Protein glycosylation modifications play a crucial role in regulating numerous biological processes, with significant advancements made, particularly in the areas of plant and animal development as well as responses to abiotic and biotic stressors. O-GlcNAcylation (O-GlcNAc) and O-Fucosylation (O-Fuc) are two important types of protein modifications. Among them, SECRET AGENT (SEC) and SPINDLY (SPY) are key homologous proteins of O-GlcNAc transferase (OGT), responsible for O-GlcNAc modification and O-Fuc modification, respectively. They play crucial roles in plant growth, development, and stress response processes. This review provides a comprehensive overview of the O-GlcNAc and O-Fuc modification processes of proteins and their associated enzymes. It analyses the evolutionary characteristics of SEC and SPY, highlights their regulatory mechanisms in plant development and stress responses, and discusses future research hotspots and challenges in plant protein glycosylation modifications.

Plant secondary metabolites are important components of regulators and defense substances that regulate growth and development, and their compound types are extremely complex. Benzoxazinoids (BXs), as one of the secondary metabolites of the alkaloid family, have natural plant active ingredients and are used in the fields of pharmaceutical ingredients and food additives. Their pharmacological and health effects have become a new research hotspot. At present, the study on BXs compounds is concentrated on gramineae corn and wheat, and the research on their regulatory mechanisms is not in-depth enough. In this paper, the secondary metabolic process of BXs compounds is introduced in detail from the composition, classification and metabolic pathways. The evolutionary characteristics indicate that they have a unique biosynthetic cluster structure. It is summarized that BXs compounds play a defensive role in plant growth and development, including defense against insects, microbial interactions and allelopathy in competing plants. Moreover, BXs serve as signaling molecules that mediate plant chelation of metal ions, respond to external environmental stimuli and participate in the regulators of plant hormones, revealing the important function of BXs compounds in the interaction between plants and environmental conditions. Concurrently, it summarizes the issues and solutions faced by BXs compounds in the current research. It is believed that with the in-depth research of BXs compounds, it will surely promote the development of new agricultural production and sustainable strategic practices, help to deeply explore their potential application value, and solve the multiple challenges facing current agricultural and forestry production.

Domestication and long-term breeding have led to the genetic bottleneck effect that significantly reduces the genetic diversity of cultivated cotton, limiting its potential for the development of new varieties and its ability to adapt to future challenges. Wild germplasm resources, however, serving as an important gene pool, may provide abundant genetic diversity for cultivated cotton improvement. This review systematically summarizes the latest advances and challenges in the study of cotton wild germplasm resources, including genetic diversity research, the identification of superior stress-resistant resources, the exploration of homoeologous tetraploid repeat genes controlling complex agronomic traits, and the creation of hybrid breeding materials. It also identifies four critical scientific issues that need to be addressed to promote the efficient utilization and breeding innovation of wild cotton resources. Future research will further leverage the superior genes of wild germplasm, combining genomics, transcriptomics, epigenomics, and other multi-omics technologies to identify key functional genes and their regulatory mechanisms, decipher the mechanisms of photoperiod sensitivity, and, in conjunction with gene editing technologies, accelerate the transfer of superior genes into cultivated species, thereby driving innovation and development in cotton breeding.

Symbiotic bacteria have developed close associations with their insect hosts during the long history of coevolution. They are distributed in multiple parts of insects, both inside and outside their bodies, and participate in the regulation of various physiological processes of insect hosts. Some insect symbiotic bacteria produce hydrolases, oxidoreductases, and transferases to degrade polyolefin plastics, polyester plastics and pesticides, and have certain application potential in environmental bioremediation. However, due to the complex structure and relatively stable chemical properties of plastics, complex and changeable degradation environment, many issues have to be overcome before popularization of insect symbiotic bacteria in environmental bioremediation, such as low activity, poor stability, narrow substrate spectrum, unknown safety, toxicity of degraded products of symbiotic bacteria and their enzymes, and their destruction on original ecological balance. This paper reviewed the mechanisms and strains of insect symbiotic bacteria degrading plastic and pesticide, methods of screening them, and application status in environmental bioremediation. This paper also analyzed the existing issues and the solutions to them and prospected the research directions. By this review, it is aimed to provide references and insights for studies in related fields and advance the application of insect symbiotic bacteria in environmental bioremediation.

Polylactic acid (PLA) is a non-natural biodegradable plastic polymerized from lactic acid. It demonstrates remarkable biodegradability and serves as a significant alternative to traditional petroleum-based plastics, particularly in the background of achieving carbon peak and carbon neutrality. As a typical carbon-neutral material, PLA is increasingly recognized as a crucial raw material for fostering national economic and social development. Currently, PLA is primarily produced through a combination of biological fermentation and chemical polymerization. However, this systhesis method is fraught with complexities, high costs, and potential risks of toxic residue accumulation. Consequently, the exploration of more environmentally sustainable and efficient production methods has become a central focus in the field of PLA synthesis. With the rapid advancements in synthetic biology, protein engineering and metabolic engineering, the key enzymes of PLA biosynthesis have been gradually identified and modified, the PLA biosynthesis pathways have been designed and assembled, leading to the establishment of cell factories for one-step synthesis of PLA utilizing microorganisms with industrial properties, thereby offering a new solution for the green synthesis of PLA. Nonetheless, the biosynthesis method is confronted with challenges, including low PLA yield and suboptimal product performance, which hinder its alignment with industrial standard. Therefore, enhancing the efficiency of PLA biosynthesis and improving product performance have emerged as critical objectives in the development of PLA biosynthesis technologies. In this paper, PLA and its synthesis methods were introduced briefly, and the advantages and disadvantages of chemical synthesis and biosynthesis were systematically analyzed. Subsequently, the pathways and key enzymes of PLA biosynthesis were summarized, and the regulatory strategies of PLA biosynthesis were summarized from the aspects of protein engineering and metabolic engineering. Finally, the key challenges and future research trends in the upgrading and development of PLA biosynthesis technology were systematically analyzed and prospected, aiming to provide useful reference for the design and development of more efficient and greener PLA biosynthesis system.

