Table of Content

26 January 2020, Volume 36 Issue 1

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Cloning and Expression Profile Analysis of NaERF1 Under Abiotic Stresses in Nicotiana alata

LU Lin, ZHAO Xi-sheng, LIU Gui-yun, LIU Wei-dong, WANG Yan-ting, WU Ling-li, REN Xue-liang, LU Li-ming

2020, 36(1):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0550

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AP2/ERF transcription factors,unique in plants and the largest family of transcription factors in plants,play an important role in plant growth and development. Exploring the physiological function of ERF transcription factors in Nicotiana alata provides useful references for the study of molecular mechanism of N. alata in resistance to abiotic stresses. Homologous cloning method was used for gene cloning,and qPCR was conducted to analyze the expression profile of NaERF1 under abiotic stress. NaERF1 belonging to ERF family was successfully cloned with ORF 819 bp,encoding 272 amino acids. Bioinformatics analysis showed that the protein encoded by NaERF1 had a molecular weight of 30.7 kD and an isoelectric point of 6.07. It had a typical conservative domain of AP2/ERF transcription factor family. NaERF1 was mainly located in the cytoplasm and contained multiple phosphorylation sites. The results of homology analysis showed that NaERF1 gene had high homology with ERF gene of Solanaceae plants,and had the closest relationship with ERF of common tobacco（Nicotiana tabacum）. The expression of NaERF1 was tissue-specific,with the highest expression in flowers,the second in stems,and the lower expression in roots and leaves. Meanwhile,the expression of NaERF1presented 5 patterns under abiotic stresses such as high salinity,drought,low temperature,ABA,low potassium and H₂O₂. Among them,the response to low potassium and ABA stress was strong. The results of this study suggest that NaERF1 gene belongs to AP2/ERF transcription factor family and may be widely involved in abiotic stress response in N. alata.

Cloning and Expression Analysis of SRPP2 Gene in Taraxacum kok-saghyz Rodin

ZHANG Rui-zhu, JIANG Yu-chen, HUANG Jun, YAN Jie

2020, 36(1):  9-14.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0684

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TkSRPP2 is a member of SRPP family in Taraxacum kok-saghyz Rodin,and plays an important role in the biosynthetics of natural rubber and stability of rubber particles. PCR was applied to clone the full length of TkSRPP2 gene from T. kok-saghyz Rodin,then the bioinformatics analysis of TkSRPP2 gene,subcellular localization and quantitative expression analysis of tobacco were conducted. The results demonstrated that the full length of TkSRPP2 gene was 633 bp,encoding 210 amino acids,the relative molecular weight of the protein was 23.15 kD,and the theoretical isoelectric point（pI）was 8.51. The phylogenetic analysis demonstrated that it was most closely related to SRPP3.The subcellular localization of tobacco revealed that TkSRPP2 protein was in cytoplasm. TkSRPP2 gene was expressed in many tissues of T. kok-saghyz Rodin,with the highest expression in 6 months roots.

Functional Analysis of Secreted Protein MoCDIE2 Inducing Plant Cell-Death in Magnaporthe oryzae

WU Hang, LI Zhi-qiang, LIU Wen-de

2020, 36(1):  15-22.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0606

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Rice blast,caused by the fungus Magnaporthe oryzae,is one of the most destructive plant diseases around the world. M. oryzae secretes many effector proteins into the interface or plant cells to suppress host immune defense response,thus helping the fungus to infect plant successfully. Using agrobacterium-mediated transient expression strategy,we obtained one putative effector protein MoCDIE2（Cell Death-Inducing Effector）that induces cell death in non-host plant Nicotiana benthamiana. Sequence alignment result indicated that MoCDIE2 gene encoded a ricin B lectin protein. Phylogenetic tree demonstrated that MoCDIE2 protein was conserved in many filamentous fungi. We used gene knock out method to generate MoCDIE2 knock out mutant,and the result showed that there were no significant differences between wild type strain Guy11 and mutants of MoCDIE2 knockout in mycelia growth and pathogenicity.

Improvement of Thermal Stability of Ganoderma lucidum Protein LZ-8 by Site-directed Mutation of Amino Acids

SUN Xi-lin, JIANG Zhen-yan, LIU Zhi-yi, DAI Lu, SUN Fei, HUANG Wei

2020, 36(1):  23-28.  doi:10.13560/J.cnki.biotech.bull.1985.2019-0529

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This work is to improve the thermal stability of Ganoderma lucidum immunoregulatory protein LZ-8 by site-directed mutagenesis of amino acids. Molecular dynamics simulation combined with temperature factor prediction were used to reasonably design LZ-8 amino acid mutation site,LZ-8 mutant protein was constructed and expressed in Pichia pastoris X33 strain,the biological activity and thermodynamic parameters of LZ-8 before and after mutation were detected and compared by HeLa cell growth inhibition experiment and differential scanning calorimetric（DSC）study. Results showed that LZ-8 N-terminal alpha helix was the temperature sensitive region predicted by theory. F8W and R9K double site mutations occurred in this region. After mutation,the thermal stability of LZ-8 was improved,the phase transition temperature Tm increased by 0.92℃,and the phase transition enthalpy ΔH increased by 23.14 kJ/mol. However,the biological activity of LZ-8 after mutation was basically unchanged. The IC₅₀ of LZ-8 and LZ-8 mutant on HeLa cell growth inhibition was 2.238 μg/mL and 2.407 μg/mL,respectively. Ganoderma lucidum LZ-8 mutant with improved stability but unchanged biological activity is obtained by rational design of amino acid mutation sites.

