； finally， we screened the best prevention and control method and concentration through the greenhouse prevention and control test. Through the test results， the bacterium was found to be 99.71% similar to Bacillus belleiensis， which can be identified as B. belleiensis； and the bacterium showed inhibition of Verticillium dahlia Kleb. in the plate standoff test； through the greenhouse efficacy test， it was found that the bacterium at the concentration of OD₆₀₀=0.6 had the preventive effect of 89.35%， which was better than other methods and treatment concentrations， and the preventive effect of 85.91% was still the best effect in the separate test again. In the comprehensive analysis， Bacillus pelliculatus BJB01 obtained from the cotton disease field in Xinjiang can inhibit Verticillium dahliae， which could be prepared as a biofungicide for cotton production.

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.

Cotton is an important cash crop and oil crop. In order to study the relationship between morphological indicators and drought resistance at the germination stage of cotton seeds， the suitable drought resistance indexes in the germination stage of cotton were screened. Took 40 cotton varieties as materials， polyethylene glycol-6000 (PEG-6000) solution simulating drought stress， and clean water as control， seven indicators （relative seed water absorption， relative germination potential， relative germination rate， relative germination exponent， radicle/hypocotyl index， relative radicle stem/fresh weight， and relative germ stem/fresh weight） related to drought resistance in the germination stage of cotton were compared by correlation analysis， and the comprehensive drought resistance evaluation value was obtained according to the membership function， and the correlation analysis was carried out with seven indicators to obtain the most suitable drought resistance index. Results showed that the relative germination potential， relative germination rate and relative germination index were screened out as important indicators for the identification of drought resistance during the germination stage of 40 cotton varieties. Under drought stress， the drought tolerance of different varieties of cotton in the germination stage can be quickly and accurately identified and evaluated by measuring these three indicators. Among the 40 cotton materials tested， ‘Zhong 85271’ had the strongest drought resistance and ‘CF75 drop 6’ had the weakest drought resistance.

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.