Table of Content

26 October 2022, Volume 38 Issue 10

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Research Progress in the Biological Functions of Plant circRNAs

ZHAO Jie, LI An, LIANG Gang, JIN Xin-xin, PAN Li-gang

2022, 38(10):  1-9.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1586

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Circular RNAs（circRNAs）are covalently closed-loop single-stranded RNA molecules. As an important member of the endogenous noncoding family，circRNA has received more and more attention in recent years. circRNAs widely exist in higher eukaryotes. The studies of circRNAs have been accelerated by the combination of next generation sequencing，chemoinformatics and some other advanced technologies. Studies have shown that circRNAs are relatively stable and conserved molecules with cell-type-，tissue-，or developmental-stage-specific expression patterns. Until now，the functions of circRNAs include acting as miRNA sponges，coping with biotic or abiotic stresses and regulating parental gene transcription，etc. Here，we summarize the classification and characteristics of plant circRNAs，and highlight the progress on their biological functions and their applications as biomarkers in plant breeding and improvement of stress resistance. This article will provide a reference for the further study of circRNAs.

Research Progress in the Functions of SINA E3 Ubiquitin Ligase in Plant

TANG Xiao-li, JIANG Fu-dong, ZHANG Hong-xia

2022, 38(10):  10-17.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1301

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The ubiquitin-proteasome system（UPS）is an important regulatory system for proteostasis in organisms. By means of degrading different target proteins，UPS participates in diverse cellular processes. SINA E3 ubiquitin ligase is a key type of E3 ubiquitin ligases of UPS，playing extensive and important biological functions. This research elaborated the functional research status of SINA E3 ubiquitin ligase since the first one was discovered in plant. Four main aspects of its functions，including in plant growth and development，plant responses under adverse environment，interactions between plant and other organisms and plant autophagy were expounded. Generally，all the reported functions of SINA E3 ubiquitin ligase in plant were summarized. Meanwhile，the existing issues and research prospects for functional study of SINA E3 in plant are proposed. Therefore，all the above would provide valuable reference for the further functional research of SINA E3 in the future.

Structure and Application of C-glycosyltransferases in Plants

ZHAO Yu-xue, WANG Yun, YU Lu-yao, LIU Jing-jing, SI Jin-ping, ZHANG Xin-feng, ZHANG Lei

2022, 38(10):  18-28.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1572

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Glycosyltransferases are a class of post-modification enzymes that specifically catalyse glycosylation reactions and are classified into 4 types of glycosyltransferases，O-，C-，N- and S-，depending on the acceptor molecule attached to the sugar donor. In recent years，along with the vigorous development of structural biology，reports on the structural elucidation and targeted modification of C-glycosyltransferases have gradually increased，providing theoretical guidance for the modification of complex natural products. This paper reviews the research progress of plant C-glycosyltransferases，discusses them from the perspectives of their substrate specificity，spatial structure of sugar donor binding and biotechnological applications，and explores the structural basis of the substrate catalytic characteristics，which may provide a reference for the subsequent heterologous synthesis of plant natural products by means of synthetic biology.

Phytochrome Interacting Factors Involving in Auxin-regulated Plant Growth and Development

QIAN Jing-jie, LIN Su-meng, ZHANG Dong-ping, GAO Yong

2022, 38(10):  29-33.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1475

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Phytochrome interacting factors（PIFs）belong to the basic helix-loop-helix（bHLH）transcription factor family，they interact specifically with the active conformer of phytochrome（PHYS）photoreceptors in the nucleus and are decomposed. PIFs participate in various signal transduction pathways to regulate plant growth and development，such as repressing seed germination，promoting seedling skotomorphogenesis and flowering time. As an important part of the regulation of intracellular signals，PIFs are widely involved in the signaling network mediated by external environmental factors（such as high temperature，light）and internal hormones（such as auxin，cytokinin，brassinolide，etc.）. When the light signal and temperature change，PIFs are involved in auxin synthesis，transport and signaling to regulate plant growth and development. This review mainly introduces the advances of PIFs involving in auxin-regulated plant growth and development in recent years，and prospects the future research direction.

Research Progress in Biological Functions of miR396-GRF Module in Plants and Its Potential Application Values

SHAN Qi, JIA Hui-shu, YAO Wen-bo, LIU Wei-can, LI Hai-yan

2022, 38(10):  34-44.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1558

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miR396 is a conserved microRNA in plant，the growth regulatory factor（GRF）gene has been proved to be their main target genes in many plant species. Current reports indicate that miR396-mediated GRF regulatory pathway（miR396-GRF）has shown an attractive application potential in improving plant varieties by molecular breeding and and promoting the regeneration efficiency of plant tissue materials. In this paper，the sequence characteristics of miR396 and GRF genes were analyzed，and the specific interaction mode of miR396-GRF modules was elucidated. In addition，the biological functions of miR396-GRF module in plant growth and development，stress response and plant tissue regeneration were reviewed. The research events of miR396-GRF module in improving plant biomass and crop yield，plant tolerance to stress and plant regeneration efficiency during genetic transformation were collected. And the current research on the molecular mechanism of miR396-GRF module were summarized. To sum up，this paper provides ideas and references for further study of miR396-GRF pathway.

Research Progress in 1, 3-Propanediol Production by Fermenting Crude Glycerol

JIANG Huan, MA Jiang-shan, ZENG Bai-quan, ZHANG Liang-bo, LI Pei-wang

2022, 38(10):  45-53.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0603

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Crude glycerol is the main by-product in biodiesel industrial. Direct bioconversion of crude glycerol into 1, 3-propanediol by microbial fermentation is promising for the value-added utilization of crude glycerol. This review outlines glycerol-based 1, 3-propanediol synthetic pathways and key enzymes，the effect of crude glycerol on microbial fermentation，the diverse 1, 3-propanediol-producing microbes involved in crude glycerol fermentation as well as improvement of crude glycerol conversion rate by microbial consortium. This review aims to provide insights for engineering 1, 3-propanediol-producing strain and improving bioconversion of crude glycerol.

