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    26 October 2023, Volume 39 Issue 10
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    Research Progress of bHLH Gene Family in Plants and Its Application Prospects in Medical Plants
    AN Chang, LU Lin, SHEN Meng-qian, CHEN Sheng-zhen, YE Kang-zhuo, QIN Yuan, ZHENG Ping
    2023, 39(10):  1-16.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0243
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    The basic/helix-loop-helix (bHLH) transcription factor represents the second largest family of transcription factors in the plant kingdom. It is widely distributed across the genomes of various plants and plays crucial regulatory roles in plant growth, development, secondary metabolism, and responses to abiotic stresses. Here, we present a comprehensive review focusing on the structural characteristics, taxonomic rules, and biological functions of the bHLH gene family, with particular emphasis on its involvement in plant growth, development, responses to abiotic stresses (such as drought, low temperature, salt, and heavy metals), and its significant role in the biosynthesis and dynamic accumulation of secondary metabolites. This thorough investigation allows for a deeper understanding of the contributions of bHLH to growth, development, stress resistance, and quality formation in plants. Moreover, it provides valuable insights for future research concerning the molecular regulatory mechanisms of bHLH in plant growth, development, stress resistance, quality formation, and the exploration of germplasm resources. Furthermore, bHLH has emerged as a prominent focus in the realms of molecular biopharmacology and ecological agriculture of Chinese medicine due to its extensive involvement in regulating the synthesis and accumulation of secondary metabolites in plants. We also summarize recent progress on the bHLH gene family and its members within two medicinal plants, namely Salvia Miltiorrhiza and Artemisia annua. This investigation aims to offer valuable references for in-depth studies on the bHLH gene family in medicinal plants and to propose novel ideas for the advancement of molecular breeding and anthropomorphic cultivation of medicinal plants, as well as the development of ecological agriculture for Chinese medicine.

    Research Advance in the Regulation Mechanism of Flower Spots Formation in Ornamental Plant
    QI Fang-ting, HUANG He
    2023, 39(10):  17-28.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0242
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    Many ornamental plants have various coloration patterns on their flower petals; these flower color variations are called flower spots. Flower spot is a very important ornamental characteristic of plants and have great significance in evolutionary biology. It could help plants to attract pollinators, defend against natural enemies and adapt to environmental changes. In this paper we reviewed recent advances in the molecular mechanism of flower spots formation, and then summarized the genetic law and influencing factors of flower spots formation. Moreover, we described the important roles of structural genes and transcription factors（TFs）related to pigment synthesis in the formation of flower spots in detail, i.e., the spatiotemporal specific expression and competition mechanism of structural genes promote the pigments differential accumulation in petals. TFs participate in the coloration patterns formation by directly or indirectly regulating the positioning expressions of structural genes. Furthermore, we introduced other regulation mechanisms, such as post-transcriptional regulation, post-translation regulation and methylation, and gathered the molecular regulatory network of flower spots formation, aiming to provide a new perspective for studying the molecular mechanism of flower spots formation in plants.

    Research Progress in Chitinase Involving in the Biocontrol of Crop Diseases and Pests
    MA Sai-mai, LI Tong-yuan, MA Yan-jun, HAN Fu-jun, PENG Hai, KONG Wei-bao
    2023, 39(10):  29-40.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0520
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    Pests and diseases seriously affect the quality, yield and safety of crops. As a green, safe and effective method to control pests and diseases, biological control has attracted more and more attention. Chitinase（EC 3.2.1.14）is a kind of glycosylated hydrolase widely existing in microorganisms and plants. It can effectively degrade the chitin in cell walls of pathogenic fungi, insects and nematodes causing diseases and insect pests, and inhibit the spore germination and mycelium growth of pathogenic fungi, and the development of insects and nematodes, which has an important role and application prospect in the biological control of crop diseases and insect pests. This paper reviewed the types and sources of chitinases, the biological control mechanism and control strategy of chitinase, and the application of transgenic technology based on chitinase in crop pest control，aiming to provide information for further investigation and application of chitinase in the fields of crop production, pests and diseases control.

    Research Progress in Bioactive Natural Products from Lysobacter
    ZHOU Shan-shan HUANG Yuan-long HUANG Jian-zhong LI Shan-ren
    2023, 39(10):  41-49.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0655
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    Microbial secondary metabolites are an important source of natural drugs and are widely used in medical and agricultural production. Actinomycetes and fungi are the main microorganisms that produce antibiotics, but it is becoming increasingly difficult to discover new antibiotics from them after a long and extensive screening. With advances in sequencing technology, more and more microbial genomes are being sequenced, and it has been found that many previously neglected microbes also have the potential to produce new antibiotics. The genus Lysobacter have antagonistic activities against a range of plant pathogenic fungi, bacteria, oomycetes, and nematodes, and several secondary metabolites with novel structures and significant biological activities have been identified from Lysobacter, which have potential applications in biocontrol agents and promising drug leads. In this paper, we review the distribution of Lysobacter resources and their secondary metabolites, aiming to provide a reference for further screening of Lysobacter from the environment and discovering more bioactive natural products.

    Research Progress in Anti-porcine Reproductive and Respiratory Syndrome Genetically Modified Pigs
    LI Shuang-xi, HUA Jin-lian
    2023, 39(10):  50-57.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0373
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    Porcine reproductive and respiratory syndrome is a viral infectious disease caused by Porcine reproductive and respiratory syndrome virus（PRRSV）. The disease is one of the three most important diseases affecting the healthy development of pig industry in China. At present, the prevention and control of the disease mainly depends on the internal and external biosecurity system of the farm, taking reasonable measures to reduce the pollution of the virus, and cutting off the circulation and transmission of the virus among pigs. From the perspective of protecting susceptible animals, breeding for disease resistance is also one of the major strategies for disease prevention and control. In recent years, with the development and maturity of gene editing technology, molecular breeding, as the core technology of pig disease resistance breeding, has shown its unique advantages. Several teams have made many breakthroughs in PRRS resistance breeding by using molecular breeding technology. This article takes the PRRSV receptor as the entry point, and reviews the current research status of pig PRRS-resistant breeding, aiming to provide reference and clues for the prevention and control of PRRS and the research of pig disease-resistant breeding.

