Table of Content

26 May 2020, Volume 36 Issue 5

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Research Progress on the Trait Value Consistency of Reference Materials for Genetically Modified Organism

WANG Hao-qian, LI Xia-ying, ZHANG Li, ZHAO Xin, CHEN Rui, CHEN Xiao-yun, GAO Fang-rui, LAN Qing-kuo, WANG Yong, ZHANG Xiu-jie

2020, 36(5):  2-8.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0013

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Reference material for genetically modified product is crucially important necessity to guarantee that results expressing the GM content are reliable,comparable and fulfill the requirements of existing legislation. The main international organizations of researching and developing the detection reference materials include European Union Joint Research Centre Geel（JRC Geel,Belgium）,and the American Oil Chemists’Society（AOCS,Urbana,IL,USA）. In recent years,China has also developed a series of plant reference materials for testing GMO ingredients. In this paper,we did the summary,statistics,and analysis on type,quantity,value expression and value conversion of the GMO reference materials,and explored their similarities and differences. In terms of domestic and international academic exchange,new product development and application of reference materials,we have put forward reasonable suggestions aiming to provide support for the subsequent research and application of reference materials in testing transgenic products in China.

Research Progress on Fatty Acid Standard Reference Material

HAN Lu, SHENG Ling-hui, GAO Yun-hua, WU Xiao, WANG Zhi-dong, LI Bao-shan

2020, 36(5):  9-15.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0995

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Fatty acid is a kind of biomolecule with important functions. It can be founded in many animals and plants,and it is also an important component of the human body. Fatty acid is closely related to many diseases in human beings. Fatty acid standard reference materials are the basis for the accuracy of detecting fatty acid,and countries in all over the world are actively developing them. Currently,the institutes of studying fatty acid standard reference materials in China mainly are National Institute of Metrology of China and Academy of State Administration of Grain. In this paper,the current status of fatty acid standard reference materials at home and abroad is reviewed,and the characteristics,composition and determination methods of fatty acid standard reference materialsare compared and analyzed. Foreign fatty acid standard reference materials generally contain a variety of characteristic components with more characteristic values,while domestic fatty acid standard reference materials are generally only developed for the measurement of fatty acids in food.The matrix is relatively single,the characteristic component is less,and the quantity is less.

Analysis on Detection Methods for Somatic Cells in Raw Milk and Necessity of Measurement Calibration

YANG Jia-yi, NIU Chun-yan, LIU Ying-ying, FU Bo-qiang, WANG Jing

2020, 36(5):  16-21.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1121

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Somatic cell number is an important indicator of indicating raw milk quality and health status of dairy cows. In 2018,China’s national food safety standards plan included somatic cell number limit grading standards in the first time,thus the accurate detection of somatic cell number will directly affect the correct grading of raw milk products. Based on the importance of somatic cells to the quality of dairy products,we summarized and compared the somatic cells limit standards of raw milk at home and abroad in this review. We analyzed the existing somatic cell detection methods and principles,as well as the key detection parameters of the method performance of different principle instruments. We also outlined the great importance of establishing measurement calibration for ensuring accurate and comparable results of somatic cell detection instruments.

Recent Progress on Isothermal Amplification Technology and Its Combination with CRISPR in Rapid Detection of Microorganisms

GAO Wei-fang, ZHANG Li-ping, ZHU Peng

2020, 36(5):  22-31.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0820

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Isothermal amplification technology is of strong suitability for rapid detection because of its low dependence on instrument and high efficiency of nucleic acid amplification. In recent years,it has been widely used in the field of rapid detection of microorganisms. This article reviewed the development of isothermal amplification technologies,as well as the application in the rapid detection of pathogenic microbial nucleic acid from the perspectives of extraction of nucleic acid,isothermal amplification technologies such as loop-mediated isothermal amplification（LAMP）and recombinase polymerase amplification (RPA),and the detection of product. The article also summarized the latest research achievements of combining isothermal nucleic acid amplification technology with gene editing technology,CRISPR（clustered regularly interspaced short palindromic repeats）,in expectation of providing new thoughts for the development of such emerging technologies in the future.

Tetra-primer Amplification Refractory Mutation System PCR and Its Application in Fauna and Flora Genetics and Breeding Research

YU Jun-jian, CHI Mei-li, JIA Yong-yi, LIU Shi-li, ZHU Jun-quan, GU Zhi-min

2020, 36(5):  32-38.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0575

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Tetra-primer amplification refractory mutation system PCR（tetra-primer ARMS PCR）is a technique of typing SNP developed on the basis of common PCR. This technique combines the advantages of amplification refractory mutation system（ARMS）and four-primer PCR（tetra-primer PCR）,is an improvement of allele specific PCR. It has the characteristics of simple operation,rapid typing and low cost and has been applied more and more widely in the field of life sciences especially in the field of breeding. In this paper,the principle and advantages of tetra-primer ARMS PCR technique,detection methods of results and improvement methods of reaction system are introduced. In addition,the applications of this technique in genetics and breeding researches are reviewed.