Using Saccharomyces cerevisiae to ferment lignocellulosic biomass, thus realizing sustainable economic and social development, and has a good application prospect. However, the carboxylic acid, furfural and polyphenols from it have a strong inhibitory effect on S. cerevisiae. These inhibitors cause severe damages to organelles such as cell membranes, mitochondria and ribosomes, which affects the utilization of fermentable sugars in the hydrolysate by S. cerevisiae. This paper systematically reviews the signal pathways and metabolic pathways by which these three types of inhibitors exert stress on S. cerevisiae. For example, weak acids can induce intracellular acidification, and furfural and phenolic compounds can reduce the activity of intracellular redox enzymes and inhibit the translation process. Based on the above stress mechanisms, this paper deeply explores positive genetic methods, such as mutagenesis breeding, laboratory adaptive evolution, cell self-flocculation, and co-culture. These methods are used to mine key genes by phenotypic mutation combined with multi-omics analysis, so as to construct efficient strains. Meanwhile, reverse genetic methods are also elaborated, that is, genomic transformation using known functional genes and strain-directed breeding based on the CRISPR-Cas system. Among them, methods such as overexpressing the target gene in S. cerevisiae, gene knockout using CRISPR-Cas9, and gene regulation using CRISPRa/CRISPRi effectively regulate the expressions of key genes, thereby enhancing the tolerance performance of S. cerevisiae. On this basis, a comprehensive strategy combining high-throughput gene library with multi-pathway gene regulation can be used for key gene screening and multi-dimensional gene regulation on a large scale. In the future, strategies such as the combined analysis of gene expression and metabolic flux can be used to monitor the activities of key enzymes and the carbon flux changes in key metabolic pathways related to tolerance in real time, so as to better cope with the synergistic effects of multiple inhibitors on S. cerevisiae, and provide references and suggestions for S. cerevisiae to efficiently utilize lignocellulose for fermentation.

Multi-omics technology refers to a combination of two or more omics, such as genomics, transcriptomics, proteomics or metabolomics, etc. By analyzing batches of data from different biomolecular levels, it establishes the internal relations among biomolecules at different them. It is an effective tool for the systematic study of complex biological systems by an approach of the systematic study of biological samples. The metabolic pathways of plants are extremely complex, including the primary metabolic pathways that provide energy and nutrients for plant growth and the secondary metabolic pathways that produce a wide variety of small molecules. The relevant metabolites not only support plant growth and development but also play a pivotal role in the plant's responses to environmental stresses. For mankind, these metabolites serve as vital sources of food, energy and bio-materials. Moreover, numerous drug molecules or lead compounds with therapeutic effects discovered from plant metabolites have contributed greatly to human health. In recent years, because of the high-throughput and comprehensive detection advantages, technology of combined omics has been more and more widely used in plant metabolic pathway analysis and have yielded many excellent results due to the advantages of its high through-put and complete detection. From three aspects, namely, resistance to environmental stress, agronomic traits research, and natural product synthesis, this paper presents a classification and elaboration of related studies on plant metabolic pathway resolution using technology of combined application of omics. Finally, this paper discusses the current challenges and future potentials of this technology.

Objective To construct a genetic transformation system and gene editing system of potato Chuanyu 50. Method Aseptic histocultured seedling stems and leaves of Chuanyu 50, the main potato (Solanum tuberosum L.) variety promoted in Sichuan province, were used as explants. The potato explants were transformed with Agrobacterium-mediated transformation containing the plant CRISPR/Cas9 vector pJCV55-StU6-200-StUBI10-T#01. Four different media with various phytohormone ratios were analyzed and screened for callus induction and differentiation. CRISRR-Cas9-based editing method was applied for creating mutants. Result 1) The optimal medium among the tested ones for genetic transformation Chuanyu 50 was B system. The medium formulation for stem and leaf pre-cultivation and callus induction, respectively, were as follows: MS basic salt+20 g/L sucrose +1 mL/L 1 000× N&N vitamins+1.0 mg/L TZR(trans-zeatin-riboside,TZR)+0.027 8 mg/L GA3(gibberellin A3,GA3)+0.02 mg/L NAA(1-naphthaleneacetic acid,NAA)+2.0 g/L phytagel, and MS basic salt+20 g/L sucrose +1 mL/L 1 000× N&N vitamins+0.5 mg/L TZR+2.5 mg/L IAA(indole acetic acid,IAA)+2.0 g/L phytagel, the callus induction rates using stem and leaf were 93% and 88%, respectively. The medium for differentiation was MS basic salt+20 g/L sucrose+1 mL/L 1 000× N&N vitamins+2.0 mg/L TZR+10 mg/L GA3+2.0 g/L phytagel. 2) It is demonstrated that the two-round rooting screening method accurately identified transgenic-positive plants with a 100% accuracy. 3) An Agrobacterium-mediated CRISPR/Cas9 gene editing system for Chuanyu 50 with 63% editing rate was established. Conclusion The genetic transformation and gene editing system of potato Chuanyu 50 are initially established and multiple factors affecting the process of callus induction and regeneration are evaluated.

Objective This study is aimed to develop and apply molecular markers to identify and analyze the patterns of genetic variation in Fruit weight 3.2 (Fw3.2) and Fruit weight 11.3 (Fw11.3), two pivotal genes involved in the regulation of tomato (Solanum lycopersicum) fruit weight, at different domestication stages. It is expected to provide effective tools for the precise identification and utilization of tomato genetic resources, as well as the rapid domestication of wild germplasm. Method Fragment length polymorphism molecular markers were designed specifically based on the copy number variation (CNV) of Fw3.2 and the 1.4 kb large fragment insertion/deletion (Indel) variation at the 3′ end of Fw11.3. These markers were used to conduct genotype analysis on a collection of 259 tomato germplasms (including 79 Solanum pimpinellifolium (PIM) accessions, 95 Solanum lycopersicum var. cerasiforme (CER) accessions and 85 big-fruited S. lycopersicum accessions (BIG)), determining variation of Fw3.2 and Fw11.3 and their distribution within different tomato populations. Result By the molecular markers developed in this study, the CNVs (fw3.2^WT and fw3.2^dup ) of the Fw3.2 gene and the large fragment Indel variants of the Fw11.3 gene (fw11.3-WT and fw11.3-D) were successfully identified. Within PIM population, the frequencies of the large-fruit alleles fw3.2^dup and fw11.3-D were 0 and 2.53%, respectively. In the CER population, these allele frequencies increased to 10.53% for fw3.2^dup and 8.42% for fw11.3-D. Notably, in the BIG population, the prevalence of fw3.2^dup and fw11.3-D alleles rose significantly to 69.41% and 92.94%, respectively. Conclusion Both fw3.2^dup and fw11.3-D alleles significantly increase tomato fruit weight. Furthermore, these alleles lead to a synergistic effect that further enhances fruit weight in tomatoes. During the domestication and genetic improvement of tomatoes, the frequency of these large-fruit alleles has progressively increased, mirroring the evolutionary trajectory toward larger fruit size.

Objective This study edited the acetyl lactate synthase (ALS) gene at designated locus of Ningjing 56, Ningxia salt-tolerant variety, and created rice germplasm resources with herbicide resistance characteristics. Method Using the cytosine base editor BE3, single base editing vector OsALS171-SpG-eBE3 and OsALS190-SpG-eBE3 were constructed with ALS gene 171 and 190 as target sites. Gene edited plants were obtained through Agrobacterium mediated transformation method and target sites were genotyped with Sanger sequencing. A rapid herbicide tolerance system was established and tolerance level was identified in edited herbicide materials. Result Through T₁ target sites sequencing, Two ALS^P171Fmutant homozygous lines and one homozygous strain of ALS^{E187K R190H}, ALS^E187K, ALS^V188I, ALS^R190H, and ALS^D201Nmutation types were obtained. Compared to wild-type material, Two ALS^P171F mutant homozygous strains have strong tolerance to bispyribac-sodium(BS), while ALS^{E187K R190H}, ALS^E187K, ALS^V188I, ALS^R190H, and ALS^D201Nmutant homozygous plants have some tolerances to BS, but the tolerance is not significant. Conclusion The gene edited materials can maintain stable inheritance, using the cytosine base editor BE3 to obtain materials with herbicide resistance characteristics, and the ALS^P171Fmutation demonstrates strong resistance to bispyribac-sodium, which is a priority site for cultivating herbicide resistant rice.