    Preparation and Application of Polyclonal Antibodies Against Beauveria bassiana Mycovirus BbPmV-4 Coat Protein
    GUO Wen-bo, LU Yang, SUI Li, ZHAO Yu, ZOU Xiao-wei, ZHANG Zheng-kun, LI Qi-yun
    2023, 39(10):  58-67.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0383
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    Beauveria bassiana polymycovirus 4（BbPmV-4）increases the virulence of host fungus Beauveria bassiana; however, the mechanism of its replication, transmission and interaction with host fungi is still unclear. In the present study, the prokaryotic expression vector of mycovirus BbPmV-4 coat protein（CP）was constructed, designated as pET-28a（+）∷BbPmV-4-CP, which was transformed into Escherichia coli BL21（DE3）for prokaryotic expression. The Japanese big ear rabbits were immunized to prepare polyclonal antibodies, which were used to detect the intracellular localization and extracellular replication of the virus by immunological methods including indirect ELISA, Western blot and immunofluorescence, respectively. The results showed that the expressed BbPmV-4-CP was a soluble protein with a relative molecular weight of approximately 28.56 kD, the titer of prepared rabbit polyclonal antibody against which was 1∶256 000. Western blot analysis confirmed that the prepared antibody recognized the corresponding antigen protein and the virus BbPmV-4 in the host strain of B. bassiana. The mycelia of nontoxic healthy strains and B. bassiana containing virus BbPmV-4 were collected for liquid culture, and the virus content in the supernatant and precipitation of the culture was detected by indirect ELISA using the prepared antibody. The results showed that there was virus in the supernatant, indicating that mycovirus BbPmV-4 replicated in the host fungal cells and dissociated outside. The indirect immunofluorescence detection results revealed that the virus was located on the fungal nucleus. In this study, highly effective and specific polyclonal antibodies against mycovirus coat protein are prepared, and a serological detection system for mycovirus is established, which may provide experimental materials for studying the replication of mycovirus in host fungi and the mechanism of its interaction with host fungi, and hypervirulent mycovirus infection and its interactive genes may be used to regulate host virulence and create high virulence strains of B. bassiana.

    Construction of a New Mini Genome Editing System Based on Csy4 and MCP
    DENG Jia-hui, LEI Jian-feng, ZHAO Yi, LIU Min, HU Zi-yao, YOU Yang-zi, SHAO Wu-kui, LIU Jian-fei, LIU Xiao-dong
    2023, 39(10):  68-79.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0248
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    MCP is the coat protein of MS2 phage, and Csy4 is a small protein involved in crRNA generation of CRISPR 1-F system, which can recognize and bind RNA with high specificity. CRISPR/Cas and other genome editing technologies have some problems, such as large molecular weight of targeted nuclease, high off-target rate, and limited by PAM site. To solve these problems and construct new mini genome editing system based on Csy4 and MCP, AlphaFold2 was used to predict the structures of MCP-FokI, FokI-MCP, Csy4-FokI and FokI-Csy4 fusion proteins. The MCP-FokI and FokI-MCP editing vectors were transformed into Arabidopsis thaliana by the flower dip method, respectively, and the CLCrV-mediated Csy4-FokI and FokI-Csy4 editing systems were delivered using A. thaliana leaf injection. The A. thaliana genomic DNA was extracted and the editing ability of the novel mini-genome editing system was tested by HI-TOM high-throughput sequencing. The results showed that each domain of MCP, FokI and Csy4 maintained their original three-dimensional structures in the fusion proteins, suggesting that they all functioned properly. The plant expression vectors of MCP-FokI and MCP-FokI binding dual target sites with four different intermediate spacer regions were constructed to target the AtCLA1 genes. Sequencing results demonstrated that neither MCP-FokI nor FokI-MCP achieved the targeted editing of the target genes. Seven CLCrV-mediated Csy4-FokI and FokI-Csy4 editing vectors with different intermediate spacer regions were constructed to target the AtCLA1 genes. Among them the CLCrV-mediated Csy4-FokI editing system was able to achieve targeted editing of the target gene, but the mutation types were all base substitution types and the editing efficiency was low. No genome editing was detected in the FokI-Csy4 system. Taken together, a novel Csy4-FokI mini-genome editing system was successfully constructed, which may provide an innovative solution to overcome the problems of CRISPR/Cas genome editing technology.

    Development of an L-tryptophan Biosensor Based on the Violacein Biosynthesis Pathway
    LI Ren-han, ZHANG Le-le, LIU Chun-li, LIU Xiu-xia, BAI Zhong-hu, YANG Yan-kun, LI Ye
    2023, 39(10):  80-92.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0271
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    High-throughput screening combining with biosensors is a powerful tool for identifying high-yield L-tryptophan producing strains, but current L-tryptophan biosensors generally have the disadvantage of low operational and dynamic ranges. By expressing the violacein biosynthetic pathway in Escherichia coli and testing different sources of VioA enzyme and RBS engineering, a novel enzyme-coupled L-tryptophan biosensor was developed. It was found that the VioA enzyme from Chromobacterium violaceum expanded detection limit of the biosensor up to 0-10 g/L of exogenously added L-tryptophan. Reducing its translation initiation rate to approximately 2 000 expanded the dynamic range of the biosensor by 55-fold. Different strains of E. coli with varying L-tryptophan yields could be visually distinguished with the naked eye. This novel L-tryptophan biosensor can play a significant role in identifying high-yield L-tryptophan and its high-value derivatives producing strains through combining methods such as high-throughput screening.

    Application of FACS Technology in the Directed Evolution of Enzyme
    LIU Jin-sheng, CHEN Zhen-ya, HUO Yi-xin, GUO Shu-yuan
    2023, 39(10):  93-106.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0486
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    Flow cytometry（FCM）is a rapid and relatively quantitative multi-parameter analytical technique for analyzing a large number of cells at the single-cell level. The constructed fluorescence activated cell sorting（FACS）based on FCM can physically separate fluorescent-labeled single cells and has been widely used in the field of directed evolution of enzyme. Directed evolution has been proven to be an effective method for obtaining industrial enzymes with excellent properties, such as high enzyme activity, strong thermal stability and solvent resistance. To conduct directed evolution, mutant library firstly needs to be constructed by random mutagenesis or DNA recombination, and then be screened under manual selection pressure. Traditional screening methods such as plate and microplate methods have limitations of low throughput, high cost, large errors, and poor accuracy, and cannot achieve high-throughput screening of large mutant library. Compared with traditional screening methods, FACS has the advantages of high throughput, low cost, small error and high precision. In this article, we first summarized the development process of FCM and FACS, as well as the composition and classification of related instruments derived from them. Then, we discussed the application status and limitations of FACS in the directed evolution of enzyme. Finally, we summarized and prospected the development direction of FACS and its application in the field of directed evolution of enzyme.

