Table of Content

26 March 2022, Volume 38 Issue 3

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Study on the Interaction of F-box Protein FKF1 and Transcription Factor FUL in Regulating Flowering in Arabidopsis

ZHOU Juan, YAN Jin-dong, LI Xin-mei, LIU Xue-qing, ZHAO Qiang, ZHAO Xiao-ying

2022, 38(3):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0499

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F-box protein FLAVIN-BINDING KELCH REPEAT F-BOX 1（FKF1）is involved in regulating photoperiod flowering in Arabidopsis;however,the underling molecular mechanism is not fully understood yet. Here,we confirmed that FKF1 interacted with transcription factor FRUITFULL（FUL）in vivo and in vitro. qRT-PCR and Western blot results indicated that FKF1 positively regulated the FUL expression in transcriptional level,but had no influence on the stability of FUL protein. Genetic analyses indicated that the flowering phenotype of 35S-FKF1-Myc/ful-8 double mutants and the transcription level of flowering integrator gene FLOWERING LOCUS T（FT）in them were consistent to that in the ful-8 mutant. Collectively,these findings indicate that FKF1 interacts with FUL and functions the upstream of FUL to promote FT expression,thereby promoting flowering.

Identification of Key Transcription Factors in Response to Salt Stress in Rice

ZHANG Bin, YANG Xin-xia

2022, 38(3):  9-15.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0802

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Salt stress is one of the most important abiotic stresses affecting rice production. In this study,the transcriptome data of rice under salt stress were used to analyze the expression changes of transcription factors at the transcriptome level by HTSeq and DESeq software. The main results were as follows：there were 26 identical transcription factors differentially expressed in rice at 1,3 and 6 h of salt stress,and an important module containing 14 genes was screened by protein interaction. GO analysis of important modules showed that 14 genes were mainly enriched in ATP binding,stress response and cytoplasmic membrane-bounded vesicle. KEGG analysis showed that it was mainly enriched in endoplasmic reticulum protein processing and endocytosis pathway. Meanwhile,the key heat shock transcription factor gene Os03g074500 and Os06g0553100 induced by salt stress were identified. It is speculated that these two genes may play a significant role in the process of rice salt stress by regulating the transcription and expression of HSP70 gene and affecting protein folding and assembly. This study provides candidate genes for subsequent genetic improvement of rice and provides scientific enlightenment for further understanding of salt tolerance mechanism.

QTL Mapping for Resistance to Rice Kernel Smut of Male Sterile Line

YANG Yi-shan, SUN Ping-yong, YU Mu-lan

2022, 38(3):  16-21.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0934

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In order to locate the resistance gene of rice kernel smut and establish the molecular breeding system for resistance to rice kernel smut,a RIL（recombinant inbred line）population consisting of 183 high-generation stable male sterile lines was constructed by hybridization and multi-year self-crossing using W9593S,a PTGMS（photo- and thermo-sensitive genic male sterile）line that has strong resistance to rice kernel smut,and Pei'ai 64S,a PTGMS line that has the same sterile allele of W9593S and is susceptible to rice kernel smut,as parent lines. With the RIL population as material,the QTL for resistance to rice kernel smut was mapped by composite interval mapping using 200 polymorphic SNP（single nucleotide polymorphism）molecular markers. The result showed that,5 QTLs with resistance to rice kernel smut were located on 4 chromosomes with phenotypic contribution rates ranging from 7.26% to 13.64%. Among them,qRRKS-11 and qRRKS-8 had the highest phenotypic contribution rates of 13.64% and 12.07%,respectively,and the genetic distances of qRRKS-11 and qRRKS-8 were 0.9 and 1.4 cM,respectively. The resistance to kernel smut is characterized by quantitative trait inheritance in rice,and the molecular marker-assisted breeding system of rice sterile lines resistant to kernel smut may be achieved by fine mapping and cloning of major QTL.

Cloning and Expression Analysis of CsCML24 Gene in Camellia sinensis

KANG Rui, LIU Chun-hui, CHEN Si-wen, ZHAO Ren-liang, ZHOU Qiong-qiong

2022, 38(3):  22-30.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0797

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Calmodulin-like protein（CML）is a type of calcium receptor protein,which mediates the interaction of Ca²⁺ with downstream target proteins,and plays an important role in plant abiotic stress response. Exploring the function of CML in tea plant(Camellia sinensis) under adversity stress could provide theoretical references for further studying the molecular regulation mechanism of CsCML24 in stress response of tea plants. The CsCML24 gene（GenBank accession number：MZ325391）of CML was cloned from the leaves of tea cultivar ‘Longjing 43’ and analyzed by bioinformatics. Meanwhile,the relative tissue-specific expressions and expression patterns under different abiotic stresses were analyzed by real-time quantitative PCR. Results of sequence analysis indicated that the full-length CDS sequence of the CsCML24 gene was 480 bp,encoding 159 amino acids. The CsCML24 had no signal peptide and transmembrane region,and belonged to hydrophilic protein,contained EF-hands,which can be combined with Ca²⁺. Tissue-specific expression results from qRT-PCR showed that the CsCML24 gene expressed in various tissues,and highly expressed in the mature leaves,and the least in the stem. The expression of CsCML24 was significantly induced under low temperature（10℃）,drought stress（20% PEG 6000）,salt stress（200 mmol/L NaCl）and ABA,and the expression varied in different stress. It is speculated that this gene regulates the low temperature and drought tolerance of plants via ABA signaling pathway.