Objective The objective of this study is to analyze the structure of ZmSTART1, clarify the expressing characteristics of ZmSTART1, analyze the function of ZmSTART1 in the process of vascular bundle construction, and thus provide a theoretical basis for the genetic improvement of corn lodging resistance and yield traits. Method Bioinformatics method was applied to analyze the structure characteristics of ZmSTART1 protein. Real-time PCR was used to analyze the temporal and spatial expression of ZmSTART1. ZmSTART1-GFP fusion technology was to transform tobacco leaves and determine the subcellular location of ZmSTART1. The overexpressing vector of ZmSTART11 was constructed, and Agrobacterium infection flocculent method was to transform Col-0 Arabidopsis thaliana. Real-time PCR technology was applied to validate the ZmSTART1 overexpressed A.thaliana positive seedlings. The vascular bundle characteristics of A. thaliana overexpressing ZmSTART1 were observedand the biological function of ZmSTART1 in regulating vascular bundle formation was clarified. Result Bioinformatics analysis revealed that ZmSTART1 was a hydrophobic protein containing only one START domain. The spatiotemporal expression characteristics of ZmSTART1 via real-time PCR technology showed that ZmSTART1 had higher expressions in the anthers, leaves, and the first internode. Subcellular localization analysis revealed that ZmSTART1 was located on the cytoplasmic membrane. Creating A.thaliana plants ZmSTART1-OE overexpressing the ZmSTART1 gene, it was found that transgenic A.thaliana overexpressing ZmSTART1-OE had more secondary and tertiary leaf veins than wild-type ones, and the number of enclosed spaces formed by the leaf veins was also significantly higher than that of the wild-type control. When ZmSTART1-OE transgenic A.thaliana leaves showed fourth order leaf veins, the wild-type control leaves only had third order leaf veins. Observing the development of stem vascular bundles, it was found that the wild-type A. thaliana had 6 vascular bundles in its primary stem structure, while ZmSTART1-OE had 7 vascular bundles in its primary stem structure. The number of fiber cell layers between bundles decreased, the safranin staining became lighter, and the lignin content decreased. Conclusion ZmSTART1 belongs to the START family and is located on the cytoplasmic membrane, playing an important role in the process of vascular bundle formation in maize.

Objective In the process of backcross transfer, the target trait of transgenic maize LD05 was accurately and quickly introduced into the conventional inbred lines of breeding by molecular detection methods, and it was determined whether there were differences in the expressions of the insect-resistant fusion gene m2cryAb-vip3A, insect-resistant and agronomic traits between homozygous and heterozygous individuals of LD05 and the control Zheng 58. Method Homozygous and heterozygous expressions of foreign target gene m2cryAb-vip3A in inbred lines were identified by PCR amplification using left and right boundary primers, and the expressions of m2cryAb-vip3A at transcription and translation levels were analyzed by RT-qPCR and ELISA. The resistances to target pests were evaluated by bioassay and field trials. Result LC915+LC966 was identified as the optimal primers for homozygous and heterozygous identification by screening and optimization. The transcription and translation levels of exogenous gene m2cryAb-vip3A were different in homozygous and heterozygous lines, and those were generally higher in homozygous lines than in heterozygous lines. The results of laboratory bioassay showed that the corrected mortality rates of Ostrinia furnacalis, Spodoptera fragiperda and Mythimna separata were 100% when feeding LD05 homozygous and heterozygous lines at the heart leaf stage, indicating high resistance level. The results of field trials showed that the resistance levels of LD05 homozygous and heterozygous lines to O. furnacalis in the heart leaf stage and silking stage, Mythimna separata in heart leaf stage and Helicoverpa armigera in silking stage were high. Agronomic character investigation showed that there was no difference between the homozygous and heterozygous lines of LD05 and Zheng 58. Conclusion The homozygous and heterozygous identification methods of target genes in LD05 transforms are established based on ordinary PCR. It is confirmed that the expression of the exogenous target gene m2cryAb-vip3A is different in the homozygous and heterozygous lines of LD05, but there is no significant difference in resistance to insect and agronomic traits.

Objective Tartary buckwheat is an important medicinal and edible crop, and flavonoids are an important bioactive component in Tartary buckwheat. The bHLH transcription factor plays an important regulatory role in the flavonoid biosynthesis. Previous studies have found that the transcription level of FtbHLH3 is significantly positively correlated with the transcription levels of most structural genes in flavonoid synthesis. Exploring the mechanism of this transcription factor in flavonoid biosynthesis in Tartary buckwheat may enrich the studies on the regulation mechanism of flavonoid biosynthesis in Tartary buckwheat, and provide excellent gene resource for breeding Tartary buckwheat cultivars with high flavoniod. Method The 35S:FtbHLH3 overexpression vector was constructed and transformed into Arabidopsis attt8 mutant to analyze its function. Result There were no pigment accumulation in the seedling of att8 mutans, and their seed coat was light yellow; while the seedlings of FtbHLH3-overexpressed line had pigment accumulation, and the seed coat color returned to the wild type's phenotype. Measurement of total anthocyanin content showed that the anthocyanins in the seedlings and seeds of the FtbHLH3-overexpressed line and the wild type were significantly higher than that of the attt8 mutant. Proanthocyanidin staining and content analysis showed that the contents of three types of Arabidopsis seedlings were all very low with no significant difference. However, the content of proanthocyadins in the seeds of FtbHLH3-overexpressed line was the highest, which was significantly higher than that of the attt8 mutant. In addition, the total flavones contents of these three types of Arabidopsis seedlings and seeds had no significant difference. RT-qPCR showed that the expressions of anthocyanin synthesis genes significantly increased in the seedlings of FtbHLH3-overexpressed lines, and the key genes AtANS and AtUFGT were significantly upregulated while compared with that of the mutant and wild-type Arabidopsis. There was no significant difference in the expressions of AtANR and AtFLS among the three type Arabidopsis seedlings. The expression of AtANR, a key gene on the branch of proanthocyanidin biosynthesis, in the seeds of the FtbHLH3-overexpressed line was significantly higher than that of wild type, but there was no significant difference in the key gene AtFLS for flavones synthesis, which was consistent with the phenotype and content. Conclusion FtbHLH3 is a positive regulator in the biosynthesis of anthocyanin and proanthocyanidin, but does not affect the synthesis of flavones.