Strategy of Screening Genetically Modified Maize

WEN Hong-tao, LI Xia-ying, YANG Yang, CHEN Zi-yan, DING Yi-jia, ZHANG Xiu-jie, ZHANG Rui-ying

2020, 36(5):  39-47.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1090

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By checking regulatory elements and marker genes in common genetic transformation vectors of GM maize lines in relevant databases and combining it with the current domestic and foreign standard methods for the detection of GM,the strategy of screening transgenic maize by real-time fluorescent PCR method was established based on the P-CaMV35S,T-NOS,Bar gene,Pat gene,Cp4-epsps gene,NPT II gene,cry1A gene,P-Rice actin,and phy structure specific fragments,and 32 varieties of transgenic maize were tested in the screening. The results showed that the screening strategy covered 30 independent trait GMO events. Meanwhile,a plasmid molecule containing maize internal standard gene and 9 target elements/gene sequences was constructed. It was proved that the plasmid molecule had favorable substitution and could be used as a positive control in conjunction with this detection method.

Analysis of Verification Results by PCR Methods for Genetically Modified Double-resistant 12-5 Event-specific Maize

WANG Hao-qian, XIAO Fang, YANG Lei, MIAO Qing-mei, ZHANG Xu-dong, ZHANG Xiu-jie

2020, 36(5):  48-55.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0299

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Insect-resistant and herbicide-tolerant genetically modified maize DR12-5 is a new transgenic strain independently researched and developed in China. It has been approved for the agricultural GMO safety certificate on January 21,2020,and thus has broad application prospects. The event-specific PCR method is one of the most effective detection methods in safety supervision for transgenic organisms,which can identify the genetically modified products. In this study,we organized 8 laboratories to verify the event-specific qualitative and quantitative PCR methods of the double-resistant（DR）12-5 transformants provided by its research and development institutions. The verified results showed that both the qualitative and quantitative PCR detection methods had good stability,strong specificity and high sensitivity. The quantitative PCR method can accurately and quantitatively detect DR 12-5 samples in mass fractions of 5% and 0.5%,and has good repeatability and reproducibility,which conforms the requirements of relevant standards. This study is conducive to the establishment and improvement of subsequent standard methods and provides technical support and decision-making basis for the safety supervision of GMOs in China.

Reverse Transcription Digital PCR Detection Method for NAi-Based Transgenic Maize

YANG Zhen-zhou, LIU Gang, XU Li

2020, 36(5):  56-63.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0897

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Aiming at the transgenic maize based on RNAi technology,this paper studied and established a set of reverse transcription chip digital PCR（dPCR）method of quantitatively detecting dsRNA in this transgenic maize. The research contents mainly included exploration and optimization of RNA extraction methods,primer probe design,reverse transcription method,dPCR reaction conditions and compositions,etc. The limit of absolute quantification of this method is 2.5 copies/μL,and the RSD is 12.4%. The relative deviation is 2.7%,and the RSD is 14.6%. When detecting the actual sample of low concentration is 21.7 copies/μL,which meets the international requirement of transgenic quantitative result RSD≤25%. Applying the method in the quantitative detection of genetically modified crops based on RNAi technology will provide accurate and reliable technical means for the safety evaluation of related genetically modified crops in China.

Qualitative PCR Assay for the Detection of GH5112E-117C Transgenic Maize Resistant to Insects and Herbicides

LI Cong-cong, Xie Ping, DONG Li-ming, XIA Wei, LAN Qing-kuo, YAN Wei, LONG Li-kun, LI Fei-wu

2020, 36(5):  64-67.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0701

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Transgenic maize GH5112E-117C is a new strain with mG2-aroA gene and mcry1Ah genes and developed by Beijing Origen Company. It is resistant to lepidopteran pests such as corn borer and armyworm,and it is tolerant to glyphosate- herbicide. Thus this strain has important industrial application prospects in China. A qualitative PCR assay for transgenic maize GH5112E-117C was developed in this paper. The assay had promising specificity to detect the transgenic maize GH5112E-117C with resistance to insects and tolerance to herbicide. At a concentration of 0.2 μmol/L primer,TaqDNA polymerase 0.625 U,DNA template 50 ng,and an annealing temperature of 58℃,the detection sensitivity of the assay stably achieved to 0.1%. This assay provides a new technical means for accurately testing insect- and herbicide-resistant transgenic maize GH5112E-117C,which provides the technical support for transgenic supervision in agriculture.

