Table of Content

26 February 2022, Volume 38 Issue 2

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Abiotic Stress Resistance of Escherichia coli Transformed with Arabidopsis thaliana AtTERT Gene

YANG Jia-hui, SUN Yu-ping, LU Ya-ning, LIU huan, LU Cun-fu, CHEN Yu-zhen

2022, 38(2):  1-9.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0498

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Telomerase is a special reverse transcriptase in eukaryotes that maintains DNA integrity at the end of the chromosome. Here we studied the effects of the AtTERT in Arabidopsis thaliana on the growth and abiotic stress response of Escherichia coli,for laying a foundation in further studying the non-telomeres function of TERT protein. The pET32a-AtTERT prokaryotic expression vector was successfully constructed after the A. thaliana AtTERT gene was transferred into E. coli. The induced conditions were optimized,the GST-AtTERT fusion protein was purified and identified,then verified by Western blotting,and the abiotic stress resistance of the AtTERT in E. coli was detected by drop plate method. The optimal conditions for the expression of AtTERT protein were as follows：the induction temperature for competent cells Transetta（DE3）was 20℃,and the concentration of inducer（IPTG）was 0.5 mmol/L. The relative molecular weight of GST-AtTERT fusion protein was 156 kD. Western blotting verification results showed that the protein bands with the expected molecular weight appeared in the induced whole bacteria,in the supernatant and precipitate. The survival rate of E. coli transformed with AtTERT was significantly higher than that of empty control E. coli under NaCl（400 and 500 mmol/L）,mannitol（400 and 600 mmol/L）and hydrogen peroxide（0.4 mmol/L）stress. With 6 freezing-thawing cycles of liquid nitrogen,the survival rate of AtTERT was significantly lower than that of the empty control bacteria. The resistance of the E. coli transformed with AtTERT under NaCl salt stress,osmotic stress and hydrogen peroxide stress was enhanced. However,the resistane of the E. coli transformed with AtTERT under low temperature was reduced, indicating that A. thaliana AtTERT has non-telomere function of resisting abiotic stress.

Proteomic Analysis of RPP1A Involved in the Seedling Growth of Arabidopsis thaliana

LI Bing-juan, ZHENG Lu, SHEN Ren-fang, LAN Ping

2022, 38(2):  10-20.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0542

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Ribosomal proteins are not only involved in protein synthesis but also regulate plant growth and development. Arabidopsis thaliana ribosomal phosphoprotein P1（RPP1）family RPP1A deletion mutant rpp1a was used to investigate the effect of RPP1A deletion on the protein profile of seedlings and to reveal the mechanism of its involvement in regulation of seedling growth. Phenotypic analysis revealed that the RPP1A deletion resulted in significant increases in biomass,total leaf surface area,and petiole length of the seedlings compared with wild-type（WT）. The label-free proteomic analysis revealed that a total of 280 differentially expressed proteins were identified in the mutant rpp1a-2 compared with the WT,with 98 proteins significantly up-regulated and 182 proteins significantly down-regulated. Differentially expressed proteins were mainly involved in cellular process,metabolic process,response to stimulus,cellular component organization or biogenesis,and oxidation-reduction process,and were significantly enriched in the metabolic pathways of ribosome,photosynthesis,mRNA surveillance pathway,RNA degradation and phenylpropanoid biosynthesis. The A. thaliana rpp1a-2 mutant promotes plant growth by inhibiting the stimulus-response and enhancing photosynthesis.

Genome-wide Identification and Expression Analysis of the bHLH Transcription Factor Family in Solanum tuberosum

FENG Jian-ying, LI Li-qin, LU Li-ming

2022, 38(2):  21-33.  doi:10.13560/j.cnki.biotech.bull.1985.2020-1327

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Genome-wide identification and expression pattern analysis of Solanum tuberosum bHLH transcription factor were conducted in order to provide reference for studying its biological function. Based on the S. tuberosum genome database and Pfam database,HMMER was used to identify the S. tuberosum bHLH family members through the whole genome. After the redundant sequences were excluded,ExPASy and MEME software were used to analyze the basic physicochemical properties and conservative element analysis of the candidate sequences. Further MEGA-X software was adapted for cluster analysis. MG2C and TBtools were employed to depict chromosome location and expression pattern respectively. As result,a total of 108 bHLH transcription factors were retrieved from the S. tuberosum genome database,the number of amino acids ranged from 62 to 694,the molecular weight was 7 527.78-75 939.94 Da,and the theoretical isoelectric point was 4.55-10.40. These bHLH transcription factors were distributed on 12 chromosomes of S. tuberosum,and all had typical HLH domain,which could be divided into 16 subgroups. Gene expression pattern analysis showed that S. tuberosum bHLH family members had tissue expression specificity and most of them responded to salt,mannitol,IAA,ABA,GA₃ and heat stress. The whole genome of S. tuberosum contains 108 highly conserved members of the bHLH gene family,whose expression is tissue specific and responds to the induction of abiotic stress.

Cloning and Expression Analysis of GhSDP1 and Its Promoter in Gossypium hirsutum

LIU Meng-meng, HAN Li-jun, LIU Bao-ling, XUE Jin-ai, LI Run-zhi

2022, 38(2):  34-43.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1077

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Triacylglycerol lipase（SDP1）is a key enzyme in the degradation of triacylglycerol and plays an important role in the regulation of vegetable oils. We focused on the cloning of the cotton SDP1 gene and tits expression analysis under three stresses,which provides basis for analyzing the biological function of the cotton SDP1 gene. The coding sequence and upstream promoter sequence of GhSDP1 were cloned in cotton variety Jifeng 1271. PlantCARE was used to analyze the cis-acting elements in the promoter region of GhSDP1,and qRT-PCR was applied to detect the expression profile of GhSDP1 under stress. The promoter activity analysis of pGhSDP1+GUS fusion vector was carried out by tobacco transient expression. The results showed that the coding sequence of GhSDP1 was 2 541 bp,and the gene presented a differential expression pattern under salt,low temperature and drought stress. pGhSDP1 not only had the basic cis-acting elements such as TATA-box and CAAT-box,but also contained a number of cis-acting elements related to light response,hormone response and adversity response. The pGhSDP1 promoter in cotton can drive the high-efficiency expression of GUS protein and has strong promoter activity. The study revealed that the cotton GhSDP1 gene participates in the corresponding new functions of the stress.

