Table of Content

26 July 2019, Volume 35 Issue 7

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Application and Research Progress on Transcriptomics

CUI Kai, WU Wei-wei, DIAO Qi-yu

2019, 35(7):  1-9.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0374

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The transcriptome is the set of all RNA molecules in the specific tissue or cell,including message RNA,ribosome RNA,transfer RNA and non-coding RNAs. The rapid development of high-throughput sequencing technology provides a fast and reliable platform for the systematic study of transcriptome. Here we reviewed the current high-throughput sequencing technology and its application in transcriptome research,and discussed some issues worthy of attention in transcriptome data analysis,as well as the application direction of transcriptome sequencing technology in biological research.

Cloning,Expression and Bioinformatics Analyses of StSRP1 Gene in Solanum tuberosum

PANG Peng-xiang, CHANG Yan-nan, YU Rui-min, GAO Gang

2019, 35(7):  10-16.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0029

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The expression of stress-related genes（StSRP1）leads to the accumulation of metabolites and changes in biochemical and physiological pathways,which are essential for plants to adapt to adverse stress conditions. By studying the structure of potato StSRP1 and its expression in stress,the foundation for verifying the role of potato StSRP1 in the process of resistance to biological stress is laid. Electronic cloning was used to clone the full-length sequence of StSRP1 gene,bioinformatics was to analyze the nucleic acid sequence,amino acid sequence,protein structure and promoter of StSRP1. Based on the NCBI database,20 strains of stress-related protein sequences highly matched with StSRP1 were selected to construct a phylogenetic tree,and SRP proteins among different species were compared. StSRP1 induced by ABA,MJ and pathogen was analyzed by qRT-PCR. The full length of the cloned sequence was 867 bp,encoding 288 amino acids,no signal peptide sequence,no transmembrane region,34 possible phosphorylation sites,hydrophilic protein,and localized to the endoplasmic reticulum. In sum,the expression of StSRP1 is regulated by ABA,MJ and pathogens. StSRP1 may be involved in the potato stress response process.

Effects of Roundup Ready Soybean AG5601 on the Functional Diversity of Microbial Community in Rhizospheric Soil

ZHANG Zhuo, LIU Mao-yan, WANG Pei, HUANG Wen-kun, LIU Er-ming, PENG Huan, PENG De-liang

2019, 35(7):  17-24.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0002

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This study aims to clarify the effects of Roundup Ready Soybean AG5601 on the functional diversity of microbial community in rhizospheric soil,for supplying basic information for the safety evaluation of transgenic soybean. BIOLOG method was applied to compare the carbon source utilization characteristics of rhizospheric microorganism among AG5601,their parent SNK500,and ordinary cultivated soybean Zhonghuang 13 at mature and stubble stage. In the mature stage,the carbon source utilization capacity of rhizospheric microbial community in AG5601 was significantly higher than that in SNK500;however,had no significant difference with that in Zhonghuang 13. At the stubble stage,there were no significant differences in the carbon source utilization capacity of rhizospheric microbial community among G5601,SNK500 and Zhonghuang 13. Functional diversity analysis found that the rhizosphere soil microbial diversity indices of Simpson（1/D）and Mcintosh（U）of AG5601 were significantly higher than those of SNK500 in the mature stage. In the stubble stage the differences in microbial diversity among 3 soybeans rhizosphere soil were insignificant. Principal component analysis indicated that there existed same in the carbon source utilization by the microbes in rhizosphere soils of SNK500 and AG5601 at the mature and stubble stage,suggesting the non-difference in the carbon source utilization pattern of the microbes during these processes. This study revealed that the effects of Roundup Ready soybean AG5601 on the diversity of microbial community in the rhizospheric soil were not significant.

Screening and Comparison of Reference Genes for microRNA Quantitative Real-time PCR in Jatropha curcas Under Chilling Stress

KONG Chun-yan, CHEN Yong-kun, WANG Sha-sha, HAO Da-hai, YANG Yu, GONG Ming

2019, 35(7):  25-32.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0042

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Screening appropriate reference gene is of essential importance for the accuracy of quantitative real-time PCR（qRT-PCR）results,and it is especially true in qRT-PCR analysis of microRNAs（miRNAs）. Screening suitable reference genes for miRNA qRT-PCR analysis in Jatropha curcas under chilling stress may provide important reference genes for qRT-PCR analysis of miRNAs in J. curcas and other species. Eleven candidate genes were selected from the chilling-treated J. curcasbased on our previous small RNA-seq results,and their expression levels in different samples were detected by qRT-PCR. The expression stability was analyzed by GeNorm,NormFinder,and BestKeeper software. The results showed that the most stable expression were miR6448 and U6 genes. miR6448 had moderate expression abundance and its Ct was 23. U6 gene had high expression abundance,and the CT was about 10. Therefore,miR6448 can be used as the moderate expression abundance reference gene for miRNA qRT-PCR analysis in J. curcas under chilling stress. U6 gene can be used as the high abundance reference gene for miRNA qRT-PCR analysis in J. curcas under chilling stress.