Research Technology and Progress in Transcriptional Regulation in Prokaryotes

CHEN Chen, HUANG Zhi-yang, YU Hai-yan, YUAN Hai-bin, TIAN Huai-xiang

2022, 38(10):  54-65.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1384

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The regulation of gene expression in prokaryotes mainly occurs at the transcriptional level. Knowledge on the regulation of transcription in prokaryotes is conducive to understanding the mechanism of gene expression regulation. In recent years，with the breakthrough of molecular biology and related technologies，the research technology of transcriptional regulation has also been continuously developed. The technical methods and new developments of prokaryotic transcriptional regulation are reviewed in the present study，including electrophoretic mobility shift assay，DNase I footprinting technology，chromatin immunoprecipitation technology，microthermophoresis technology，isothermal titration calorimetry and bacterial one hybrid system，aiming to systematically understand the advantages/disadvantages and suitability of these methods，help researchers to better use the transcriptional regulation of prokaryotes for bringing great benefits to human life.

Determination of Lignin Monomer Crosslinking Structures in Wheat Stems by UPLC-MS/MS

SUN Shu-fang, LUO Yong-li, LI Chun-hui, JIN Min, XU Qian

2022, 38(10):  66-72.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0002

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A method for the qualitative and quantitative determination of lignin monomer crosslinking structures in wheat stems by UPLC-MS/MS was established，providing a scientific basis for the development and utilization of lignin. The chromatographic separation was carried out on an ACQUITY UPLC^® BEH C18（2.1 mm×100 mm，1.7 μm），ultrapure water and acetonitrile were used as the mobile phase to conduct gradient elution，and MRM（multiple reaction monitoring）were employed for detection. The results showed that the crosslinking structures of 9 lignin monomers had good linearity in the linear correlation range（R²=0.998 9），the recovery was 91.67%-100%，and the relative standard deviations was 0.24%-2.10%. The method was applied to detect the crosslinking structure characteristics of 9 lignin monomers in wheat stems 14 d after jointing. The results revealed that the retention times of the crosslinking structures of 9 lignin monomers in the sample were the same as that of the standard one. G（8-8）G was the crosslinking structure of lignin monomers with the highest mass concentration，and the mass concentration range was 9.53-10.12 ng/g. In conclusion，the method is of strong specificity and high sensitivity. It is suitable for accurate qualitative and quantitative analysis of the crosslinking structures of nine lignin monomers in the wheat stems.

Construction of Yeast One-hybrid Bait Vector of Tobacco NtCBT Gene Promoter and Screening of Interacted Proteins

YU Jing, YANG Hui, YU Shi-zhou, ZHAO Hui-na, ZHENG Qing-xia, WANG Bing, LEI Bo

2022, 38(10):  73-79.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1597

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To screen the upstream regulatory transcription factors of cembratrienol synthase（NtCBT）in the cembranoids synthesis pathway of tobacco（Nicotiana tabacum L.），the promoter of NtCBT gene was cut into P1 to P6 regions，six yeast one-hybrid bait vectors pAbAi-Px were constructed and transformed into Y1H competent yeast cells，bait strains were constructed，and a self-activation experiment was completed. Furthermore，a screening was finished from yeast cDNA library of tobacco trichomes in order to obtain the transcription factors that interacted with P5 regions（-279 - -119 bp）. The results showed that，the growths of the strains containing P1 to P5 regions bait strain were inhibited on the medium added with 200 ng/mL AbA. A total of 49 positive colonies were obtained through Y1H screening when P5 regions as bait strain，among them，35 colonies were non-repeating sequences，and 3 of colonies were annotated as transcription factors of ANL2，ML1 and NF-Y. Above results lay a foundation for further study of gene expression and regulation mechanism of NtCBT.

Reference Genes Selection and Validation for RT-qPCR in Sapindus mukorossi

XU Yuan-yuan, ZHAO Guo-chun, HAO Ying-ying, WENG Xue-huang, CHEN Zhong, JIA Li-ming

2022, 38(10):  80-89.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1616

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The roots，stems，leaves，flowers，and pericarps of Sapindus mukorossi Gaertn. are rich in bioactive triterpenoid saponin. In order to understand the expressions of related genes in triterpenoid saponin biosynthesis pathway，it is necessary to screen the stably-expressed reference genes. In this study，8 traditional reference genes including 18S rRNA（guanine1575-N7）-methyltransferase（Sm18S），actin-related protein 8（SmACT），elongation factor 1-alpha（SmEF-1α），large subunit ribosomal protein L1（SmRPL1），small subunit ribosomal protein S26e（SmRPS26），tubulin-specific chaperone C（SmTBCC），ubiquitin-conjugating enzyme E2 M（SmUBC12），E3 ubiquitin-protein ligase BAH（SmUBP）were selected as candidate reference genes based on the RNA-seq data of roots，stems，leaves，buds，male flowers，female flowers，and developing pericarps of S. mukorossi. The expressions of these candidate reference genes were detected by RT-qPCR in the root，stem，leaf，bud，male flower，female flower，and pericarps at different developmental stages，and the stability of them were then evaluated by geNorm，NormFinder and BestKeeper software and RefFinder online analysis tool. The results showed that the expressions of 8 candidate reference genes differed in the variations among all samples. And the optimal reference genes screened by geNorm，NormFinder and BestKeeper were also slightly different. The results of comprehensive analysis showed that SmACT，SmRPL1 and SmUBP expressed quite stably，while the stability of SmEF-1α was the lowest among all candidate reference genes. The RT-qPCR results of 8 triterpenoid saponin biosynthesis related genes using SmACT，SmACT+SmRPL1 and SmACT+SmRPL1+SmUBP as the reference genes respectively were in accordance with the results of RNA-seq. Thus，it is concluded that SmACT and the reference gene combinations of SmACT+SmRPL1 or SmACT+SmRPL1+SmUBP can be used as reference genes for gene expression analysis of triterpenoid saponin biosynthesis pathway in S. mukorossi and other biological processes in S. mukorossi and related plants.