Objective GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) is a domain commonly found in animal and plant proteins and plays an important role in plant growth and development and response to stress. The GRAM gene family members were identified in the whole genome of potato, the expression pattern of potato GRAM family genes under salt stress was analyzed, and the role of GRAM family in potato salt stress was explored. Method Bioinformatics methods were used to identify the GRAM family members in potato (Solanum tuberosum L.), and analyze the physicochemical properties, chromosome localization, subcellular localization, gene structure, motif and collinearity of the protein. The expression patterns of the family members under salt stress were studied by transcriptome sequencing and real-time quantitative PCR (RT-qPCR). Result A total of 26 GRAM family genes were identified in potato, which were unevenly distributed on 7 chromosomes. Physicochemical properties analysis showed that all StGRAM proteins were hydrophilic proteins and most of them were alkaline proteins. Subcellular localization predicted that most of the StGRAM proteins were present in chloroplasts and nuclei. According to the phylogenetic analysis, the StGRAM family was divided into three subfamilies, and the members of the same subfamily had similar gene structure and motif distribution. Through the intra-species collinearity analysis of potato, it was found that StGRAM had only one pair of collinearity genes, and the inter-species collinearity showed that StGRAM had 5 pairs and 3 pairs of homologous genes in rice and Arabidopsis thaliana, respectively. A large number of hormone response elements and stress response elements were found in the promoter region of StGRAM gene. Transcriptome sequencing analysis and RT-qPCR analysis showed that StGRAM gene was induced by salt stress, which may be involved in the response of potato to salt stress. StGRAM25 gene may have different response patterns to neutral salt and alkaline salt. Conclusion The StGRAM gene family plays an important role in potato salt stress response and signal transduction.

Objective DABB (dimeric α+β barrel domain protein) is a kind of protein containing 1‒2 dimerized α+β barrel domains, which plays roles in the responses of various plants to stress. This work aims to predict the properties of DABB proteins in Chinese cabbage (Brassica rapa ssp. pekinensis), investigate the expression changes of these genes in response to salt-alkali stress in Chinese cabbage varieties with different salt-alkali stress tolerances, and provide theoretical evidence for studying the resistant functions of DABB proteins in Chinese cabbage. Method The DABB gene family of Chinese cabbage was identified genome-widely by bioinformatics methods, and the genetic relationship, physical and chemical properties of the encoded proteins, chromosome localizations and gene structures were analyzed, real-time fluorescence quantitative PCR was used to analyze the expression patterns of these genes in different Chinese cabbage varieties under salt-alkali stress. Result A total of 7 DABB genes were identified in the genome of Chinese cabbage, distributed in 5 chromosomes, the DABBs with close relatives were similar in structure; the amino acid number of the encoded protein was 79-495 aa, the relative molecular weight was 8.94-54.19 kD, most of them were stable proteins, and subcellular localization prediction showed that most of the DABB proteins were located in the plasma membrane. Under salt-alkali stress treatments, 6 DABB genes of Chinese cabbage performed differentially expression response, but the expression patterns were different in the Chinese cabbage cultivars with different salt-alkali stress tolerance. Conclusion Totally 7 genes encoding the protein containing DABB domains are identified in the Chinese cabbage genome, 6 of them show differentially expression response under salt-alkali stress treatments, the different expression patterns of these genes may be related to the salt-alkali stress tolerance of Chinese cabbage.

Objective The gene structure and expression analysis of GASA in pepper (Capsicum annuum L.) were studied to provide reference for elucidating the biological function of CaGASA gene and further cultivating pepper varieties with strong stress resistance. Method Bioinformatics method was applied to identify eight GASA family members in pepper, and systematically analyze their physical and chemical properties, phylogenetic relationships, gene structures, conserved motifs, chromosomal locations, gene synteny analyses, and promoter regions. Real-time fluorescence quantitative PCR (RT-qPCR) was used to verify the expression patterns of GASA gene family members in the pepper under different tissues, abiotic stresses and hormone treatments. Result Eight CaGASA proteins were distributed unevenly on the five chromosomes of pepper, and all CaGASA proteins had a common domain. The prediction of subcellular localization indicated that CaGASA protein existed in the extracellular region. The results of co-linear analysis of GASA gene in pepper, Arabidopsis, tomato (Solanum lycopersicum L.) and rice (Oryza sativa L.) showed that there were 14 co-linear events between pepper and other three species. CaCAGA5 was found to be co-linear with that of the three species. According to the cis-elements analyses, CaGASA genes may be induced by a variety of phytohormones and stresses. Transcriptome data analysis showed that the expression of CaGASA in the flower and fruit was higher than that in the leaf. RT-qPCR analysis showed that CaGASA gene was induced by low and high temperature stress and abscisic acid, gibberellin, indole acetic acid and jasmonic acid hormones. Conclusion The GASA gene family may be involved in pepper flower development, response to hormones and response to high and low temperature stress.

Objective To identify MdPLATZs transcription factor families from the whole apple (Malus domestica) genome, to analyze their expression characteristics, and to lay the foundation for the subsequent in-depth study of the biological functions ofMdPLATZstranscription factors. Method Seventeen members of the PLATZ gene family were identified from the whole apple genome by bioinformatics methods and divided into four subfamilies based on their sequence features, and their physicochemical characteristics, phylogenetic relationships, gene structures, chromosomal localisation and promoter cis-acting elements were analyzed. The expressions of PLATZ family members in different tissues of apple were determined, and the gene functions of MdPLATZ9 by heterologous expression and drought stress treatment were identified. Result Bioinformatics analyses showed that the molecular weight of MdPLATZs ranged from 17.23-29.39 kD, the isoelectric point ranged from 8.24-9.51, and most of the members were localised in the nucleus. These genes were distributed on 13 chromosomes of apple, and members of the same subfamily had similar gene structures and high sequence homology, with obvious covariance between groups. The PLATZ structural domains were highly conserved in the MdPLATZs family. Gene expression pattern analysis showed that there was tissue expression specificity among MdPLATZs family members, with MdPLATZs being expressed in flowers, and MdPLATZ7 being the most highly expressed in flowers and stems. Further analysis showed that peroxidase and catalase activities were significantly enhanced in Arabidopsis plants overexpressing MdPLATZ9, while malondialdehyde and hydrogen peroxide levels reduced, ascorbic acid levels increased, and the rate of superoxide anion production was slowed down, which indicated that the overexpression of MdPLATZ9 improved drought tolerance in Arabidopsis. Conclusion Seventeen members of the MdPLATZs transcription factor family are identified from the whole apple genome, and the members of this family show high similarity and homology in gene structure, protein motifs, and significant segmental duplications. Arabidopsis plants overexpressing MdPLATZ9 demonstrates significant drought resistance.