Cloning and Expression Analysis of Different-length Fragments of Oidium heveae（HO-73）Promoter WY172

YIN Jin-yao, WANG Yi, XU Liang-xiang, ZHU Li, WANG Chen, LIU Wen-bo, MIAO Wei-guo

2020, 36(1):  29-36.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0728

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The aim of this study is to clone 4 different-length deletion fragments of Oidium heveae promoter WY172 and its upstream 2K sequence in order to analyze the expression activity of each fragment of the promoter. Based on the prior research in our laboratory,the 2K sequence upstream of WY172 was used as the research object to conduct the gradual deletion mutation,and 4 fragments in different lengths which might have promoter activity were obtained. In combination with WY172,the pBI121 vector was used as the backbone,and the CaMV35S promoter before the GUS gene was replaced,then the recombinant expression vector was constructed respectively,and the Agrobacterium was transformed by ATMT method. The enzyme activities of the WY172 promoter and fragments of different lengths were analyzed by GUS staining and enzyme activity assay. Five recombinant plant expression vectors including pBI121-WY172,pBI121-WY172Q,pBI121-WY172Q1,pBI121-WY172Q2 and pBI121-WY172Q3 were constructed. GUS gene was expressed in all of the recombinant plant expression vectors,and its expression（blue occurred）was stronger than the positive control CaMV35S promoter. Moreover,the transient expression of pBI121-WY172Q3 recombinant vector was the most intense. The results of GUS enzyme activity assay showed that all deletion mutant fragments presented promoter activity regulating gene expression,and the activation activity was stronger than that of CaMV35S promoter. Among them,the GUS enzyme activity regulated by WY172Q3 was the highest. Therefore,we infer that WY172 and its four 2K-length deletion fragments on the upstream 2K sequence have promoter activity,and WY172Q3 promoter fragment has the strongest expression activity.

Isolation and Identification of a Root Rot Pathogen of Rehmannia glutinosa and Its Characterization

WANG Ya-li, KANG Chun-xiao, YANG Chuan-zhen, WEI Yi-xuan, WANG Rui-fei, LI Ming-jun, YANG Qing-xiang

2020, 36(1):  37-44.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0771

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We aimed to isolate and identify the pathogens resulting in Rehmannia glutinosa root rot as well as study its pathogenicity and biological characteristics. In this study,streak plate method was used to isolate and purify the pathogens of rot root in R. glutinosa. ITS sequencing and genetic evolution analysis were used to identify the isolated strains. Inoculation experiment was used to confirm the pathogenic of the isolated potential pathogens. The growth test was performed to analyze physiological characteristics of the isolated stain. One of the obtained strains presented the colony morphology of orange color,the neat edge,the smooth and moist surface,and microscopic morphology with oval shape,suspected to be a fungus with buds. The strain was clustered together with the Rhodotorula paludigena at confidence of 91 and they displayed the close genetic relation. R. glutinosa roots inoculated with the strain and the incidence was 100% at day 20. The optimal growth temperature and pH of the strain was 28℃ and 5.0,respectively. The strain grew well in the medium with 0.5 g/L ferulic acid,vanillic acid and p-hydroxybenzoic acid,i.e.,the secretion in the root of R. glutinosa that was closely related to the root rot of R. glutinosa. It is preliminarily verified that R. paludigena DH5 may cause the root rot of R. glutinosa and well adapts to the rhizosphere environment containing a large content of phenolic compounds.

Medium Optimization for the Laccase Production by White Rot Fungus Porodaedalea laricis and Its Dye Decolorizing Capacity

WU Yi, MA Hong-fei, CAO Yong-jia, SI Jing, CUI Bao-kai

2020, 36(1):  45-59.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0974

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In this study,Plackett-Burman design,path of the steepest ascent,and Box-Behnken design were used to optimize the liquid medium composition for laccase production of white rot fungus Porodaedalea laricis with the laccase activity set as a response. The optimal compositions for production of P. laricis laccase was obtained as follows：peeled potato 365.61 g/L,peptone 5.0 g/L,glucose 20.0 g/L,KH₂PO₄ 1.0 g/L,MgSO₄·7H₂O 0.5 g/L,MnSO₄·H₂O 0.15 g/L,CaCl₂·2H₂O 0.03 g/L,ammonium tartrate 6.68 g/L,sodium succinate 1.5 g/L,Tween 80 0.48 mL/L,corncob 46.43 g/L,and vitamin B1 0.01 g/L. Under the optimized conditions,the laccase activity was 3.29 U/mL,which was 2.81-fold of that given in the basal medium,and this yield was very in good agreement with the theoretical result of 3.32 U/mL,indicating the model was accurate and reliable. Moreover,the laccase exerted notably decolorizing capacity for many synthetic dyes including Reactive Brilliant Blue X-BR,Remazol Brilliant Blue R,Acid Black 172,Congo Red,Methylene Blue,Neutral Red,Indigo Blue,Naphthol Green B,and Crystal Violet,with the respective decolorization rate of 95.64%,97.21%,36.11%,91.63%,61.42%,74.65%,48.60%,25.13%,and 68.80% after 168 h.

