Table of Content

26 June 2019, Volume 35 Issue 6

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Establishment of a CRISPR/Cas9-mediated Gene Cluster-knockout System in the Endophytic Streptomyces SAT1

TIAN Wen-jia, DOU Gui-ming, WANG Sha, SUN Jing-ya, MA Yu-chao

2019, 35(6):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1080

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The hyBl（hygB-like）gene cluster,a secondary metabolic gene cluster in endophytic Streptomyces SAT1,is 52% similar to the hygromycin B gene cluster,and may play a key role in SAT1 inhibiting Cryphonectria parasitica. In order to further study the antibacterial mechanism of the strain and the function of the hyBl gene cluster,hyBl gene cluster was used to establish the CRISPR/Cas9-mediated gene cluster-knockout system in SAT1. A series of conditions were optimized,including the screening of protospacers,the construction of knockout vectors,the conjugation and transformation of donor bacteria,the selection of Ca²⁺ and Mg²⁺,and conjugation time,etc.,and the knockout system of gene cluster for SAT1 was established. Finally,a mutant SAT1△hyBl（14 kb deleted）was obtained with Escherichia coli ET12567（pUZ8002/pCRISPomyces2-CB）as a donor and conjugating on the MS（containing 10 mmol Mg²⁺）for 16-18 h. The antibacterial activity of SAT1△hyBl was 77% lower than that of wild strain,indicating the synthesized secondary metabolites guided by hyBl gene cluster indeed played an important role in inhibiting chestnut blight. This study demonstrates that the CRISPR/Cas9 system can be used for gene cluster editing in endophytic Streptomyces SAT1,and provides promising technical support in further studying the antimicrobial mechanism in SAT1 and verifying the functions of unknown gene clusters.

Construction of Processing Tomato α-Man Mutant Based on CRISPR/Cas9

ZHANG Yuan-yuan, SHAO Dong-nan, CUI Bai-ming

2019, 35(6):  9-16.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0134

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In order to cultivate tomato variety with low-softening-degree and long-shelf-duration fruit,the first exon of the N-glycan processing enzyme gene（α-Man）was edited directly by the hCas9 nuclease combined with 21 bp guide RNA（gRNA）and while driven by tomato U6 promoter. Firstly,the plant expression vector based on CRISPR/Cas9 system was constructed,and the transgenic lines of tomato were obtained by Agrobacterium-mediated genetic transformation. Then the DNA fragment near α-Man’s editing site from the genomic DNA of transgenic tomato leaves was detected and sequenced by restriction endonuclease and PCR amplification. The results showed that 2 of 14 transgenic tomato strains were detected to have mutations. The sequencing results of the α-Man mutant TA clone suggested that there were 2 types of editing,one was a 52 bp deletion mutation and the other was a single base mutation. This indicates that the editing of tomato α-Man gene is achieved.

Cloning and Expression Analysis of the PpNAC72 in Peach

WU Xiao-qi, FENG Tao, LIU Yan-jun, JIAO Si-peng, SUN Pan, YANG Jing-hui

2019, 35(6):  16-23.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0891

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The aims are to clone the PpNAC72 from the peach sepecies “Jinliuzaohong”,to analyze the expression profile in different development stages,and to explore its role in the peach growth. RT-PCR was used to clone the PpNAC72,and subsequently to quantitatively analyze its expression profile and bioinformatics. The results showed that the PpNAC72（GenBank accession number：MH627940）had an open reading frame sequence of 1 050 bp,encoding 349 amino acids,an isoelectric point of 8.34,and a NAM domain with typical NAC family structure. Amino acid homology analysis demonstrated that PpNAC72 had high similarity to the reported NAC of other plants,and the NAC protein had the highest homology with sweet cherry. The results of qRT-PCR revealed that PpNAC72 was expressed in different degrees in peach organs,and the expression level was the highest in fruits. The expression level was also significantly varied in different periods,and the expression level was the highest in the middle and late growth stages of peach fruit. The PpNAC72 might be involved in the maturation of peach as a transcription factor.