Objective The ABA receptors PYRl/PYLs/RCARs play an important role in the ABA signal transduction pathway. The interaction proteins of VvPYL4 were screened by yeast two-hybrid technique to explore signaling pathway of the VvPYL4 responding to Plasmopara viticola infection in grape. Method The cDNA library was constructed using the Beta grape leaves infected with P. viticola. The bait expression vector pGBKT7-VvPYL4 was constructed to facilitate the identification of proteins that interact with VvPYL4 from the cDNA library by yeast two-hybrid technology. The expression patterns of candidate interacting protein genes induced by P. viticola were analyzed using real-time PCR and the interactions of the proteins were verified using bimolecular fluorescence complementation technology. Result The results titer of the cDNA library was 7.16×10⁷ CFU/mL, the recombination rate was 100%, and the average length encoded by the inserted cDNA was around 1 000 bp. The bait expression vector pGBKT7-VvPYL4 was successfully constructed. Result showed that there was no autoactivation activity in the yeast cells. The cDNA library plasmid and bait vector pGBKT7-VvPYL4 were co-transformed into yeast AH109 strain. After several screening, sequencing, BLAST alignment and rotation verification, 53 candidate proteins were obtained, which were involved in signal transduction, plant growth and development, and environmental stress response, respectively. Based on real-time PCR analysis, the expressions of genes encoding four proteins were induced by P. viticola. During a bimolecular fluorescence complementation assay, the robust yellow fluorescence signals were detected in the leaves of T. benthamiana that were co-transformed with the pSPYCE-PP2C24 and pSPYNE-PYL4 expression vectors, suggesting an interaction between the PYL4 and PP2C24 proteins. Conclusion A cDNA library is constructed from grapevine leaves infected with P. viticola, leading to the identification of 53 candidate proteins that interact with VvPYL4. Among these, the genes encoding four proteins are found to respond to stress induced by P. viticola. Furthermore, the interaction between VvPYL4 and the PP2C24 protein is confirmed.

【Objectve】This study aims to clarify the functional mechanism of the transcription factor VcMYB17 in blueberry drought stress response, providing potential genetic resources for blueberry cultivar improvement. Method Bioinformatics methods were employed to characterize the basic properties of VcMYB17. RT-qPCR was used to analyze the tissue specificity of VcMYB17, the response of VcMYB17 to different hormones and drought stress, and the effect of overexpression of VcMYB17 on drought resistance genes in Arabidopsis thaliana. Subcellular localization of VcMYB17 was examined in transiently transformed tobacco, and its transcriptional activating activity was verified in yeast. The VcMYB17-OE transgenic blueberry callus and A. thaliana were obtained through Agrobacterium infection. Drought stress phenotypes were evaluated, including measurements of survival rate, fresh weight, water loss rate, relative conductivity, malondialdehyde (MDA) content, and peroxidase (POD), and superoxide dismutase (SOD) activities. Result VcMYB17 is an R2R3 type MYB transcription factor that is highly expressed in the leaves of blueberry. Exogenous treatment with ABA, GA, MeJA, SA and PEG causes the expressions of VcMYB17 to be downregulated. VcMYB17 is localized in the nucleus and has transcriptional activating activity. Under drought stress, the overexpression of VcMYB17 inhibits plant growth, makes plants accumulate more MDA content and decrease POD and SOD activities. Furthermore, the expressions of drought response genes such as AtERD1, AtERD15, AtP5CS1, AtRD29A, AtRD22 and AtDREB1A in VcMYB17-OE plants are significantly lower than that in wild type plants. Conclusion VcMYB17 is an R2R3 MYB transcription factor localized in the nucleus. The expression of VcMYB17 is inhibited by ABA, GA, MeJA, SA and PEG. Overexpression of VcMYB17 decreases the expression of drought response genes, resulting in reduced drought resistance in A. thaliana and blueberry callus.

Objective The black spot disease caused by Marssonina rosae is the most prevalent fungal disease of garden roses, severely impacting the ornamental and economic value. Previous studies have shown that the xylan hydrolase gene RcXYNC was differentially expressed in susceptible and highly resistant rose strains; however, the regulatory mechanism remains unclear. The objective of our research is to investigate the function of RcXYNC and its resistance mechanism to black spot disease. Method Firstly, the coding and promoter sequences of RcXYNC were cloned from the susceptible rose strain R12-26 and highly resistant strain R13-54. The relative expression of RcXYNC of M. rosae-infected leaves at different stageswas detected by quantitative real-time PCR (RT-qPCR). Then, the function of RcXYNC was analyzed by transient overexpression and virus-induced gene silencing (VIGS) and the H₂O₂ accumulation, transcription levels of defense-related genes in the salicylic acid and jasmonic acid/ethylene signaling pathways were measured. Finally, the target proteins interacting with RcXYNC were identified by yeast two-hybrid system. Result The coding and promoter sequences of RcXYNC were consistent in highly resistant and susceptible strains, both containing a conserved domain of Glyco-10 and stress responsive elements. Transient overexpression of RcXYNC led to a significant increase in black spot areas on rose leaves while silencing of RcXYNC resulted in a significant reduction in black spot areas. Transient overexpression or silencing of RcXYNC significantly increased the expressions of most salicylic acid and jasmonic acid/ethylene signaling pathway defense-related genes in rose leaves, while the rest genes showed no significant changes. Total 20 proteins interacting with RcXYNC were discovered, and most of them were involved in plant cell metabolism processes. Conclusion RcXYNC is a negative regulator to defense against M. rosae, potentially reducing resistance of roses to black spot disease by affecting the salicylic acid and jasmonic acid/ethylene signaling pathways.

Objective Investigate the role of expansin (EXPs) of snapdragon (Antirrhinum majus) in response to Sclerotinia sclerotiorum infection, identify the EXPs gene family of snapdragon, and mine the key candidate genes against S. sclerotiorum. It provides a basis for further investigation of the biological function of the EXPs gene family in snapdragon. Method Based on the whole snapdragon genome data, bioinformatics methods were used to analyze the physicochemical properties, secondary and tertiary structures, gene structure, protein domains, phylogenetic evolution, collinearity relationships and cis-acting elements of the EXPs family proteins in snapdragon. The transcriptome sequencing (RNA-seq) and quantitative real-time PCR (RT-qPCR) were applied to examine the expression patterns of AmEXPs during S. sclerotiorum infection while using resistant and susceptible A. majus materials. Candidate genes were identified. These genes were further investigated by constructing overexpression vectors and transiently transformed into Nicotiana benthamiana for preliminary resistance screening. Result Thirty-two AmEXPs were identified in the snapdragon, all of them have conserved structural domains DPBB-1 and Pollen allerg1, and they are divided into four subfamilies (EXPA, EXPB, EXLA, and EXLB). Thy encoded 211-297 amino acids, with molecular weights of 22 018.69-31 787.97 Da, and isoelectric points ranging from 5.01-9.83. There are highly similar structures and conserved motifs among the genes of various subfamilies. Each gene's promoter contains cis-acting elements related to hormones or stress. Ten candidate genes were identified according to their expression patterns. These genes were further investigated by constructing overexpression vectors and transiently transformed into N. benthamiana for preliminary resistance screening. It was found that AmEXP13 and AmEXLB1 genes significantly enhanced the resistance of N.benthamiana to S. sclerotiorum compared to the control, while AmEXPA17, AmEXPA21, AmEXPA9, and AmEXPA16 genes made N. benthamiana more susceptible, and AmEXLB2, AmEXPB1, AmEXPA2, and AmEXPA12 genes showed no significant differences. Conclusion Thirty-two EXPs gene family members were identified from the snapdragon genome, among which six EXPs genes respond to resistance to S. sclerotiorum. AmEXPA13 and AmEXLB1 are the key candidate genes for positive regulation of resistance, while AmEXPA17, AmEXPA21, AmEXPA9, and AmEXPA16 are the key candidate genes for negative regulation of resistance.