Isolation and Characterization of Heterotrophic Nitrification-Aerboic Denitrification Diutina rugosa

DU Quan-neng, ZHU Wen-juan, LAN Shi-le

2020, 36(1):  60-65.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0685

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In order to obtain a heterotrophic nitrification-aerobic denitrification strain,a yeast with heterotrophic nitrification-aerobic denitrification ability was isolated and screened from the sludge of aquaculture pond,named as DW-1. The strain was identified as Diutina rugosa by morphological observation and 26S rDNA sequence analysis. The effects of carbon sources,C/N,initial pH value,cultural temperature and shaking speed on the nitrogen removal performance of strain DW-1 were preliminarily discussed with ammonia nitrogen as the sole nitrogen source. The results showed that the degradation rate of ammonia nitrogen and removal rate of total nitrogen by strain DW-1 were 94.94% and 48.69% respectively,under the conditions of sodium acetate as the sole carbon source,C/N 25,pH 6.0,appropriate culture temperature 32℃ and rotate speed 170 r/min;while the accumulation of nitrite nitrogen in the whole process was only 0.067 mg/L. The heterotrophic nitrification-aerobic denitrification performance of D. rugosa DW-1 indicates that it has a promising application prospect in the treatment of nitrogen-containing wastewater.

Effects of Modification of Alanine Aminotransferase on Synthesis of L-tryptophan in Escherichia coli

MENG Shuai-shuai, HUANG Qin-geng, WU Song-gang, LIU Feng

2020, 36(1):  66-72.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0864

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This work is to explore the effects of transaminase for L-alanine synthesis in Escherichia coli on the metabolism and L-tryptophan synthesis of the strain. Three genes（alaA,alaC and avtA）encoding L-alanine aminotransferase were knocked out by Red recombination technique. Fermentation experiments of shaking flask and 50 L fermenter were used to investigate the accumulation of L-tryptophan,L-alanine metabolism and cell growth. Results showed that the growth of the cells and L-tryptophan synthesis were strongly inhibited in these 3 genes deficient engineering strain E. coli FS-T0ΔalaAΔalaCΔavtA;however,the influences on the growth of single or both L-alanine aminotransferase-deficient strains were very little,but L-tryptophan synthesis varied significantly. By E. coli FS-T4ΔalaAΔalaC engineering strain,the yield of L-tryptophan was 6.08 g/L while the L-alanine production was only 0.16 g/L,which increased by 26.7% and decreased by 91.0%,respectively compared with the original strain. In the 50-L fermenter,the yield of L-tryptophan further increased to 41.9 g/L,and glucose conversion rate was 20.5%,which was 13.8% and 5.1% higher than the original strain,respectively. Deficiency of both AlaA and AlaC transaminase will not affect the needs of the whole cell amino acid pool,and is conducive to reduce the accumulation of miscellaneous acids,thus leading more carbon sources into L-tryptophan synthesis metabolic flux.

Enantioconvergent Hydrolysis of m-Nitrostyrene Oxide by Phaseolus vulgaris Epoxide Hydrolase

LI Chuang, WEN Zheng, LIU Chang, WU Min-chen

2020, 36(1):  73-80.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0624

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This work aims to provide a superior biocatalyst for producing optically pure（R）-m-nitrophenyl-1,2-ethanediol（mNPED）via studying the catalytic properties of PvEH2,a Phaseolus vulgaris epoxide hydrolase,in the enantioconvergent hydrolysis of racemic（rac-）m-nitrostyrene oxide（mNSO）. The engineered Escherichia coli strain expressing the recombinant PvEH2,designated E. coli/pveh2,was constructed. Using rac-mNSO as substrate,the EH activity and regioselectivity of E. coli/pveh2 were assayed by chiral chromatography. Then,according to the effects of several organic solvents and their dosages on enzymatic activity,the buffer/co-solvent reaction system was screened and optimized. Results showed that the transcription level of PvEH2 gene in P. vulgaris might be affected by the induction factors in vitro. The EH activity of E. coli/pveh2 was 15.4 U/g _{dry cell},and its regioselectivity coefficients,α_S and β_R,were 90.3% and 96.4%,respectively. In the optimized phosphate buffer containing 5%（V/V）glycerol,the maximum allowable substrate concentration（40 mmol/L）was 4 times higher than that in sole buffer.（R）-mNPED with 84.9% ee_p and 90.2% yield was obtained at 12 h reaction. E. coli/pveh2 or PvEH2 demonstrates an excellent regioselectivity in the hydrolysis of rac-mNSO,thus making it a promising biocatalyst to prepare optically pure（R）-mNPED.