Identification and Analysis of Alternative Splicing in Longissimus dorsi of Sheep at Different Development Stages

BAO Jing-jing, PU Ya-bin, MA Yue-hui, ZHAO Qian-jun

2019, 35(7):  33-38.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1078

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The alternative splicing（AS）is an important regulatory mechanism of protein diversity and genetic diversity in animals and plants.The objective of this study was to identify and analyze alternative splicing in the longissimus dorsi of sheep at different developmental stages. The transcriptomic profiling of the longissimus dorsi of 90-day-old fetus（F90）,30-day-old lamb（L30）and adult 3-year-old sheep（A3Y）were sequenced,and the alternative splicing event（AS）and differential splicing gene（DSG）were identified using rMATS software. The GO function enrichment and KEGG pathway analysis were performed for DSG. The results showed that 13 923,11 959 and 12 164 alternative splicing events were identified in genome of longissimus dorsi tissues in the F90,L30 and A3Y,respectively,and the proportion of skipped exons（SE）was the highest,about 70.69% of the total number of AS. The proportion of 5' splice sites（A5SS）,3'splice sites（A3SS）,mutually exclusive exons（MXE）and retained introns（RI）was about 7.28%,11.18%,5.96%,and 4.84%,respectively. 2 545,2 689,and 1 701 significant differentially spliced genes（P<0.05）were identified in the F90_vs_L30,F90_vs_A3Y and L30_vs_A3Y groups,respectively（P < 0.05）. The GO and KEGG analysis showed that differentially spliced genes were significantly enriched in striated muscle development,muscle structure development,myocyte differentiation,sarcolemma,insulin signaling pathway,Wnt signaling pathway,MAPK signaling pathway and other pathways closely related to muscle development. These results suggest that alternative splicing plays an important role in the development of the longissimus dorsi.

Comparison of Biological Characteristics of Feline Mesenchymal Stem Cells Derived from Four Different Tissues

LUO Dong-zhang, LUO Hui-na, ZHAN Xiao-shu, LI Xin-yi, HUANG Man-qing, LIN Jie-wei, HUANG Qi-liang, HONG Chun, LIN Hao-mei, CHEN Sheng-feng, WANG Bing-yun

2019, 35(7):  39-45.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0149

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To compare the biological characteristics of Mesenchymal Stem Cells（MSC）derived from adipose,bone marrow,amniotic membrane and umbilical cord,whole blood culture was used to isolate bone marrow-derived MSC and digestion to isolate others-derived MSCs. Then the morphology,growth characteristics,differentiation capacity,and cell surface markers were compared and analyzed. The results showed that feline MSC derived from 4 different tissues had common characteristics of adherent growth,spindle-shaped and polygonal-shaped. They all had the ability of adipogenic and osteogenic differentiation. The cell proliferation rate of adipose derived-MSC and bone marrow derived-MSC were faster than amniotic membrane derived-MSC and umbilical cord derived-MSC,and the doubling time of MSC from adipose and bone marrow was shorter. The adipose derived-MSC demonstrated the best growth activity and potential application prospect at clinic,because it maintained fine proliferative activity when they passed to the 9th generation. Cell surface markers of CD44,CD90 and CD105 were higher expressed,and CD34 were less expressed in four kinds of MSC.

Cloning,Expression and Comparative Analysis of Two Progesterone Receptor Genes in Cyprinus carpio

ZHU Rui, YE Yu-qing, WANG Ya-xin, YANG Chen-ru, WANG Hong-wei, SUN Xiao-qing, ZHANG Yan, LI Shang-qi, LI Jiong-tang

2019, 35(7):  46-53.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1089

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Progesterone receptor（Pgr）is a receptor mediating the regulation of progesterone on fish gonad development and germ cell maturation. To study the expression pattern and differential expression tendency of two common carp（Cyprinus carpio）Pgr genes,Pgr1 and Pgr2 were cloned using rapid amplification of cDNA ends. The full-length cDNA sequence lengths of Pgr1 and Pgr2 were 2 713 bp and 2 730 bp,both of which encoded proteins of 628 amino acids. They had a nucleotide identity of 91% and a protein identity of 90%. They both contained two domains,zinc finger domain and nuclear hormone receptor domain homologous superfamily. In the phylogenetic analysis,common carp Pgr1 and goldfish Pgr1 were clustered together and common carp Pgr2 and goldfish Pgr2 were grouped together. These two branches were then clustered with zebrafish Pgr. The ratio of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site between Pgr1 and Pgr2 was 0.360,suggesting that they were under negative selection. qRT-PCR analysis demonstrated that Pgr1 and Pgr2 had higher expression levels in the gonads than in the other tissues,revealing that both genes might be associated with the gonad development of common carp. They had higher expression levels in sperms and oocytes than in embryos after induced by spawning drugs,indicating that they might be involved in germ cell maturation. In the most examined cases,significantly higher Pgr1 expression level was revealed compared with Pgr2,suggesting that Pgr1 plays the dominate role in common carp gonad development and germ cell maturation.