Methodological Research on Rapid Detection of CRISPR/Cas9-Mediated Gene Mutations in Cells Based on High-resolution Melting Technique

CHEN Li, LU Xi, YANG Hong-lan, ZHANG Peng, HE Zhi-xu

2022, 38(10):  90-96.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1477

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This study is aimed to establish a method for rapid detection of CRISPR/Cas9-mediated gene mutations in cells using high-resolution melting（HRM）assay. The condition of HRM analysis was optimized and perfected using wild-type mouse embryonic stem cells（mESCs），homozygous mutation mESCs and heterozygous mutation mESCs. In order to verify the feasibility and accuracy of HRM method，the established HRM analysis was used to detect 30 monoclones of CRISPR/Cas9-edited mESCs with known their sequencing results. According to the difference of melting temperature and melting curve，wild-type mESCs，homozygous mutation mESCs and heterozygous mutation mESCs were distinguished. The results showed that the optimized HRM assay identified wild-type mESCs，homozygous mutation mESCs and heterozygous mutation mESCs. Thirty monoclones of mESCs edited by CRISPR/Cas9 technique were analyzed using the established HRM method. The results indicated that 14 monoclones were heterozygous mutation mESCs，2 monoclones were homozygous mutation mESCs and 14 monoclones were wild-type mESCs. These results were consistent with the sequencing results at accuracy of 100%.The high-resolution melting assay established in this study is a sensitive，accurate，simple and high-throughput method，and may rapidly scan CRISPR/Cas9-mediated gene mutations in cells.

Mito-OS-Timer：A Targeted Fluorescent Stopwatch for Monitoring Mitochondrial Oxidative Stress

ZHANG Xiao-ni, WENG Yi-chun, FAN Yi-hao, WANG Xiao-juan, ZHAO Jia-yu, ZHANG Yun-long

2022, 38(10):  97-105.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1607

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Mitochondrial oxidative stress（Mito-OS）is a kind of imbalance between reactive oxygen species production and antioxidant system in mitochondria. And oxidative stress induced by reactive oxygen species（ROS）is considered as important factors for aging and disease. At present，the evaluation methods of oxidative stress in cells or animal models are mainly based on cell morphology，animal phenotype or production of characteristic metabolites，et al. It is unable to monitor dynamic changes in real time. This study established a mitochondrial oxidative stress fluorescent protein monitoring system，named Mito-OS-Timer，which can monitor the dynamic changes of mitochondrial oxidative stress in real time. Based on the mechanism that dsRed1-E5 red/green fluorescence conversion rate was positively correlated with oxygen concentration change，dsRed1-E5 gene was fused with ATP5PB fragment of ATP synthase subunit located in mitochondrial inner membrane. Then，we established a recombinant plasmid pMito-OS-Timer and stably expressed cell line HEK293T. After being treated with 0-300 μmol/L H₂O₂ and 0-5 μmol/L rotenone，the results showed that there was a significantly positive correlation between the rate of red-green fluorescence transformation and the degree of mitochondrial oxidative stress. In addition，Mito-OS-Timer was used to detect the enhancement of oxidative stress induced by folliculin（FLCN）gene silencing by pLVX-shFLCN. This system provides a new visualization method for studying mitochondrial oxidative stress.

G-Triplex Visualization Nucleic Acid Sensor for the Detection of Tetracycline

LIU Ning-ning, WANG Xin-xin, LAN Xin-yue, CHU Hua-shuo, CHEN Xu, CHANG Shi-min, LI Teng-fei, XU Wen-tao

2022, 38(10):  106-114.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1172

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Tetracycline is widely used in animal husbandry and aquaculture to prevent bacterial infection and improve growth rate because of its broad-spectrum activity and low cost. Excessive use of tetracycline leads to antibiotic residues in food，which seriously threatens human health. Thiophenin T（ThT）can be embedded into a special nucleic acid structure，so that the fluorescence intensity is strongly excited to achieve signal output. In this paper，tetracycline aptamer can bind with G-rich sequence to form a double chain，but tetracycline preferentially binds with the aptamer when it exists. The G-rich sequence after unbound can fold itself to form a G-triplex structure，which enhances the fluorescence intensity of ThT. According to the good linear relationship between the target concentration and the fluorescence intensity of ThT，the quantitative detection of tetracycline can be acheieved. The method offers good linearity in the range of tetracycline concentration from 1 nmol/L-1 μmol/L 1 μ m，R² is as high as 0.99，detection limit is 0.07 nmol/L，and detection can be completed within 25 min. Therefore，a visualization method based on G-triplex for double-stranded competitive internal cleavage has been developed and simple，rapid，sensitive，specific and low-cost detection of tetracycline is achieved.

Functional Analysis of ZmNF-YB13 Responding to Drought and Salt Stress

ZHANG Tong-tong, ZHENG Deng-yu, WU Zhong-yi, ZHANG Zhong-bao, YU Rong

2022, 38(10):  115-123.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0066

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Maize NF-Y family is a class of important transcription factors，they play an important role in regulating plant development and stress response. Exploring the gene function of this family will provide important gene resources for maize stress-resistant breeding. In this study，ZmNF-YB13 gene was cloned，and its basic characteristics，the relative tissue-specific expressions and expression patterns under different stresses were analyzed by bioinformatics and real-time quantitative PCR. The gene was 537 bp in length and encoded 178 amino acids. The molecular weight of ZmNF-YB13 protein was 18.9 kD，and the theoretical isoelectric point was 6.83. ZmNF-YB13 protein had a unique conserved domain of NF-Y family. qPCR analysis showed that ZmNF-YB13 gene expression was the highest in maize silk；meanwhile ZmNF-YB13 gene expression was up-regulated under different abiotic stress and hormone stress. On 1/2 MS medium containing mannitol，NaCl，ABA and JA，the root length of ZmNF-YB13 transgenic Arabidopsis thaliana was significantly longer than that of the wild type. Under drought and high salt treatments，transgenic A. thaliana grown in soil had more green leaves and lower MDA content than wild-type. Concurrently，the POD activity of transgenic plants was significantly higher than that of wild type under drought treatment. It is speculated that ZmNF-YB13 gene may be involved in response to drought and salt stress in maize.