Objective The C3H family genes play a significant role in regulating plant growth and development. Investigating the characteristics and functions of the C3H gene family in Grona styracifolia will lay a foundation for studying its roles in the physiological and morphological formation of this species. Method Based on the comparison of transcriptome data between ‘Guangyao Da 1’ and wild G. styracifolia, the C3H family genes were identified. Bioinformatics techniques were utilized to analyze the physicochemical properties, chromosomal positions, phylogenetic relationships, conserved motifs, and domains of these genes. real-time quantitative PCR (RT-qPCR) was applied to examine the expression patterns of the C3H family genes in different tissues and varieties of G. styracifolia. The correlation between C3H genes and key genes involved in flavonoid biosynthesis was analyzed. Additionally, the total flavonoid content in the stem tips and bases of different varieties were measured. Result The G. styracifolia C3H gene family consists of 27 members distributed across 9 chromosomes, primarily encoding basic, unstable, and hydrophilic proteins. Phylogenetic analysis shows that the G. styracifoliaC3H family genes are divided into three groups, with members in the same group being relatively conserved. Analysis of conserved motifs and domains indicates that the most frequently occurring motif is related to auxin regulation, and there is cooperative interaction with domains such as ANKYR among the G. styracifolia C3H family members. Expression pattern analysis reveals that the G. styracifoliaC3H family genes mainly function in the stems of it and are highly expressed at the stem tips of the ‘Guangyao Da 1’ variety. Correlation analysis found that the expressions of G. styracifolia C3H family genes are often positively correlated with key genes in flavonoid biosynthesis. Total flavonoid content measurement showed that at the stem tip, the total flavonoid content of ‘Guangyao Da 1’ is significantly higher than that of wild creeping G. styracifolia, while the opposite is true at the stem base. Conclusion The GsC3H family genes may indirectly influence the agronomic traits of ‘Guangyao Da 1’ G. styracifolia by regulating the biosynthesis of flavonoids in the stem segments.

Objective PIFs (Phytochrome interacting factors) are the core components in the photopigment-mediated photothermal signaling pathway. Studying the function of PIF3 in tea plant (Camellia sinensis (L.) O. Kuntze) provides important theoretical guidance for using light signaling pathways to avoid or mitigate the harm caused by adverse conditions. Method The CsPIF3a gene was cloned from tea plants through homology cloning and subsequently analyzed using bioinformatics tools. Expression patterns of CsPIF3a across various tissues and under different light and temperature conditions were assessed using real-time quantitative PCR (RT-qPCR). Additionally, tobacco transient expression assays and yeast two-hybrid assays were conducted to determine the protein localization and transcriptional activity of CsPIF3a. Result The open reading frame (ORF) of CsPIF3a was 2 163 bp in length, encoding a protein of 721 amino acids. The predicted molecular mass of the encoded protein was 76.97 kD. The theoretical isoelectric point was 5.70, having hydrophilic properties. The phylogenetic tree analysis showed that CsPIF3a was closely related to Actinidia eriantha Bentham. and contained a basic helix-loop-helix (bHLH) domain, an active phytochrome binding (APB) domain, and an APA domain. The secondary structure analysis of CsPIF3a revealed that it comprised 18.17% α-helix, 6.80% β-sheet, 1.53% β-turn, and 73.51% random coil, with 46.87% of the regions being disordered. Subcellular localization assays demonstrated that CsPIF3a was localized in the nucleus and presented transcriptional activity. Reverse transcription quantitative PCR (RT-qPCR) analysis indicated that CsPIF3a was predominantly expressed in the old leaves and stems of tea plants. Additionally, its expression was found to be responsive to temperature stress and correlated with light intensity. Conclusion CsPIF3a is localized in the nucleus and exhibits transcriptional activity. In response to temperature stress, the expression level of CsPIF3a gene was related to light intensity.

Objective ESR2 gene in the AP2/ERF (APETALA2/ethylene-responsive factor) transcription factor family plays an important role in the regulation of plant leaf bud development. To clone the tobacco NtESR2 gene and analyze its function, we aimed to provide a target gene for the regulation of tobacco leaf bud development. Method NtESR2 gene was cloned from cultivated tobacco (Nicotiana tabacum L.) by homologous cloning, and its bioinformatics, gene expression, hormone response, and subcellular localization analysis were performed. Meanwhile, CRISPR/Cas9 editing technology was used to create the NtESR2a and NtESR2b double mutant ntesr2, and the phenotype of the ntesr2 mutant was observed. Result NtESR2 has two copies in cultivated tobacco, NtESR2a and NtESR2b, with CDS lengths of 1 188 bp and 1 200 bp, encoding 395 and 399 amino acids, respectively. NtESR2a protein is located in the nucleus, while NtESR2b protein is located in the nucleus and cell membrane. Both proteins have only one AP2 domain and belong to the ERF subfamily, of which NtESR2a is from Nicotiana sylvestris and NtESR2b is from Nicotiana tomentosiformis. NtESR2 gene is expressed in the roots, stems, leaves, terminal buds, axillary buds and flowers of tobacco, among which the expression in terminal buds and axillary buds is higher, indicating that NtESR2 gene may have an important regulatory role in these tissues. Cis-acting element analysis and exogenous hormone treatment showed that the gene is regulated by a variety of plant hormones. The ntesr2 mutant showed the characteristics of cotyledon fusion and meristem loss, and the expression of NtCUC2, NtLAS, NtREV, NtYUCCA and NtPIN1 genes was significantly downregulated. Conclusion Two members of the ERF transcription factor NtESR2 are cloned from tobacco. They are highly expressed in apical buds and axillary buds and are regulated by a variety of plant hormones such as auxin. NtESR2 regulates the transport of auxin to affect the morphological establishment process of tobacco leaf buds. The research results provide a theoretical basis for regulating the development of tobacco leaf buds.