Recombinant Expression of Mytilus coruscus Mytilin-1 Mature Peptide in Pichia pastoris and Its Antibacterial Activity

ZHANG Ya-li, TAO Yan, XIE Jing, QIAN Yun-fang

2019, 35(7):  54-60.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0104

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Mytilins are small cationic antibacterial peptides expressed predominantly in the haemocytes of shellfish,including multiple isoforms,and they play an important role in the innate immune system of shellfish. Mytilin-1 is one of the isoforms. The primary structure of Mytilus coruscus mytilin-1 is composed of signal peptide,mature peptide and C-terminal extension peptide. The mytilin-1’s mature peptide consisting of 34 amino acid residues determines its biological activity;8 conserved cysteine residues in the mature peptide may form 4 pairs of disulfide bond to stabilize the structure of mytilin-1 mature peptide. In the present study,having mytilin-1 mature peptide from M. coruscus as the recombinant DNA-expressed protein,a codon-optimized sequence（named as “mMy1”）corresponding to the cDNA encoding mytilin-1 mature peptide was synthesized based on the preferential codon usage of Pichia pastoris,and ligated to the expression vector pPICZαA and electronically transformed into competent P. pastoris X-33. The positive transformants containing multi-copy gene insertions were screened using high-concentration zeocin. The recombinant mMy1 was induced for 96 h with 1.0% methanol at 29℃,250 r/min and pH6.0. Tricine-SDS-PAGE and Western blot analysis demonstrated that the recombinant mMy1 with a molecular mass of 4.8 kD was expressed successfully in P. pastoris X-33. Bacteriostatic test indicated that the culture medium supernatant containing recombinant mMy1 showed antibacterial activity against Gram-positive（i.e.,Staphylococcus aureus and Bacillus subtilis）and Gram-negative（i.e.,Escherchia coli）bacteria.

Screening and Identification of High Uricase-producting Strain from Marine and the Enzymatic Properties

ZHANG Qing-fang, PANG Fei, YU Shuang, XIAO Jing-hui, DOU Shao-hua, CHI Nai-yu

2019, 35(7):  61-69.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1057

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Uricase is used in routine clinical analysis and clinical medicine widely. Its importance in medical treatment and diagnosis is increasing. A high uricase-producting strain Z7 was screened from the Dalian Huanghai Sea mud and seawater by transparent circle method and enzyme coupling spectrophotometry,and identified as Bacillus fastidiosus by 16S rDNA phylogenetic analysis,morphology and physiological and biochemical characteristics. Results showed that the molecular weight of the protein was about 33 kD. The optimum temperature for uricase was 25℃,and specific activity was 673.7 U/mg . The optimum pH for uricase was 8.0,and it was stable in the pH range of 8.0 to 9.0. Its relative enzyme activity could still maintain more than 90% after 1 h’s incubation. The activation of Fe³⁺ and Ca²⁺ on uricase was stimulative effects,while Ag⁺,Hg²⁺ showed strong inhibitory effects. Owing to the excellent chill enzyme activity and stability,Z7 can lay a theoretical foundation for the industrialized production of uricase and has potential development value.

Identification of Bacillus cereus Strain Producing Fibrinolytic Enzyme from Sipunculus nudus

LI Xiao-mei, ZHOU Zong-hui, YIN Xiu-hua, JIANG Hong-rui, LIU Xiao-ling

2019, 35(7):  70-75.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0109

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Screening and isolating the fibrinolytic strains of Sipunculus nudus in the North of Guangxi by a combination of skim milk plate and fibrin plate assay,the specificity and fibrinolytic mechanism of the strain were analyzed. As a result,5 strains isolated and screened from Sipunculus nudus showed ability of fibrin degradation. The crude enzyme fermented from the strain GXUSP-1 presented the maximal fibrinolytic activity as 303.2 U/mL（equivalent to urinary kinase activity unit）. GXUSP-1 was identified as Bacillus cereus,and belonged to marine symbiotic bacteria. Meanwhile,thrombolytic effect in vitro of the crude enzyme fermented from GXUSP-1 was significant,which dissolved thrombus both directly and indirectly. GXUSP-1 demonstrates promising potential industrial application.