Identification and Gene Mapping of a Seedling Lethal Mutant in Rice

TANG Yue-hui, ZHAO Yu-fan, LIN Jin, WANG Yin, CAO Bo-yuan, CHE Yi-fan, YANG Wen-jie, BAO Xin-xin, YANG Tong-wen

2022, 38(10):  124-131.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0069

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Peptide release factor 1（RF1）plays a key role in the regulation of the translation termination by recognizing stop codons. Rice seedling lethal mutant was obtained，the expression profile of OseRF1-1 was clarified，and a basis for further function analysis of OseRF1-1 was provided. Using the Japonica rice cv. Huazhiwu and T-DNA insertion mutant as materials，Tail-PCR，paraffin section technology，Southern blotting and Northern blotting technologies were used to identify a T-DNA single-copy insertion mutant of the peptide release factor eRF1-1，named ls. DNAMAN6.0 software and SMART were applied to analyze the OseRF1-1’s bioinformatics characteristics，and RT-qPCR was used to analyze the tissue-specific expression of OseRF1-1. Transforming Arabidopsis thaliana by a PEG-mediated approach was to clarify the subcellular localization of OseRF1-1. The results suggested that the ls mutant rapidly died at the 3rd to 5th leaf tillering stages. Paraffin section showed that the junction region between leaf sheath and tiller node in ls mutant was browning，and the symptoms were more serious in the lower part of tiller nodes. Tail-PCR analysis showed that the right border of the T-DNA and the NOS terminator sequence in ls mutant were lost，resulting in a fusion transcript of GUS and OseRF1-1 in ls mutant. However，the stop codon remained in GUS，which led to the OseRF1-1 not translated normally. OseRF1-1 was expressed in all tissues tested，especially highly expressed in panicles. This OseRF1-1 gene was localized in nucleus and cytoplasm. Above results indicate that ls mutant causing rice seedlings lethal may be resulted from the abnormal transcription of OseRF1-1 gene by T-DNA insertion.

Effects of Blend Seeding on the Yield，Purity and Yield Advantage of F₁ in Three-line Hybrid Wheat

KONG De-zhen, NIE Ying-bin, XU Hong-jun, CUI Feng-juan, MU Pei-yuan, TIAN Xiao-ming

2022, 38(10):  132-139.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1493

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In order to explore the effects of different ratios of restoring lines on seed production，purity and heterosis of F₁ yield in hybrid wheat production with strong heterosis combinations，three AL（Alborubrum Korn）-type CMS（cytoplasmic male sterility）wheat hybrid combinations were used as materials in the sterile line（female parent♀）. The seeds were mixed with different proportions of restoring line（male parent♂）seeds for sowing. The yield of hybrid seed production，the purity of seed production，and the change of hybrid F₁ yield heterosis were studied. The results showed that the two hybrid combinations 36A×2014AR5 and 002A×99AR142-1 were mixed with different proportions of restoration line seed production，the yield of hybrid seed production increased with the increase of the mixed male parent ratio，and the seed purity of the hybrids gradually decreased. When the male parent’s mixed sowing proportion was 5%，it reached the requirement of 96% of field seed purity，and the seed production yield was also higher. The yield of the F₁ generations produced by the two hybrid combinations with different mixed sowing ratios was compared，and the mixed seeding production. The seed yields of mixed sowing and mixed harvesting were higher than those of the hybrid control of the same combination and row ratio seed production，and the yield advantage of F₁ with mixing ratios of 12% and 9% was more significant. The SSR molecular markers Wmc474，which were linked to the restorer gene（qRf-2A-1）and located on chromosomes 2A，were used to predict the seed purities via mixed sowing and mixed harvesting under different mixing ratios. Concurrently，the prediction results of the regression equation for purity detection of AL-type three-line hybrid wheat hybrids established by the SSR marker Xbarc8 linked to the restorer gene（qRf-1B-1）on chromosome 1B were compared，showing that the two regression equations established with the two molecular markers can be used in the identification of the purity of the seed production of mixed restorer lines.

Expression Profile Analysis of Thionin-like Gene Family in Barley

YANG Hong-liang, YUAN Zhen, QIAN Xu-jia-zhi, XU Da-wei

2022, 38(10):  140-147.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0163

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Thionin-like（Thil）genes possesses activity against pathogenic microorganisms in plant defense system；however，the function of Thil genes is not clear. In order to provide reference for studying its biological function，this study analyzed the sequence composition，gene structure，chromosome distribution，protein conserved domains，phylogenetic tree，gene promoter elements，subcellular localization and expression profile of Thil gene family in barley. Seven barley Thils genes were identified，and they were distributed on chromosome 1, 2, 4, 5 and 6. The gene structure analysis showed that the sequences of the family members were conservative except Thil4. The phylogenetic tree revealed that barley Thil genes were divided into four subfamilies. The results of expression profile analysis demonstrated that the expression of Thil gene varied in different tissues of barley. In general，the expression of Thil gene was very low in the roots and seeds，and the highest in the young leaves. The subcellular localizations of Thil proteins in different members of the family are also different. Thil1 and Thil2 were mainly located in the cell membrane，nucleus and cytoplasm，Thil3 and Thil7 were located in the cell membrane，and Thil6 was located in chloroplast. The Thil gene was associated with plant protection and defense mechanisms. The elements that can stimulate plant protection mechanisms responded most in young leaves. Quantitative RT-PCR（qRT-PCR）results showing the expression of the Thil gene was the highest in young leaves. It can be found that the same members are involved in different developmental processes of plants in the evolutionary process from the subcellular localization experiment.