Effects of Low Temperature Stress on Different Clones of Cryptomeria fortunei and Evaluation of Their Cold Resistance

CUI Jie-bing, ZHANG Meng, ZHANG Ying-ting, XU Jin

2022, 38(3):  31-40.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0615

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This study is aimed to assess the cold resistance of Cryptomeria fortunei by analyzing the difference of cold resistance among its different clones,providing theoretical basis for the introduction,cultivation and cold resistance breeding of C. fortunei. The study subjects were 10 2-year branches from different clones,which were treated at 4℃,0℃,-4℃,-8℃,-12℃,-16℃ and -20℃ for 12 h,respectively. They were then measured in terms of the contents of malondialdehyde（MDA）,the activity of superoxide dismutase（SOD）and the parameters of chlorophyll fluorescence. Combined with principal component analysis and membership function calculation,comprehensive evaluation and cold resistance ranking were carried out. The results showed that：as the temperature decreased,the content of MDA fluctuated within a certain range of temperature,but generally it exhibited a tendency to increase first,then decrease,and finally increase. In contrast,the activity of SOD decreased shortly at first,and then increased and finally decreased after cold acclimation. The maximum photochemical efficiency（Fv/Fm）and the actual photosynthetic quantum yield（Y（II））of PS II in the different clones presented a similar trend,reaching their peak at 0℃ and decreased as the temperature futher decreasing. The photochemical quenching coefficient（qP）did not change significantly at the beginning of cold stress,but it decreased as the low temperature stress aggravated to below 0℃. There were significant differences in cold resistance among different clones of C. fortunei,among which 57#,68# and 32# clones were of stronger cold resistance,while 3#,25# and 42# clones were of weaker cold resistance.

Transcriptome Identification of Terpenoid Synthase Genes in Jasminum sambac and Their Expressions Responding to Exogenous Hormones

HONG Ya-ping, CHEN Xue-jin, WANG Peng-jie, GU Meng-ya, GAO Ting, YE Nai-xing

2022, 38(3):  41-49.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0716

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The objective of this work is to explore the role of terpenoid synthase（TPS）in the synthesis of terpenoid in jasmine and its expression under exogenous hormones treatments,which will lay a theoretical basis for further study of the function of J. sambac TPS（JsTPS）family and its role in exogenous hormones stress. Based on the transcriptome data of jasmine in this study,bioinformatics was applied to identify and analyze the JsTPS family. Real-time fluorescence quantitative detection was to analyze the expressions of JsTPS family in different tissues and exogenous hormones treatments. The result showed that there were 8 TPS genes of having both Terpene_synthase and Terpene_synthase_C conserved domain in JsTPS family,named JsTPS1 - JsTPS8 respectively. The protein lengths of JsTPS genes ranged from 163 to 844 amino acids. The molecular weights of JsTPS genes ranged from 19 103.6 to 96 887.4 kD. And they were mainly located in cytoplasm and chloroplast. The JsTPS genes were divided into 4 subfamilies（a,b,e/f,and g）based on phylogenetic relationships. The analysis of qRT-PCR showed that the expressions of gene JsTPS1,JsTPS2,JsTPS3,JsTPS5,JsTPS6,and JsTPS7 were high in petals;also the expressions of the JsTPS1,JsTPS2,JsTPS3,JsTPS5,JsTPS6 and JsTPS7 were induced by IAA,GA,SA and MeJA. The MeJA significantly induced the expressions of 6 JsTPS genes,and the relative expression reached the maximum at 4-9 h. The induction from high to low was MeJA > IAA > GA > SA. J. sambac contained 8 TPS family members which were mainly expressed in petals and closely related to the formation of aroma in J. sambac. They were up-regulated under IAA and MeJA inductions. GA and SA treatments may inhibit the expressions of partial JsTPS genes.

Effects of FPS and SS Co-overexpression in Panax notoginseng Cells on Saponins Synthesis

YANG Yan, YU Long-feng, WANG Shao-mei, LI Wei-na, GE Feng

2022, 38(3):  50-58.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0706

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This work aims to study the effect of FPS（farnesyl pyrophasphate synthase）and SS（squalene synthase）co-overexpression in Panax notoginseng cells on saponins synthesis. The pCAMBIA1300S-FPS overexpression vector was transferred into the P. notoginseng cells in which SS was overexpressed in advance by Agrobacterium tumefaciens EHA105. Transgenic cell lines were screened through double antibiotic culture and genomic PCR. Relative expressions of FPS and SS in the transgenic cell lines were analyzed by qRT-PCR. Contents of P. notoginseng saponins（PNS）and monomer saponins were detected by spectrophotometry and chromatography,respectively. In four transgenic cell lines with overexpression of both FPS and SS,relative expression levels of FPS were all higher than those of two control groups of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 3.26 times and 3.74 times of the above two control lines;relative expressions of SS showed no significant difference compared with the line in which only SS was overexpressed,but were higher than the non-transgenic line. Contents of PNS were all higher than those of the non-transgenic line and the line in which only SS was overexpressed,and the highest level was respectively 2.46 times and 1.73 times of the above two control lines. Contents of monomer saponins Re,Rh1,Rd and Rg1 were mostly higher than those of the control lines. Overexpression of FPS and SS in P. notoginseng cells may enhance the accumulation of PNS and monomer saponins. In addition,co-overexpression of FPS and SS may further improve PNS content compared with the overexpression of only SS.

Isolation,Identification and Symbiotic Matching of Soybean Rhizobia from Shanxi Province

WANG Xiao-li, QIN Jie, WANG Min, WANG Li-xiang, DU Wei-jun

2022, 38(3):  59-68.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1315

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As one of the origin of soybean,Shanxi is rich in rhizobia resources. In order to further excavate soybean rhizobia resources in Shanxi province and screen the rhizobia of having good symbiotic nitrogen fixation effect with the local main soybean varieties. In this experiment,root nodules of different soybean varieties were collected in Guangling of Datong,Qingxu of Taiyuan and Taigu of Jinzhong. After multiple streaking,the colony morphology,Congo red staining,BTB staining phenotype identification and 16S rDNA,nodA,nifH genotype identification of the isolated and purified single colonies were performed. BOX-PCR was used for electrophoresis detection of all rhizobia. The strains with completely consistent bands were excluded and the bacteria with inconsistent bands were clustered and analyzed in Bionumerics ^@ 7.5 software. Then the representative strains were selected and inoculated into two soybean materials Jinda 88 and Kefeng 1 to screen strains with good matching. A total of 203 strains were isolated,from 187 strains of them the nodA and nifH were amplified. While 172 fast-growing strains belonged to Sinorhizobium fredii and slowly-growing 15 strains belonged to Bradyrhizobium diazoefficiens and Bradyrhizobium daqingense,respectively. The 85 rhizobia strains with inconsistent bands were divided into 4 groups by BOX-PCR,which were divided into 16 subgroups with 70% as the limit. Rhizobium GL11 and GL43 were selected in better match with Jinda 88. Compared with USDA110 inoculation,the shoot dry weight increased by 38.5% and 30.1%,respectively;the root dry weight increased by 22.2% and 27.8%,respectively;the fresh weight of nodules increased by 24.3% and 41.5%,respectively;the dry weight of nodules increased by 36.6% and 31.0%,respectively. Rhizobium TG37 was selected as a good match with Kefeng 1 and its plant height,shoot dry weight,dry root weight,fresh nodule weight,dry nodule weight and number of nodules increased by 8.1%,15.0%,28.6%,27.3%,44.3% and 60.6%,respectively,compared with USDA110 inoculation. Shanxi province has rich resources of rhizobia. S. fredii is the dominant species. In addition,two types of slow-growing rhizobia B. diazoefficiens and B. daqingense were isolated,which enriches the rhizobia resources. The screening of rhizobia with good symbiosis and matching lays a material and theoretical basis for soybean production practice.