Nematicidal Activity of Bacteria against Bursaphelenchus xylophilus and Its Fermentation and Culture Characteristics

ZHANG Wan-jun, WU Xiao-qin, WANG Ya-hui

2019, 35(7):  76-82.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0010

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The aim of this study is to screen bacteria with efficient nematicidal activity against Bursaphelenchus xylophilus. Immersion test was to assess the nematicidal activities of 3 Bacillus strains,then the morphological depiction on the polypides of nematodes killed by fermentation filtrate of the selected efficient strains were observed,as well as the culture condition and the stability of substances with nematicidal activity were also determined. The results revealed that the corrected mortality of nematodes was 100% after B. xylophilus were treated for 48 h with fermentation filtrate by 3 bacteria strains. The corrected mortality of nematodes was still up to 99.55% and 88.32%,respectively when they were treated for 48 h with B. cereus JK-XZ3 in fermentation filtrate diluted for 4 times and 8 times. It was significantly higher than the corrected mortality of nematodes treated with B. pumilus HR10 and B. velezensis YH-20 fermented filtrate. Moreover,the polypides of nematodes treated with the fermentation filtrate of JK-XZ3 were broken,and contents overflowed,and body wall was dissolved. The filtrate of JK-XZ3 fermented in 30℃ for 4d presented the highest nematicidal activity,and the corrected mortality of nematodes,when treated for 48h with the fermentation filtrate diluted for 4 and 8 times,were both up to 100%,respectively;the corrected mortality of nematodes was more than 84% when the nematodes were treated for 48 h in the 8 times-diluted fermentation filtrate processed at 40、60、80 and 100℃;at pH of 8-10,the corrected mortality of nematodes was > 88% when they were treated for 48 h in 8 times-diluted fermentation filtrate. It indicated that the fermentation filtrate of the strain demonstrated the characteristics of high temperature and alkali resistances. B. cereus JK-XZ3 is a highly virulent nematicidal strain with potentials of research and application.

Isolation of Antifungal Substances from Bacillus amyloliquefaciens BA-26 and Its Antifungal Activity against Botrytis cinerea

LIU Chao, LIU Hong-wei, WANG Bu-qing, ZHAO Wen-ya, WANG Ya-na, ZHANG Li-ping

2019, 35(7):  83-89.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0055

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The objective of this study are to analyze the antifungal substances produced by Bacillus amyloliquefaciens BA-26,to clarify the antifungal effects of these active substances,and to explore the mechanisms of their inhibition to plant pathogenic fungi. Antifungal substances produced by B. amyloliquefaciens BA-26 were isolated and purified by ammonium sulfate deposition,macroporous resin and RP-HPLC methods. Using the plate diffusion method,the inhibitory spectrum and the antifungal activities of these components were detected. The minimum inhibitory concentration of each purified component was determined by double dilution method,and their antifungal mechanisms to hyphae and spore germination were studied. The crude extract from strain BA-26 fermentation presented a broad inhibitory spectrum,and had significant inhibitory effect on various plant pathogenic fungi,such as Rhizoctonia solani and so on. Eleven antifungal components were isolated from the antifungal crude extract. The antifungal activity of A6-19 and A6-20 components showed the strongest antifungal activity against Botrytis cinerea,and the minimum inhibitory concentration was 7.81 μg/mL. These components inhibited the growth of B. cinerea mycelium,and resulted in them enlarged and thickened;in addition,they destroyed cell membrane,and inhibited spore germination. In this study,antifungal substances of B. amyloliquefaciens BA-26 were isolated and their mechanisms were preliminarily explored. These results laid the foundation for the application of these antimicrobial substances,especially in the field of anti-pathogenic fungi.

Pan-genome Sequencing and Comparative Genomic Analysis of Atrazine-degrading Bacteria

WANG Ya-li, ZHU Shan-shan, YANG Feng-shan, MA Yu-kun, FU Hai-yan, LIU Chun-guang

2019, 35(7):  90-99.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0360

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Arthrobacter aurescens TC1 and Pseudomonas sp. ADP are currently the model strains of atrazine-degrading bacteria. Microbacterium sp. HBT4 was screened in this experiment for discovering the similarities and differences of biological information among the 3 bacterial genomes and predicting important genes. In this study,pan-genome sequencing was carried out by using small DNA library preparation and sequencing technology on Illumina Hiseq 4000 sequencing platform. Genome composition analysis,gene function annotation,gene mutation detection and comparative genomics analysis were carried out by using related software. The nucleotide composition,collinearity and variation differences between the isolated Microbacterium HBT4 and model strains were analyzed. The genome size of the strain was about 3.53 Mb. It was predicted that there were 3 397 coding genes,1.33% repetitive sequences and 63 non-coding RNA in the strain HBT4. There were 3 324 annotations of gene function in general database and 1 149 annotations of gene function in special database. SNP,Small InDel and horizontal transfer genes were found through analyzing variance among strains and no structural variation genes were found. The number and proportion of GO-annotated genes in the specific genes of the strain in cell components,molecular functions and biological processes were obtained. From KEGG metabolic pathway enrichment map,it was found that the dihydrothiosyllysine residue succinyltransferase encoded by the specific gene was located between the metabolic pathway of α-ketoglutaric acid and succinyl coenzyme A in the tricarboxylic acid cycle. The proportional distribution,phylogenetic tree and collinearity of the core genome and non-essential genome of the three strains were obtained. It was found that there were 986 common gene families among the three strains,and 1171 specific gene families strain HBT4. Compared with the two model strains,the strain HBT4 obtained in this study had both similarities and differences in gene family.