    Research Progress in Microfluidic Technology in the Detection of Pathogenic Microorganisms
    WAN Qi-wu, BAO Xu-dong, DING Ke, MOU Hua-ming, LUO Yang
    2023, 39(10):  107-114.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0479
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    Rapid and accurate detection of pathogenic microorganisms is of great significance for epidemic prevention and control as well as the protection of people's lives and health. In recent years, researchers have developed a variety of technical methods for the detection of pathogenic microorganisms by reasonably designing microfluidic chips and combining microfluidic technology with various detection technologies. Compared with traditional pathogenic microorganism detection technology, microfluidic detection technology has outstanding advantages, i.e., low technical requirements for operators, less sample demand and high degree of automation, and is suitable for accurate and rapid detection of pathogenic microorganisms in various complex environments. In this paper, the application of microfluidic technology in the detection of pathogenic microorganisms such as viruses, bacteria, fungi, chlamydia and mycoplasma was reviewed, aiming to provide research ideas for the detection of pathogenic microorganisms, promote the development of microfluidic technology in the detection of pathogenic microorganisms, and improve the prevention and control ability of diseases.

    Cloning, Functional Identification and Expression Analysis of FAH, a Key Gene for Tyrosine Metabolism in Brassica napus L.
    ZHI Tian-tian, ZHOU Zhou, CHEN Ji-peng, HAN Cheng-yun
    2023, 39(10):  115-127.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0270
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    The objective of this work is to clone the tyrosine metabolism key gene FAH in Brassica napus L., identify its function and analyze its expression, so as to provide theoretical evidence for further understanding the role and function of FAH in B. napus L. Two FAH genes BnaA06g38260D（BnaA06FAH）and BnaC05g49430D（BnaC05FAH）, which was the highest protein homology to AtFAH in Arabidopsis, were cloned from the B. napus L. variety‘westar’, the phylogenetic relationship was analyzed by bioinformatics, and the overexpression vector was constructed and transformed into Arabidopsis mutant sscd1 for function verification; the promoter sequences of two FAH genes BnaA06FAH and BnaC05FAH were cloned. PlantCARE was used to analyze the promoter cis-acting elements, and the fusion expression vector of gene promoter and GUS was constructed to analyze its expression patterns. As results, the amino acid sequence similarity of BnaA06FAH, BnaC05FAH and AtFAH was 93.11% and 92.40% respectively, the overexpressions of BnaA06FAH and BnaC05FAH completely inhibited the mimic lesion in sscd1 under short-day condition, suggesting both BnaA06FAH and BnaC05FAH had high structural and functional similarity with AtFAH. BnaA06FAH and BnaC05FAH promoters had the core elements of eukaryotic promoter TATA-box and CAAT-box, and also contained several cis-acting elements related to stress, hormone, stress response and a variety of cis-acting elements related to disease resistance, however, BnaC05FAH promoter shared more cis-acting elements with Arabidopsis AtFAH than with BnaA06FAH. GUS activity assays indicated BnaC05FAH promoter drove the expression of GUS gene stronger than BnaA06FAH, and the site of GUS gene expression drove by two different promoters were not completely consistent. Conclusively, there were differences in the sites and intensity where BnaA06FAH and BnaC05FAH promoters functioned. The results showed that both BnaA06FAH and BnaC05FAH played a role in regulating lesion mimic in the sscd1 mutant, but the expression of gene downstream and the expression site drove by BnaA06FAH and BnaC05FAH promoters was different.

    Cloning，Expression and Functional Identification of PRR5 Gene in Pakchoi
    HOU Rui-ze BAO Yue CHEN Qi-liang MAO Gui-ling WEI Bo-lin HOU Lei-ping LI Mei-lan
    2023, 39(10):  128-135.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0338
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    Cloning BrcPRR5 and studying its expression pattern and function in different tissues and different developmental stages may lay a foundation for understanding the effect of PRR5 on flowering transformation of pakchoi. The homologous gene BrcPRR5 of PRR5 was cloned by RT-PCR and analyzed by bioinformatics. The relative expressions of the gene in different tissue sites and different developmental stages were determined by fluorescence quantitative PCR. The overexpression vector was constructed and transformed into Arabidopsis for functional verification. The results showed that the full-length CDS of BrcPRR5 was 1 701 bp, encoding 566 amino acids. Through multiple amino acid sequence alignment with homologous proteins of other species, the obtained sequence belonged to PRR5 homologous gene of pak choi. The expression of BrcPRR5 in the stem and flower was higher than that in the leaf and pod, and the highest expression was in S0 stem tip, indicating that the up-regulated expression of S0 promoted floral transformation during flowering transition of pakchoi. The flowering stage of overexpressed transgenic plants was earlier, and the plant height and stem diameter of transgenic plants were significantly better than those of wild type. BrcPRR5 may promote plant bolting and flowering earlier.