A Quantitative Method for Genetically Modified Soybean LineMON89788 Using Microchip Digital PCR

YANG Zhen-zhou, LIU Gang, LIANG Wen

2020, 36(5):  68-73.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1002

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A microchip digital PCR（dPCR）quantitative method for insect-resistant and herbicide-resistant genetically modified soybean lineMON89788was developed and several critical experimental conditions were studied,including genomic DNA extraction,quality and concentration control of nucleic acid template,screening of the primer probes,PCR program,etc.The reproducibility and quantitative detection limit of the method were also investigated.The quantitative repeatability RSD ranged from 1.17% to 9.97% when a 5% GM soybean（MON89788）sample was analyzed,andall of them met the international requirement that the RSD of transgenic quantitative results should be < 25%. The quantitative results of MON89788samples containing 5%,1% and 0.1% GM were 5.20%,0.94% and 0.11% and the RSD was 6.2%,3.6% and 15.2%,respectively. The limit of quantificationwas 0.1%,which fully satisfiedthe EU’s GM quantitative labeling limit（0.9%）. Applying the method established in this experiment in the quantitative detection of transgenic soybeans may provide strong technical support for standardizing the implementation of transgenic supervision in China.

Establishment and Performance Evaluation of Nano-fluorescence Strip for Detecting CSFV Antibody

WU Hao-xing, LIU Bai-hong, LIU Xue-wei, ZHOU Jing-yun, MA Yong-ying, YANG Xin-yan, LI Bao-chun, CHEN Xi-zhao

2020, 36(5):  74-79.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0986

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An immunochromatography method for the detection of the antibodies against classical swine fever virus（CSFV）was established based on lanthanide europium microsphere labeling technology. By optimizing the coating concentration,resolving concentration and reaction time in the reaction system,the optimal reaction conditions were determined and the detection method was established. Then the performance of the system was evaluated from sensitivity,specificity,precision and clinical evaluation. The optimized reaction system was as that the concentration of coating was at 0.1 mg/mL,the dilution parameter was 6 times,and the detection time was 15 min. From the performance evaluation of the strips,the sensitivity of the strip for the detection of CSFV antibody was that the diluted 128 times of national reference samples of swine fever positive serum were still detected,and no cross-reactions were observed with the positive sera against common CSFV,for example PPRSV,PRV,FMDV,PCV,PEDV,BVDV,and BDV. The coefficient of variation was < 10%. Compared with the commercial ELISA kit of CSFV antibody,the coincidence rate for positive was 90% and that for negative was 100%,and the total coincidence rate was 97.7%. Compared with FVNT,the coincidence rate for positive were 93% and that for negative was 100%,and the total coincidence rate was 98.5%. According to the above comprehensive evaluation,the fluorescence detection method of CSFV antibody established in this study conforms to all the performance parameters. It can be used for rapid,economical and convenient detection of CSFV antibody in pigs,and can be widely applied to pig farm management at the grass-roots units.

Establishment and Application of PRRSV NADC30-like SYBR Green I qPCR Detection Method

XIANG Ming-yuan, LIAO Chang-yu, JIANG Di-ke, ZHANG Peng-fei, WANG Yin, LUO Yan, YANG Ze-xiao, YAO Xue-ping

2020, 36(5):  80-85.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0885

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The objective of this study is to establish an efficient and rapid real-time PCR for the detection of PRRSV NADC30-like. Specific primers were designed based on the conserved sequence of Nsp2 gene of NADC30 strain,and the optimal reaction conditions were determined by optimization,and sensitivity,specificity,reproducibility experiments and detection of clinical samples were performed. The results showed that standards at the concentration of 10⁷ copies/μL to 10² copies/μL had a fine linear relationship,and the minimum detectable concentration was 2.25×10 copies/μL. There was no cross reaction with HP-PRRSV,PCV,PEDV,TGEV,PRV,CSFV,and PoRV. All coefficient of variation（CV）of intra-groups and inter-group were < 1.9%. The detection rate was higher than normal PCR in the detection of clinical samples. In conclusion,a quantitative PCR method for the detection of PRRSV NADC30-like strain is established,which has the advantages of high sensitivity,specificity,stability,accuracy and rapid detection. It can be used for the early diagnosis of infection of PRRSV NADC30-like,as well as rapid detection and quantitative analysis of samples.