Screening,Identification of Naphthalene Degrading Bacteria at Low Temperature and Optimization of Their Degradation Conditions

GUO Ya-nan, ZHANG Xin-yu, XU Meng, WANG Ji-hua

2019, 35(7):  100-107.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0362

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The enrichment culture method was used to screen low temperature strains using naphthalene as the sole carbon source and energy source from the contaminated soil in Daqing Oilfield,Heilongjiang Province. The gas chromatography-mass spectrometry（GC-MS）was employed to study the degradation of naphthalene by degrading bacteria in naphthalene-inorganic salt medium. The single-factor test and orthogonal test were applied to determine and optimize the culture conditions of the degrading bacteria,and the main controlling factors in the degradation stage were analyzed. The results showed that 2 strains efficiently degrading naphthalene under low temperature condition were screened and named as GN1 and GN2. GN1 and GN2 rapidly degraded naphthalene under low temperature conditions. On the basis of the non-biological factors of the control group,the degradation rate of naphthalene（300 mg/L）reached 94.43% and 95.47% in 4 days,indicating that they had obvious advantages in tolerance and degradation rate. The 2 strains were identified as Pseudomonas by morphological observation,physiological and biochemical characteristics and 16S rDNA gene. The strains GN1 and GN2 grew best in naphthalene-inorganic salt medium（Naphthalene concentration 300 mg/L）,culture temperature 15℃,initial pH 6.0,culture rotation 180 r/min,and culture time 7 d. The growth of the 2 bacteria was significantly related to 5 environmental factors.

Preliminary Study on the Regulation of Mxr1 Phosphorylation by Ptp

HOU Cheng-lin, YANG Yan-kun, CHEN Jia-li, BAI Zhong-hu

2019, 35(7):  108-113.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0020

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The small molecule tyrosine phosphatase Ptp interacting with Pichia pastoris transcriptional activator Mxr1 was found by PULL-DOWN,and its function of dephosphorylation was verified,resulting in the hydrolysis of phosphate group in phosphorylated Mxr1 215th serine. Phosphorylation of Mxr1S215 in different media was detected by Western blot of Mxr1S215th specific site phosphorylation antibody. It was found that Mxr1in the wild strain was not phosphorylated in the glycerol medium,while phosphorylated in the methanol medium. In the Ptp-highly expressed strain,no phosphorylation occurred in Mxr1 in either glycerol or methanol medium,but phosphorylation was significantly higher in the Ptp-knockout strain than in the wild type one,indicating that Ptp indeed regulated Mxr1 phosphorylation. The expression in Escherichia coli was conducted,then the proteins were purified,and the Ptp enzymatic properties were determined. The P. pastorisMxr1S215A mutant strain was constructed,and several genes related to methanol metabolism were found to change at the transcriptional level,suggesting that it was regulated by Mxr1 phosphorylation at 215th site.

Expression and Enzymatic Characterization of Codon-optimized FAD-dependent Glucose Dehydrogenase in Pichia pastoris

DONG Cong, GAO Qing-hua, WANG Yue, LUO Tong-yang

2019, 35(7):  114-120.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0941

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The objective of this work is to obtain high-yield FAD-dependent glucose dehydrogenase（FAD-GDH）. First,by optimizing the codons of FAD-GDH gene according to the codon preference of Pichia pastoris,the gene fragment was artificially synthesized,then the recombinant vector pMD-GDH containing FAD-GDH e gene was constructed and transferred into P. pastoris（X33）,and the secretory expression by methanol induction was achieved. Results showed that a stable recombinant strain with high enzymatic activity was obtained by screening positive transformants at test-tube level. In the 10 L fermenter after 136 h induction culture,enzyme activity reached 257 600 U/L. The analysis of enzymatic characterization demonstrated that the optimal pH and temperature while using glucose as substrate were 7.0 and 55℃,respectively. The initial enzyme activity still remained 70% after 150 min treatment at 50℃. In the range of pH 4-7,FAD-GDH still retained over 50% activity after incubated at 37℃ for 4 h. Cu²⁺ presented relatively large inhibition to enzyme activity. FAD-GDH harbored a relative high substrate specificity and D-glucose was the optimal substrate. The efficient expression of FAD-GDH in P. pastoris is achieved by optimizing the codon preference of P. pastoris,which provides a theoretical basis for the detection of blood glucose.

Probe on the Catalytic Mechanism of Glycosyltransferase in Grapefruit for Quercetin and Its Analogues

YE Jing-si, DOU Wen-fang, WANG Cheng

2019, 35(7):  121-128.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0057

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Using Glycosyltransferase（FGT）in grapefruit as catalytic enzyme,UDP-glucose,UDP-N-acetyl-D-glucosamine,and UDP-mannose as glycosyl donor,quercetin and naringin as glycosylation receptor,glycosylation was studied. The reaction of the product was detected by high performance liquid chromatography,mass spectrometry and nuclear magnetic resonance（NMR）hydrogen spectrum. The glycosylation reaction of quercetin with UDP-glucose and UDP-N-acetyl-D-glucosamine was confirmed. The molecular weight of quercetin glycoside was 464 and 505,respectively. According to the results of nuclear magnetic resonance,the structures of the products were quercetin-3-O-beta-D glucoside and quercetin-3-O-beta-D-N-acetyl-D-glucosamine. It was verified that FGT enzyme was used to modify quercetin glycosylation.