    Functional Analysis of WRKY6 Gene in Tomato Under Low-phosphorus Stress
    CHEN Hao-ting, ZHANG Yu-jing, LIU Jie, DAI Ze-min, LIU Wei, SHI Yu, ZHANG Yi, LI Tian-lai
    2023, 39(10):  136-147.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0339
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    The purpose of this study is to analyze the role of tomato transcription factor SlWRKY6 in response to low-phosphorus stress, and to lay a theoretical foundation for studying the molecular mechanism of low-phosphorus tolerance in tomato and exploring tomato germplasm resources of improving low-phosphorus tolerance and phosphorus utilization. Wild-type tomato Ailsa craig was used as the material and its cDNA was used as the template to clone SlWRKY6, and the RNAi-SlWRKY6 and OE-SlWRKY6 transgenic tomato plant lines were constructed by agrobacterium tumefacien-mediated transformation. Tomato plants with three different genotypes of wild type, RNAi-SlWRKY6 and OE-SlWRKY6, were subjected to low phosphorus. On the day 18 of low-phosphorus treatment, the results of phenotype and physiological and biochemical indexes in the leaves and roots were determined. Phenotypic observation showed that OE-SlWRKY6 transgenic tomato plants were of thicker and shorter after low phosphorus treatment while compared with RNAi-SlWRKY6 transgenic tomato plants, they had a higher number of leaves, and were less affected by low-phosphorus stress, and showed stronger low-phosphorus tolerance. The analysis results of physiological and biochemical indexes showed that the organic phosphorus and total phosphorus content in the roots and leaves of transgenic tomato plant OE-SlWRKY6 significantly increased compared with wild type, while the acid phosphatase activity and some organic acid contents in the roots significantly increased. The expressions of phosphorus transporters also significantly reduced. However, the trend of changes in these indicators in the transgenic tomato plant RNAi-SlWRKY6 under low-phosphorus stress was opposite to that in transgenic tomato plant OE-SlWRKY6. After low-phosphorus stress, the relative expressions of LePT1 decreased in the roots of OE-SlWRKY6 plants, which was opposite to that of RNAi-SlWRKY6 plants. The relative expressions of LePT2 in the roots of wild type, RNAi-SlWRKY6 and OE-SlWRKY6 plants showed an overall increasing trend, while the relative expressions of LePT3 showed an overall decreasing trend. According to the above results, SlWRKY6 responded to low-phosphorus stress and its expression was positively correlated with the low-phosphorus tolerance of tomato plants, and might affect the response of phosphorus transporter genes to low phosphorus. The results of this study may provide certain theoretical basis for further revealing the function of tomato WRKY transcription factor family and its response mechanism to low-phosphorus stress.

    Effects of Hormonal and Adversely Stress on Vitamin E and γ-TMT Gene Expression in Soybeans
    BAI Miao, TIAN Wen-qing, WU Shuai, WANG Min, WANG Li-xiang, YUE Ai-qin, NIU Jing-ping, ZHANG Yong-po, GAO Chun-yan, ZHANG Wu-xia, GUO Shu-jin, DU Wei-jun, ZHAO Jin-zhong
    2023, 39(10):  148-162.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0183
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    The response of vitamin content and synthesis-related gene γ-TMT to hormonal and stress stress was clarified, which opened up new ideas for vitamin E to participate in the study of adversity stress. Taking Jinda 88, the main cultivars of Shanxi province, as the experimental materials, the vitamin E component content, physiological indexes and γ-TMT expression patterns in different tissues were analyzed under hormonal and adversity stress. The results showed that vitamin E was mainly present in the leaves, a small amount in stems, and absent in roots, and the total vitamin E content in leaves and stems was smaller than that in control under stress conditions such as hormones and salts, but the content of γ-tocopherols under NaCl, H2O2 and ABA stress was higher than that of the control. Under hormonal and adversity stress, the growth of soybean plants was significantly inhibited, and the plant height, fresh weight, dry weight, SPAD value and water content were all lower than those of the control. Genomic data were mined to 3 γ-TMTs, and there were many stresses response and hormone response elements in the promoters, and the expression of γ-TMT1-γ-TMT3 in roots and leaves was upregulated or down-regulated under hormonal and adversity stress. Under NaCl stress, the content of γ-tocopherol in soybean leaves increased, while the expression of γ-TMT2 and γ-TMT3 was significantly down-regulated, and it was speculated that γ-TMT2 and γ-TMT3 may affect the content of vitamin components under salt stress. Hormonal and adverse stress significantly inhibited the contents of vitamin E in soybean stems and leaves, which in turn inhibited plant growth, i.e.,affected soybean γ-TMT expression and vitamin E components.

    Cloning of Sugarcane ShPR10 Gene and Study on the Interaction Between ShPR10 Protein and P1 Protein Encoded by Sugarcane Streak Mosaic Virus
    HUANG Jia-yan, FENG Xiao-yan, SHEN Lin-bo, WANG Wen-zhi, HU Hai-yan, ZHANG Shu-zhen
    2023, 39(10):  163-174.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0403
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    Pathogenesis related protein 10（PR10）plays an important role in plant resistance to viral infection. In the early stage, the RNA silencing suppressor P1 encoded by sugarcane streak mosaic virus（SCSMV）was used as bait to screen and obtain a sugarcane ShPR10 protein. In order to explore the function of ShPR10 in sugarcane response to SCSMV infection, the sugarcane ShPR10 gene was cloned by homologous cloning technology, and its coding protein was analyzed via bioinformatics. The subcellular localization of ShPR10 protein was analyzed by fusion expression with green fluorescent protein. The interaction between ShPR10 and SCSMV P1 was validated by yeast two hybrid and bimolecular fluorescence complementation techniques. The effect of ShPR10 on P1 silencing suppressor activity was analyzed using the Agrobacterium tumefaciens co-infiltration transient expression system and Western blot technology. The results showed that the open reading frame of sugarcane ShPR10 gene was 570 bp, which encoded an unstable hydrophilic protein with a molecular weight of 21.17 kD, an isoelectric point of 4.77, one P-loop motif, and no transmembrane domains and signal peptides. The secondary structure of ShPR10 contained 51.85% random coil, 35.98% α-helix, 7.41% extended-strand, and 4.76% β-turn. The amino acid sequence similarity between ShPR10 protein and ZmPR10 protein of Zea mays was as high as 91.53%, and the two proteins were clustered into one branch on the evolutionary tree. ShPR10 was located in the cytoplasm and nucleus, and interacted with SCSMV P1 in yeast and tobacco cells. ShPR10 itself did not have silencing suppressor activity, and its expression weakened the silencing suppressor activity of P1, but had no significant effect on the content of P1 protein. In summary, ShPR10 may weaken the silencing suppressor activity of P1 by binding to P1, thereby improving the resistance of sugarcane to SCSMV.