Screening of Antagonist Against Tomato Fruit Rot and Their Preservation Qualities on Tomato

ZHANG Hong-yan, LIN Guo-li, LI Ru-lian, JI Xiao-qi

2022, 38(3):  69-78.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0521

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A strain of fruit rot pathogen was isolated from storing tomatoes. In order to clarify the taxonomic status and investigate the suitable biocontrol strain,its morphology and phylogenetic analysis were conducted. The antagonistic Streptomyces was isolated and identified,and its effect on the preservation of tomato was studied. The results showed that the pathogen Qyg16-2 that caused the fruit rot of tomato was Botryosphaeria dothidea. The actinomecetes X13 with good antagonistic effects from healthy tomato rhizospheric soil on Qyg16-2 was identified as Streptomyces sampsonii by morphology,physiological and biochemical characteristics and 16S rDNA sequence. X13 significantly inhibited the colony extension and spore germination of pathogen B. dothidea,its cell-free fermentation filtrate significantly increased the activity of PPO,POD and PAL,decreased the decay index of stored tomato,and delayed the decline of post-harvest tomato fruit firmness,soluble solid content,titration acid content and vitamin C content. Conclusively,S. sampsonii X13 has significant antagonistic effects on B. dothidea,and has promising preservation effect in storing tomato fruit.

Effects of Chemical Fertilizer Reduction and Application of Plant Growth Regulators from Traditional Chinese Medicine on the Quality and Its Bacterial Community in Rhizosphere Soil

XIE Tian-peng, LIU Na, LIU Yue-min, QU Xin, BO Shuang-qin, JING Ming

2022, 38(3):  79-91.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0467

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In order to achieve the green development goal of “reducing chemical fertilizer and increasing efficiency” in the cultivation of Angelica sinensis, the effects of chemical fertilizer reduction and application of plant growth regulators from traditional Chinese medicine on the quality and bacterial community in rhizosphere soil of A. sinensis were investigated, so as to provide scientific basis for the mechanism of improving the quality of A. sinensis. A. sinensis was taken as the research object. Four treatments were set up, including chemical fertilizer（CK）, Chinese medicine source regulator combined with chemical fertilizer（T1）, Chinese medicine source regulator combined with 80% chemical fertilizer（T2）,and Chinese medicine source regulator combined with 60% chemical fertilizer（T3）. The growth indexes,disease indexes,yield,rhizosphere soil physical and chemical indexes of A. sinensis in the 5 growth stages were determined,and the changes of bacterial community in the rhizosphere soil were analyzed by 16S rDNA amplification sequence. Results showed as below：1）The root length,main root diameter and root biomass of A. sinensis in treatment T1 and T2 were significantly higher than those in other treatments at early growth stage. The aphid emergence rate, aphid density,disease grade and incidence rate of A. sinensis in CK were higher than those in other treatments, and the yield was the lowest in CK,the highest in T2, which was 2.61 times higher than that of CK. 2）There was no significant difference in the salinity,ammonium nitrogen and available potassium among the treatments in whole life growth period. The organic matter in CK at seedling stage was higher than that in other treatments, but there was no difference among treatments at other growth stages. The available phosphorus changed greatly in the whole growth period, and the pH value increased gradually with the reduction of chemical fertilizer. 3）There was no difference in the diversity of bacterial community in the rhizosphere soil of A. sinensis during the whole growth period. The community structure was consistent at phylum level. Proteobacteria, Bacteroidetes, Firmicutes,Actinobacteria, Gemmatimonadetes and Acidobacteria were the dominant phylum. 4）At the genus level, the dominant genera were Flavobacterium, Sphingomonas, Pseudomonas, Bacteroides, MND1, Pedobacter, Citrobacter, Ellin6067, Gemmatimonas, Massilia, Sphingobiu, Lysobacter, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Haliangium, and Nitrospira. The abundance of Pseudomonas, Citrobacter, Ellin6067 and Massilia were significantly different among treatments in different growth stages of A. sinensis. However,only the abundance of Pseudomonas was positively correlated with the root growth index of A. sinensis,and negatively correlated with the disease index. The abundance of Pseudomonas in CK was significantly lower than that in other treatments in the early stage and drug preparation stage. Chemical fertilizer reduction and application of plant growth regulators from traditional Chinese medicine may promote the root growth of A. sinensis at early growth stages,improve the disease condition of root rot in the proprietary period,and increase the yield. The Chinese medicine source regulator may improve the quality of A. sinensis by increasing the abundance of Pseudomonas in the rhizosphere soil.