Heterologous Expression and Application of Phosphatidylinositol-specific Phospholipase C

LIN Mei-xuan, ZHOU Xiao-man, GUAN Feng, CUI Wen-jing

2020, 36(1):  81-87.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0630

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This work aims to optimize the fermentation conditions and purify the phosphatidylinositol-specific phospholipase C（PI-PLC）exogenously expressed in Escherichia coli,and also to evaluate the enzymolysis effect of PI-PLC on glycosylphosphatidylinositol-anchored protein（GPI-AP）. In this study,we synthesized the codon-optimized gene sequence of PI-PLC from Bacillus cereus in the NCBI database,according to the codon preference of E. coli,and inserted the sequence into plasmid pGEX-6P-1. The recombinant plasmid pGEX-6P-1-PI-PLC was transformed into E. coli BL21（DE3）,and expression of PI-PLC was induced by isopropylthio-β-D-galactoside（IPTG）. SDS-PAGE analysis revealed that the GST-tagged PI-PLC fusion protein of was presented as a soluble protein in the supernatant of cell lysate,its molecule weight was about 61 kD,which was consistent with the predicted. The optimized induction conditions for PI-PLC expression were obtained as follows：5% of inoculum size,induced 24 h with 0.3 mmol/L IPTG at 16℃ when the OD_600nm reached 0.5. The protein was further purified with GST purification kit,the concentration of purified PI-PLC was 0.52 mg/mL,and the specific enzyme activity was 1322.5 U/mg. The PI-PLC enzymatic solution significantly digested the model GPI-anchored protein CD59 on the surface of mammal cells. Therefore,PI-PLC obtained in this study can be used to study and identify the GPI-Aps in cytobiology in the future.

Construction of Fsp27 Gene Silencing Vector and Its Effect on Cell Lipolysis

XU Xiang, DONG Wei-peng, ZHANG Shao-hua, FENG Chen-yi, LIU Tian-fu, YAN Jiong

2020, 36(1):  88-94.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0369

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This work is aimed to construct the fat-specific protein of 27（Fsp27）gene silencing vector and to investigate the effect of Fsp27 gene expression on the lipolysis of 3T3-L1 cells as well as explore its mechanism. RNAi technology was used to construct the eukaryotic interference vector of Fsp27 gene to down-regulate the expression of the Fsp27 gene. The “cocktail” method induced differentiation of 3T3-L1 preadipocytes into mature adipocytes. Liposomes were transfected into adipocytes,oil red O stained lipid droplets,and enzymatic method was employed to determine the content of glycerol and triglyceride in cells. Western blot analysis was used to detect the protein expression of Fsp27,HSL,ATGL and PPARγ in cells. The results from Western blot showed that the positive sh-Fsp27 vector down-regulated the expression of Fsp27,while the expression of ATGL and PPARγ increased（P<0.05）,and the silencing results by sh-Fsp27-2 was the best. The results of enzymology demonstrated that the content of triglyceride in the positive sh-Fsp27 interference group decreased,and the content of glycerol increased（P<0.05）. The results of oil red O staining showed that there were large lipid droplets accumulated in the blank control group and the negative control group,while the distribution of small lipid droplets in the positive sh-Fsp27 group was major,and no obvious big fat drops was observed. In conclusion,the silencing effect by sh-Fsp27-2 is the best,Fsp27 gene silencing may accelerate the lipolysis rate of 3T3-L1 cells,which is mainly achieved by inhibiting the fusion among small lipid droplets and increasing the ATGL-mediated hydrolysis .

Selection of Winter Rape Varieties Suitable for Cold and Dry Areas in Hebei Province

LI Ai-guo, LI Ji-ming, LI He-ping, LIU Gui-hua, SONG Cong-min, WU Jun-yan

2020, 36(1):  95-100.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0890

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Screening rape varieties suitable for planting in Hebei low plain area may allow ecological and economic benefits simultaneously achieved,which may provide theoretical basis for the selection of rape varieties and the adjustment of planting structure in similar climate areas. Through the analysis of variance,correlation and cluster,the variations in agronomic traits of 16 rape varieties were analyzed. There were the most obvious differences in branching parts among varieties,with the variation coefficient of 61.82%,the lowest branching parts for variety JR5（1.2 cm）,and the highest branches for variety 14T×38（15.9 cm）.The second significant difference was in yield and secondary branching number,with variation coefficients of 26.69% and 24.89%,respectively. The yield of variety 14T×38 was the highest,and that of the 20SY-13 was the lowest. The varieties with the largest and smallest number of secondary branches were 20SY-13 and Tianyou 142,respectively. The correlation between rapeseed yield and a number of primary branches and the main inflorescence pod number were very significantly positive（P<0.01）,plant height was significantly positive correlated with branch height,length of main inflorescence and the number of main inflorescence pod,and a number of primary branches and the main inflorescence pod number was significantly positive correlated（P<0.05）,while secondary branch number was very significantly negative correlation with the main inflorescence pod number. When the Euclidean distance was 5,16 varieties can be grouped into 5 categories. The higher yield could be obtained while cultivars with higher plant height,more primary branches and pods in the main inflorescence,less secondary branches and normal overwintering were selected for planting in the local area. The safely overwintering should be considered if 20SY-13 is planted in autumn in this area.