    Optimization of Sweet Potato Genetic Transformation System Mediated by Agrobacterium rhizogenes
    TAO Na, LI Mao-xing, GUO Hua-chun
    2023, 39(10):  175-183.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0369
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    Genetic transformation system is of great significance for the verification of plant gene function. In order to establish Agrobacterium rhizogenes mediated genetic transformation system in sweet potato（Ipomoea batatas L.）, a three-factor design of 3×3×5 was used to study the effects of infection time, explant type, and A. rhizogenes strains on the induction rate of hairy roots in sweet potato. The appropriate explant type and infection time were determined, and the 5×5 two-factor design was used to study the effects of five A. rhizomei strains and sweet potato varieties（lines）on the induction rates of sweet potato. The detection of induced hairy roots by PCR confirmed that the rolB gene of the A. rhizogenes Ri plasmid was integrated into the genome of sweet potato cells. The results showed that the infection time, explant type, A. rhizogenes strain, and sweet potato variety all affected the induction rate of hairy roots, and there was an interaction between the factors. Stem segment was the most suitable explants for hair roots, and the optimal infection time was 20 min. MSU440 was the suitable strain for ‘Xushu 22’. The suitable strain for ‘Taizhong 6’ ‘YS’ were K599. The suitable strain for‘1610’ was C58C1. The suitable strain for ‘Huishu’was C58C1. Using the A. rhizogenes strain MSU440, stem segments of ‘Xushu 22’were used as explants and Agrobacterium infection for 20 min, and the highest induction rate of hairy root was 79.63%. The transgenic efficiency of the hairy root of sweet potato was found to be 38.1% by PCR amplification of the target gene. A genetic transformation system of sweet potato mediated by A. rhizogenes was established, which lays the foundation for further transgenic breeding of sweet potato.

    Identification of the Cysteine Protease Family and Corresponding miRNAs in Nicotiana tabacum L. and Their Responses to PVY
    YIN Guo-ying, LIU Chang, CHANG Yong-chun, YU Wang-jie, WANG Bing, ZHANG Pan, GUO Yu-shuang
    2023, 39(10):  184-196.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0297
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    Plant cysteine proteases（CPs）widely affect plant physiological processes and disease resistance regulation. To study the functional mechanism of tobacco cysteine proteases in regulating potato virus Y（PVY）, the CPs family genes of Nicotiana tabacum L. at the genome-wide level were identifed. The evolutionary relationships, motifs and promoter cis-acting elements of CPs family genes were analyzed by bioinformatics. Then the miRNAs corresponding to CPs were screend, the expression characteristics of CPs genes and their corresponding miRNAs after PVY infection were analyzed. The results showed that a total of 70 CPs genes were identified in Nicotiana tabacum L. genome, which were divided into five subfamilies, of which C1A contained 39 CPs, C2A contained 2 CPs, C12 contained 5 CPs, C13 contained 8 CPs and C14A contained 16 CPs. Motif analyses showed that the CPs of subfamily had similar motif distribution. Promoter cis-acting elements analysis showed that several CPs family gene promoters contained light response elements, hormone response elements and low temperature, drought, high temperature, salt stress response elements. Based on the sequencing results of transcriptome and small RNA, it was found that 38 CPs were up-regulated while 18 CPs were down-regulated after PVY infection.Total 51 miRNAs were found to be targeted to 28 members of cysteine protease gene family, 38 miRNAs were up-regulated while 13 were down-regulated after PVY infection,7 of them were negatively correlated with miRNAs.The results of RT-qPCR showed that 15 CPs genes were significantly up-regulated after PVY infection. This study is conducive to better understanding the functions of CPs genes and their corresponding miRNAs in regulating tobacco PVY responses, which may provide evidence for the molecular mechanism of anti-PVY in Solanaceae crops.

    Regulation of Leaf Bud by REVOLUTA in Tobacco Based on CRISPR/Cas9 System
    WANG Bing, ZHAO Hui-na, YU Jing, CHEN Jie, LUO Mei, LEI Bo
    2023, 39(10):  197-208.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0364
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    The leaf buds of crop are regulated by the meristem, which is one of the effective measures to increase crop yield. At present, there are few studies on the molecular mechanism of meristem regulation in tobacco, and there is a lack of germplasm resources that can be used for plant type improvement. In this study, two different REVOLUTA（REV）single-target sequences, C15NtREV and C16NtREV, were constructed respectively by targeting mutation of REV gene through CRISPR/Cas9 editing system, and regenerated seedlings were obtained by Agrobacterium-mediated leaf disc transformation method, and the transgenic plants were identified by PCR sequencing. The sequencing results showed that the Ko-C15Ntrev mutant had a shift mutation after amino acid position 26 of NtREV, while the Ko-C16Ntrev mutant had a shift mutation after amino acid position 60 of NtREV. In addition, the apical bud phenotypes of two single-target homozygous mutants were observed separately by scanning electron microscope, the results showed that the double-copy homologous mutants of Ko-C15Ntrev had apical bud deletion and leaf deformities, while the single-copy homologous mutants of Ko-C16Ntrev did not have the absence of apical buds, but the apical buds development delayed. Compared with wild type, the natural plant height of the Ko-C16Ntrev mutant increased by 3.76%, while the number of leaf blades and fresh axillary bud weight of the Ko-C16Ntrev mutant decreased by 21.47% and 23.41%, respectively, and both reached extremely significant differences, indicating that NtREV is involved in the development of shoot apical meristem in tobacco, which in turn regulates the development of leaf and axillary bud. These mutants provided important research materials for the subsequent study of the molecular mechanism of bud development in tobacco.

    Isolation and Identification of Apple Brown Rot Pathogen in Parts of Gansu and Screening of Antagonistic Bacteria
    JIANG Jing-jing, ZHOU Zhao-xu, DU Hui, LYU Zhao-long, WANG Chun-ming, GUO Jian-guo, ZHANG Xin-rui, LI Ji-ping
    2023, 39(10):  209-218.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0234
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    This work aims to clarify the pathogenic species of apple brown rot in Jingning county and Yuzhong county of Gansu province and to screen high-quality pathogen antagonistic bacteria for providing resources for biological control of apple postharvest diseases. The pathogens were isolated and purified by conventional tissue isolation method, verified by Koch's postulates, and identified by molecular biology method combined with morphology. The antagonistic bacteria were screened by plate confrontation, identified by morphological, physiological, biochemical and molecular biological methods, and the disease control effects of the antagonistic bacteria were determined by in vitro fruits. The results showed that the pathogens causing apple brown rot in Jingning county and Yuzhong county of Gansu province were Monilia yunnanensis. The optimal antagonistic bacterium X2-2 was Bacillus amyloliquefaciens, which inhibited（81.29±0.57）% of the mycelium of M. yunnanensis and 98.8% of the control effect of the antagonistic bacteria on the isolated fruits. It significantly inhibited the expansion rate of M. yunnanensis lesion spots of apple fruits. M. yunnanensis was the main pathogen of apple brown rot in some areas of Gansu province. The endogenous antagonistic bacterium X2-2 screened in this study had significant antibacterial effect and had the potential of being a high-quality biocontrol strain.