Copy Number Variations of Mitochondrial DNA and Genomic DNA from Different Tissues of Duck Based on Digital PCR

JI Yi, XU Xiao-li, JIANG Yuan-yuan, WANG Xiao-fu, XU Jun-feng, LI Yue-ying, CHEN Xiao-yun

2020, 36(5):  86-91.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0934

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Meat and meat products are the important nutrition sources for human being life,but adulteration of meat products occur frequently on the market in recent years,which makes the quality and safety of meat and meat products has become a hot topic all over the world. The identification of animal sources targeting nucleic acids is a widely used method at present. In nucleic acid detection,mitochondrial gene or nucleic genes are often used as targets,and there is a lack of unified standards. In this study,Shaoxing duck and Beijing duck and other different breeds and fresh tissues（duck blood,duck breast,duck liver,duck skin,duck heart and duck leg meat）were used as experimental materials,DNA was extracted from them and microdrop digital PCR was used to compare the copy numbers between mitochondrial DNA（mtDNA）and nucleic DNA. The variation coefficient of the copy number and their ratios were used as judgment basis. Results demonstrated that the copy number of nucleicDNA in various duck tissues were relatively stable,and the variation coefficient in nucleic DNA was smaller than that in mtDNA,suggesting that the nucleic DNA was the most suitable DNA source for the quantitative detection of adulteration of duck meat and products. Notably,the variation coefficient of the ratio of mtDNA copy number to nuclear DNA copy number in the duck leg meat was the smallest,indicating that duck leg meat was the best choice while we used mtDNA as the target gene for adulteration detection.

Optimization of Quantitative Determination of Bacitracin Based on Turbidimetric Method

WANG Zhi-xin, LIU Yang, ZHOU Jing-bo, HONG Dan, LU Lei-zhen, NING Ya-wei, JIA Ying-min

2020, 36(5):  92-97.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0994

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The quantitative determination methods of bacitracin in the research and application are not standardized,and the results cannot be used for reference. In order to standardize the determination methods,the linear relationship between the logarithmic value of bacitracin concentration and OD₆₀₀ was established,and the initial concentration of indicating bacteria,the ratio of bacitracin solution to bacterial suspension and the culture time were optimized with repeatability and precision as indicators. The optimal determination conditions of bacitracin against bacteria by turbidimetric method were as follows：the initial concentration of indicating bacteria were 10⁷ CFU/mL,the ratio of bacitracin solution to bacterial suspension was 1∶9,and the culture time was 4 h. Under these conditions,the linear relationship was fine with R² above 0.99 and the repeatability was well. Furthermore,Escherichia coli and Staphylococcus aureus were selected to verify the feasibility of the method,and this method presented favorable repeatability and high precision. The results would provide reference and basis for the further application of turbidimetric method and the quantitative determination of bacitracin in the test and production process.

Screening and Identification of Phosphate Solubilizing Bacteria from Maize Rhizosphere Soil and Its Growth Promoting Effect

WAN Shui-xia, WANG Jing, LI Fan, JIANG Guang-yue, XU Wen-jing, LIU Zuo-jun

2020, 36(5):  98-103.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0678

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In order to obtain plant growth promoting rhizobacteria（PGPR）in maize and clarify its growth-promoting characteristics,PGPR strain were screened from maize rhizosphere soil samples. Then the P-solubilizing characteristics and IAA-producing ability of preferred strains were studied using soil pot experiment. The strains were further identified and their growth-promoting effects were investigated in pot experiment. As results,2 excellent PGPR strains were isolated and named as CH07 and FD11,and their solubilized phosphorus reached 368.5 mg/L and 321.5 mg/L,the produced IAA（indole acetic acid）were 30.93 mg/L and 15.93 mg/L,respectively. According to the results of morphologic characteristics,physiological biochemical properties and phylogenetic analysis of 16S rRNA genes,CH07 was identified as Bacillus aryabhattai and FD11 as Streptomyces maritimus. Finally,the growth promoting effects of the two bacterial isolates were compared by pot experiment. The results showed that CH07,FD11,especially the mixture of CH07 and FD11,had a positive effect on the plant height and shoot fresh weight of amaranth,and could be used as an excellent strain resource for the development of biological fertilizer.