Genetic Analysis of FBA Trait in Upland Cotton with Major Gene Plus Polygenes Mixed Genetic Model

MA Qi, LI Ji-lian, XU Shou-zhen, CHEN Hong, LIU Wen-hao, NING Xinzhu, LIN Hai

2022, 38(10):  148-158.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1606

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Cotton plant architecture is closely related to cultivation mode，mechanical collection efficiency and yield. Fruit branch angle（FBA）is one of the key factors that determine cotton plant architecture. Exploring the genetic characteristics on FBA of upland cotton（Gossyp-ium hirsutum）will provide important guidance for genetic breeding of cotton plant architecture. In this study，a natural population composed of 418 accessions of upland cotton from different regions，were used as the research object. Then，measuring the FBA with digital display protractor，we performed the variability and correlation analysis of FBA in different positions of cotton main stem，as well as the phenotypic description and statistical analysis of FBA of natural populations in different ecological environments. Simultaneously，the combined populations four generations（P₁，P₂，F₁ and F₂）constructed by parents with extremely different fruit branch angles were selected as the research objects，the multi-generation joint genetic analysis of FBA phenotypic traits of the four generations was carried out，and the genetic effect and heritability of major genes were estimated using the mixed genetic model of major gene plus polygenes of plant quantitative traits. Phenotypic identification analysis showed that the coefficient of variation for FBA in the natural population of upland cotton was relatively small，and the average coefficient of variation under the four environments was 5.63%. Furthermore，the middle fruit branch（the 4th-6th position from the base）angles best represented the level of FBA of the whole cotton plant. Additionally，mixed genetic model for major gene plus polygene demonstrated that the optimal model for controlling FBA was two major genes with equal additive effect，the additive effect value of major gene was 3.65，and the heritability was 90.22%. These results revealed that the FBA of upland cotton was mainly controlled by the main gene，and the heritability of the main gene was high. The above results are conducive to clarifying the genetic law of FBA of upland cotton，which is of important practical significance for molecular genetic analysis of FBA and plant architecture genetic breeding of upland cotton.

Production of Marker-free Mutants of Brassica campestris Mediated by CRISPR/Cas9 Through Vacuum Infiltration

ZONG Mei, HAN Shuo, GUO Ning, DUAN Meng-meng, LIU Fan, WANG Gui-xiang

2022, 38(10):  159-163.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0042

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The objective is to acquire non-transgenic Brassica campestris mutants via CRISPR/Cas9 gene editing system，for achieving the application of CRISPR/Cas9 in B. campestris in planta transformation method and providing reference for application of marker-free gene editing technique. Using ‘49 Caixin’ as plant material and dehydrogenase gene（PDS）as target gene，total of 25 plants were transformed by in planta transformation via vacuum-infiltration method. The results showed that 3 of the 2 032 transformed seedlings were identified to have PDS gene editing，one of which was detected to have exogenous vector insertion and heterozygous editing of PDS gene，and the phenotype was consistent with that of the wild type. The other two seedlings had a dwarf，albino phenotype，and there was no insertion of foreign vector in their genomes. Different types of targeted mutagenesis of PDS gene were detected in these two seedlings compared with the wild type. In conclusion，gene editing can be achieved in B. campestris by CRISPR/Cas9 through in planta tissue culture-independent transformation，and particularly non-transgenic editing mutants without exogenous insertion can be obtained directly.

Cloning，Expression and Functional Analysis of Potassium Channel Gene MiSPIK in Mangifera indica

HAN Lei, LI Jun-lin, GAO Ai-ping, HUANG Jian-feng, LI Jian-zhao, SONG Zhi-zhong

2022, 38(10):  164-172.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0055

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Shaker-type potassium（K⁺）channels play important roles in plant growth and development. However，their biological function in fruit crops is still unknown. We focused on the cloning and functional verification of SPIK channel gene in mango（Mangifera indica），which provide gene resources and theoretical foundation for the biological function analysis of Shaker type K⁺ channels in tropical fruit plants. The Shaker type K⁺ channel gene MiSPIK（GenBank ID：OM179914）was isolated by RACE-PCR technology from ‘Guire 82’ mango. The sequence characteristics of this gene and its coding protein were analyzed via bioinformatics. The tissue-specific expression characteristics and its responses to 5 abiotic stresses，including K⁺ deficiency，high K⁺ stress，low temperature，NaCl and PEG treatment，were further detected in the transcriptional level via qRT-PCR. MiSPIK-overexpressed transgenic Arabidopsis line was created. Results showed that MiSPIK protein had a molecular weight of 90 006.83 kD with the isoelectric point（pI）of 7.59，which possessed 6 transmembrane domains and belonged to unstable hydrophilic protein. The amino acid sequence identity was 59.46% among MiSPIK，pear PbrSPIK，and Arabidopsis AtSPIK，and phylogenetic tree analysis demonstrated that these three SPIK homologs were tightly clustered together and had the closest genetic distance. In the transcriptional level，MiSPIK was specifically expressed in mango pollens，and its expression in the roots of annual grafted seedlings significantly reduced by K⁺ deficiency or NaCl treatment，and significantly enhanced by high K⁺ stress，PEG or low temperature treatment. In addition，over-expression of MiSPIK in Arabidopsis significantly enhanced the K⁺ accumulation status and induced earlier bolting and flowering of transgenic plants. MiSPIK was a pollen-specifically expressed Shaker type K⁺ channel gene in mango，whose expression was prone to be regulated by distinct abiotic stresses，and may take part in the high efficient utilization of K⁺ nutrition in mango.

Transcriptome Analysis of Lycium ruthenicum Murr. Shoots in Anthocyanin Biosynthesis Response to Salt Stress

GUO Ai, JIANG Mu-yan, Ha Li-ma-ti·Ba He-tai-li, LIU Yu-yuan, WANG Jing

2022, 38(10):  173-183.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1576

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In order to analyze the process of Lycium ruthenicum Murr.（L. ruthenicum）shoots in anthocyanin biosynthesis response to salt stress，L. ruthenicum seedlings were treated with 300 mmol/L NaCl，and the shoots were collected at 3 d after treatment，and the anthocyanin content and transcriptome were analyzed by LC-MS/MS and RNA-seq. Combined with the analysis of species and contents of anthocyanin and differential expressed genes（DEGs）in GO and KEGG pathway，DEGs related to anthocyanin biosynthesis induced by salt stress were mined，and the results of transcriptome data were verified by RT-qPCR. The results showed delphinidin-3-O-rutinoside in L. ruthenicum shoots was the highest upregulated anthocyanin（11.9 times），and the content of petunia-3-O-rutinoside was the highest anthocyanin（5.313±0.286）μg/g. Total 1 416 DEGs（P<0.01）were screened，including 867 up-regulated and 549 down-regulated DEGs，which were classified into GO terms，such as catalytic activity，photosystem and single-organism metabolic process. DEGs were significantly enriched in 14 KEGG pathways. Meanwhile，there were 7 DEGs involved in anthocyanin biosynthesis and all of them were up-regulated，and27 DEGs were involved in phytohormone signal transduction，in which 15 DEGs were related to ABA signal transduction pathway. The expressions of 11 MYB and 7 bHLH transcription factors were significantly changed. Sum up，the results of this study suggested that DEGs related to ABA pathway，MYB，bHLH transcription factor and anthocyanin biosynthesis play important role in anthocyanin biosynthesis in L.ruthenicum shoot responses to NaCl stress.