Screening,Identification and Antagonism of Phosphate-Solubilizing Bacteria from the Compost Chinese Medicinal Herbal Residues

HU Shan, LIANG Wei-qu, HUANG Hao, XU Cong, LUO Hua-jian, HU Chu-wei, HUANG Xiao-yan, CHEN Shi-li

2022, 38(3):  92-102.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0571

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To develop new,safe and efficient microbial fertilizer,Chinese medicine residues after two months composting was used as test material to screen phosphate-solubilizing and antagonistic bacteria. Organophosphorus bacteria medium was used to preliminarily screen 5 strains of phosphate-solubilizing bacteria. Phosphorus solubilizing ring method and phosphorus-molybdenum blue colorimetry were employed to determine phosphorus solubility of the strains. The strain with the best phosphorus solubility was YP5. Based on the colony morphology,physiological and biochemical characteristics,and 16S rDNA sequence analysis,YP5 was identified as Pseudomonas aeruginosa. The results of promoting and antagonist factors showed that YP5 secreted iron carrier and a large amount of extracellular protease. YP5 was highly sensitive to norfloxacin,chloramphenicol,gentamicin,enrofloxacin and amikacin. The results from the plate confrontation method and the virulent medium method revealed that YP5 fermentation broth and cell-free filtrate presented promising antagonism on 9 plant pathogens such as Fusarium solani,and the maximum inhibition rate was > 80%. The highest yield of phenazino-1-carboxylic acid of YP5 in KB medium at 28℃was 17.73 mg/L by high performance liquid chromatography.

Screening of Potential PGPR Strains Producting Growth-promoting Volatile Compounds and Study on Their Growth-promoting Characteristics

GAO Ya-hui, JIANG Ming-guo, FENG Jing, ZHOU Gui

2022, 38(3):  103-112.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0619

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The aim of this work is to screen the potential plant growth-promoting rhizobacteria（PGPR）whose volatile organic compounds（VOCs）can promote plant growth,and to investigate the growth-promoting effect of the VOCs on plants and other growth-promoting functions,which provides new ideas and reliable materials for the research and development of microbial fertilizers. Using 48 functional bacteria isolated from marine samples and preserved in our laboratory as test strains,two-compartment plate test and VOCs pot experiment were used to screen them,16S rRNA to identify them,and GC-MS to analyze the VOCs components produced by the strains. Finally plate activity test was applied to detect the activities of the bacteria for nitrogen fixation,phosphorus solubilization and IAA production. The results showed that a strain of potential PGPR GX14001 was obtained. The VOCs produced by PGPR-GX14001 showed obvious growth-promoting effects on both Nicotiana benthamiana and Brassica chinensis L. seedlings. The strain GX14001 was identified as Microbacterium aurantiacum by 16S rRNA. Seven specific compounds were obtained by GC-MS analysis of VOCs. The GX14001 had strong dissolved organic/inorganic phosphorus activity and general nitrogen fixation activity,but weak IAA production capacity,which was 1.737 μg/mL. PGPR promoted the growth of plants through different growth-promoting mechanisms. The results showed that the VOCs produced by GX14001 had obvious growth-promoting effect on plants,but not high in the other aspects of growth-promoting activity;and there was no high positive correlation between them,indicating that the most important growth-promoting effect was via the produced VOCs.

Screening and Degradation Effect of Lignin-degrading Bacteria in Termite Nurseries

HAN Dong-jing, WANG Zhi-hua, ZHOU Ning, LIU Guo-qing, YANG Shao-hua, WANG Wen-jun

2022, 38(3):  113-120.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0626

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In order to select strains that can effectively degrade lignin,waste termite nests were used as a source for screening and re-screening was conducted with nitrogen-free medium,alkaline lignin medium,aniline blue medium and guaiacol medium. The screened strains were identified by 16S rRNA as well as morphological analysis. The alkaline lignin before and after degradation was observed in a characterized structure using scanning electron microscopy and Fourier infrared spectroscopy. The results showed that a strain of lignin-degrading bacteria was identified and named R. ornithinolytica RS-1. The lignin degradation rate reached a maximum of 24.21% in the first 3 d of fermentation with alkaline lignin as substrate,while the enzyme activities of 3 lignin-degrading enzymes（peroxidase,manganese peroxidase,and laccase）reached a maximum of 177.42,1466.67 and 6.94 U/L,respectively. Scanning electron microscopy and infrared spectroscopy revealed significant structural changes after treatment,indicating that the bacteria can be used as an effective strain for lignin degradation.

Gene Cloning and Enzymatic Properties of the Short Chain Dehydrogenase SDR-X1 from Pseudomonas citronellolis SJTE-3

FU Ya-li, PENG Wan-li, LIN Shuang-jun, DENG Zi-xin, LIANG Ru-bing

2022, 38(3):  121-129.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0471

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Pseudomonas citronellolis SJTE-3 can efficiently degrade 17β-estradiol as the sole carbon source,but the key enzyme catalyzing the transformation of estradiol remains unclear. The short chain dehydrogenase SDR-X1（WP_043267487.1）in strain SJTE-3 degrading estradiol was identified and its function was studied. First fluorescence quantitative PCR was used to detect the transcription levels of gene sdr-x1 under different carbon sources. Gene sdr-x1 was cloned and over-expressed in Escherichia coli BL21（DE3）strain,and the recombinant protein SDR-X1 was purified by affinity chromatography. The catalytic properties of protein SDR-X1 to estrogen were characterized and the conversion products of 17β-estradiol were identified by high performance liquid chromatography. The transcription of gene sdr-x1 was induced by 17β-estradiol. The results of multiple sequence alignment showed that protein SDR-X1 contained the conserved motifs and amino acid residues of short chain dehydrogenase. Using NAD⁺ as its co-factor,17β-estradiol was oxidized into estrone. Its Km value was（0.039 86±0.004 061）mmol/L and V_max value was（3.168±0.135）mmol/L/min/mg,and 61.75% of 17β-estradiol was converted within 15 min. The enzyme SDR-X1 had certain tolerance to temperature and its optimal reaction temperature was 50℃. The alkaline pH promoted the enzymatic reaction of SDR-X1. Different divalent metal ions had different effects on the enzymatic activity,and Mg²⁺ and Mn²⁺ enhanced the activity of the enzyme SDR-X1. The enzyme SDR-X1 in P. citronellolis SJTE-3 effectively catalyzed the transformation of 17β-estradiol and was involved in the estrogen-degrading process. Its characterization can promote the mechanism study of estrogen metabolism in bacteria.