Molecular Cloning and Expression Analysis of a F-box Protein Gene FnFBOX1 and Its Promoter from Fragaria nilgerrensis

SHI Ya-qian, SHEN Ya-ru, CHEN Man-ying, HE Shu-min, LIU Yu-han, HE Tian-nan, CHEN Qing-xi, WEN Zhi-feng

2022, 38(2):  44-56.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0295

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This work is to explore the involvement of Fragaria nilgerrensis Schidl. FnFBOX1 gene in plant-pathogen defense response during pathogen infection of Colletotrichum gloeosporioides and transcriptional activity of pFnFBOX1 promoter,thus laying a foundation for further revealing that FBOX1 gene in the regulation of resistance to anthracnose in strawberry. Having F. nilgerrensis as the experimental material,the cDNA sequence of a F-box protein gene FnFBOX1 was cloned by RT-PCR. Then amino acid sequences alignment,phylogenetic tree,subcellular localization,promoter clone and construction of transient expression vector,and tissue specific expression analysis were conducted. And the expression patterns of FnFBOX1 in F. nilgerrensis and infected ‘Miaoxiang 3’ in response to stresses of salicylic acid and C. gloeosporioides were detected by real-time quantitative PCR. The results showed that the ORF of FnFBOX1 was 1 227 bp length,encoding 408 amino acids. Analysis from amino acid sequence alignment and phylogenetic tree showed that FnFBOX1 had the highest homology with the FvFBOX1 of Fragaria vesca. Subcellular localization assays showed that the FnFBOX1 was located in the nucleus. The 821 bp region of promoter pFnFBOX1 was cloned,and the transient expression vector pFnFBOX1∷GUS was generated. Histochemical staining analysis and GUS activity determination showed that pFnFBOX1 had the activity of driving downstream gene transcription. C. gloeosporioides infection and SA treatment promoted the increase of GUS activity derived by pFnFBOX1. RT-qPCR analysis showed that FnFBOX1 gene was expressed in different tissues of F. nilgerrensis. Both the disease-resistant F. nilgerrensis and the susceptible ‘Miaoxiang 3’ strawberry were treated with C. gloeosporioides and SA,and the result showed that the FnFBOX1 gene expression in the F. nilgerrensis reached the highest at 12 h after inoculation with C. gloeosporioides,which was 6.3 times over than that of 0 h. At 24 h after SA treatment,its expression reached the maximum,which was 7.9 times over than that of 0 h. ‘Miaoxiang 3’ strawberry also responded to C. gloeosporioides and SA treatment,but the response degree was not as high as that of F. nilgerrensis.

Analysis of Antimicrobial Active Metabolites from Antagonistic Strains Against Fusarium solani

YANG Rui-xian, LIU Ping, WANG Zu-hua, RUAN Bao-shuo, WANG Zhi-da

2022, 38(2):  57-66.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0337

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Previous studies revealed that two stains Bacillus amyloliquefaciens md8 and md9 have strong antagonism to Fusarium solani causing tree peony root rot;however,the components of antimicrobial substances are not clear yet. First,the biosynthesis gene fragments of 3 lipopeptides were identified. Acid precipitation and the Sephadex gel chromatography column were used to separate and purify the antimicrobial substances. Then Oxford Cup method was adapted to detect the inhibitory activity of the crude lipopeptides and separation components. RT-qPCR was employed to detect the expression variations of lipopetide biosynthesis genes in the strains md8 and md9 during inhibiting F. solani process in the dual culture. And finally MALDI-TOF-MS technique was used to analyze the types of antimicrobial substances. The results showed that the crude lipopeptide extracts and separation components via Sephadex gel chromatography had favorable effect against the pathogen of root rot. The RT-qPCR results indicated that gene ituD of md8 and md9 for iturin biosynthesis was constantly expressed in high amounts throughout the antagonism. The gene fenA for fengycin biosynthesis were up-regulated when md8 and md9 were confronted with F. solani. The MALDI-TOF-MS results further verified that iturin B and bacillomycin D were the main antimicrobial components of md8 and md9 against F. solani. Combined with RT-qPCR results,it was speculated that iturin produced by the strains md8 and md9 played an important role in the inhibitory effect on F. solani.

Composition Features of Microbial Community in the Rhizospheric Soil of Bletilla striata with Root Rot

ZHAO Lin-yan, GUAN Hui-lin, XIANG Ping, LI Ze-cheng, BAI Yu-long, SONG Hong-chuan, SUN Shi-zhong, XU Wu-mei

2022, 38(2):  67-74.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0757

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Analyzing the composition features of microbial community in the rhizospheric soil of Bletilla striata with root rot would provide the scientific basis for understanding the microbial mechanisms leading to the root rot of B. striata and taking corresponding prevention and control measures. The Illumina HiSeq high-throughput sequencing was used to compare and analyze the microbial community structure in the rhizospheric soils of root-rot and healthy B. striata. Also,the soil physicochemical properties and enzyme activities were quantitatively analyzed. The dominant fungi were Ascomycota and Mortierellomycota,and the dominant bacteria were Proteobacteria and Bacteroidetes in the rhizospheric soil of B. striata. Compared with the healthy individuals,the relative abundance of Ascomycota significantly increased,while the relative abundance of Mortierellomycota decreased. The relative abundance of pathogenic Fusarium and Erysiphe significantly increased in the rhizospheric soil of root-rot B. striata（P<0.05）. Multivariate statistical analyses showed the significant differentiation of bacterial and fungal community in the rhizospheric soils between healthy and root-rot B. striata. In addition,the activities of urease,protease and sucrase,and available potassium content were higher in the rhizospheric soil of healthy individuals;while soil pH and the contents of organic matter,NH₄⁺-N,and NO₃^--N were lower. The root-rot disease of B. striata could be related to the increased relative abundance of pathogenic microorganisms,the changes of microbial community structure,the decreased soil enzyme activity,and the imbalance of plant nutrients.