Establishment of NF-κB Luciferase Reporter Cell Line with Commonality

ZHENG Hai-liang, WANG Dong-fang, FANG Ya, DIAO Xiao-wei, WEN Yong, YUAN Yun-xia

2019, 35(7):  129-133.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0058

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This work is aimed to establish luciferase reporter gene system with commonality. Lentivirus containing NF-κB-RE-Luc2P was transduced into HEK293 and monoclonal HEK293 cell line expressing luciferase reporter gene under control of NF-κB response element was established and verified with TNF-α stimulation. To demonstrate the cell line capacity of commonality and modification,FGF family receptor FGFRⅡwas further transduced into this cell line and the screened FGFRⅡ/NF-κB-RE-Luc2P HEK293 single clones was further validated by FGF analogue stimulation. Dose-response effect and more than 100 fold of signal to noise ratio were observed in this cell line in TNF-α stimulation,indicating the cell line was generated successfully. In response to FGF analogue stimulation,dose-response effect was fitted as the “S” curve,implying that the cell line can be applied to the activity detection of FGF-type medicine. The results showed that NF-κB-RE-Luc2P HEK293 and modified one FGFRⅡ/NF-κB-RE-Luc2P HEK293 was established. that the cell line has commonality regarding NF-κB signaling.

Antigenic Epitope Analysis and Preparation of Monoclonal Antibody of MMP-9

XU Ying-xue, YIN Huan-cai

2019, 35(7):  134-140.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1059

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In this study,we used bioinformatics analysis to predict antigenic epitope region of MMP-9,which was designated as Δmmp9. Analyzing the nucleotide sequence of the Δmmp9,the primers were designed to amplify the target fragment,and the recombinant pET28α（+）-Δmmp9 expression vector was constructed,and transferred into Escherichia coli competent cell BL21（DE3）for inducing the recombinant protein Δmmp9. Then recombinant protein Δmmp9 was purified,and then was used to immunize BALB/c mice as antigen. Furthermore,7 hybridoma cell lines steadily secreting monoclonal antibodies（MAbs）were screened,and titers of seven MAbs all reached 1：240 000. In summary,MMP-9 antigenic epitope region is screened by bioinformatics methods and its MAbs are prepared successfully,its promising specificity is confirmed by antibody pairing test,and 3 captured antibodies and corresponding labeled antibodies are screened out.

Construction of TOX3 Gene Lentiviral RNA Interference Vector and Effect on Proliferation of Human Breast Cancer Cells ZR-75-1

HAN Cui-cui, LIU Li-kun, WANG Yu-chun, YANG Ying, LIU Ji-cheng, ZHOU Zhong-guang

2019, 35(7):  141-147.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0868

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This work aims to construct TOX3 gene lentiviral RNA interference（RNAi）vector and observe its effect on the proliferation of human breast cancer ZR-75-1cells. First,designing interference sequence targeting the TOX3,constructing vectors and then packaging lentiviral vectors and measuring the virus titer after sequence identification were conducted. Then the recombinant lentiviral vector was transfected into human ZR-75-1cells,the number of cells expressing GFP was observed under fluorescence microscope,and then the expression of TOX3 in the transfected ZR-75-1cells was detected by real time-PCR and Western blot. Finally,the effect of TOX3 on ZR-75-1 cells proliferation was measured by MTT assay and colony formation experiment. The results showed that the vector sequence of each group was confirmed correctly by gene sequencing,the virus titer was > 2×10⁸ TU/mL,and over 95% cells expressed GFP under the fluorescence microscope after being transfected,the expression of TOX3 mRNA and protein significantly reduced in the interference groups,particularly the interfere effect of TOX3-shRNA-3 group was the best,the proliferation and monoclonal ability of ZR-75-1cells decreased after the transfection. In sum,the study successfully constructed TOX3 gene lentiviral RNA interference vector,and the proliferation of ZR-75-1cells decreased after TOX3 was silenced.

Research Progress in Na⁺ Regulation Mechanism of Plants Under Salt Stress

LI Xiao-yuan, XIE Li-nan

2019, 35(7):  148-155.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0088

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Salt stress is one of the main abiotic factors limiting crop production. Excessive soluble salt（mainly Na⁺）in soil leads to secondary stress such as osmotic stress,ion stress and oxidative stress in plants. Under high-salt stress,plants rely on Na⁺ efflux or ion compartmentation strategies to reduce cell Na⁺ concentration in cytoplasm and then reconstruct or maintain the ions homeostasis in plants. This paper mainly reviews the progress in pathway and regulation of Na⁺ ion dynamic balance in plant cells under salt stress. A thorough understanding of the ion dynamic equilibrium process is conducive to creating more stress-tolerant crop varieties and achieving sustainable agricultural development.