Isolation and Identification of a Secretory Siderophore Fungus,and Its Biological Activity

ZOU Xue-feng, LI Ming-gang, BAO Ling-feng, CHEN Qi-bin, ZHAO Jiang-yuan, WANG Lin, PU Yong-yu, HAO Da-cheng, ZHANG Qing, YANG Pei-wen

2022, 38(3):  130-138.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0394

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A secretory siderophore producing fungus was isolated from the soil environment of Ailao Mountain primeval forest in Yunnan National Nature Reserve,and its effects on the physiological and biochemical functions of soil microorganisms infected with tobacco bacterial wilt were studied,which provides a theoretical basis for the development and utilization of the strain. Dilution coating method was used to isolate and purify the fungus producing the secretory siderophore from soil sample,the taxonomic status of the strain was identified by morphology and rDNA-ITS gene sequence. Chromazurol S（CAS）solution detection combined with full band ultraviolet absorption method was to determine the chemical structure type and activity of siderophore. DNS colorimetric method and Biolog-ECO analysis method were used to analyze the effects of strain secondary metabolites on the enzyme activity and microbial metabolism of tobacco bacterial wilt soil. The secretory siderophore-producing fungus was identified as Aspergillus welwitschiae based on morphological observations and sequence alignment analysis;the chemical structure type of the secretory siderophore of the strain was carboxylates,and its siderophore activity was 78.79%. The secondary metabolites of the strain significantly increased the activities of soil invertase and urease（P<0.05）by 13.7% and 14.46% respectively. The secondary metabolites of the strain also significantly improved the carbon source utilization capacity：Average color change rate（AWCD）and diversity index（Shannon index and richness index）increased by 86.15%,50.43% and 66.67% respectively. In conclusion,siderophore microbial resources are stored in primeval forest soil environment. The improvement of physiological and biochemical functions of the soil microbial community mediated by strain-secreted siderophore is an important technical means to optimize the management of the soil ecosystem.

Screening and Identification of Strains Producing 7-β-xylosyltaxanes Glycoside Hydrolases from the Periplaneta americana Gut

TANG Bin, LIU Wen-bin, LI Xiao-bo, WANG Ning, JIN Xiao-bao

2022, 38(3):  139-148.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0620

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The objective of this work is to screen endophytes that can produce 7-β-xylosyltaxanes glycoside hydrolases and to identify its biological characteristics. Taking total 178 strains of intestinal endophytes from Periplaneta americana preserved in the laboratory as the research object,the Congo red transparent circle method and fluorescent color method were used to screen the xylanase-producing strains,and the thin-layer chromatography was further used to screen strains producing the 7-β-xylosyltaxanes glycoside hydrolases. On the basis of this,a strain producing high 7-β-xylosyltaxanes glycoside hydrolases was selected for morphological,physiological,biochemical and molecular biological identification,and the conversion rate of 7-β-xylosyl-10-deacetylpaclitaxel by the strain was measured by high performance liquid chromatography. Among the 178 strains of intestinal endophytes in P. americana,total 55 endophytes produced xylanase,of which 8 strains of Streptomyces and 2 strains of Cupriavidus produced 7-β-xylosyltaxanes glycoside hydrolases. The high-yield strain WA11-2-9 was identified as Streptomyces enissocaesilis by bacterial morphology,physiological and biochemical experiments,combined with 16S rDNA sequence analysis. WA11-2-9 converted 7-β-xylosyl-10-deacetylpaclitaxel to 10-deacetylbaccatin III and 10-deacetylpaclitaxel. The conversion rate of the main product 10-deacetylpaclitaxel was 21.52%. In conclusion,the intestinal endophytes WA11-2-9（GenBank accession number：MZ411692）from P. americana secreted 7-β-xylosyltaxanes glycoside hydrolases,which lays a solid foundation for the subsequent biotransformation of paclitaxel.

Study on Antibacterial Stability of Musca domestica Cecropin-MDC Against Salmonella typhimurium

WANG Zi-yan, WANG Jian, ZHANG Lun, GUI Shui-qing, LU Xue-mei

2022, 38(3):  149-156.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0847

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This work aims to investigate the antibacterial stability of the housefly antimicrobial peptide（Musca domestica cecropin,MDC）against Salmonella typhimurium under different conditions. The Oxford cup method was used to determine the influences of pH,temperature,light,protease,metal ions,surfactants and organic reagents on the antibacterial activity（bacteriostatic ring size）of MDC against S. typhimurium. The results showed that the activity of MDC decreased slightly under acidic condition（remained 85% of that under the normal condition）,but not changed under alkaline condition with a good stability. Under low or high temperature treatment,no obvious difference was observed in its antibacterial activity. In addition,the activity of MDC still remained stable after exposed to darkness,normal light or ultraviolet for 30 min. MDC was sensitive to trypsin and its activity completely disappeared,but good stability to pepsin. MDC was still stable in the Ca²⁺ and K⁺,but it was sensitive to Mg²⁺. Interestingly,the antibacterial activity of MDC under Fe³⁺increased compared with the control group（P<0.05）. Moreover,the antimicrobial activities of MDC treated with surfactant and organic reagents significantly reduced but still partially preserved. To sum up,MDC can significantly inhibit the growth and reproduction of S. typhimurium,and its activity is almost not affected by the change of pH value/temperature/light and different pepsin and metal ions. Ferric ion can synergistically enhance the antibacterial activity of MDC,but Mg²⁺,surfactant,organic reagent and trypsin hydrolysis caused its antibacterial activity partially or completely lost,respectively.

Expression and Activity Identification of SARS-CoV-2 S1 Protein

WANG Qiao-ju, HU Yu-meng, WEN Ya-ya, SONG Li, MENG Chuang, PAN Zhi-ming, JIAO Xin-an

2022, 38(3):  157-163.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1076

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This work aims to obtain SARS-CoV-2 S1 protein with high immunoactivity,and to provide a good candidate antigen for the immune prevention and clinical diagnosis of COVID-19. PCR was adapted to amplify the S1 subunit of S protein,and the homologous recombination to clone the target sequence into the cold shock expression vector pCold I,then the recombinant plasmid was transformed into competent cells Escherichia coli BL21 and induced to express S1 protein. After purification by Ni ²⁺column affinity chromatography,SDS-PAGE and Western blotting were used to identify the expressions of the target protein. The results showed that the S1 protein containing His tag was expressed successfully,also,S1 protein with high concentration and purity was obtained,which reacted with S1 polyclonal antibody. In addition,the results of cell stimulation by protein demonstrated that S1 protein by prokaryotic expression presented promising immunogenicity,and it significantly increased the expression of cytokines and chemokines in mice macrophages,thus promoting the immune response of RAW264.7 cells. In sum,this study successfully constructed the recombinant plasmid pCold I-S1,and obtained S1 protein that could be a good candidate antigen for SARS-COV-2.