Anthocyanin Accumulation and Its Gene Expression Induced by Low Nitrogen Stress in Cassava Seedlings

ZOU Liang-ping, GUO Xin, QI Deng-feng, ZHAI Min, LI Zhuang, ZHAO Ping-juan, PENG Ming, NIU Xing-kui

2022, 38(2):  75-82.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0441

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In order to clarify the relationship between the nitrogen and the anthocyanin in cassava,we studied the agronomic traits and the inductions of anthocyanin synthesis-related genes and anthocyanin accumulation differences of cassava Arg7 sterile seedlings growing in the modified MS media for 40 d with 1）40 mmol/L NO₃^- + 20 mmol/L NH₄⁺,2）40 mmol/L NO₃^-,3）20 mmol/L NH₄⁺,4）0.4 mmol/L NO₃^- + 0.2 mmol/L NH₄⁺,5）0.4 mmol/L NO₃^-,6）0.2 mmol/L NH₄⁺,7）1 mmol/L（N）,8）5 mmol/L（N）,9）9 mmol/L（N）,and 10）13 mmol/L（N）,based on the previous research results of that nitrogen deficiency accelerates anthocyanin synthesis and accumulation in Arabidopsisetc. Results showed that no anthocyanin was observed in the seedlings of cassava variety Arg7 in any of the three modified MS media containing 40 mmol/L NO₃^- alone,20 mmol/L NH₄⁺alone or their combination for 40 d. Compared to cassava seedlings in the combination of 40 mmol/L NO₃^- and 20 mmol/L NH₄⁺,the plant height and length of primary roots under 40 mmol/L NO₃^- alone treatment were not significantly different,but the number of primary roots was significantly reduced. And the plant height,the length and number of primary roots of the seedlings in media with 20 mmol/L NH₄⁺ were significantly inhibited. The anthocyanins accumulated significantly in the stem and petiole of seedlings in any of three MS media supplemented with 0.4 mmol/L NH₄⁺,0.2 mmol/L NH₄⁺,or combination of 0.4 mmol/L NO₃^- and 0.2 mmol/L NH₄⁺ for 40 d,as well as genes related to anthocyanin synthesis were induced to be expressed. Compared to the combination of 40 mmol/L NO₃^- and 20 mmol/L NH₄⁺,the plant height,the lengths and the number of primary roots under MS media with 0.4 mmol/L NO₃^-,0.2 mmol/L NH₄⁺,or combination of 0.4 mmol/L NO₃^-,and 0.2 mmol/L NH₄⁺ significantly decreased,but the anthocyanin content significantly increased. Furthermore,there was a negative correlation between anthocyanin content and nitrogen concentration（R²=0.96）. This indicates that the induced expression of anthocyanins in cassava Arg7 seedlings was only related to nitrogen concentration,but not to the form.

Transcriptome Analysis of Response to Heavy Metal Copper Stress in Setcreasea purpurea Root Tissue

PENG Guo-ying, HU Liang, HUANG Chao, YANG Kun, WAN Wei, HUANG Chang-gan

2022, 38(2):  83-94.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0664

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Setcreasea purpurea is a highly copper（Cu）tolerant and hyperaccumulator plant. It is the first time that RNA-Seq technology was applied to profile the transcriptome of it in this study. Candidate genes related to copper tolerance of S. purpurea were assembled based on de novo transcriptome analysis. A total of 82 471 high-quality unigenes with an N50 length of 2 299 bp were obtained through transcriptome profiling,which provided abundant data for future studies on S. purpurea. The sequencing data of the control group（CK）,300 mol/L stress group（CT1）,and 1 000 mol/L stress group（CT2）were deposited in the NCBI sequence read archive database under the accession number SAMN11265427. After comparing CT1 with CK,a total of 5 028 unigenes were significantly differentially expressed in the roots,accounting for 6.10% of all unigenes. Among them,the significantly up-regulated unigenes and down-regulated unigenes were 3 138 and 1 890,respectively. After comparing CT2 with CT1,a total of 6 813 unigenes were significantly differentially expressed in the roots,accounting for 8.26% of all unigenes. Among them,the significantly up-regulated unigenes and down-regulated unigenes were 2 555 and 4 258,respectively. The results were consistent with Illumina sequencing data based on the quantitative fluorescence analysis of 10 randomly selected genes by qRT-PCR,which confirmed the validity of differentially expressed gene data. The above experiment provides a theoretical basis for examining the molecular mechanism of copper tolerance under copper stress on the molecular level.