Transcriptome Sequencing and Analysis of Xanthoceras sorbifolia Bunge Fruit

ZHAO Yang-yang, GUO Yu-xiao, ZHANG Ling-yun

2019, 35(6):  24-31.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1101

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In order to study the expressions of functional genes in the transcriptome of Xanthoceras sorbifolia Bunge fruit,the RNA-Seq technology was used to sequence Xanthoceras sorbifolia Bunge fruit in different stag. The sequencing results showed that a total of 68 298 Unigene sequences after sequencing and assembly were obtained from the samples in 2 stages,and 24 691 Unigene sequences were annotated,accounting for 36.15%. Based on GO database,the 14 766 Unigene sequences were divided into 3 categories of cellular component,molecular function and biological process and 54 function groups. The 13 994 Unigene sequences were grouped into 25 functional categories based on KOG database. Based on KEGG metabolic pathway database,8 284 Unigene sequences of X. sorbifolia Bunge fruit may be classified into 127 metabolic pathways. And 11 732 SSR（Simple Sequence Repeats）loci were discovered in X. sorbifolia Bunge fruit transcriptome. Most of them were single nucleotide SSR,accounting for 60.85%. This study provides the foundation and reference for molecular biology study in X.sorbifolia Bunge fruit.

Effects of Shading and Phosphorus Application on Growth and Chlorophyll Fluorescence Parameters of Fagopyrum dibotrys（D. Don）Hara

WANG Rui-jie, GUAN Ping, LIU Xiao, YANG Shu-jun, JI La-la, DENG Xiao-hong, WANG Jian-jian

2019, 35(6):  32-38.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0079

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The effects of shading and phosphorus application on the growth and chlorophyll fluorescence parameters of Fagophyrum dibotrys（D.Don）Hara（Abbreviated as FD）were studied for laying a theoretical foundation for setting light and phosphorus during the cultivation of FD. Setting 3 shading strengths（no shading;light shading：shading 40%-45%;severe shading：shading 90%-95%）and 2 phosphorus application tests（not added and add 14.4 g/pot）. Shading increased the morphological indicators of FD（such as leaf area,height,root length and other morphological indicators）,as well as chlorophyll content and chlorophyll fluorescence parameters,but reduced leaf thickness. Phosphorus application increased the number of FD leaves,leaf area,height,biomass accumulation,photochemical quenching coefficient（qP）and other indicators. Compared with the control,shading 40%-45% promoted the morphology,biomass,and photosynthetic efficiency（Yield）of the FD more largely than shading 90%-95% while applying phosphorus. In sum,appropriate shading and phosphorus application in actual production are beneficial to the reduction of photosystem damage by the FD,thus promoting the growth of morphological indicators and increasing yield.

Molecular Cloning and Prokaryotic Expression Analysis of Protein Kinase SnRK1 Subunit α Gene from Jatropha curcas

WANG Hai-bo, GUO Jun-yun, TIAN Xue-lian

2019, 35(6):  39-47.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0948

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The SnRK1（Sucrose non-fermenting-1 related protein kinase 1）in plant is ortholog of yeast SNF1（Sucrose non-fermenting-1）and mammalian AMPK（AMP-activated protein kinase）. These evolutionarily conserved kinases are involved in metabolic sensors and cellular signaling caused by energy depletion and low-carbon status. Based on the complete genome data,bioinformatics was used to identify a protein kinase SnRK1 subunit α gene from Jatropha curcas,it was named JcSnRK1α and then cloned and characterized. The results showed that the full-length cDNA of JcSnRK1α was 1700 bp with 11 exons and 10 introns,containing a 1545 bp open reading frame（ORF）. The ORF encoded 514 amino acids with the molecular weight 58.8 kD and the pI value 8.57. Domain analysis by CDD revealed that the JcSnRK1α composed of catalytic kinase domain with T-loop region,together with an ubiquitin-associated domain（UBA）and a kinase-associated domain（KA1）. TATA box,CAAT box,heat,low temperature-,drought-,wound-,and gibberellin-responsive elements were identified in the promoter of JcSnRK1α. qRT-PCR analysis showed that JcSnRK1α expressed specifically in different organs,abundantly in stems and roots,but scarcely in leaves. Meanwhile,JcSnRK1α gene was of remarkably cold-induced expression in stems and roots,which reached the highest after 3 h and 0.5 h chilling stress,respectively,increasing 2.04 and 3.16 times compared to the control. A prokaryotic expression vector pGEX-4T-1-JcSnRK1α was constructed and transformed into Escherichia coli Rosetta,and SDS-PAGE results showed that the molecular weight was 84.8 kD,which was consistent with the expected weight. This study laid a foundation for further studies on the gene function and the mechanism in signaling transduction and stress response in J. curcas.