miRNA-mediated Regulation Involved in Plant Pathogen

YANG Li-juan, LI Shi-fang, LU Mei-guang

2020, 36(1):  101-109.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1045

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MicroRNA（miRNA）is a kind of small single-stranded RNA molecule with a length of about 22 nt,which is ubiquitous in eukaryotes. A large number of studies have found that miRNA is widely involved in the growth and development of plant,metabolism,material transport,stress response,pathogen defense and other biochemical processes. At present,a large number of miRNAs have been identified from plants,while there are few studies on miRNA involved in the regulation of plants pathogens. As an important transcriptional regulator,miRNAs can be involved in the regulation of PAMP-triggered immunity and effector-triggered immunity. In Arabidopsis,miRNA393 negatively regulates auxin by targeting the auxin receptor gene,thereby plays a role in resisting bacterial infection. In rice,miRNA528 can be in response to the infection of rice stripe virus（RSV）,and the increase of miRNA528 level using artificial means will help maintain the rice resistance to RSV. This paper describes the mechanism of action of miRNAs,the progress of miRNAs related to plant bacterial,fungal,viral infections,and summarizes the research on pathogen-regulated miRNAs in fruit trees with important economic value such as grapes,apples,pears,and peaches,aiming at providing a comprehensive theoretical basis for miRNA research in plants,especially in fruit tree.

Mutagenesis Breeding in DHA Production by Oleaginous Microorganisms

LI Jian-tao, LIU Xian-hua, HE Yao-dong, WANG Guang-yi

2020, 36(1):  110-115.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0637

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Docosahexaenoic acid（DHA）is essential in the maintenance of normal physiological function of human body and thus has significant biomedical value. Oleaginous microorganisms possess strong capacity to synthesize fatty acids and maintain relatively high percentage of DHA in total fatty acids. Mutagenesis is an effective and prospective breeding approach for the selection and improvement of oleaginous microorganisms. This review summarizes the methods and mechanisms of breeding the high-yield DHA strains by physical（γ-ray,ultraviolet,ion beam,and ARTP）or chemical（ethyl methanesulphonate,N-methyl-N’-nitro-N-nitrosoguanidine,and diethyl sulfate）agents. Also the paper introduces the research achievements on oleaginous microorganisms yielding DHA via varied mutation breeding methods,discusses the trends of mutation breeding,and put forward the idea of hybrid breeding by combining different breeding methods,its hoped that the study of DHA production by oleaginous microorganisms will be enlightened in the future.

Advances in Restorer Genes for Fertility on Cytoplasmic Male Sterility in Major Crops

ZHAO Guo-long, LIN Chun-jing, JIN Dong-chun, ZHANG Chun-bao

2020, 36(1):  116-125.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0900

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Heterosis is one of the main ways that can improve the crop yield,stress resistance and quality. The sterile/recovery（CMS/Rf）system is widely used in crop heterosis. Studying the recovery mechanism of cytoplasmic male sterile lines is an important basis of molecular genetic in the selection of the ‘three lines’ breeding program. Currently,different types of sterile lines have been created in major crops. Aiding of molecular technology,genomics and sequencing technology,many fertility-restoring genes have been located,and some of them have been cloned and functionally identified. This paper systematically summarizes the genetic model and molecular marker localization,cloning and application of CMS/Rf system in hybrid breeding in the main crops. It is aimed that this paper may provide ideas for molecular marker assisted selection,transgenic or gene editing methods to create new restorer lines in the future.

Research Progress on the MBW Complexes in Plant Anthocyanin Biosynthesis Pathway

GAO Guo-ying, WU Xiao-fang, ZHANG Da-wei, ZHOU Ding-gang, ZHANG Kai-xuan, YAN Ming-li

2020, 36(1):  126-134.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0738

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Anthocyanin is one of the key substances in the plant coloration,which can not only endue plants with rich colors,but also have a wide range of biological functions,such as anti-oxidation,anti-ultraviolet,resistance to diseases and insect pests. The plant anthocyanin biosynthesis pathway is completed while synergizing by a series of structural and regulative genes. The structural genes are regulated and expressed by MYB,bHLH and WD40 transcription factors and the MBW complexes composed of them. This paper reviews the recent research progresses in functions of MYB,bHLH and WD40 transcription factors and MBW complexes in anthocyanin synthesis in plants,and summarizes various biological processes and regulatory networks in anthocyanin synthesis.

Advances in the Maintenance and Termination of Floral Meristem Regulated by C-type Floral Organ Gene AGAMOUS（AG）

WEI Ming-ming, ZENG Xia, AN Ze-wei, HU Yan-shi, HUANG Xiao, LI Wei-guo

2020, 36(1):  135-143.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0877

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The maintenance and termination of floral meristems plays key role in the floral organogenesis and metagenesis of plant. Successful floral meristems determinacy ensures the normal reproductive development and life cycle progression of plants. Many studies have shown that AGAMOUS（AG）gene,as a master regulator of both floral organ differentiation and floral determinacy,can coordinate various cell fate decisions in flower development. However,the molecular mechanism of AG involvement in the regulation of plant metagenesis and floral meristem maintenance and termination remains unclear. This paper summarized such research progress and current status of AG gene in regulating the floral meristem maintenance and termination in recent years,with a view to providing a reference for further study of the maintenance and termination of stem cells during floral organ differentiation as well as the molecular regulation between stem cell activity and other developmental processes.