Isolation and Identification of a Cellulase-producing Endophytic Fungus in Paris polyphylla var. yunnanensis

ZHANG Gong-you, WANG Yi-han, GUO Min, ZHANG Ting-ting, WANG Bing, LIU Hong-mei

2022, 38(2):  95-104.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0266

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This paper aims to isolate and identify an endophytic fungus with cellulase activity in Paris polyphylla var. yunnanensis. Surface disinfection method was applied to isolate the endophytic fungi from P. polyphylla var. yunnanensis rhizome. CMC plate assay was used to detect the cellulase activity of the isolated strains. Morphological features and sequence characters based techniques were applied to identify the strains producing high-yield cellulase. The factors affecting the cellulase activity were also explored. Finally,plate activity screen method was used to test the activity of other extracellular hydrolases production. A total of 41 fungal endophytes were isolated from 3 origins of P. polyphylla var. yunnanensis. Among the 41 isolates,strains named as AS-5,AS-7,AS-9 and AS-18 were found to be able to produce cellulase,and strain AS-9 showed the strongest cellulase production activity. AS-9 was identified as Setophoma terrestris based on the morphological observations and ITS,LSU sequences analysis. AS-9 presented maximal cellulase activity at pH7.0 and 28℃. UV exposure did not have a significant effect on cellulase production of AS9. Meanwhile,S. terrestris AS-9 also demonstrated the activities of producing caseinase,lipase,asparaginase,glutaminase and urease production. To the best of our knowledge,S. terrestris AS-9 is first detected as an endophytic fungus in P. polyphylla var. yunnanensis and shows a better cellulase production capacity. It is worth for further study.

Effects of a Xylanase-producing Bacillus cereus on the Composition and Fermented Products of Cigar Leaves

LI Zhi-hao, ZHANG Ge, MO Zhi-jie, DENG Shuai-jun, LI Jia-yi, ZHANG Hai-bo, LIU Xiao-hui, LIU Hao-bao

2022, 38(2):  105-112.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0349

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Hemicellulose in cigar wrapper leaves plays an important role in the characteristics of vein and leaf toughness. The thick vein and high hemicellulose content in cigar leaves lead to poor toughness and usability of cigar wrapper. A xylanase-producing strain Bacillus cereus was preliminary screened in the previous stage of our lab,it was found that its tolerance to nicotine was fine. Then the liquid fermentation experiment was used to determine the optimal fermentation condition when the strain was using cigar leaves as nutrient sources to produce xylanase. Under the optimal fermentation condition,the degradation effect of hemicellulose in the tobacco leaves was measured,and finally the effects of the strain on the composition and contents of aroma materials in the cigar leaves were compared and analyzed. The results showed that the growth of the strain was inhibited little in the medium of ≤ 1.0 g/L nicotine,i.e.,the strain had the favorable tolerance to nicotine. When the initial pH of the culture medium was 6.7,the inoculation amount was 5%,and the concentration of cigar leaf substrate was 20 g/L,and fermentation for 24 h,the xylanase production of the strain was the highest,reaching（4.04±0.18）U/mL. Under the optimal fermentation conditions,the hemicellulose content in the cigar leaves was 3.38%,which decreased by 6.63%,and the cellulose content was 10.54%,which decreased by 8.19%, meanwhile,the contents of cellulose and hemicellulose in the cigar leaves decreased under the fermentation. The analysis of aroma materials in the fermented products revealed that phyllol alcohol（2.85%）,oleic acid（0.62%）,n-hexadecane（1.75%）and hentriacontane（14.47%）were enhanced after fermentation by the strain. It is expected that B. cereus can be used in the solid fermentation of leaves for cigar wrapper to improve the physical properties of tobacco leaves,thus laying a foundation for the development and utilization of this strain.

Isolation and Identification of Nicosulfuron Degrading Strain Chryseobacterium sp. LAM-M5 and Study on Its Degradation Pathway

MA Qing-yun, JIANG Xu, LI Qing-qing, SONG Jin-long, ZHOU Yi-qing, RUAN Zhi-yong

2022, 38(2):  113-122.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0243

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In order to obtain more nicosulfuron-degrading bacterial resources,the activated sludge samples from a nicosulfuron manufacturer in Hefei city were used for study object,from which an isolated and screened strain grew well with glucose as sole carbon source and nicosulfuron as sole nitrogen source. Based on its phenotypic characteristics,the 16S rRNA gene sequence similarity,the values of DNA-DNA hybridization and average nucleotide identity,it was identified as Chryseobacterium lacus LAM-M5. This strain degraded 92.39% of 50 mg/L nicosulfuron within 7 d. Liquid chromatography mass spectrometry was used to detect and identify the metabolites after mixing culture of LAM-M5 and nicosulfuron,and 5 major substances were detected. The metabolic pathway of LAM-M5 degrading nicosulfuron was primarily speculated based on the chemical structure and characteristics of intermediates. High performance liquid chromatography was employed to isolate and identify the acids generated during the degradation of nicosulfuron by strain LAM-M5,and the results showed that L-malic acid was predominant. It was deduced that strain LAM-M5 metabolized the glucose into L-malic acid when its growth was stressed by the nicosulfuron,from which the environmental pH decreased and the nicosulfuron hydrolyzed,thus the stress was released. Through the analysis of the whole genome sequence of strain LAM-M5,7 annotated genes that may contribute to the degradation of nicosulfuron were predicted. The results reveal that strain LAM-M5 has excellent ability of degrading nicosulfuron and may have promising application potential in the microbial remediation of nicosulfuron -polluted environment.

Heterologous Expression and Characterization of Endo-type Alginate Lyase from Yeosuana marina sp. JLT21

CHANG Qing, SHU Yue-rong, WANG Wen-tao, JIANG Hao, YAN Quan-de, QIAN Zheng, GAO Xue-chun, WU Jin-hong, ZHANG Yong

2022, 38(2):  123-131.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0539

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Alginate oligosaccharides（AO）have rich biological activity,preparing AO using enzymatic approach is of important,practical and applicable value. To mine highly active and stable enzymes for preparing AO,a gene from an alginate lyase YAM-1 of Yeosuana marina sp. JLT21 was expressed in Escherichia coli,and the enzyme was purified and characterized. As one of polysaccharide lyase family 7（PL7）members,YMA-1 consisted of 306 amino acids. The recombinant YMA-1 showed the optimal activity of 1.3×10⁴ U/mg at pH 9.0 and 55℃,and Cu²⁺ efficiently stimulated its activity. YMA-1 also demonstrated a highly catalytic activity towards sodium alginate,poly G and poly M,and their specific activities were（5 201.21±86.46）,（6 399.73±253.12）and（3 751.68±116.25）U/mg,respectively under 37℃ and pH 9.0. The end-products from the enzymolysis of sodium alginate were mainly unsaturated trisaccharides and tetrasaccharides. Duo to its broad substrate spectrum,high activity and thermostability,YMA-1 as endo-type alginate lyase in PL7 family has potential applications in green and efficient production of alginate oligosaccharides.