Isolation and Screening of Plant Growth-promoting Rhizobacteria in Pepper and Their Disease-resistant Growth-promoting Characteristics

YANG Mo, GAO Ting, LI Yan-jing, WEI Chong-yao, GAO Miao, MA Lian-ju

2020, 36(5):  104-109.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0840

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In order to obtain plant growth-promoting rhizobacteria（PGPR）of pepper and to explore their disease-resistant and growth-promoting characteristics,the PGPR were isolated and screened from pepper rhizosphere soil in Xuzhou city of Jiangsu province,using nitrogen fixation,inorganic phosphorus and organic phosphorus media. The strains were identified by morphological characteristics and 16SrDNA. Their abilities of fixing nitrogen,releasing phosphorus,secreting IAA and resistances to 4 pepper pathogenic diseases were investigated. The results showed that 13 PGPR strains of pepper were obtained and identified as Bacillus,Pseudomonas,Lelliottia,Siccibacter,Achromobacter,Microbacterium and Paenibacillus. All of them had function of fixing nitrogen,7 of them dissolved organic phosphorus,belonging to Lelliottia,Bacillus,Siccibacter,Microbacterium and Paenibacillus;5 dissolved solubilized inorganic phosphorus,belonging to Lelliottia,Bacillus,Siccibacter,and Pseudomonas;3 presented the ability of secreting IAA,belonging to Lelliottia,Siccibacter,Bacillus;and 5 demonstrated the resistances to diseases,belonging to Bacillus,Lelliottia,and Siccibacter. Pepper rhizosphere soil contains multi-functional PGPR,which has potential application value in agricultural production.

Screening and Performance Analysis of High-temperature Phosphate-solubilizing Microorgams in High Temperature Compost with Phosphate Rock and Discarded Vinasse Lost Grains

ZHANG Rui-rui, QIU Shu-yi, ZHOU Shao-qi, WANG Xue-li

2020, 36(5):  110-119.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1150

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In order to solve the problem of the phosphorus dissolution in the high temperature environment,the phosphate-solubilizing microorgams were screened from high temperature compost. Using inorganic phosphorus selective medium,5 strains of high temperature resistant and phosphate-solubilizing microorganisms（bacteria GDB1-2;fungus GDF1-3）were isolated and screened from the high-temperature compost samples added with phosphate rock and lost grains. The 5 strains were identified by morphological and molecular biology and solubilized-phosphorus capability. The results showed that the 5 isolates were Bacillus subtilis,Bacillus licheniformis,Lichtheimia ramosa,Aspergillus fumigatus and Aspergillus nidulans. The phosphorus solubilization curve of the 5 strains showed that the highest phosphorus content of each strain ranged from 136.85 to 174.33 μg/mL. It was found that each strain had a wide range of high temperature resistance and grew between 40-60℃,but its ability to dissolve phosphorus reached a maximum at 50℃. Under the initial pH of 5-9,all the 5 strains maintained a certain ability of dissolving phosphorus. The research results provide materials for the subsequent development of high-temperature environmental microbial resources,and have a good application and promotion prospects.

Cloning, Expression Analyses of HbAIH Gene from Hevea brasiliensis

YANG Hong, YUE Yi-fan, HU Yan-ling, DENG Zhi, DAI Long-jun, LI De-jun

2020, 36(5):  120-129.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0940

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Agmatine iminohydrolase（AIH）in plant is one of the most important enzymes in the arginine decarboxylase pathway in which arginine is subsequently converted to poluamine. This study is the first time for an AIH gene，HbAIH，to be cloned from Hevea brasiliensis. The full-length sequence of HbAIH was 1 462 bp with a 1 137 bp open reading frame，encoding a sequence with 378 amino acid residues. HbAIH was a hydrophilic protein，with a calculated molecular weight of 42.28 kD and an isoelectric point of 5.09. Sequence alignment revealed that the deduced amino acid sequence of HbAIH presented the high similarity with those of MeAIH in Manihot esculenta，RcAIH in Ricinus communis and JcAIH in Jatropha curcas from Euphorbiaceae，and also contained 11 highly conservative amino acid residues that formed the active sites and participated in the binding with the substrate. qRT-PCR analyses demonstrated that HbAIH expressed with no tissue specificity，the highest in female flowers，followed by barks，stem apexes，latex，leaves and male flowers. HbAIH expression significantly varied in different developmental stages of rubber tree leaves，with the highest expression in light young stage and the lowest one in mature stage，suggesting that HbAIH might be associated with leaf development. In addition，the expression of HbAIH was induced by wounding，low temperature，drought，high salt，hydrogen peroxide，ethrel，MeJA treatments，and tapping panel dryness. All these results suggest that HbAIH is likely involved in growth and development of H. brasiliensis，regulating latex biosynthesis and latex flow，as well as stress responses.