Cloning and Response Analysis to Abiotic Stress of GbR2R3-MYB1 Gene from Ginkgo biloba

LUO Ying, TANG Zhi, WANG Fan, LIU Xiao-xia, LUO Xiao-fang, HE Fu-lin

2022, 38(10):  184-194.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1546

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R2R3-MYB transcription factor is one of the subfamily members having big number in MYB family. It plays an important role in growth and development，hormone signal transduction，secondary metabolite and stress regulation of plant. In this study，the GbR2R3-MYB1 gene from Ginkgo biloba was cloned，and bioinformatics software was applied to analyze the physicochemical properties，structure and function of the GbR2R3-MYB1 proteins. After the plant fusion expression vector pCAMBIA1300-R2R3MYB1-GFP was constructed and injected to tobacco epidermal cells through agrobacterium-mediated method，the subcellular localization of GbR2R3-MYB1 gene was observed. Finally RT-qPCR was sued to analyze the GbR2R3-MYB1 gene expressions in G. biloba leaves under abiotic stress. The results showed that the total length of GbR2R3-MYB1 coding region was 819 bp，encoding a 272 amino acid protein. The relative molecular weight of GbR2R3-MYB1 protein was about 30 001.60 Da and its theoretical isoelectric point（PI）was 6.59，which was an unstably hydrophilic protein. In the secondary structure of GbR2R3-MYB1 protein，there were 28.31% of α-helix，4.78% of beta turn，61.03% of random coil and 5.88% of extended strand. GbR2R3-MYB1 protein had the highest amino acid sequence similarity with R2R3-MYB of Pinus taeda and Picea glauca，and genetic relationships were close among them，which were basically consistent with phylogenetic evolution tree analysis. Subcellular localization results showed that GbR2R3-MYB1 was located on the cell nucleus. The RT-qPCR results demonstrated that GbR2R3-MYB1 gene was induced by salt，drought，cold and heat stress，and its relative expression were up-regulated first，then down-regulated under salt and drought stress，respectively，while down-regulated first then up-regulated，finally down-regulated under cold and heat stress. The cloning and functional analysis of GbR2R3-MYB1 gene will help to clarify the molecular mechanism of stress resistance in G. biloba，and provide resources and basis for the variety improvement of other plants.

Effects of Ralstonia solanacearum Infection on Soil Fungal Community Diversity

LI Ting-ting, DENG Xu-hui, LI Ruo-chen, LIU Hong-jun, SHEN Zong-zhuan, LI Rong, SHEN Qi-rong

2022, 38(10):  195-203.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1595

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The soil fungal community plays an important role in maintaining soil fertility and plant health，however，the effect of bacterial wilt from Ralstonia solanacearum infection on the soil fungal community still is little. Real-time quantitative PCR and high-throughput sequencing technology of MiSeq were used to study the fungal community composition in the bulk and rhizosphere soils cropped with diseased and non-diseased tomato. The results showed that the occurrence of R. solanacearum infection significantly affected the fungal community composition of bulk and rhizosphere soil. Compared to the non-diseased tomato rhizosphere soil，the fungal abundance，alpha diversity of Chao1 and Shannon in the diseased tomato rhizosphere soil significantly reduced. And the relative abundances of indigenous beneficial fungi such as Gliocladium，Mortierella，Acremonium and Trichoderma significantly reduced whereas the relative abundance of the harmful fungus of Fusarium significantly increased. In summary，the occurrence of R. solanacearum infection altered the composition of the soil fungal community. The fungal abundance，alpha diversity，and the relative abundance of indigenous beneficial fungi depleted while the relative abundance of harmful fungi enriched. The results may provide the elucidation of the microecological mechanism of outbreak of soil-borne bacterial wilt.

Effects of Rhizobium Seed Dressing on the Soil Microbial Community of Grass-legume Mixtures in Alpine Regions

YAN Hui-lin, LU Guang-xin, DENG Ye, GU Song-song, YAN Cheng-liang, MA Kun, ZHAO Yang-an, ZHANG Hai-juan, WANG Ying-cheng, ZHOU Xue-li, DOU Sheng-yun

2022, 38(10):  204-215.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1066

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Rhizobium seed dressing is one of the effective measures to improve the nitrogen fixation efficiency of plants. Our study investigated the effects of rhizobium seed dressing on the structure and diversity of soil microbial community in typical grass-legume mixtures in alpine regions，aiming to provide a theoretical basis for the application in forage production. Taking the mixed sowing of Medicago sativa cv.‘Beilin201’，Elymus sibiricus L.cv.‘Chuancao No.2’，and Elynus nutans Griseb.cv.‘Aba’ as the research object，2 treatments of rhizobium seed dressing and no seed dressing were set. The bulk and rhizosphere soil were collected，and the prokaryotic biodiversity and community structure were analyzed based on 16S rRNA gene high-throughput sequencing technology，the functional prediction was calculated by FAPROTAX tools and the classification and changes of prokaryotes microbial high-level phenotypes were compared by BugBase analysis. A total of 8 814 OTUs were detected from 32 soil samples，belonging to 3 577 genera in 6 phyla，while the dominant phyla were Actinobacteria（26.11%-46.20%）and Proteobacteria（20.61%-32.91%）. Rhizobium seed dressing changed prokaryotic community structure and decreased diversity（Observed_richness，Shannon index，and inverse Simpson index），but increased the abundance of digestion and denitrification taxa. The population belonging to aerobic，anaerobic，facultative anaerobic，gram-negative，and gram-positive prokaryotes in the rhizosphere soil of Gramineae increased after seed dressing，while the results of Legumes were the opposite. After seed dressing，the abundance of nifH gene in the legume rhizosphere significantly increased. In summary，seed dressing reduced the α-diversity of community，changed the structural composition，but increased the relative abundances of digesting and denitrifying populations，and increased the abundance of nifH genes. This study provides a scientific basis for the cultivation of rhizobia seed dressing in the grass-legume mixtures.