    Physiological and Metabolitic Mechanisms of Different Pineapple Cultivars Responding to Low Temperature Stress
    LIU Chuan-he, HE Han, HE Xiu-gu, CHEN Xin, LIU Kai, SHAO Xue-hua, LAI Duo, QIN Jian, ZHUANG Qing-li, KUANG Shi-zi, XIAO Wei-qiang
    2023, 39(10):  219-230.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0071
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    The aim of this trial is to explore the relevant mechanisms of two different pineapple cultivars responding variously to low temperature stress. Having the pineapple cultivars ‘Yuetian’ with stronger chilling-resistance and ‘Comte de Paris’ with weaker chilling-resistance as materials, the physiological aspects, relative expressions of genes and differentially accumulated metabolites（DAMs）of the pineapple leaves were determined and compared under low temperature（LT, 5℃）and normal temperature（NT, 25℃）treatments for 48 h. The obtained results indicated that the contents of soluble protein, soluble sugar and proline, as well as the activities of POD, SOD and CAT enzymes in ‘Yuetian’ were significantly higher than those in ‘Comte de Paris’ after LT treatment, while the content of MDA was significantly lower than that in ‘Comte de Paris’, respectively. After LT treatment, the relative expressions of POD and CAT genes in ‘Yuetian’ were significantly higher than those in ‘Comte de Paris’, respectively; whereas MDA content was significantly lower than that of ‘Comte de Paris’, and the relative expressions of POD and CAT genes was significantly higher than that of ‘Comte de Paris’. The contents of phytohormones of auxin, cytokinin, jasmonic acid, salicylic acid and strigolactone in ‘Yuetian’ were higher than those in ‘Comte de Paris’, nevertheless the contents of gibberellin and abscisic acid were lower than those in ‘Comte de Paris’ after LT treatment, respectively. Metabolomics analysis showed that a total of 262 DAMs were detected between ‘Yuetian’ and ‘Comte de Paris’ after LT treatment, which predominantly included flavonoid metabolites. KEGG analysis indicated that the DAMs were principally enriched in the three pathways including biosynthesis of secondary metabolites, flavonoid biosynthesis, as well as flavone and flavonol biosynthesis. Further, 110 DAMs were screened between ‘Yuetian’ and ‘Comte de Paris’ e induced by LT treatment, and the accumulating levels of these screened metabolites in ‘Yuetian’ were correspondingly observed to be significantly higher than those in ‘Comte de Paris’. Among the 110 DAMs, 41 had significant differences at NT condition, and the fold changes increased after LT treatment. Besides, 69 had no significant differences at NT condition and had significant differences after LT treatment. Summarily, the diversity in osmotic adjustment and anti-oxidized activity, as well as endogenous hormones and metabolites accumulation might be the vital aspects that result in the various responses to low temperature stress for the two involved pineapple cultivars.

    Transcriptome Analysis of Rhizome Development in Chrysanthemum× × morifolium
    XU Jun, YE Yu-qing, NIU Ya-jing, HUANG He, ZHANG Meng-meng
    2023, 39(10):  231-245.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0315
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    In order to explore the molecular mechanism of chrysanthemum rhizome formation, we performed transcriptome analysis of eight tissues（RH: rhizome apical; RT: middle of rhizome; RB: bottle of rhizome; SA: shoot apical; L: leaf; S: shoot; R: root; F: ray floret）of chrysanthemum. Approximately 159.51 gigabase（GB）paired-end clean reads were obtained and assembled into 100 235 Unigenes. Among these Unigenes, 64 956（64.80%）were annotated in seven public protein databases. In order to find the key genes of rhizome development, the highly expressed genes in the rhizome tip and rhizome were screened by weighted correlation network analysis（WGCNA）, K-means and screening differential genes based on Venn. Finally, a total of 20 highly expressed genes in the rhizome tip and 36 highly expressed genes in the rhizome were obtained. These genes include a large number of genes related to plant abiotic stress, the ABA metabolic gene. In addition, the phytochrome gene PHYB, the UV-B photoreception gene UVR8, and the photoperiod core transcription factors were also uncovered, six differentially expressed genes were selected for RT-qPCR analysis, and the results were consistent with RNA-seq data, verifying the validity of RNA-seq data. And these differentially expressed genes were also expressed differentially in the rhizomes of chrysanthemum strain‘2005042’.Taken above together, the formation and development of chrysanthemum rhizomes may be affected by plant hormones including abscisic acid and photoperiod. This study provides important clues to further explore the molecular mechanism of the development of chrysanthemum rhizomes.

    Identification, Biocontrol and Plant Growth-promoting Potential of Endophytic Bacterial Strain JY-3-1R from Aconitum carmichaelii Debx.
    ZOU Lan, WANG Qian, LI Mu-yi, YE Kun-hao, HUANG Jing
    2023, 39(10):  246-255.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0590
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    In order to provide promising microbial resource for the control of soil-borne diseases in Aconitum carmichaelii Debx., endophytic bacterial strains with biocontrol and plant growth promoting potential were isolated and screened from healthy A. carmichaelii plants. Pure culture method was used to isolate endophytic bacteria, and dual-culture method was to check the antagonistic activity of candidate strains against pathogens. Multi-locus housekeeping gene sequence analysis was to investigate phylogeny of the strain. Enzyme- and siderophore-producing capacities of bacterial strain were measured on plates. Salkowski method was applied to measure indoleacetic acid（IAA）-producing ability of the strain. Field experiment was employed to demonstrate plant growth promoting ability and biocontrol potential against southern blight in A. carmichaelii. One strain JY-3-1R screened from 111 A. carmichaelii endophytic bacteria showed significant antagonistic activity against Sclerotium rolfsii and Fusarium oxysporum in vitro. The JY-3-1R was identified as Bacillus amyloliquefaciens. Cell-free culture filtrate of JY-3-1R presented 100% inhibition on the hyphal growth and sclerotia germination of S. rolfsii. JY-3-1R was able to produce IAA, siderophore, protease, cellulase, glucanase, also had the functional genes of synthesizing iturin, fengyin, surfactin, bacillomycin, bacilysin, bacillaene, and macrolactin. Under field conditions, JY-3-1R inoculation reduced around 40% of southern blight occurrence, whose biocontrol rates ranged from 61.53% to 84.61%. This biocontrol efficiency lasted at least for 30 d. Meanwhile, JY-3-1R inoculation promoted 34.34%, 82.59% and 56.08% of stem, main root and lateral root dry weight, respectively of A. carmichaelii. Altogether, JY-3-1R demonstrated both plant growth promoting and biocontrol potential, and could be used as good candidate of biofertilizer and biocontrol agent of A. carmichaelii.