Production of Prodigiosin Using Waste Oil from the Processing of Sophora flavescens Seeds

NI Liang, ZHANG Sen, GUO Sheng, ZENG Fei, XU Ming-ming, DUAN Jin-ao

2020, 36(5):  130-138.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1161

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To improve the utilization efficiency of Sophora flavescens resources,the strains that can use the waste oil after extracting alkaloids from S. flavescens seed to produce prodigiosin were screened,and its fermentation parameters were optimized by single factor investigation and response surface optimization. UPLC-Q-TOF-MS/MS was used to analyze the purified fermented products. As the results,a strain L9 was screened and identified as Serratia marcescens by morphology and 16S rDNA sequencing. The optimal parameters were as follows：the optimal concentration of S. flavescens seed oil,beef extract and calcium chloride were 13 g/L,9.5 g/L and 0.3 g/L,respectively,and the fermentation temperature was 30℃. Under the optimal fermentation conditions,the maximum yield of prodigiosin was about 317.21 mg/L,and the yield was increased by 3.2 times. In this paper,the by-products produced in S. flavescens seed processing was taken as the research object,and its oil components were used as resources,i.e.,producing the high value-added product prodigiosin while disposing the wastes from processing S. flavescens seed,which provides a reference for the resource utilization of solid waste in the processing of seeds medicinal materials.

Generation of a Vero Cell Line Stably Expressing African Swine Fever Virus P54 Protein

WANG Cai-xia, DU Fang-yuan, LIN Xiang-mei, Grzegorz Wozniakowski, WANG Qin, FENG Chun-yan, WU Shao-qiang

2020, 36(5):  139-144.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1099

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In order to establish the Vero cell lines that may stably express African swine fever virus（ASFV）,the fusion gene fragment from the ASFV-P54 gene with green fluorescent gene Azami Green was cloned into the lentivirus vector pLV-puro. The resulting plasmid,pLV-ASFV-P54-AG,was then co-transfected HEK-293V cells together with packaging plasmids pH1 and pH2 for packaging recombinant lentiviruses. The generated lentiviruses were then utilized to infect Vero cells mediated in the presence of polybrene. A Vero cell line,constitutively expressing the ASFV-P54 protein,was screened and designated as Vero-AG-ASFV-P54. Moreover,indirect immunofluorescence assays further demonstrated that the generated cell line reacted with P54 polyclonal antibody. The results further verified by National Veterinary Research Institute in Poland showed that the cell line reacted with the positive sera of ASFV antibody,but did not react with the negative sera. The above results reveal that the Vero-AG-ASFV-P54 cell line generated in this study may stably and efficiently express ASFV-P54 protein with biological activity.

Polymorphism of OLR1 Gene and Its Association with Meat Quality Traits in Sujiang Pigs

REN Shan-mao, WANG Wen-hui, TAO Yong

2020, 36(5):  145-149.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0827

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This work is to investigate the polymorphism of lipoprotein-receptor-l（OLR1）gene and its association with the meat quality traits in Sujiang pigs. The PstⅠ genetic polymorphism of OLR1 gene in Sujiang pigs was detected by PCR-RFLP,and the association between OLR1 genotype and meat quality traits was analyzed with one-way ANOVA. The results showed that Pst Ⅰ polymorphism in the intron 5 of OLR1 gene in the experimental population of Sujiang pig was found,and three genotypes（CC,CD and DD）were detected. The polymorphism information content was moderate. The difference of the water loss rate and marbling between CC and DD genotypes of OLR1 gene were significant（P<0.05）,and the b value of meat color of CD genotype by color difference meter was significantly higher than that of DD genotype（P<0.05）. Therefore,the detected PCR-RFLP-Pst I polymorphism of OLR1 gene is significantly correlated with some meat traits such as marbling,and it could be used as a potential candidate gene of meat quality traits in the continuous breeding of Sujiang pigs.

Heterologous Expression of Cyclina sinensis Mytimacin Antibacterial Peptide Based on Recombinant Pichia pastoris

ZHAO Zhen, WANG Sha-sha, LÜ Xing-xing, TAO Yan, XIE Jing, QIAN Yun-fang

2020, 36(5):  150-158.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0700

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Mytimacin is a member of the Macin antimicrobial peptide family expressed in invertebrates. It is a favorable candidate to develop natural antimicrobial agents by recombinant DNA technology because of its strong antimicrobial activity against pathogens. RT-PCR was used to clone the gene encoding mytimacin mature peptide from the adductor muscle of Cyclina sinensis;the Xho I restriction site and the signal peptidase recognition site were added to its 5' end,and the Xba I restriction site and a 6×His-tag to its 3' end after three times of PCRs the acquired target gene was named “CsMm”. The recombinant Pichia pastoris X-33/pPICZαA-CsMm was constructed using pPICZαA as an expression vector and P. pastoris X-33 as an engineering strain. The yeast transformants containing multicopy gene insertions screened by high-concentration zeocin were induced for 72 h with 1.5% methanol at 28℃ and 250 r/min,and the acquired culture medium supernatant was purified by immobilized metal affinity chromatography（IMAC）;the purified protein was identified by MALDI-TOF-TOF mass spectrometry analysis. In addition,the antibacterial activity of the recombinant CsMm was detected by coating method and turbidimetric method. The results showed that the heterologous expression based on the recombinant P. pastoris X-33/pPICZαA-CsMm a recombinant protein with an expression level of 25.6 mg/L;MALDI-TOF-TOF mass spectrometry analysis demonstrated that the purified recombinant protein was the expected recombinant CsMm with a molecular weight of 7.8 kD. The bacteriostasis test showed that the recombinant CsMm had obvious antibacterial activity against Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Vibrio Parahemolyticus. The recombinant P. pastoris X-33/pPICZαA-CsMm constructed in the present study can effectively synthesize the bioactive recombinant C. sinensis Mytimacin,which provides a technical approach for the development of natural small molecule antibacterial agent from shellfish.