Construction of Vps28 Knock-out Mice and Model Study of the Impact on Lactation and Immune Traits

DING Ya-qun, DING Ning, XIE Shen-min, HUANG Meng-na, ZHANG Yu, ZHANG Qin, JIANG Li

2022, 38(3):  164-172.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0446

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Although many candidate genes for milk production traits have been mined based on QTL mapping and association analysis,only a few genes have been verified in vivo and in vitro. The results of our previous genome-wide association study（GWAS）showed that Vps28 was significantly associated with milk production traits in dairy cows. In addition,Vps28 was highly expressed in the mammary gland tissue of dairy cows. To further explore the role and function of Vps28 on milk production traits,CRISPR/Cas9 technology was used to construct Vps28 knockout mice,and its functions in vivo were studied. The results showed that Vps28 knockout homozygous mouse（Vps28-/-）were very likely to cause early embryonic lethality. Thus only heterozygous knock-out mice（Vps28+/-）were obtained in this study. Our results showed that the mRNA and protein expression in the mammary gland and spleen of Vps28 knockout mice（Vps28+/-）significantly decreased if compared with wild-type mice. Furthermore,Vps28 knockout mice had nipple dysplasia and reduced milk production. Also the white blood cells count,neutrophilic granulocyte count,lymphocyte count and intermediate cell count in the Vps28 knock-out mice were significantly lower than those in wild-type mice. These results suggest that Vps28 not only affects lactation,but also related immune traits in mice.

Construction of Homozygous Plin1-knockout Mouse Model and Phenotype Analysis Based on CRISPR/Cas9 Technology

YAN Jiong, FENG Chen-yi, GAO Xue-kun, XU Xiang, YANG Jia-min, CHEN Zhao-yang

2022, 38(3):  173-180.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0647

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The objectives are to construct a homozygous Plin1-knockout mouse model by CRISPR/Cas9 technology and to preliminarily analyze their phenotypic changes. A pair of sgRNA based on the sequence before and after exon 2 of Plin1 gene was designed and its corresponding plasmid expression vectors were constructed. The sgRNA,obtained by in vitro transcription of the expression vector,mixed with Cas9 protein was delivered to the mouse fertilized egg by microinjection technology,and then the embryo transfer of the fallopian tube was conducted. F₀ generation positive mice were obtained by gene sequencing and PCR genotype identification and screening after the first generation of mice was born. Then F₀ generation mice were crossed with wild-type mice to obtain F₁ generation heterozygous mice. The F₁ generation of heterozygous mice was inbred between male and female,and the F₂ generation homozygous mouse model was obtained. Routinely feeding homozygous Plin1-knockout mice,heterozygous and wild-type mice in the same litter,their physical parameters such as body weight and length of the mice were measured. Quantitative Real-time-Polymerase Chain Reaction and Western blot were to detect the expressions of Plin1 at mRNA and protein levels in each tissue respectively. As results,741bp（including exon 2）fragment of Plin1 gene in the mice was knocked out. Compared with wild-type mice,the PLIN1 mRNA and protein expressions of the Plin1-knockout mice significantly reduced（P <0.05）. After 4 weeks of routine feeding,compared with wild-type and heterozygous mice,the weight of homozygous Plin1-knockout mice significantly reduced（P < 0.05）. In conclusion,a mouse model of Plin1-knockout mice is successfully constructed,and preliminary phenotypic analysis found that Plin1-knockout mice were thinner than wild-type ones.

Improvement Effect of Bifidobacterium lactis V9 on NAFLD Rats Induced by High-fat Diet

ZHONG Ming-yue, LIU Chun-yan, YAN Yan, ZHANG Xiao-hui, YUAN Hai-sheng, XU Guo-quan, ZHANG He-ping, WANG Yu-zhen

2022, 38(3):  181-187.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0630

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This work aims to study the effect of probiotic Bifidobacterium lactis V9（V9）on lipid metabolism and intestinal injury in non-alcoholic fatty liver disease（NAFLD）rats induced by high-fat diet,and to explore its mechanism. Male Wistar rats were randomly divided into 5 groups（n=8）：blank control group（Control）,high-fat diet model group（HFD）,berberine-positive drug group（Berberine）,the probiotic B. lactis V9 treatment group（HFD/V9）,and the probiotic B. lactis V9 control group（V9）. Except Control and V9 were given ordinary maintenance feed,others were given a high-fat diet for 6 weeks to construct a NAFLD model. After 6 weeks,the high-fat diet was replaced with ordinary maintenance feed,and the rats were given probiotic B. lactis V9（1×10 ⁹CFU/mL）or berberine（50 mg/kg）by gavage. After 4 weeks,blood,liver and colon tissues were collected for follow-up testing. Compared with the Control group,the mRNA expressions of liver non-esterified fatty acids（NEFA）,triglycerides（TG）,liver fatty acid synthase（FAS）and liver fatty acid binding protein 1（Fabp1）in the HFD group significantly increased（P<0.01）. At the same time,mRNA expressions of the intestinal cytokine interleukin 1β（IL-1β）,tumor necrosis factor α（TNF-α）,Toll-like receptor 2（TLR2）and Toll-like receptor 9（TLR9）in the HFD model group also significantly increased（P<0.01）. On the contrary,the expression of tight junction protein ZO-1 and occludin mRNA in the HFD model group significantly decreased（P<0.01）. After treatment with probiotic B. lactis V9,the contents of NEFA and TG in the liver significantly reduced（P<0.01）,the mRNA expressions of FAS and Fabp1 in the liver also significantly reduced（P<0.01）. Both probiotic B. lactis V9 and berberine treatment reduced the transcription levels of intestinal IL-1β,TNF-α,TLR2 and TLR9（P<0.01）,which increased the expressions of intestinal tight junction proteins ZO-1 and occludin mRNA. HE staining showed that there was no significant difference in colonic pathology among the above groups. Probiotic Probiotics B. lactis V9 can improve NAFLD induced by high-fat diet by alleviating lipid metabolism disorders,reducing intestinal inflammation and improving mulosal barrier.