    Cloning and Function Analysis of Gene UGTPg17 and UGTPg36 in Lonicera macranthoides
    YANG Min, LONG Yu-qing, ZENG Juan, ZENG Mei, ZHOU Xin-ru, WANG Ling, FU Xue-sen, ZHOU Ri-bao, LIU Xiang-dan
    2023, 39(10):  256-267.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0194
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    This work is to clone the genes of glycosyltransferases UGTPg17 and UGTPg36 from Lonicera macranthoides, and analyze the correlation between their expression and saponin content at different flowering stages. According to the Unigene sequence of L. macranthoides transcriptome, specific primers were designed to clone UGTPg17 and UGTPg36. The physical and chemical properties, protein structure and evolutionary relationship of the cloned UGTPg17- and UGTPg36-encoding proteins were analyzed using the bioinformatics websites; quantitative reverse-transcription polymerase chain reaction（RT-qPCR）was used to analyze gene expressions at different flowering stages. The content of L. macranthoides saponin was determined by HPLC; and the correlation between gene expression and saponin content was analyzed by SPSS. The open reading frame length of UGTPg17 and UGTPg36 genes was 1 410 bp and 1 428 bp, respectively, encoding 469 and 475 amino acids, containing 4 and 7 glycosylation sites, respectively, which belonged to unstable and hydrophilic proteins without transmembrane region. UGTPg36 protein did not contain signal peptide sequence, and the average S value of UGTPg17 protein was >0.5, suggesting that it may contain signal peptide. The expressions of UGTPg17 and UGTPg36 in different flowering stages showed an upward trend, and the saponin content fluctuated in different flowering stages. The correlation analysis between the two showed that the two UGT genes were significantly negatively correlated with the saponin content. In this study, UGTPg17 and UGTPg36 genes were successfully cloned from L. macranthoides, and their expressions variedt at different flowering stages. Correlation analysis showed that UGTPg17 and UGTPg36 genes were related to saponin biosynthesis of L. macranthoides, which lays a foundation for further exploring their specific functions.

    Sequencing Analysis of the Whole Genome of Streptomyces sp. FXP04
    TANG Bi-yao FU Xue-peng
    2023, 39(10):  268-280.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0178
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    Streptomyces sp. FXP04 is a gram-positive actinomycete isolated from the rhizosphere soil of healthy potato in Keshan county, Qiqihar, which has obvious antagonistic effect against Phytophthora infestans. This work aims to analyze the whole genome sequence information of strain FXP04, to explore the mechanism of action as well as the gene resources for the biosynthesis of secondary metabolites. The whole genome of Streptomycessp. FXP04 was sequenced by Illumina and PacBio platform, and gene prediction, functional annotation, comparative genomics analysis and synthesis gene cluster prediction of secondary metabolite were carried out. The genomic size of strain FXP04 was 4 535 201bp, encoding a total of 5 037 genes, with a GC content of 72.95%, including three 5S rRNA, three 16S rRNA, three 23S rRNA, 33 tRNA and 23 sRNA. It contained 1 699 tandem repeats，1 284 microsatellite DNA,188 microsatellite DNA. It was annotated to 2 088, 2 319, 1 530, 73, 139 and 14 genes in GO, COG, KEGG, CAZy, VFDB and ARDB databases, respectively. At the same time, it was predicted that there were 10 secondary metabolite synthesis gene clusters in strain FXP04, which encoded bacteriostatic substances such as piericidin, youssoufene and so on. The internal mechanism of strain FXP04 was analyzed by genome sequencing, which provides further theoretical basis for the development and utilization of strain FXP04.

    Affecting Mechanism of Loop B3 on the Function of GH7 Endoglucanase
    YANG Jun-zhao, ZHANG Xin-rui, SUN Qing-yang, ZHENG Fei
    2023, 39(10):  281-291.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0307
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    Glycoside hydrolase family 7（GH7）contains two kinds of cellulases, cellobiohydrolase and endoglucanase, of which the study of cellobiohydrolase is more mature, however few studies on endoglucanase. A thermophilic endoglucanase of GH7 from the genome of the M. thermophila, MtCel7b, was identified and characterized with the maximum activity at 60℃, pH 5.0. After 1 h incubation at 90℃, MtCel7b retained over 40% activity. The loop B3 of MtCel7b, can be divided into short- and long-chains based on amino acid sequence comparison. In order to explore the effect of loop B3 on the structure and function of, endoglucanase, the mutant B3cut was formed by truncating 14 amino acids on long-chain B3 loop of MtCel7b. At high temperatures, the activity of B3cut was 9%-44% higher than that of MtCel7b; however, the hydrolysis ability of B3cut to different substrates was reduced by 34%-74%. Additional analysis with the assistance of molecular dynamics simulations demonstrated that in the mutant B3cut, the truncation of its B3 loop led to a significant displacement of loop A3 and loop B1 at both ends of the catalytic cleft, narrowing the spatial structure of the catalytic pocket and strengthening the network of hydrogen bonding interactions around the catalytic site, resulting in a more stable enzyme at high temperatures. This study sheds light on the role of loop B3 in GH7 endoglucanase and provides insight into the improvement of the enzyme molecule.