Research Progress on Drought Stress Response Mechanism in Camellia oleifera

DONG Bin, LI Rong-xi, HONG Wen-hong, HUANG Li-ying, HUANG Yong-fang, LAI Qiao-hui

2020, 36(1):  144-149.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0689

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Camellia oleifera is a unique woody edible oil tree in China. In recent years,China is making huge efforts to improve the self-sufficiency of cooking oil. Though regarded as a drought-resistant plant,C. oleifera is mainly cultivated in hilly lands of southern China,however,the serious water shortage and drought environment are still the major obstacles of limiting C. oleifera growth. This paper reviewed the the morphological indexes,yeld and quality,osmotic adjustment substances,protective enzymes,photosynthetic characteristics and chlorophyll fluorescence,and molecular level of C. oleifera under drought stress. We also discussed the current issues and the focuses in the future studying the drought stress of C.oleifera. In order to provide some suggestions for the research of drought resistance mechanism,drought resistant cultivation and drought resistance breeding in C. oleifera.

Research Advances in Functional Mechanisms of Bacillus amyloliquefacien

WANG Shi-wei, WANG Qing-hui

2020, 36(1):  150-159.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0786

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Bacillus amyloliquefaciens belongs to the genus Bacillus,it can be widely used in agricultural production,food industry,environmental protection,cosmetics industry,medicine,desert management and so on. These applications are closely related to the characteristics and functions of B. amyloliquefaciens. It is because B. amyloliquefaciens has many physiological functions,such as forming biofilm,colonizing plants,promoting plant growth and so on,that the wide application of B. amyloliquefaciens becomes feasible. Here the functional mechanism and important physiological characteristics of B. amyloliquefaciens are summarized,aiming at providing scientific theoretical data for better developing and utilizing this probiotics.

A General Overview of Nanomaterials Immobilized Lipases for Biodiesel Production

ZHANG Wei-wei, YANG Hui-xia, XUE Ping

2020, 36(1):  160-166.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0618

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With the increasing global demand for energy and environmental pressure,the application of immobilized lipase in the sustainable biodiesel production has received extensive attentions. Nanomaterials,including nanoparticles（magnetic and non-magnetic）,carbon nanotubes and electrospun nanofibers,with the advantages of large surface area,stability and easy modification,have been considered as important supports for lipase immobilization. The application of nanomaterials as enzyme support in lipase immobilization is presented. Moreover,the current status of biodiesel production using a variety of nano-immobilized lipase is discussed,and the outlook of nano-immobilized lipase is also prospected. It is aimed at laying a foundation for further research and industrial application of immobilized lipase.

Anti-aging Effect and Molecular Mechanism of Probiotics：A Review

YU Jie, LIU Xin, ZHANG Chi, ZHAO Ji-chun, LI Fu-hua, MING Jian

2020, 36(1):  167-174.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0635

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Aging can cause many adverse physiological changes in individuals and increase their susceptibility to disease. Clasrifying the mechanism of aging is crucial for seeking intervention approaches. Current research findings suggest that free radical oxidative stress,inflammatory aging,immune aging,and intestinal flora imbalance are the main mechanisms of aging. Besides,probiotics have been reported to have potential anti-aging effects. This paper compares and analyzes the anti-aging evaluation models of probiotics,and focuses on the effects of probiotics on senescence-induced alterations of intestinal flora and anti-aging related signaling pathways,thus providing new ideas for further studying the anti-aging effect of probiotics.

Progress of Immunocyte in the Thermogenesis of Brown Adipose Tissue and Browning of White Adipose Tissue

SUN Wen-yang, LIN Jian-chun, GUN Shuang-bao, WANG Jin-yong

2020, 36(1):  175-181.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0542

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Obesity has become a global prevalent disease that threats human health. Brown adipose tissue（BAT）and beige adipocytes have attracted considerable attention as a novel therapeutic intervention in the treatment of obesity,due to the features of increasing energy consumption via thermogenesis. M2 macrophages（alternatively activated macrophages,M2 type）was recently found to have an unexpected involvement in promoting the thermogenic activity of BAT and browning of white adipose tissue（WAT）,i.e.,the processing of beige adipocyte biogenesis,but later researches had come to the opposite conclusion. Whether M2 macrophages involved in browning of WAT is still controversial. Herein,we review the promoting effect of M2 macrophages,type II innate lymphoid cells and eosinophils on BAT thermogenesis and browning of WAT. Also we summarize the studies about the M2 macrophages having no effect and inhibiting effect on browning of white adipose tissue.