Functional Study of BbRho5 on the Growth Rate of Beauveria bassiana

GUAN Yi, LI Xin, WANG Ding-yi, DU Xi, ZHANG Long-bin, YE Xiu-yun

2022, 38(2):  132-140.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0210

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In order to clarify the role of BbRho5 in the biocontrol potential of B. bassiana,a Bbrho5 single gene knockout strain ΔBbrho5 was constructed. The colony growth rate was measured on different media while the wild-type strain WT was used as the control. In addition,carbendazim-tolerance of WT and ΔBbrho5 and their virulence to Galleria mellonella larval body wall were also tested. The transcriptome data of ΔBbrho5 and WT were further obtained and analyzed. The results showed that the functional defect of BbRho5 protein significantly inhibited the hyphal growth rate of B. bassiana;meanwhile BbRho5 showed mild effect in carbendazim-tolerance and virulence in B. bassiana. Compared with WT,there were 770 differentially expressed genes（DEGs）in ΔBbrho5,of which 395 were up-regulated and 375 were down-regulated. GO analysis showed that DEGs in ΔBbrho5 VS WT were mainly enriched in oxidoreductase activity and monooxygenase activity. KEGG pathway enrichment showed that DEGs were mainly enriched in nitrogen metabolism and various amino acid metabolism pathways. Seven functional genes were enriched in the nitrogen metabolism pathway,of which 5 were up-regulated and 2 were down-regulated,indicating that ΔBbrho5 enhanced nitrogen source utilization and glutamate synthesis to cope with the growth retardation caused by Bbrho5 deficiency. The above results reveal that the small GTPases BbRho5 has an important effect on the growth rate of B. bassiana,and nitrogen metabolism and amino acid metabolism may be important metabolic pathways.

Effects on the Biosynthesis of Ethanol by Promoters PpetE and Pcpc560 in Synechocystis sp. PCC 6803

YE Peng-lin, Kwasi Kyere-Yeboah, GAO E-bin

2022, 38(2):  141-149.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0241

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To increase the synthesized ethanol yield of engineered Synechocystis sp. PCC 6803,the strong promoter Pcpc560 was selected to drive and enhance the expression of exogenous ethanol-producing genes（pdc,and yqhD）,thus ethanol yield was enhanced. The specific methods were that by homologous double exchange,the pyruvate decarboxylase gene（pdc）derived from Zymomonas mobilis and NADPH-dependent aldehyde reductase（yqhD）derived from Escherichia coli were introduced and driven by different promoters. By reverse transcription quantitative PCR analysis,the expressions of exogenous ethanol-producing genes（pdc and yqhD）and the ethanol yields of corresponding engineered strains under the driven of different promoters were detected and compared. The results showed that the light intensity promoter,Pcpc560 derived from the Synechocystis sp. PCC 6803 significantly enhanced the expressions of the exogenous ethanol-producing genes（pdc and yqhD）and increased synthesized ethanol output compared with the medium copper ion-induced promoter PpetE. The combined expression of super-strong promoter Pcpc560 with pdc and yqhD has significantly improved the ethanol production of the engineered strain.

Effects of Dendrobium officinale Polysaccharides on the Intestinal Mucosal Structure and Microbiota in Mice Fed a High-fat Diet

XIE Guo-zhen, TANG Yuan, NING Xiao-mei, QIU Ji-hui, TAN Zhou-jin

2022, 38(2):  150-157.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0390

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To investigate the effects of Dendrobium officinale polysaccharides（DOP）on the intestinal mucosal barrier of mice fed a high fat diet,DOP were extracted by water extraction and alcohol precipitation method. DOP were gavaged with the mice fed a high fat diet for 8 weeks,and the intestinal mucosal structure and intestinal mucosal microbiota of mice were observed. The results showed that high fat diet significantly destroyed the intestinal mucosal structure,which was characterized by intestinal mucosal atrophy,epithelial cells shedding and inflammatory exudation. Moreover,infection-associated and inflammation-related bacteria,for example,Corynebacterium_1 and Staphylococcus proliferated greatly. DOP protected intestinal mucosal structure. In addition,DOP reduced the abundance of Corynebacterium_1,increased the abundance of Candidatus_Arthromitus,and enhanced the proliferation of bacteria related to carbohydrate metabolism and short-chain fatty acid production,i.e.,Muribaculaceae,Bacteroides and Lachnospiraceae_NK4A136_group. The results showed that DOP could protect the intestinal mucosal barrier by maintaining the structural integrity of intestinal mucosa,regulating the composition of intestinal mucosal microbiota,promoting carbohydrate metabolism,and producing short-chain fatty acids.

Purification of Antioxidant Peptides from Natto Supernatant and Study on Its Activity

ZHANG Feng-wen, ZHOU Li-ya, DONG Chao, SHI Yan-mao

2022, 38(2):  158-165.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0379

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High antioxidant polypeptides were extracted from fermented natto,the supernatant of the natto were isolated and purified by SephadexG-50 and RP HPLC,and their structures were identified by electrospray ionization tandem mass spectrometry. The results showed that 3 components（F1,F2 and F3）from the polypeptide mixture of fermented natto were obtained after the separation and purification of the antioxidant peptide via Sephadex G-50 gel. The antioxidant activity of the component F3 was the strongest,the total reduction power reached（8.4+0.6）mmol/g,higher than that of other components. F3 was separated and purified by semi preparative RP-HPLC,and 4 components（G1,G2,G3,and G4）were obtained. The G1 and G3 in the highest contents were selected for determining DPPH scavenging rate,and G1’s DPPH free radical scavenging rate reached 32.67% The antioxidant peptides in G1 were sequenced and identified by ESI-MS/MS. and the 10 antioxidant peptides with the highest content and the strongest activity were obtained. The activity of peptide SFEWVLEH was the strongest verified by chemical synthesis method. The DPPH scavenging rate was 46.66% ± 0.96%.