Objective The regulatory mechanism of NtMYC2 transcription factor in JA-mediated on tobacco trichomes density improvement was studied. Method NtMYC2 gene was cloned from Nicotiana tabacum L. by homologous cloning method. Bioinformatics analysis and subcellular localization were performed to analyze NtMYC2 genetic characterization. Fluorescence quantitative PCR technology was used to detect its expressions files in different tissues and responses to different concentrations of MeJA treatment. NtMYC2 knockout materials were created to verify the function of regulating glandular trichomes. The yeast two-hybrid system was adopted to clarify the protein interaction between NtMYC2 and NtJAZ11. Result NtMYC2 had two homologous sequences, NtMYC2a and NtMYC2b, with open reading frame lengths of 2 040 and 2 046 bp, and coding protein lengths of 679 and 681 amino acids. The homology evolution analysis found that NtMYC2a was highly homologous to Nicotiana tomentosiformis and NtMYC2b was highly homologous to Nicotiana sylvestris, respectively. Both of them had the close relationship with homologous genes from Solanum lycopersicum, Solanum tuberosum and Capsicum annuum, but the distant relationship from Artemisia annua and Arabidopsis thaliana. The protein sequence alignment found that NtMYC2 had JID, TAD and bHLH domains and belonged to the bHLH gene family. Expression analysis clarified that both NtMYC2a and NtMYC2b had the highest expressions in leaves and the lowest expression levels in roots. Both of them were strongly induced by MeJA. NtMYC2 fusion protein was localized in the nucleus. Moreover, long and short glandular trichomes density ofthe leaf surface of NtMYC2 gene knockout lines decreased significantly, non-secretory trichomes disappeared. Yeast two-hybrid results verified that both NtMYC2a and NtMYC2b interacted with NtJAZ11. Conclusion NtMYC2 is involved in the regulation of JA-mediated tobacco trichomes development with protein interacting with NtJAZ11.

Objective This study aims to identify efficient phosphate-solubilizing growth-promoting strains to increase the available phosphorus content in soil and promote the growth and healthy development of plants and their root systems. Method A phosphate-solubilizing growth-promoting strain capable of thriving in an alkaline environment was isolated from a long-term rice-oilseed rape rotation in Ningbo, Zhejiang province, China. The strain was identified using a combination of the morphological, physiological and biochemical characteristics, along with 16S rDNA molecular analysis. The incubation and phosphate solubilizing conditions were optimized, and the growth promotion effects on rapes were verified through a pot experiment. Subsequently, high-throughput sequencing techniques were also employed to analyze the structure of the bacterial communities in the rhizosphere soil of oilseed rapes (Brassica napus). Result The isolated strain LM1-2 showed a strong capacity for phosphorus solubilization. The amounts of solubilized phosphorus and indole-3-acetic acid (IAA) synthesized were 493.23 and 9.41 μg/mL, respectively. Strain LM1-2 was identified as Kluyvera intermedia. The shake flask experiments demonstrated that the optimal conditions were an initial pH of 10, a liquid volume 30% of the container's volume, and a culture period of 7-9 d at 25℃, with glucose, tryptone, and tricalcium phosphate serving as carbon, nitrogen, and phosphorus sources, respectively. Strain LM1-2 had the strongest phosphorus-solubilizing ability, reaching 587.03 μg/mL. The pot experiment revealed that, compared to the control group, strain LM1-2 increased available phosphorus by 45.29%. The root length, root diameter, root forks and root crossings of oilseed rape treated with LM1-2 significantly increased by 40.78%, 18.97%, 55.30%, and 87.82%, respectively. Additionally, the fresh and dry weights of the oilseed rape increased by 15.51% and 26.82%, respectively. Inoculation with LM1-2 changed the structure of the rhizosphere bacterial community and significantly increased the Simpson index of alpha diversity, with Acidobacteriota being the dominant phylum and its relative abundance increased by 23.66%. Conclusion Strain LM1-2 demonstrates the excellent performance in phosphorus solubilization and growth promotion of oilseed rape, indicating its potential for diverse applications in agricultural production.

Objective Isolation of endophytic bacteria from blueberry and investigation of their mitigating effects on aluminum stress in Brassica chinensis L. growth are aimed to provide strain resources for improving the acid and aluminum tolerance capacity of plants. Method Tissue block culture method was used to isolate the endophytic bacteria from blueberry leaf buds, and determine their aluminum tolerance characteristics by shaker culture. The dominant strains were identified by combining morphological characteristics, physiological and biochemical characteristics, 16S rDNA and gryB genome sequencing. The toxicity of the strains was verified by hemolysis test. Result Three endophytic bacteria (Y01, Y02, and Y03) were isolated from the leaf buds of blueberry, strains Y01 and Y03 showed fine tolerance to of <1 000 μmol/L aluminum concentration. Under hydroponic conditions, Brassica chinensis L. grew best at 100 μmol/L aluminum concentration, but 300 μmol/L aluminum concentration caused severe impact on B. chinensis L., and the biomass significantly reduced. Three strains of endophytic bacteria alleviated the aluminum toxicity to varying degrees, and the best effect was achieved by strain Y01, which increased the height, root length, fresh weight and chlorophyll content of B. chinensis L. by 95.6%, 40.2%, 594.7%, 22.2%. The activities of SOD, POD and CAT, and the contents of soluble sugar and soluble protein in the leaves of B. chinensis L. significantly increased by 30.8%, 7.8%, 78.3%, 91.7% and 202.5%, respectively. The activity of SOD, POD and CAT and that content of soluble sugar and soluble protein in root system increased by 118.2%, 101.2%, 59.6%, 79.2% and 74.3%, respectively,and maintained at a high level. Strain Y01 was identified as Bacillus cereus, and the hemolytic test was positive for this strain. Conclusion Endophytic bacteria Y01 isolated from blueberry alleviates the toxic effects of aluminum stress on cell membranes and other organs of B. chinensis L. by increasing chlorophyll content, antioxidant enzyme activities and release of soluble substances. This strain may improve the acid and aluminum resistance of plants as a new strain resource.

Objective Continuous planting of tobacco for many years will lead to the deterioration of soil microbial ecological environment, the aggravation of diseases and the reduction of quality, while corn-tobacco rotation can effectively alleviate the obstacles by continuous cropping. It is aimed to clarify the effects of rotating cropping and continuous cropping on rhizosphere soil and microecology of Jiyan 9, and to provide theoretical support for alleviating the obstacles by tobacco continuous cropping and farmland soil detection and restoration. Method The experiment was carried out in the tobacco planting demonstration field of Sanyuanpu town, Liuhe county, Tonghua city, Jilin province in 2023, and the rhizosphere soil of maize-tobacco rotation and tobacco continuous cropping was taken as the research object. The soil nutrients and enzyme activities of rotating croppng and continuous cropping were determined, and the microbial community structure and diversity were analyzed based on 16S rDNA and ITS sequences. Result Rotating cropping significantly increased the total nitrogen, available potassium, organic matter content, urease and protease activities in rhizosphere soil, and significantly reduced peroxidase activity. Compared with continuous cropping, rotating cropping significantly increased the diversity and richness of rhizosphere soil bacterial community. There was no significant difference in fungal community diversity between the two planting modes, and continuous cropping significantly increased the richness of rhizosphere soil fungi (P<0.05).The relative abundance of Gematimonas in the rotating cropping mode was significantly higher than that in continuous cropping, and the relative abundance of fungi such as Penisilium, Astrostroma and Fusariumsignificantly increased in continuous cropping compared with rotating cropping. Continuous cropping induced the enrichment of oxidative phosphorylation,pyruvate metabolism related microbial communities in the rhizosphere of tobacco. Rotating cropping can promote the aggregation of functionally related microorganisms such as nitrate reduction and aromatic compound degradation, promote soil nutrient cycling, and inhibit potential pathogenic bacteria. Gemmatimonas was positively correlated with total nitrogen, available potassium, organic matter content, urease and protease activities. Penicillium, Asterostroma, Fusarium were positively correlated with total phosphorus content and peroxidase and phosphatase activities. Conclusion Maize-tobacco rotation can effectively improve soil fertility, make bacteria more abundant, and increase the proportion of indicator beneficial bacteria, while continuous cropping leads to a large number of pathogenic bacteria. The increase in the relative abundance of pathogenic fungi may be the main factor leading to the occurrence of tobacco continuous cropping disorder. This study provides theoretical support for alleviating tobacco continuous cropping obstacles and soil detection and remediation in farmland.