Research Progress on RNA-cleaving DNAzyme in the Detection of Pathogens

WANG Xin, ZHU Long-jiao, XU Wen-tao, ZHAI Chen, WANG Shu-ya, HUANG Wei-xia

2020, 36(1):  182-192.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0568

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DNA is an important carrier of genetic information,and its folding property of spatial conformation makes it have many functions. Using a RNA-cleaving DNAzyme to recognize a specific single-stranded DNA molecule and to cleave one of the single strands,a signal output sensor can be constructed,which converts the specific recognition process into the signal outputs of gel electrophoresis character,fluorescence,and colorimetric,and it can be combined with amplification reaction to achieve signal amplified. RNA-cleaving DNAzymes are obtained by in vitro screening techniques that specifically bind to target substances（such as small molecules,proteins,and even whole cells）. Due to its advantages of convenient preparation,easy modification and fine stability,RNA-cleaving DNAzymes are used to construct biosensors to detect pathogenic microorganisms,for example in-situ detection and even in-vivo detection in medical treatment. In the future,it will be combined with mature detection,for instance,equipment blood glucose meter,cross-flow chromatography strips for microbiological testing. Furthermore,it can be used in important areas such as biosensing,food safety,and medical care. Here we review the application of RNA-cleaving DNAzymes in microbial detection. In addition,we discuss the mechanism,products,targets and characterization of RNA-cleaving DNAzymes in microbial detection,as well as the significance and problems of RNA-cleaving DNAzymes in the actual detection of microorganisms. Finally,we prospect the further application of this technology,aiming at the better development of nucleic acid cleavage enzymes in microbial detection.

Research Progress of Nucleic Acid Aptamer Optical Biosensor in Kanamycin Detection

WU Ya, XU Zhi-hui, ZHANG Biao, ZHAO Dong-fang, CAO Wen-xin, ZHANG Xing-ping

2020, 36(1):  193-201.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1008

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Kanamycin is an aminoglycoside antibiotic,and it is one of commonly used veterinary drugs in China because of its good effect and low price. However,if kanamycin is used in over-dosage,it will remain in the animal body largely;then,it is enriched into the human body by the food chain;and subsequently,the toxic side effects such as ototoxicity,nephrotoxicity and even death may be caused. Thus quantitative detection of kanamycin is very important. In recent years,a large number of optical methods for detecting kanamycin based on nucleic acid aptamers have been developed to ensure testing requirements. Firstly,the related concepts of nucleic acid aptamers and optical biosensors are clarified. Then,according to their different reaction mechanisms,the optical biosensors are classified,and the basic principles and detection ranges of various sensors are also described. Finally,the advantages,disadvantages and development directions of these sensors are summarized and prospected. The exploration of these methods will provide new technical support for food safety testing.

Application of PCR-membrane Chip Technology in the Identification of Yak and Yak-cattle Meat

ZHAO Rui-xiao, HAN Fei, JIANG Ming-feng, ZENG Lian, HAN Jia-yu, HUANG Ying, ZHOU Hang

2020, 36(1):  202-208.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0607

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This paper aims to quickly and efficiently identify the authenticity of yak and yak-cattle meat products by PCR-membrane chip technology. We collected samples（8 fresh meat and 19 dry meat samples of fresh pig,goat,oxen,buffalo,yak,chicken,duck,rabbit,dog,and yak-cattle）,extracted and purified DNA,used 11 specific primers to perform multiplex PCR. Further we hybridized the amplified products with a membrane chip containing 12 probes via reverse dot blot,measured the accuracy and sensitivity of the membrane chip,and identified the authenticity of the commercially available yak meat products. The results demonstrated that the membrane chip was of high accuracy,strong specificity,no cross-reaction,and high detection sensitivity（low to 0.1 ng）. Detection results of the commercial fake yak meat products showed that almost all of them were made from yellow cattle and buffalo meat. In conclusion,the application of PCR-membrane chip technology can quickly and accurately identify the real and fake yak meat products,which is conducive to combating counterfeit products and maintaining market balance.

Expression Vector Adaptation of Valencene-producing Saccharomyces cerevisiae and Optimization of Fermentation Carbon and Nitrogen Sources

CHEN He-feng, ZHU Chao-yi, LI Shuang

2020, 36(1):  209-219.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0564

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Valencene,a terpenoid existing in various citrus fruits,was widely used in perfume,soap,food,and beverage industry. However,the concentration of valencene in nature is extremely low,and current strategy to obtain valencene is complicated and expensive,thus construction of cell factory for valencene biosynthesis will be a more efficient and environmentally friendly. Saccharomyces cerevisiae was chosen as host to construct cell factory in this study. Firstly,the valencene synthase from Callitropsis nootkatensis（CnVS）was introduced into S. cerevisiae strain,resulting in the preliminary synthesis of valencene,and the initial yield of this strain was 4.16 mg/L. Then the gene erg9 and rox1 in Mevalonate（MVA）pathway of S. cerevisiae were knocked out using CRISPR/Cas9 system,improving carbon flux towards valencene synthesis. The results of different carbon/nitrogen source concentrations in fermentation indicated that the high accumulation of cell growth may not be conducive to valencene accumulation. Finally,the influences of different CnVS expression vectors on valencene yield were examined and the highest yield of 17.54 mg/L was obtained,which was 4.2 times higher than the original strain.