Study on Urine Metabolomics in Rats of Different Ages

YANG Yu-ping, ZHANG Xia, WANG Chong-chong, WANG Xiao-yan

2022, 38(2):  166-172.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0331

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This work aims to explore the age-related changes of urine metabolites in healthy rats during growth,development and aging. Urine samples were collected at 3,5,7,9,12,56,and 111 weeks after birth,and the GC/TOF-MS platform was employed for metabolomics detection. The results showed that all urine samples appeared in-group clustering and between-group separation in principal component analysis and partial least square discriminant analysis. Amino acids,organic acids,and carbohydrates were the dominant metabolite types in the rat urine samples. The levels of urine metabolites such as 4-hydroxyproline,L-lysine,and uric acid gradually decreased with age. D-ribose,aconitic acid,and fumaric acid increased in old age,while nicotinic acid and 5-hydroxyindole decreased in old age. The above results revealed that age affected urine metabolism in rats,and a variety of urine metabolites appeared age-related changes during growth,development and aging.

Fermentation Optimization for PQQ Synthesis Based on the Genome-scale Metabolic Model of Methylovorus sp. J1-1

KOU Hang, WANG Yan-mei, LI Tong, BO Ming-jing, ZHANG Wei-cai, XIONG Xiang-hua, LI Ming

2022, 38(2):  173-183.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0345

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The aim of this study is to explore the fermentation strategies and related target genes that can increase PQQ production by constructing genome-scale metabolic model of Methylovorus sp. J1-1. A genome-scale metabolic model（GSMM）of Methylovorus sp. J1-1 was constructed based on its annotated genome and biochemical information. Subsequently,amino acids and several potential genetic targets that may increase PQQ yield were predicted by COBRApy and validated. The GSMM iKH584 of J1-1 was constructed,containing 584 genes,779 biochemical reactions,121 exchange reactions and 765 metabolites,and it may be used in follow-up simulation. According to the iKH584 simulation,addition of glutamic acid,glutamine and proline,overexpression of the glyA and hps1,and knockout of hps2 all promoted PQQ synthesis. The results showed that the addition of glutamic acid and proline increased PQQ production by 10.4% and 22.9% respectively,overexpression of the glyA and hps1 increased the extracellular concentration of PQQ by 20.6% and 14.6% respectively,and hps2 knockout enhanced PQQ concentration up to 140.84 mg/L and increased by 8.0%,which were consistent with the simulated results,indicating that the metabolic model iKH584 of J1-1 was basically correct. Finally,fed-batch fermentations of J1-1△hps2 in 5 L fermentor were carried out,and the PQQ product reached to 812.64 mg/L,improved 11.1% compared with the parent strain J1-1. Conclusively,the established metabolic model iKH584 of J1-1 can be used to guide the fermentation and strain modification of J1-1 for improving the PQQ yield.

Regulation Mechanism of Phosphate Homeostasis in Plants

LIU Chao, CHU Hong-long, WU Li-fang, TANG Li-zhou, HAN Li-hong

2022, 38(2):  184-194.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0472

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Phosphorus is one of the essential plant macronutrient,which is an important component of life macromolecules,and plays an indispensable role in the process of plant life cycle. Maintaining phosphate homeostasis is critical for plant growth and environmental response. Phosphate homeostasis uptake and transport in plants involves multiple signaling molecules. The process of plant maintaining phosphate homeostasis mainly includes the activation of soil phosphate homeostasis,the uptake and transportation of phosphate homeostasis,the allocation and remobilization of phosphate in plants. It involves phosphate homeostasis mechanisms such as response to low phosphate homeostasis stress,transcription factor regulation,miRNA regulation,mycorrhizal symbiosis,inter organelle transfer regulation and so on. The research of phosphate homeostasis nutrition mechanism in the future needs the integration of interdisciplinary knowledge,from model plant research to commercial crops. In this paper, we summarized the latest research progress on the core molecules and their mechanisms of uptake and transport of phosphate in plant cells,aiming to provide an important reference for crop engineering and breeding strategies.

Gibberellin Metabolism Regulation and Green Revolution

LI Yi-dan, SHAN Xiao-hui

2022, 38(2):  195-204.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0389

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Gibberellins（GAs）are very important plant hormones that control diverse aspects of plant growth and development. Some important regulatory genes involved in gibberellin biosynthesis and signaling transduction pathway positively impact plant architecture,yield and quality of cereal crops,which has been applied in agricultural production. The alleles of Rht-1 and sd-1,that confer a semi-dwarf feature to wheat and rice,respectively,underpin the‘Green Revolution’in the latter half of the twentieth century. This paper reviewed the research achievements on the GA metabolism regulation in impacting the height,yield,nutrition usage of‘Geen Revolution’varieties,and prospected how to develop and utilize GA regulation genes for breeding more‘Green Revolution’varieties in the future.

Research Progress in Molecular Genetic Manipulation Technology of Bacillus amyloliquefaciens and Its Application

QIU Yi-bin, MA Yan-qin, SHA Yuan-yuan, ZHU Yi-fan, SU Er-zheng, LEI Peng, LI Sha, XU Hong

2022, 38(2):  205-217.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0447

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Bacillus amyloliquefaciens is a Generally Recognized as Safe（GRAS）strain recognized by the FDA. It has outstanding advantages in the production of industrial enzyme preparations,high molecular polymers,bulk chemicals,and green biological pesticides. In recent years,as the molecular genetic manipulation technology of B. amyloliquefaciens has become more and more mature,there has been a more urgent need for the use of the bacteria to develop microbial fermentation platform strains for use in the field of synthetic biology manufacturing. In this paper,the genetic manipulation tools,metabolic modification applications and future development prospects of B. amyloliquefaciens are reviewed in detail,which will provide reference for further promoting the innovation and development of B. amyloliquefaciens on synthetic biology technology.