Objective To explore the microbial characteristics in the Morchella spp. soil and provide scientific guidance for the rational cultivation of Morchella spp. Method High-throughput sequencing technology was applied to determine the communities and diversity indices of bacteria and fungi in the soils growing diseased and healthy Morchella spp., and further analyze their roles for transformation of soil substances, nutrient uptake and disease resistances of plant. Result The contents of available phosphorus, pH and the activity of catalase in the diseased Morchella spp. soil were significantly higher than those in healthy Morchella spp. soil, increasing with 13.22%, 6.18%, and 41.01%, respectively, while the activities of urease, acid phosphatase and sucrase significantly decreased with 58.58%, 31.85%, and 74.01% respectively. The bacterial indices of Chao1, ACE and Simpson in DM significantly decreased with 32.45%, 32.43%, and 20.42%, respectively compared to those in HM, and there was no significant change for fungal indices. The results of principal component analysis indicated that the community structure of bacteria (R²=0.195, P=0.028) and fungi (R²=0.17, P=0.001) in DM were significantly different from that in HM. The abundance of beneficial bacteria of Haliangium, Gemmatimonas and Flavobacterium in DMwere significantly lower than that in HM, which decreased by 92.85%, 90.48%, and 81.67%. The abundance of Enterobacter and Polaromonas in DMwere significantly higher than that in HM, which increased with 40.33 and 8.13 times, respectively. The abundance of beneficial fungus Schizothecium in DMwas significantly lower than that in HM, decreasing by 90.39%, and the abundance of Fusarium in the DM were significantly higher than that in HM, increasing with 2.51 times. The results of functional prediction showed that the metabolic pathways of bacteria in diseased Morchella spp. soil were different with that in healthy Morchella spp. soil, and the metabolic type of fungi was mainly saprotrophic. The results of Mantel analysis showed that the pH was the key factor affecting the fungal microbial community. The analysis of co-occurrence network showed that the average degree, total module number and average clustering coefficient of the bacterial and fungal network in the diseased Morchella spp. group were lower than those in the healthy group. Conclusion The richness and diversity of soil bacteria in the diseased Morchella spp. significantly decrease, the diseased Morchella spp. soil abundance of pathogen increase and that of the beneficial microbe decrease, and the complexity and stability of microbial network reduce, which are the important factors for the diseased Morchella spp.

Objective To isolate and screen protease-producing bacteria from sauerkraut juice, clarify the species classification status of the strains, optimize the fermentation enzyme production process, enrich the resources of protease strains, and improve the efficiency of fermentation enzyme production. Method Using the sauerkraut juice of Chinese cabbage in a sauerkraut workshop as the isolation source, the ten-fold layer dilution method was used to screen the casein plate, and the folinol method was further re-screened to obtain the protease-producing strains. Morphological observation and 16S rRNA gene phylogenetic analysis were combined to complete the species identification. Single factor test was to optimize the composition and conditions of the fermentation medium, and the response surface method was further to optimize the enzyme-producing process for improving the efficiency of enzyme production. Result A total of 15 protease-producing strains were obtained by initial screening, and strain P-133 with acidic, neutral and alkaline protease abilities was obtained by re-screening with forinol method. The acidic, neutral and alkaline protease activities were 1.00, 52.50 and 48.50 U/mL, respectively, and were identified as Pseudomonas sp. The results via single factor and response surface tests showed that the optimal conditions for enzyme production of strain P-133 were soluble starch 5.0 g/L, rapeseed meal 5.9 g/L, NaCl 3.3 g/L, K₂HPO₄ 5.0 g/L, pH 7.8, temperature 28℃, inoculation volume 5%. and liquid-loading capacity 75 mL/250 mL. Under these conditions, the acidic, neutral and alkaline protease activities were 4.14, 185.19 and 177.30 U/mL, respectively, which were 4.14, 3.53 and 3.66 times higher than those before optimization. Conclusion Pseudomonas sp. P-133 with acidic, neutral and alkaline proteases is isolated and screened from sauerkraut juice. The activities of acidic, neutral and alkaline proteases were 4.14, 3.53 and 3.66 times higher than those before optimization, respectively.

Objective To explore the impact of continuous high-dose intake of doxycycline (Dox) in the Tet-On system on the health status of experimental pigs. Method Nine 6-month-old Diannan miniature pigs were selected as model animals, and the recommended dose groups of 3 (30 mg/kg) times and 5 (50 mg/kg) times and blank control group were set up. The experimental pigs in each group were intramuscularly injected with corresponding drugs or physiological saline for 30 d. During the experiment, blood was collected on the day 0, 10, 20, and 28 for blood routine and liver and kidney function index measurements. The experimental pigs’ livers and kidneys at 2 h after last injection were subjected to morphological observation, pathological examination and disease detection. Result Compared with the control group, Lym# and MPV decreased, while PLT increased in the 30 mg/kg dose group; while the changes in these indicators were more significant in the 50 mg/kg dose group, and RDW-SD significantly reduced, meanwhile ALT, MG, P and other indicators increased, and NA, CO2, CYSC and other indicators decreased, showing more obvious damages to liver and kidney function. Histopathological examination results showed that the livers and kidneys of the experimental pigs in the 30 mg/kg dose group and the 50 mg/kg dose group had varying degrees of lesions, and the liver inflammatory cells and red blood cell infiltration in the 50 mg/kg dose group were more severe. The kidneys showed more obvious atrophy and structural tissue damage. The pathogenic infection detection results excludes the interference of viral infection on the experimental results. Conclusion This study preliminarily proves that long-term use of high-dose Dox can cause significant damage to experimental pigs' liver and kidney functions, and the degree of damage is positively correlated with the dose of Dox. This study may provide references for the research of conditional induced expression in large gene editing animals.