Effect of Co-expression of HAC1 and Molecular Chaperone Genes on the Expression of Mannanase in Pichia pastoris

MIN Qi, GAO Zi-han, YAO Yin, ZHANG Hua-shan, XIONG Hai-rong, ZHANG Li

2020, 36(5):  159-168.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0718

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For the higher activity of expressed mannanase ManA in Pichia pastoris,the unfolded protein response（UPR）activation factor HAC1 and 5 reported endoplasmic reticulum protein folding related chaperones ERO1,PDI,PDI1,CPR5,and BiP in P. pastoris were constructed to have 6 intracellular-expressing recombinant plasmids,then each was electro-transformed into recombinant strain P. pastoris secreting ManA for intracellular co-expression respectively,and the influence of the recombinant strain on the expression of ManA during the shake-flask fermentation were analyzed. The results showed that the enzyme activity of ManA in the supernatant of the recombinant strain with co-expressing HAC1,ERO1 and PDI increased by 26%,15% and 20%,and the enzyme activity reached 1 014 U/mL,925 U/mL and 965 U/mL at the shake-flask fermentation. By analyzing the data of enzyme activity in supernatant,intracellular-retention enzyme activity,and protein concentration in supernatant as well as further selecting HAC1,ERO1,PDI to have 2 genes or 3 genes combinations,which then co-expressed in the recombinant strains secreting and expressing ManA,while the enzyme activity in the fermentation supernatants of each co-expressed recombinant strain was not further increased. Co-expression of HAC1 or molecular chaperone ERO1,and PDI separately enhanced the correct folding of ManA and thus increased the expression of the protein.

Isolation and Identification of the Growth-promoting Bacteria from Pbycosphere of Dunaliella salina

DUAN Lu-lu, HANG Wei, CHENG Yu-jiao, CUI Hong-li, WANG Ji-ping, LI Run-zhi

2020, 36(5):  169-175.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1264

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Through exploring the influence of phycosphere microorganisms on the microalgae growth and metabolite accumulation,the predominant growth-promoting strains were screened. The symbiotic bacterial strains from the phycosphere of Dunaliella salina Ds-SXYC-2 were isolated and identified,and 1∶1 algal-bacteria co-culture system was constructed,and the growth of D. salina and the accumulation of its metabolite were measured. The results showed that 5 symbiotic bacterial strains were isolated from the phycosphere of D. salina,and they belonged to 3 genera,including Nesterenkonia（Strain B1 and B2）,Halomonas（strain B3 and B4）,and Marinobacter（Strain B5）. All the 5 strains promoted the growth of D. salina. Among them,strain B3 significantly promoted the growth and the accumulation of metabolites. After 15 d of co-culture,the biomass of D. salina reached 2.3 g/L,which was higher than that of the control without bacteria and increased by 28.9%. The content of chlorophyll a（4.61 mg/L）and beta-carotene increased by 36.3% and 56.4%,respectively,in contrast to the control. The contents of intracellular polysaccharide,proteins and total lipids increased by 34.8%,71.2% and 37.6%,respectively,compared with the control group. Halomonas strain B3 could be used as the dominant strain to promote the growth and metabolite accumulation of D. salina,and the co-culture system could be constructed and applied to the commercial production of D. salina.