    Whole Genome Sequencing Analysis of a Phenol-degrading Strain Alcaligenes faecalis JF101
    ZHANG Ao-jie, LI Qing-yun, SONG Wen-hong, YAN Shao-hui, TANG Ai-xing, LIU You-yan
    2023, 39(10):  292-303.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0281
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    The genus Alcaligenes shows extensive degradation capacity for aromatic compounds. In the present study, the phenol-degrading genes in Alcaligenes faecalis JF101 were analyzed and the potential functions were explored. The whole genome of strain JF101 was sequenced, assembled and annotated for analyzing the genes involved in phenol degradation. Moreover, the comparative genomics with five related strains was carried out. Results showed that the whole genome size of strain JF101 was 4 143 816 bp, and the GC content was 57.44%, with 3 804 protein-coding genes, 55 tRNAs genes, 9 rRNAs genes and 5 sRNA. There were 3 040, 2 529 and 2 415 annotated genes in COG, GO and KEGG databases, respectively. Eleven genes involving in phenol degradation were identified. Comparative genomics revealed that strain JF101 had no plasmid, and shared 2 008 common homologous gene families and 5 specific gene families with the five related strains. In addition, the putative gene Trk and Kdp involved in compatible solute transport, were found in strain JF101, and the salt-tolerant ability was confirmed by experiments. The whole-genome analysis of strain JF101 will benefit the further elucidating mechanism of phenol degradation and developing engineering applications.

    Allogeneic Expression of Cholesterol 7α-hydroxylase in Pichia pastoris
    ZHAO Xin, DU Yu-yao, YIN Zi-yang, MAO Shu-hong
    2023, 39(10):  304-310.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0387
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    Cholesterol 7α-hydroxylase（CYP7A1）is a rate-limiting enzyme that breaks down cholesterol into bile acids, which catalyzes cholesterol to produce 7α-hydroxycholesterol, it is an important steroidal drug and intermediate. Taking human CYP7A1 as the research object, firstly, recombinant Pichia pastoris/pPIC3.5K-CYP7A1 was constructed, and then the yield of the target product 7α-hydroxycholesterol increased by site-directed mutation and adaptation to NADPH cytochrome oxidoreductase（CPR）from different sources. Among them, the mutant G485A increased the yield of 7α-hydroxycholesterol by 8.70%. The co-expression of P. pastoris with five different sources of CPR and CYP7A1 increased the yield of 7α-hydroxycholesterol, and the CPR（SgCPR）derived from Siraitia grosvenorii was the most compatible with CYP7A1, which increased the yield of 7α-hydroxycholesterol by 82.26%. Based on the above research, the co-expression of G485A and SgCPR in P. pastoris increased the yield of 7α-hydroxycholesterol by 133.79%, and the yield was 0.25 mg/L. The human cholesterol 7α-hydroxylase was successfully expressed in P. pastoris, and the yield of 7α-hydroxycholesterol increased based on molecular modification of CYP7A1 and adaptation of CPR from different sources.

    Preparation of NH3 and H2S Deodorizing Microbial Agents and Their Deodorizing Effects and Mechanisms on Kitchen Waste Composting
    CUI Ruo-qi, ZHANG Ling-yue, JIANG Hai-rong, ZHANG Yu-ling, ZHANG Ming-lu, REN Lian-hai
    2023, 39(10):  311-322.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0277
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    In order to alleviate the pollution caused by the odor gas generated by the degradation or decay of kitchen waste, eight strains with deodorizing effect on NH3 and H2S were isolated and screened from sludge and kitchen waste compost, and mixed them to prepare a composite microbial deodorant. The deodorization effect, physicochemical properties and microbial community changes during composting were studied. The results from response surface analysis showed that the degradation rate of NH4+-N was 92.55% under optimal temperature, pH and inoculum for NH4+-N degradation rate were 27℃, 6.21 and 14%, respectively. The maximum yield of SO42- was 173.57 mg/L when the optimal temperature, pH and inoculum for SO42- production were 29℃, 6.61 and 5%, respectively. The removal rates of H2S and NH3 reached 61.72% and 55.32%, respectively after adding microbial agents in kitchen waste composting. Meanwhile, it reduced the water content, C/N and pH. Besides, it promoted the composting temperature rising, and extended the time of high temperature. In addition, the addition of bacterial agent is beneficial to the change of microbial community structure, increases the number of bacteria that remove ammonia and hydrogen sulfide, accelerates the metabolic transformation process of hydrogen sulfide and ammonia, and suppresses the amount of ammonia and hydrogen sulfide released, which has promising application potential for the control of kitchen waste odor gases.

    Screening, Expression, and Validation of Nanobodies with FLAG Tag
    WANG Xin-yi, WANG Xiao-qian, WANG Hong-jun, CHAO Yue-hui
    2023, 39(10):  323-331.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0291
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    Nanobodies are a novel type of engineered antibodies with small size, high stability, and strong affinity, offering new possibilities for scientific research. The FLAG tag is a short peptide tag widely used in biological research, playing a significant role in various studies. In order to prepare nanobodies against the FLAG tag, high-affinity nanobodies were screened using yeast two-hybrid technology and the prepared FLAG nanobodies were subsequently tested for performance. Through DNA recombination technology, a bait vector containing the FLAG tag was constructed. Using yeast two-hybrid technology, nanobodies against the FLAG tag were screened from a camel-derived nanobody yeast library. Five single candidate antibody DNA sequences were screened from the yeast library. To exclude interference from the expression of protein sequences in the carrier itself on antibody screening, the possibility of non-specific hybridization was ruled out through a ‘point-to-point’ verification method. This operation confirmed that all five screened nanobodies specifically reacted with the FLAG tag. For the production of nanobodies, five nanobody prokaryotic expression vectors were constructed and expressed using the Escherichia coli system. SDS-PAGE and Western blot（WB）analysis showed that two soluble anti-FLAG tagged protein nanobodies were successfully obtained. The effects of these two nanobodies were compared with commercially available conventional FLAG tag antibodies. The results showed that both the prepared nanobodies and the commercial antibodies recognized FLAG peptides and fusion proteins with FLAG tags, and there were no significant differences in specificity, indicating that the prepared FLAG nanobodies had good application prospects. Based on yeast two-hybrid technology, FLAG tag nanobodies were successfully screened and prepared. This achievement not only enriches the types of nanobodies but also provides a new approach for the development and application of antibodies. This study provides strong support for further research on the application of FLAG tags in biological research, as well as the application of nanobodies in bioengineering.