Whole Genome Sequencing and Comparative Genomic Analysis of a High-yield Lipase-producing Strain WCO-9

ZHANG Ze-ying, FAN Qing-feng, DENG Yun-feng, WEI Ting-zhou, ZHOU Zheng-fu, ZHOU Jian, WANG Jin, JIANG Shi-jie

2022, 38(10):  216-225.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0099

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Acinetobacter junii WCO-9 is a high-yield lipase-producing strain isolated from oil-contaminated soil. The lipase produced by this strain has significantly better hydrolysis activity than that of the control strain ATCC 17908. In order to explore the efficient enzyme production mechanism of WCO-9 strain and to discover specific lipase functional genes，ONT（Oxford Nanopore Technologies）was used to complete the whole genome sequencing of WCO-9 strain，and the comparative genomic analysis of the same strain was carried out. The results showed that the genome size of lipase-producing WCO-9 strain was 3.19 Mb，GC content was 38.62%，and there were 2 929 coding genes，including 11 lipase-coding genes（1 specific gene）and 3 lipase molecular chaperones. The collinearity analysis also revealed that the WCO-9 strain was significantly different from the control strain ATCC 17908. The whole genome sequence analysis of WCO-9 strain will be of great significance for the study of high-efficiency enzyme production mechanism and the discovery of new lipase-specific genes，which provides a theoretical basis for the creation and industrial application of high-yield lipase engineering bacteria in the future.

Biosafety and Nitrogen Removal Performance of a Safe and Efficient Aerobic Denitrifying Pseudomonas stutzeri DZ11

ZHAO Yang, SUN Hui-ming, LIN Hao-peng, LUO Ping-ting, ZHU Ya-ting, CHEN Qiong-hua, SHU Hu

2022, 38(10):  226-234.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1084

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The aerobic denitrifying bacteria separated and screened from the bottom sludge and sewage of the aquaculture farm in Minzhong town，Zhongshan city，were studied，aiming to lay the foundation for the development of the denitrification process of aquaculture wastewater. The high-performance bacterium DZ11 was selected from the samples，and its morphological observation，physiological and biochemical determination and 16S rDNA gene sequence analysis were performed to determine the species of the strain. Besides，drug susceptibility test and biological safety test were conducted to confirm the safety of strains. Then the influences of carbon source types，temperature，pH value and carbon-nitrogen ratio on the nitrogen removal efficiency were studied to improve the denitrification efficiency of strains’ performance. The identification results showed that the strain DZ11 was identified as Pseudomonas stutzeri and named Pseudomonas stutzeri DZ11. Drug susceptibility test and biological safety test demonstrated that the strain had no obvious drug resistance and had high biological safety. The result of single factor optimization is that the optimal carbon source of strain DZ11 was sodium citrate，the optimal temperature was 30.0-35.0℃，the optimal pH was 7，and the optimal C/N was in the range of 10-20. When NH₄-N，NO₃-N，and NO₂-N were the only substrates，the transformation rates were 84.39%，99.0% and 97.65%，respectively，indicating that the strain had a good denitrification effect. In sum，the strain DZ11 has high biological properties and high-efficiency denitrification performance，and has great application potential in the denitrification process of aquaculture wastewater.

Nicotinic Acid Promotes γ-aminobutyric Acid Biosynthesis in Lactobacillus brevis

ZHANG Jian, CAI Heng, SHEN Zhen-hao, LIU Zhong-hao, ZHAO Juan, ZHANG Qiao-zhen, GAO Qiang

2022, 38(10):  235-242.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1618

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Adding 0.15% nicotinic acid to medium significantly increased the γ-aminobutyric acid（GABA）production of Lactobacillus brevis. In order to clarify the role of nicotinic acid，L. brevis was inoculated in the medium with（experimental group）and without nicotinic acid（control group）for fermentation. The differential metabolites were studied by liquid chromatography-mass spectrometry（LC-MS），and the integrity of cell membrane was analyzed using propidium iodide（PI）single staining method. The results showed that 33 metabolites were significantly different between the experimental group and the control group. The unsaturated fatty acids and some amino acids including glutamate，proline，aspartate，serine，histidine，and ornithine，significantly increased in the experimental group，whereas nicotinic acid nucleotide，2-phosphoglycerate，adenosine monophosphate and tyramine significantly decreased. PI single staining experiments demonstrated that the cells with red fluorescence in the experimental group were significantly more than those in the control group. Therefore，the addition of nicotinic acid has a broad impact on the physiological metabolism of L. brevis，and its promoting effect on GABA biosynthesis may be related to increasing the permeability of the cell membrane of L. brevis，resulting in easier substrates entry into the cells and easier products discharge from the cells.

Porcine Kobuvirus Structural Proteins VP0 and VP1 Prokaryotic Expression and Establishment of Indirect ELISA Method

SHEN Jun-qiang, ZHANG Li-ping, YU Rui-ming, WANG Yong-lu, PAN Li, LIU Xia, LIU Xin-sheng

2022, 38(10):  243-253.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1550

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In this study，the indirect-ELISA method was established based on the structural proteins VP0 and VP1 of porcine Kobuvirus（PKV）. The genes of structural proteins VP0 and VP1 of PKV were synthesized and ligated into the prokaryotic expression vector pET-32a，and then transferred into competent cells BL21. The expressions of two proteins were induced by IPTG and then were purified by Ni column. The two indirect ELISA assays were established while recombinant protein pET-32a-VP0 and pET-32a-VP as coating antigen through a checkerboard titration method for repeatability，sensitivity，specificity for experiments，and clinical testing. The results showed that the optimal coating conditions of recombinant protein pET-32a-VP0 and pET-32a-VP1 antigen were 2.0 mg/mL and 2.5 mg/mL at 37℃ for 1 h，respectively. The optimal conditions for the blocking solution were 5% skimmed milk，37℃，2 h；the optimal incubation conditions for the serum were 1∶200，37℃，1 h；the optimal incubation conditions for the secondary antibody were 1∶20 000 and 1∶15 000，37℃，1 h；optimal colour development time was 10 min. The critical values of the two indirect-ELISA assays were 0.306 and 0.277 respectively. The results of experiment indicated that the indirect-ELISA methods were successfully established，which are accurate and efficient for detecting serum specific antibody of PKV. The methods are characterized as good reproducibility，sensitivity，specificity and stability. It is very helpful for future clinical diagnosis and the development of antibody detection kits of PKV.