Advances in Transcriptome Research on Plant Response to Waterlogging

WANG De-yun, LUO Guang-ming, LIU Pei-pei, ZHOU Li, XU Yue-ying, CHEN Yun-ting

2019, 35(7):  156-161.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0312

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Transcriptome sequencing technology allows the study of the species gene function and specific physiological processes at the mRNA level. This technology can be used to study the expression levels of all genes under drowning stress,which is conducive to revealing the response mechanism of plants to waterlogging. We reviewed the differential gene expressions in the pathways of signal transduction,endogenous hormones,photosynthesis,respiratory metabolism and reactive oxygen scavenging in plants under waterlogging stress when using transcriptome sequencing technology,as well as in various transcription factors and functional proteins. Besides,we gave the prospect in the future research so as to provide a reference for the study of plant under waterlog condition.

Research Progress of Fe³⁺ Reductase Genes（FRO）in Plants

QIAO Meng-xin, LI Su-zhen, CHEN Jing-tang

2019, 35(7):  162-171.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1012

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Fe is an essential microelement for plants’ growth,and the appropriate iron content is beneficial to the normal growth and development of plants. In terms of the physiological process of absorbing iron,the monocotyledon plants and the non-monocotyledon plants are different in valence state,and the transformation between Fe²⁺ and Fe³⁺ exists in the transport process of iron in plants. Fe³⁺ chelate reductase gene FRO have the function of reducing Fe³⁺ to Fe²⁺,thus it is of great significance to study the specific function and regulation mechanism of FRO at the molecular level. In this paper,subcellular localization,reduction objects,induction conditions of FRO in Arabidopsis thaliana,tomato,soybean,rice,peanut and Tribulus terrestris are reviewed aiming at providing a theoretical basis for studying Fe uptake mechanism.

Effects of Nanomaterials on Plant Growth and Development

GAO Meng-di, SHENG Mao-yin, FU Ji-feng

2019, 35(7):  172-180.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1022

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Nanotechnology has been widely concerned since its birth in the late 1980s. Because of its special properties that many large-size materials do not have,and with the emergence of a variety of new types of nanomaterials and their excellent performance in soil improvement and plant growth,nanotechnology has shown great application prospects in the fields of forestry and agriculture. In order to make full use of nanomaterials and explore the regulation,mechanism and risk of plant growth and development,the application of nanomaterials in plant growth and development is reviewed in this paper,and the future research direction is also prospected.

Genetic Diversity of Aegilops tauschii Coss. and Its Utilization in Improving Common Wheat

ZHAO Xin-peng, ZHOU Yun, LÜ Lin-lin, LI Suo-ping, ZHANG Da-le

2019, 35(7):  181-189.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1103

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As the donor species of D genome in common wheat（Triticum aestivum）,Aegilops tauschii has abundant genetic diversities,which provides valuable genetic resources for improving common wheat since the genetic background of T. aestivum is becoming increasingly consistent. This paper systematically summarizes the genetic information of A. tauschii based on the recent researches of domestic and overseas,including its genetic diversity and utilization in wheat breeding. This review is aimed to provide references for better utilizing the germplasm resource of A. tauschii.

Research Advances on Phytase and Prospect of Applying Soil Phytase

DING Rui, CHEN Xu-hui, LI Bing-xue

2019, 35(7):  190-195.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1027

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Phosphorus is a major nutrient-limiting factor in crops,and it is significant to solve the phosphorus limitation in crop by developing and utilizing the resources of soil organic phosphorus. Phytic acid acts as the major form of soil organic phosphorus,the process of its mineralization and releasing soil available phosphorus is an enzymatic reaction,and phytase is a key in this process. In present,phytase has showed great application potential in the sustainable development of agriculture. The advances of high-throughput sequencing technologies have provided new ideas and strategy on the study of soil phytase. Here,the diversity,production technique,the strategies of improving production as well as application situation in agriculture are summarized,the issues and the trends for future development of soil phytase are analyzed,and its application prospect is carried out as well.

Research Progress on the Mitochondrial Pyruvate Carrier（MPC）

XIE Wen-ya, ZOU Shi-ying, Gao Ru-xin, HE Xiao-yun

2019, 35(7):  196-201.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0926

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The mitochondrial pyruvate carrier（MPC）in mitochondrial inner membrane transports the pyruvate from the cytoplasm to the mitochondrial matrix. The pyruvate involves in the tricarboxylic acid cycle,gluconeogenesis,and the metabolic processes such as lipids and amino acids,which provides energy to the body. Therefore,MPC regulates energy metabolism by controlling the flux of pyruvate into the mitochondrial matrix. This paper summarized the biological characteristics of MPC and its relationship with metabolic diseases. It has been found that inhibition of MPC activity may lead to a decrease in hepatic gluconeogenesis,which in turn affects glucose homeostasis and reduces the incidence of typeⅡdiabetes. It also prevents the transmission of insulin signaling by affecting glucose-stimulated insulin secretion,resulting in that body is not sensitive to external glucose uptake;thus the MPC complex is essential to the glucose homeostasis. However,the activity of MPC is inhibited in most cancer tissues,specifically activating the MPC function should be conducted to enhance its aerobic metabolism,to inhibit that the cancer cells use lactic acid to supply energy and subsequently to hinder the proliferation of cancer cells. MPC is considered to be an initial point for studying energy metabolism regulation and potential therapeutic target sites,and also provides new therapeutic ideas for studying metabolic diseases such as obesity,diabetes and cancers.