Analysis of Fresh-cut Shiitake（Lentinula edodes）and Autolysis During Storage

ZHAO Shuang, SU Zhe, GU Tong-tong, GAO Qi, RONG Cheng-bo, SONG Shuang, LIU Yu

2020, 36(5):  176-182.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0781

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This study is aimed to analyze irritable physiological change and cellular structure variation of Lentinula edodes after fresh-cut process. The fruiting body of shiitake 0912 was chosen as object in this study. The changing trend of physiological indexes at low temperature storage and room temperature storage conditions were investigated. The variation of cellular structure in aging tissue was detected by microtechnic including optical microscopy,fluorescence microscopy and transmission electron microscopy. The results showed that low temperature storage was beneficial to reduce the rate of browning,softening and weight losing,also to slow down the decline of antioxidant capacity. Microstructure analysis indicated that autolysis phenomenon occurred in the tissues after mechanical damage,such as cell wall breakage,membrane ablation,self-phagocytosis,DNA fragmentation,nucleus disappearance,and cell apoptosis. The autolysis phenomenon in fresh-cut shiitake occurs,and the damage of cellular structures is one of the important factors that induce the deterioration of shiitake,and accelerates the rotten progress with storage environment.

Application of Multigene Model in Prognosis Prediction of Hepatocellular Carcinoma

WEI Zhi-han, FA Bo-tao, YU Zhang-sheng

2020, 36(5):  183-192.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1254

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Predicting the types of hepatocellular carcinoma（HCC）while using little molecular information may provide patients with more personalized therapy. Here,known HCC prognosis-related pathways were investigated,and 41 dominant genes were identified. Based on these genes,a risk prediction model was constructed using machine learning and validated with 4 HCC datasets. The results revealed that the model was proven to efficiently divide HCC patients into 2 subgroups with significantly different prognosis：average log rank P-values of cross-validation within TCGA dataset was 0.03,and the log rank P-values of validation on other external datasets were 0.000 38,0.002 1 and 0.01,respectively. After analyzing the HCC subgroups with bioinformatics pipeline,the prognosis of HCC significantly was related to signal pathways such as cell cycle,and 12 potential biomarkers of HCC were screened. In sum,the 41-gene based stratification model may robustly and accurately predict prognosis among HCC patients.

Laccase-Mediated Oxidative Coupling of Phenolic Compounds in vivo：from Fundamentals to Multifunctional Applications in Green Synthesis

CHEN Hui-ling, ZHANG Qing-yun, SUN Kai

2020, 36(5):  193-204.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1007

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Laccases（p-diphenol dioxygen oxidoreductases,EC 1.10.3.2）are polyphenol oxidoreductase containing trinuclear copper cluster sites in nature and are presented widely in fungi,bacteria,plants,and insects. These enzymes can not only promote the bio-decomposition of polymer lignin and humus in ecosystem,but also catalyze the participation of monophenols and polyphenols in the biosynthesis of functional phenolic polymers such as melanin,lignin,flavonoids and keratin. The decomposition metabolism and anabolism mediated by laccases are beneficial to global carbon cycling and morphogenesis variations in ecosystem. In organisms,laccase catalyzes the single electron oxidation of natural phenols to form phenoxy active radicals or quinones intermediates,which then undergo self-coupling or cross-coupling reactions to form a variety of complex macromolecules C-C,C-O-C or C-N-C functional polymerization products. Thus,through mimicking laccase-catalyzing biosynthetic pathways in laboratory,we intend to reasonably design and directly modify the structures and functions of polymerization products by laccase-mediating the radical coupling of phenolic substrates in vitro synthetic processes,aiming at providing a reference and novel ideas for expanding and developing the multifunctional applications of laccase in green chemosynthesis.

A Sequencing Strategy for Inverted Repeats in RNAi Vectors

YANG Wen-wen, NI Jia-yao, HU Rui-jie, WANG Hua-zhong

2020, 36(5):  205-210.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0977

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Adjacent inverted repeats in DNA tend to form secondary structure in a single strand,which are difficult templates for sequencing. The objective of this work is to solve the difficulty in sequencing inverted repeats inserted in RNAi vectors,and thus to lay a foundation for the verification of such vectors through DNA sequencing. A routine method of molecular cloning was adopted to construct an RNAi vector expressing tandem reverse repetitive fragments of wheat TaATG2 gene. Colony PCR was used to preliminarily identify the constructed vector,and 2 sequencing strategies were designed in this study：One was to use intact plasmids as sequencing templates,and the other was to use linearized plasmids as sequencing templates in which one of the reverse repetitive fragments was removed by digestion. The results showed that the sequencing reaction of the first strategy was influenced by secondary DNA structures formed within the region of reverse repetitive fragments. The sequencing signals from the reverse repetitive fragments were very weak or displayed chaotic peaks. The second strategy eliminated the interference between the two reverse repetitive fragments. Under this strategy,clear sequencing signals and accurate sequences were obtained for each fragment retained on the vector. The two reverse repetitive fragments inserted in an RNAi vector can be separately and effectively sequenced by 2 times of digesting and sequencing under a strategy of sequencing one fragment in the vector after removing the other by digestion.