Research Progress in Phosphorylation Modification of Antiviral Nucleotide Analogs

LIANG Xing-xing, WANG Jia, XU Wen-tao

2022, 38(2):  218-226.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0565

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Nucleoside and nucleotide analogs are a class of antiviral prodrugs. After entering the human cells,they are gradually phosphorylated to generate nucleoside triphosphate analogues to serve as the antimetabolites. The resulted triphosphate analogues achieve the effect of disease treatment mainly by inhibiting virus replication and promoting apoptosis of infected cells. During the phosphorylation,the metabolic conversion process of nucleoside analogs activated by kinases in the cell is usually insufficient,resulting in a lower concentration of the final nucleoside triphosphate analogues,thus reducing the antiviral effect. Therefore,by directly preparing nucleotide analogs as antiviral prodrugs,the lipophilic and cleavable groups are modified on the phosphate groups to increase its stability and enhance the absorption capacity of cells,which can avoid the rate-limiting step of phosphorylation in the cell. In the end,it can effectively increase the final active nucleoside triphosphate concentration and exert a better antiviral effect. This article summarizes the development process of nucleotide analogs,and lists and analyzes the synthesis and modification methods of different types of nucleotide analogs,aiming to provide useful references for the further development of efficient and safe antiviral nucleotide analogs in the future.

Research Progress of the Toxic Effects and Detoxification Measures of Engineered Nanoparticles in Biological Nitrogen-removing Process

ZHANG Man-man, HE Teng-xia, DING Chen-yu, CHEN Meng-ping, WU Qi-feng

2022, 38(2):  227-236.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0470

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Nitrogen compounds play an important role in the metabolism of life. However,excessive inorganic nitrogen may cause water deterioration and affect human health. Biological nitrogen-removing technology can efficiently remove inorganic nitrogen from the environment without causing secondary pollution. With the wide application of engineered nanoparticles in life,a large number of them have been released into the soil and water bodies,which greatly hinder the biological nitrogen-removing process in wastewater treatment process. Therefore,the toxic effect of engineered nanoparticles and the detoxification measures during microbial nitrogen-removing process have become the research hotspots in recent years. In this paper,the way of engineered nanoparticles entering water environment is described carefully. Thereafter,the impact of engineered nanoparticles on wastewater treatment systems and biological denitrification are systematically analyzed. Additionally,the toxic effects of engineered nanoparticles on denitrifying microorganisms and the detoxification mechanism of denitrifying microorganisms are carefully analyzed. The prospects for future research trends are proposed. It is aimed to have a theoretical significance for improving the biological nitrogen-removing efficiency in the presence of engineered nanoparticles. Meanwhile,it may promote the exploration of the toxicity and stress mechanism of nanoparticles to heterotrophic nitrification and aerobic denitrification bacteria.

Research Progress in the Post-translational Modification of Superoxide Dismutase

JIA Hai-hong, LI Bing-qing

2022, 38(2):  237-244.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0611

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Superoxide dismutase（SOD）is a kind of antioxidant metal enzyme in living organisms,which can catalyze the disproportionation of superoxide anion radicals to produce oxygen（O₂）and hydrogen peroxide（H₂O₂）,and plays an important role in the balance of oxidation and antioxidant in the body as well as is closely related to the occurrence and development of many diseases. The regulation of SOD enzyme activity has always been the focus of research,most of which focus on two aspects：transcription level（gene expression）and translation level（enzyme protein synthesis）.With the development of research,it is found that post-translational modification of protein（PTM）has an important effect on the enzyme activity of SOD. In recent years,more and more attentions have been paid to the influence of protein post-translational modification on SOD enzyme activity. In this review,we summarized several ways of SOD post-translation modification,such as nitration,phosphorylation,S-glutathione,glycosylation,acetylation,sulfonic acid modification,sulfonic acid modification,SUMO modification,etc.,and discussed the effect of modification on SOD enzyme activity and its physiological significance. Also we prospected the development and challenges of SOD post-translational modification. It provides a theoretical basis for the research,treatment and development of targeted drugs of related diseases.

Research Progress in NAD⁺ Cap Modification at the 5' End of RNA

DONG Hai-jiao, YANG Xiao-yu, MO Bei-xin, CHEN Xue-mei, CUI Jie

2022, 38(2):  245-251.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0975

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The m7G cap,which has long been considered as a hallmark of eukaryotic mRNAs,protects RNAs from degradation and recruits the machinery for splicing,polyadenylation,nuclear export,and translation. The nucleotide-containing metabolite,nicotinamide adenine dinucleotide（NAD⁺）,has recently been detected at the 5' end of RNAs in several species including prokaryotes as a new cap. Now research on the biological functions of NAD⁺-capped RNAs（NAD-RNAs）is still at the initial stage. This review summarizes the discovery of NAD-RNAs as well as the development of its detection and identification technology,discusses the regulatory functions of the NAD⁺ cap on RNA as well as the factors affecting RNA NAD⁺ capping and decapping,and speculates on the potential functions of NAD-RNAs in growth,development and environmental responses. Finally,we propose future research directions and topics in order to garner attention on the outstanding questions regarding the molecular and biological functions NAD-RNAs.