Research Advance for the Hazard Risks and Antimicrobial Bioactive Peptides of Food-borne Pathogenic Microorganisms

XU Chong-xin, ZHANG Cun-zheng, LIU Yuan, ZHAG Xiao, ZHONG Jian-feng, LIU Xian-jin

2019, 35(7):  202-212.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1010

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Food-borne pathogenic microorganisms are the main biological hazards that contaminate food and edible-agricultural products and seriously threaten the health of humans and animals. At the same time,they also cause huge economic losses to food and edible-agricultural products industry,which is the key factor of restricting their development. Traditional dependence on antibiotics has a significant effect on pathogenic microorganism prevention and control. However,due to long-term unreasonable use or even abuse of antibiotics,the drug resistance of pathogenic microorganisms,as well as potential harmfulness to normal functions of internal organs,intestinal tissues,nervous and metabolic systems in humans and animals,are also prominent. Antimicrobial bioactive peptides,as biological peptides with antimicrobial functions,are considered as antimicrobial substances friendly to humans,animals and ecological environment,and they expected to replace antibiotics in pathogenic microorganism prevention and control. Now,antimicrobial bioactive peptide has become a hot spot to explore new and safe antibacterial drugs. In this paper,we comprehensively sort out their types,pollution characteristics and main hazard risks of food-borne pathogenic microorganisms,and systematically summarize their main types and source of antimicrobial bioactive peptides and their creation techniques. Then we emphatically introduce the current research status of antimicrobial bioactive peptides for food-borne pathogenic microorganism prevention and control. Finally,we discuss their application prospect,existing issues and countermeasures. To sum up,this paper is to provide the latest references or innovative idea for screening as well as prevention and control of food-borne pathogenic microorganisms in food and edible agricultural products.

Research Progress in the Quantitative and Unitive Detecting Technologies Based on Functional Nucleic Acid and Labeled Fluorescence

XIAO Bing, LUO Yun-bo, HUANG Kun-lun, ZHANG Yuan, XU Wen-tao

2019, 35(7):  213-221.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0592

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Functional nucleic acid is a natural or an artificial nucleic acid sequence with specific spatial conformation and biological function. It has advantages such as easy to be modified,low cost,high stability and strong specificity. After combined with fluorescence biosensors,the functional nucleic acid-based fluorescent biosensors have been widely used in the detection of environmental pollutants and risk factors of food as well as disease diagnosis. First,we clarify the concepts of functional nucleic acid and quantitative fluorescence detecting technology,and elaborates in detail the features of several key fluorescent materials,as well as their biological molecular recognition,the reaction principles and the luminous models with functional nucleic acid. Then,we describe,assess,and compare the quantitative and unitive detecting technologies based on functional nucleic acid and labeled fluorescence and their applications. Finally,we discuss the research significance and issues while applying the quantitative and unitive detecting technologies of functional nucleic acid and labeled fluorescence in varied detections and prospect the direction of future development and application.

Advances in the Techniques of Studying Translatome

WEI Kang-ning, CUI Jun-xia, WANG Meng-lei, WANG Li, LI Yong-fang

2019, 35(7):  222-229.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0006

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High-throughput sequencing-based RNA-Seq has been widely used to detect the expression and regulation mechanism of genes in tissues and cells. However,published literature have shown that mRNA level cannot reflect the real abundance of the related protein,while translatome that studies the being-translated mRNAs is more correlated to the actual abundance of proteins than the transcriptome. Therefore,studying translatome is more theoretical and practical meaning than studying transcriptome. In this paper,we reviewed the main techniques in studying translatome,from the most classical polysome profiling to the recently developed ribosome profiling,ribosomal affinity-purification,and RNC-RNA-Seq,and further compared the advantages and limitations of each technique. We also briefly discussed the prospect of studying translatome for providing a reference in studying translatome.

“Past and Present”Species Name of Bacillus velezensis

LIU Yang, LIU Xiao-kun, CHEN Wen-hao

2019, 35(7):  230-232.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0600

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Bacillus velezensis is a common functional bacterial species in the environment,and its taxonomic status and species name determination has always been the focus of debate among bacterial taxonomists. Until recently,the effective species name of B. velezensis was determined. In this paper,the evolution process of the species name of B. velezensis is systematically expounded in a time-smooth manner,in order to give readers a clear understanding of the origin and alternation of the species name of B. velezensis.