Establishment and Preliminary Exploration of in vitro Pig-a Gene Mutation Assay Based on L5178Y Cells

WANG Ya-nan, WEN Hai-ruo, WANG Xue

2020, 36(1):  220-228.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0855

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The objective of this work isto establish and validatea method of detecting Pig-a gene mutation in mammalian cells based on mouse lymphoma cells L5178Y tk^+/--3.7.2C with negative compounds Glc,NaCl and positive compounds EMS,ENU,4-NQO,and B（a）P. Cytotoxicity evaluation was performed by calculating the relative doubling rate of the cells,and the antibody-labeled mutant cells were used to determine the flow detection template. The frequency of Pig-a gene mutation was detected on the 4th,8th,12th,16th and 20th day after EMS treatment,and the time point of the maximum mutation frequency was determined. The location of CD90 protein in the cells was detected by immunofluorescence technique,and the PCR was used for mutation site analysis. Results are as following：1）The RPD of the concentration groups set by Glc,NaCl,EMS,ENU,B（a）P,and 4-NQO were all > 50%. 2）The frequency of mutation of the Pig-a gene peaked on the 8th day after administration. The mutation frequency of Glc and NaCl in Pig-a gene was < 200×10^-6. There was no significant difference between each concentration group and solvent control group（P>0.05）. EMS,ENU,B（a）P,and 4-NQO resulted intheincrease in the mutationfrequency of the Pig-agene,and there was a significant difference compared with the solvent control group. 3）Immunofluorescence imaging showed that there was no CD90 protein on the surface of mutant cells,and wild type cells normally expressed CD45 and CD90 proteins. 4）Detection of gene mutation sites revealed the presence of 3mutation types：G→C,A→C,and C→T. In conclusion,this study successfully establishes a method of detecting the genotoxicity of in vitro Pig-a gene mutation in mouse lymphoma L5178Y cells with/without of S₉ metabolic activation,providing new approach in vitro genotoxicity evaluation or screening genotoxicity in the early development stage of drugs.

Effects of TET2 on T Cell Proliferation by Electroporation

YANG Lei, YE Zhou-jie, LI Zhao-long, SHEN Yang-kun, FU Ya-juan

2020, 36(1):  229-237.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0891

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Gene editing of T cells using recombinant viruses for immunotherapy has attracted widespread attentions. However,the application of recombinant viruses was constrained due to the disadvantages of random integration,long-time preparation and high cost. However,the application of electroporation technique can quickly bring exogenous DNA into cells,which is conducive to improving gene editing efficiency of T cells. The TET（Ten-eleven translocation）family proteins,such as DNA demethylase,can catalyze the conversion of 5-methyl cytosine（5mC）to 5-hydroxymethyl cytosine（5hmC）and play an important regulatory role in cell genome epigenetics. The researches demonstrated that the CAR-T cells lacking TET2 gene proliferated more rapidly and functioned more powerfully. In this study,the CRISPR/Cas9 technology was used to knock out the TET2 gene. First,sgRNA was transcribed in vitro and incubated with the Cas9 protein expressed by Escherichia coli expression system to form Cas9：sgRNA ribonucleoprotein complex（RNP）,and the activity of sgRNA was verified by in vitro enzyme digestion. Then,RNP was introduced into cells by electroporation,and the gene editing efficiency of T cells was tested. Finally,the proliferation of T cells was detected by flow cytometry. The results of gene sequencing and T7E Ⅰ enzyme digestion showed that the TET2 gene in the T cells was successfully knocked out,while the activity and function of the T cells were not affected. The results of flow cytometry analysis,CCK-8 and trypan blue cell viability test showed that the proliferation of T cells lacking TET2 gene was significantly faster than wild-type T cells. This study provides a basis for the application of non-viral vector replacing traditional recombinant virus used in the preparation of chimeric-antigen-carrying T cells,and it characterizes with short preparation cycle and high safety. Meanwhile,TET2 gene deficiency promotes the proliferation of CAR T cells,and enables them to initiate effective anti-tumor responses,which provides a new insight into CAR T cell immunotherapy.

Research Progress of Fuel Ethanol Fermentation Technology

GUO Zhen-qiang, ZHANG Yong, CAO Yun-qi, LIU Yun-yun, ZHAO Yu, WU Ai-min

2020, 36(1):  238-244.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0610

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Based on the analysis of the principle of fuel ethanol production from lignocellulose biomass,the fermentation process of fuel ethanol production is mainly discussed. At present,the fermentation process mainly includes direct fermentation,separate enzymatic hydrolysis and fermentation,simultaneous saccharification and fermentation,simultaneous saccharification and co-fermentation,and consolidated bioprocessing. The research status of these technologies is analyzed and their development trends are prospected. Consolidated bioprocessing conducted by highly efficient fermentation strains constructed by genetic engineering will be the development trend of high-efficiency fermentation technology in the future. The aim of this paper is to provide an important reference for effectively improving the substrate metabolic ability of fermentation strains and obtaining high ethanol yield.