Regulation of miR-301b on Goat Intramuscular Adipocyte Differentiation

ZHANG Hao, HE Chang-sheng, LI Yan-yan, WANG Yong, ZHU Jiang-jiang, Emu Quzhe, LIN Ya-qiu

2022, 38(10):  254-261.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0015

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The purpose of this experiment is to clarify the regulatory effect of miR-301b on goat intramuscular adipocyte differentiation and to explore the possible mechanism of its regulatory effect. Chemically-synthesized miR-301b mimics and miR-301b siRNA were used to overexpress and interfere miR-301b in goat intramuscular adipocytes by liposome transfection，and the regulatory effect of miR-301b on goat intramuscular adipocyte differentiation was clarified from the cell morphology and mRNA level by oil red O staining，qPCR and bioinformatics analysis，and its possible mechanism was explored preliminary. The results showed that the overexpression efficiency of miR-301b was about 29 290 times. The results of cell morphology showed that the accumulation of lipid droplets and triglyceride synthesis of goat intramuscular adipocytes decreased after the overexpression of miR-301b. qPCR results showed that expressions of lipogenic marker gene CEBPα，PPARγ，SREBP1 and LPL in the overexpressed miR-301b significantly reduced（P<0.01）. The interference efficiency of miR-301b was about 60%. After the expression of miR-301b was interfered，the results of cell morphology showed that the accumulation of lipid droplets in goat intramuscular adipocytes increased and the synthesis of triglycerides increased. qPCR results showed that interfering miR-301b significantly promoted expressions of adipogenic marker gene AP2，CEBPα，CEBPβ，PPARγ，SREBP1 and LPL. Through bioinformatics analysis of miR-301b，it was speculated that KLF3 was the target gene of miR-301b. qPCR verification found that overexpression of miR-301b significantly inhibited the mRNA expression of KLF3，and interference to miR-301b significantly promoted the expression of KLF3. In conclusion，overexpression of miR-301b inhibited the differentiation of intramuscular preadipocytes in goats，and down-regulated the expression of KLF3；interfering miR-301b promoted the differentiation of intramuscular preadipocytes in goats，and the expression of KLF3 was up-regulated. Thus it is speculated that miR-301b may inhibit the differentiation of goat intramuscular adipocytes through KLF3.

Identification of Hair Follicle Cell Subsets and Bioinformatics Analysis of Characteristic Genes in Tianzhu White Yak During Catagen

ZHENG Qing-bo, YE Na, ZHANG Xiao-lan, BAO Peng-jia, WANG Fu-bin, REN Wen-wen, LIAO Yue-jiao, YAN Ping, PAN He-ping

2022, 38(10):  262-272.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1556

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The main cell types in the hair follicle development process of Tianzhu white yak during the catagen were analyzed at the single cell level，aiming to perform functional prediction and bioinformatics analysis on the characteristic genes of the main cell groups，and to explore the hair follicle development mechanism during catagen. Single cell RNA sequencing was used to analyze the heterogeneity of degenerative hair follicles during catagen，and known marker molecules were used to screen and identify cell groups. GO and KEGG analysis were performed on the main identified cell groups，and immune tissue analysis was performed on the key molecules involved in hair follicle morphogenesis. The results showed that catagen of Tianzhu white yak was involved in IFE-DC cells，epidermal cell lines，melanocytes，INFU and other cell groups. Its characteristic genes were mainly involved in biological processes such as epidermis development，epithelial cell differentiation，super fiber tissue development，tissue morphogenesis and cell morphology. KEGG enrichment analysis discovered that the characteristic genes were mainly enriched in adhesion connection，cell cycle，RNA transport and other pathways related to hair follicle development. The different involved cell types contained 28 common genes such as GJA1，FKBP4，KRT1，KRT80 and FGFR2. This study successfully identified the main cell groups of Tianzhu white yak during catagen，obtained the enrichment pathways of characteristic genes，and revealed the hair follicle development mechanism during catagen.

Construction of Coagulation Factor 8 Gene Knockout Mouse Model Based on CRSIPR/Cas9 Technique and Verification of Phenotype

WANG Hai-jie, WANG Cheng-ji, GUO Yang, WANG Yun, CHEN Yan-juan, LIANG Min, WANG Jue, GONG Hui, SHEN Ru-ling

2022, 38(10):  273-280.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1564

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A coagulation factor 8（F8）gene knockout mouse model was constructed based on CRISPR/Cas9 technique and its phenotype was verified. sgRNA target sites were designed according to exon 1 non-coding region to exon 26 downstream sequences of F8 gene，the sgRNA was obtained from in vitro transcription and mixed with mRNA of encoding Cas9. F0 generation positive knockout mice were obtained by microinjection of fertilized eggs. F8 ^-/- gene knockout homozygous mice（F8^-/- mice）were obtained by breeding and genotype identification. The expression of F8 gene at mRNA level in main tissues was detected by reverse transcription PCR（RT-PCR），the protein expression of F8 gene in plasma was detected by enzyme-linked immunosorbent assay（ELISA），and the coagulation function of F8 knockout mice was detected by activated partial thromboplastin time（APTT）and fibrinogen content（FIB）. PCR and sequencing results showed that F8 gene was knocked out successfully in mouse genome，and RT-PCR and ELISA results demonstrated that the expression of F8 in F8^-/- mice was significantly lower than that in wild-type mice（WT mice）（P < 0.0001，P<0.01）. APTT，FIB and drop blood test results revealed that the plasma coagulation time of F8^-/- mice was significantly longer than that of WT mice. F8^-/- mice had severe coagulation disorder phenotype，which restored to normal levels after administration of human factor VIII. The F8 gene knockout mice model is successfully constructed by CRISPR/Cas9 technique and its phenotype is preliminarily verified，which confirms that the deletion of F8 gene can lead to coagulation dysfunction.