Research Progress in the Biosynthesis and Metabolic Engineering of Hyaluronic Acid

MA Yan-qin, QIU Yi-bin, LI Sha, XU Hong

2022, 38(2):  252-262.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0443

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Hyaluronic acid（HA）,a functional glycosamine polymer widely existing in organism,presents broad potential applications in daily-use chemicals,medicine and food. With the development of synthetic biology technologies such as genetic engineering and metabolic engineering,some new challenges are emerging while the biosynthesis process and mechanism of HA have been studied in depth. This review summarizes the key genes and pathways of HA synthase from the perspective of molecular biology,aligns and analyses the amino acid sequence of HA synthase from different sources. It also describes in detail the bottleneck issues such as the safety of HA production,substrate imbalance and low oxygen solubility in fermentation,and introduces the development of strategies on corresponding synthetic biology in commonly used microbial hosts,thereby induces the effective strategies for high-yield HA by microorganism. This review may provide an important support for further promoting the green biomanufacturing of HA.

Establishment of an Efficient in planta Transformation Method for Camellia sinensis

ZHOU Cheng-zhe, CHANG Xiao-jun, ZHU Chen, CHENG Chun-zhen, CHEN Yu-kun, LAI Zhong-xiong, LIN Yu-ling, GUO Yu-qiong

2022, 38(2):  263-268.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0635

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To address the lack of an efficient and stable genetic transformation system for tea trees,an in planta transformation transgenic method based on Agrobacterium tumefaciens-mediated transformation of tea trees was established,laying a solid foundation for gene function research and germplasm innovation of tea tree. The seedlings of tea cultivar ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ were used as receptor plants,and the apical bud and axillary bud were removed. Using the A. tumefaciens broth infecting the wounds of the seedling,regenerative buds were obtained through resistance screening. After molecular biological identification,transgenic plants were obtained by short-ear cutting method. The results demonstrated that the GUS（-glucuronidase）and HYG（hyglymycin）labeled genes were tested to be positive with multiple PCR,and the PCR product sequence was consistent with the labeled gene sequence. The transformation efficiency of ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ was 8.14%,2.99%,and 2.53%,respectively. This study establishes an in planta transformation method of tea plant that does not rely on tissue culture,and the transformation protocol characterizes of simple operation,high transformation rate,low cost,and short experiment cycle.

Applications of Recombinase Polymerase Amplification in Plant Virus Detection

LUO Xue-cong, AN Meng-nan, WU Yuan-hua, XIA Zi-hao

2022, 38(2):  269-280.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0453

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With the development of molecular biology technology,a number of nucleic acid isothermal amplification technologies have been developed gradually. Recombinase polymerase amplification（RPA）,as a rapid and sensitive detection technique,has great advantages. At present,RPA has been applied in many fields on detections,such as genetically modified organism,a variety of pathogens and food safety. As an emerging technology,RPA is developing rapidly in plant virus detection. The RPA technique requires only a pair of primers and is finished in only 30 min at constant temperature（37-42^oC）,which has high sensitivity and specificity. As a result,the RPA technique is rapidly becoming a very effective tool for detection of plant viruses in under-equipped laboratories or in on-site facilities. In this review,the detection principle,primer design and application for RPA are introduced,and the recent research progress and existing problems of RPA in plant virus detection is reviewed,aiming to provide references for the application of RPA in te detection of plant detection.

Discovery of Viruses from Small Brown Planthopper by Small RNA Deep Sequencing

PIAO Jun, ZHANG Lu-Jie, PIAO Jing-Ai, ZHOU Yi-Jun, LI Shuo

2022, 38(2):  281-288.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0461

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Small brown planthopper（SBPH）（Laodelphax striatellus Fallén）is an important agricultural pest. As a virus vector,it can transmit some plant viruses,which cause viral diseases of grain crops,including rice,wheat and maize. At present,there is no comprehensive understanding of insect virus species in SBPH,thus it is difficult to conduct relevant research on SBPH management by exploiting insect viruses. In this paper,in order to excavate insect virus resources,the virus species in SBPH were identified by small RNA deep sequencing technology. The results showed that 13 viruses were found via sequencing data alignment,involving in 8 families and 2 unclassified viruses. Besides the dominant rice stripe virus,the rest were specialized parasitic insect viruses,including 5 positive-stranded RNA viruses,2 single-stranded DNA viruses and 5 double-stranded DNA viruses. Among them,a new virus similar to Drosophlia A virus was identified,and the 1-1 932 nt nucleotides of viral RNA-dependent RNA polymerase（RdRp）gene was obtained. After Blast and phylogenetic analysis,the unusual “C-A-B” permutation was found in the palm subdomain conserved region of RdRp,which was a unique arrangement pattern of Permutotetraviridae viruses,thus it was considered that the virus was a novel permutotetra-like virus,temporarily named Laodelphax striatellus permutotetra-like virus（LsPLV）. A permutotetra-like virus was first identified in Hemiptera insect. The study suggests that insect virus species are abundant in SBPH,which lays the foundation for biological control of SBPH by exploiting insect viruses in the future.

Analysis of the Development Trend in the Field of Plant Synthetic Biology

GUO Xiao-zhen, ZHANG Xue-fu

2022, 38(2):  289-296.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1600s

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Synthetic biology,as a disruptive technology,can be applied to solve the bottleneck problem in current agricultural disciplines. In this study,bibliometrics is used to analyze the basic situation of synthetic biology from the time series,number distribution and topic distribution of published papers in the field. Based on the topic distribution in the field,it can be seen that the topic of plant synthetic biology is steadily increasing. Focusing on the topic of plant synthetic biology,the main path analysis was used to explore the important knowledge nodes in the development process of plant synthetic biology from the perspective of knowledge flow. Based on the main path analysis method the representative content glucosinolate as the representative secondary metabolite,including the mesotrioside biosynthetic pathway,the functional analysis of important catalytic enzymes,the regulation of transcription factors,the application of omics method,and the use of microbial yeast for biomass synthesis,which respectively characterized the core theory and technology of synthetic biology.