Inhibition of AAV-mediated RNAi to SARS-CoV-2 S Gene Expression

LIU Xiao-mei, WANG Dong-xin, ZHANG Chun, WEI Shuang-shi

2022, 38(3):  188-193.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0596

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The purpose of this study is to investigate the inhibitory effect of AAV-delivered shRNA on the expression of SARS-CoV-2 S gene in vitro and in vivo,and to provide reference for further research on the inhibition of SARS-CoV-2 infection. Seven shRNAs targeting SARS-CoV-2 S were designed. The expression vectors of shRNA and S gene were co-transfected into cultured cells,and the inhibition effect on S gene expression was analyzed by qRT-PCR and Western blot,and shRNA1 presented the highest inhibition. The rAAV vector of expressing shRNA1 was constructed. When rAAV-shRNA1 and S gene expression plasmid were co-transfected into 293T cells,the inhibition rate of S gene expression was 65.6%. Mice were injected nasally or intramuscularly with rAAV-shRNA1 and S gene expression plasmid,and the inhibition rate of the S gene expression was 36.8% and 90.3%,respectively.

Expression and Purification of the MLL3_SET Protein with a Site-directed Mutation of an Unnatural Amino Acid

WANG Xiao-qin, HUANG Yin-ping, WANG Wei-qian, WU Ping, QUAN Shu

2022, 38(3):  194-202.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0465

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This work aims to introduce the unnatural amino acid,N-propargyl-lysine（PrK）,into the catalytic domain（MLL3_SET）of histone H3K4 methyltransferase MLL3,to express and purify the mutant protein（MLL3_SET*）,and evaluate its enzyme activity,which will lay the foundation for further single-molecule fluorescence resonance energy transfer（smFRET）experiments to characterize the mechanism of MLL3. MLL3_SET was linked into pET-28b（+）to construct an expression vector. Residue N4905 was selected for PrK introduction upon MLL3_SET crystal structure analysis. The commercial E.coli strain（C321. ΔA. exp）was genetically modified to integrate the T7 RNA polymerase gene. Further,MLL3_SET* was expressed and purified in the modified strain,and finally the enzyme activity was determined. The results showed that the constructed C321. ΔA. exp lacZ∷T7p07 strain harboring the pET28b-MLL3_SET* plasmid normally expressed the target protein after co-transformation with the aaRS-tRNA orthogonal system and the addition of PrK. The MLL3_SET* was purified successfully by Ni-NTA affinity chromatography and gel filtration chromatography. The results of the multi-component enzyme-coupled reaction showed that although the enzyme activity of MLL3_SET* was lower than that of wild-type MLL3_SET,it retained about 43% of the wild-type enzyme activity. This study successfully achieves prokaryotic expression and purification of PrK labeled MLL3_SET protein retaining certain enzymatic activity,which lays a foundation for the subsequent in-depth study of the molecular mechanism of MLL3_SET.

A Review of WRKY Mediated Regulation of Sugar for Cold Acclimation in Horticultural Crops

PAN Ying-jie, ZHANG Ying, WU Qi-man, LI Zheng-qing

2022, 38(3):  203-212.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0691

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Sugar in plant can not only promote the cellular carbon metabolism and energy metabolism,also act as a signal molecule to promote plant growth and participate in the regulation of plant response to adversity stress. Currently,a number of studies reveal that the accumulation of soluble sugar is conducive to protecting plants from freezing injury in cold hardening,it has been shown that the cold resistance of horticultural crops such as cucumber has been improved under exogenous sugar application,however,the specific mechanisms are not clear yet. Sugar can promote plants’ responses to abiotic stress through different regulatory mechanisms,such as physiology and biochemistry,and different molecular regulatory mechanisms. WRKY,as a core transcription factor in the ABA-related adversity response signaling pathway,is also an important factor in the sugar metabolism regulation mechanisms,and may play an important role in the related metabolic mechanisms in response to cold stress. Here the response and regulatory mechanisms of cold acclimation in horticultural crops by sugar are reviewed,and the role of WRKY family in the process is analyzed and discussed.

Research Progress in the Mitigative Effects of Rhizosphere Microorganisms on Heavy Metal Stress in Plants and Their Mechanisms

YANG Lu, XIN Jian-pan, TIAN Ru-nan

2022, 38(3):  213-225.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0811

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Phytoremediation technology has become an important means in green remediation of heavy metal pollution. Under heavy metal stress,rhizosphere microorganisms can effectively improve plant growth and physiological metabolism,and enhance the absorption and enrichment of heavy metals in plants. This paper first discusses the rhizosphere microbial composition and root-microbe interaction,and analyses the mitigative effects of plant rhizosphere growth-promoting bacteria and mycorrhizal fungi on plant resistance to heavy metal stress. Meanwhile,the article also discusses the mechanism of the rhizosphere microbes alleviating heavy metal toxicity from the aspects of microorganism,plant root exudates,plant ethylene synthesis,plant photosynthesis,plant antioxidant defense system,water and nutrient absorption and rhizospheric soil microenvironment. Finally,this paper combines the current research status and draws some conclusions. Such as,finding new strains of beneficial to plant growth under heavy metal stress,exploring the interaction between different microorganisms in the rhizosphere under heavy metal stress,and revealing the physiological and molecular mechanisms of plant root-microbial interaction system under heavy metal stress,aiming to provide guidance for the theoretical research and practice of microbial assisted phytoremediation of heavy metal pollution in the future.

Application of Single-cell Transcriptome Sequencing in Animals

XIONG He-li, SHA Qian, LIU Shao-na, XIANG De-cai, ZHANG Bin, ZHAO Zhi-yong

2022, 38(3):  226-233.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0523

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Cell is the basic structural component and functional unit of organism. Cell type and function are determined by its whole transcriptional expression profile. Single-cell transcriptome sequencing can be used to obtain the transcriptional expression profile of a single cell,so as to identify cell types,cell states and rare types with high-precision resolution,thus to analyze the dynamic changes of cells and the relationship between cells at the single cell level,and further to decipher the molecular cellular mechanisms behind cell changes and cell abnormalities. With the stability and throughout improvement of single-cell sequencing,as well as the reduction of sequencing cost,single-cell transcriptome sequencing has been widely used in the fields of developmental biology,tumor,immunity and disease. However,these researches are mainly focused on human and model organisms,there are a few researches on animals. Therefore,the objective of this paper is to introduce the single-cell transcriptome sequencing and its biological application,and review some of its pioneering research in animals,aiming to provide a method reference for better application of single-cell transcriptome sequencing in animals.

Regulation Mechanism of Ribonucleases in Escherichia coli

SUN Man-luan, GE Sai, BU Jia, ZHU Zhuang-yan

2022, 38(3):  234-245.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0341

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Ribonucleases（RNases）involve in almost every aspect of RNA metabolism,and play important regulating role in the physiological functions of bacteria. Bacteria have evolved multiple strategies to regulate RNases to avoid unnecessary RNA degradation. To date,the regulation mechanisms include post-transcriptional regulation,post-translational modification,cellular localization and related trans-acting inhibitors. Here,we systematically describe and explain the classification,function and regulation mechanism of the RNases in E. coli,and summarize the response mechanism of E. coli adaptive regulation to its RNases under different environmental pressures as well as existing issues.

Application of Cyclodextrin Glucosyltransferase in the Glycosylation Modification of Natural Products

ZHANG Guo-ning, FENG Jing-xian, YANG Ying-bo, CHEN Wan-sheng, XIAO Ying

2022, 38(3):  246-255.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0642

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Cyclodextrin glucosyltransferase（CGTase）is a kind of α-amylase that can catalyze the cleavage and cyclization of α-1, 4 bonds in starch or polysaccharides to form cyclodextrins（CDs）. CGTase is mainly used in industry to produce cyclodextrins. In recent years,There has been remarkable progress in the use of the transglycosylation effect of CGTase to modify the properties of natural products,which is becoming a promising development direction. This review introduces the source,protein structure and catalytic mechanism of CGTase,and focuses on its application in glycosylation modification of natural products in recent years,aiming to provide references for the in-depth research and application of CGTase in this field.

Microbial Enrichment on Leaf Surface and DNA Extraction Method Based on the Metagenomics Sequencing

ZHANG Yu-han, FAN Yi, LI Ting-ting, PANG Shuang, LIU Wei, BAI Ke-yu, ZHANG Xi-mei

2022, 38(3):  256-263.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0550

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Obtaining high-quality total genomic DNA of microbial community is the basis and difficulty in the study of metagenomics of complex microbial communities. The plant leaf surface is a complex ecosystem with abundant microbial diversity,and the microbial community mediates leaf functional traits and affects plant adaptability. An in-depth understanding of the basic structure and functional principle of the microbial community on leaf surface will contribute to the applications of promoting plant growth and plant protection. Due to the harsh living environment of leaf surface,it is difficult to enrich microorganisms on leaf surface,which severely limits the extraction of high-quality genomic DNA of microorganisms on leaf surface. Based on the existing DNA extraction method,the surfactant Silwet L-77 was added for pretreatment,and the eluent was recycled,which increased the enrichment of microorganisms on leaf surface. The genomic DNA with high purity and concentration was extracted in combination with commercial kit methods. After quality control and library building sequencing as verification,DNA quality met the requirements for metagenomic library construction. This method may improve the enrichment and collection efficiency of microorganisms on leaf surface,increase the success rate of DNA extraction of microorganisms on leaf surface,and provide a reference for applying high-throughput sequencing technology to study the composition of microorganisms on surface and molecular biology research of plants.

Development of Fluorescence Quantitative Lyophilized Detection Kit Based on Escherichia coli O157∶H7

SU Yuan, ZHU Long-jiao, CAO Ji-juan, LIU Jian-long, XU Wen-tao

2022, 38(3):  264-275.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0768

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Escherichia coli O157∶H7,as a food-borne pathogen,is widely distributed and causes great harms. To develop a kit based on E. coli O157∶H7 that can not only achieve rapid,simple and sensitive detection,but also achieve room temperature storage and transportation,this study utilized real-time fluorescence quantitative polymerase chain reaction（qPCR）,combinig with vacuum lyophilizing technology,and developed an E. coli O157∶H7 fluorescence quantitative lyophilizing detection kit. This kit retains nucleic acid detection performance,and is easy to store and transport at room temperature,and reduces aerosol pollution. The developed kit adopts lyophilizing technology to retain the detection performance of nucleic acid amplification reagents. After rehydration,the accumulation of fluorescent signals is monitored in real time through cyclic amplification under the action of Taq DNA polymerase to achieve quantitative fluorescence detection. By the proposed method 2.1 copies/μL of eaeA gene can be detected within 40 min. This technology provides a fine research foundation and technical reference for the detection of E. coli O157∶H7,and fills the lack of detection kits with high sensitivity and easy storage in the market.

Tetracycline Bivalent Aptamer Non-enzyme Label-free Sensor

LAN Xin-yue, LIU Ning-ning, ZHU Long-jiao, CHEN Xu, CHU Hua-shuo, LI Xiang-yang, DUAN Nuo, XU Wen-tao

2022, 38(3):  276-284.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1138

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As a general antibiotic,tetracycline（TC）residue in milk and dairy products is often caused by improper use or abuse during the livestock breeding and processing,which poses a serious threat to human health. Chromatography,enzyme-linked immunosorbent assay and other traditional detection methods are highly dependent on the instrument,as well as the procedure is complicated,the analysis time is prolonged,cannot well meet the requirements of rapid detection on site. Therefore,a non-enzyme-labeled sensor based on bivalent aptamer was established for TC detection,which responds the detection results with the change of thioflavin T（ThT）fluorescence signal. The logarithmic value in the TC concentration range of 10 nmol/L-1 μmol/L showed a good linear relationship,R ² was over 0.99,and the detection limit was as low as 96 pmol/L. At the same time,it also allowed rapid and specificity detection of TC in a milk samples within 20 minutes.