Cloning and Expression Analysis of SikCML7 Gene from Saussurea involucrata

LIU Zhao-shu, LIU Yuan-yuan, QIN Yu-jie, BI Quan, WANG Sai-sai, ZHU Jian-bo

2019, 35(6):  48-54.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1056

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Calmodulin-like protein is one of the most important Ca²⁺ receptors and participates in the adaptation process of plants to environment. In order to explore whether or not calmodulin-like proteins are involved in the plant response to stress,the SikCML7 gene was cloned based on the low temperature transcriptome of Saussurea involucrata. Bioinformatics analysis indicated that the open reading frame of SikCML7 was 450 bp and encoded 149 amino acids,and there were 4 calcium binding regions（EF-hands）in the encoded proteins. Phylogenetic tree analysis indicated that this gene was close to Arabidopsis AtCML9 in the evolutionary affinity. The results of real-time quantitative PCR showed that the expression of SikCML7gene was up-regulated after treatment with two different low temperatures（4℃/-2℃）,drought and salt stress respectively;however this gene demonstrated significantly different expression patterns under chilling stress and freezing stress as well as the certain responses to drought and salt stress. In sum,SikCML7 indeed participates in the responses process of S. involucrata to low temperature,draught,and salt stresses.

Functional Analysis of Calcium-dependent Protein Kinase CPK14 in Pollen Tube Growth

ZHOU Li-ming, LU Xin-rui, MA Sheng-wei, FANG Wei

2019, 35(6):  55-61.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0219

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The purpose of the study is to reveal the biological function of CPK14（calcium-dependent protein kinase 14）in pollen tube polar growth. Based on the available microarray database,the expression heat-map of pollen-specific CPK was depicted. The constitutively active CPK14（CA-CPK14）was constructed by removing the auto-inhibitor and Ca²⁺ binding domain at the C-terminal and retaining the complete kinase and N-terminal variable regions. The resulting construct（full-length CPK14,CA-CPK14 or GFP alone）was transiently expressed in tobacco pollen tubes via particle bombardment,and the overexpression phenotypes of transformed tubes were compared and analyzed. The effect of CA-CPK14 on the activity of ROP1 was analyzed by co-expression experiment of CA-CPK14 and GFP-RIC4ΔC（a marker of active ROP1）. The results showed that eight pollen-specific CPKs（including CPK14）were identified,and they were highly expressed in mature pollen grains and less expressed in other tissues. CPK14 overexpression caused a swelling tip of pollen tubes,while CA-CPK14（continuous activation and lack of Ca²⁺ regulation）inhibited the germination and growth of pollen tube. ROP1 was a key regulator of pollen tube growth. The expression of CA-CPK14 inhibited the accumulation and distribution of active ROP1 maker（GFP-RIC4ΔC）at the top of pollen tube. These results suggest that CPK14 may play a role in the ROP1-mediated regulation mechanism of pollen tube growth.

Growth,Lipid Accumulation,and Fat Composition of Eustigmatos sp. Under Different Nitrogen Source and Concentration

ZHAO Wei, LI Tao, WU Hua-lian, CHEN Hao, LIU De-Hai, XIANG Wen-zhou, WU Hou-bo

2019, 35(6):  62-68.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0009

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Eustigmatophyceae can accumulate high content of eicosapntemacnioc acid（EPA）,which is of great value for development and utilization. The aims of the present work are to investigate the effects of different nitrogen sources and concentrations on the growth,fat accumulation and fatty acid composition of Eustigmatos sp. tolerating high salt. Using G-11 as basic medium,3 nitrogen sources（sodium nitrate,urea,and bicarbonate）were selected and 3 nitrogen concentrations（3.5 mmol/L,5.9 mmol/L and 17.6 mmol/L）for each nitrogen source were set,the time-course changes in biomass,total lipids content,fat classification and fatty acid composition of Eustigmatos sp. were traced and determined. The results showed that the highest biomass concentration of Eustigmatos sp. was 7.07 g/L while ammonium bicarbonate（17.6 mmol/L）as nitrogen source. The total lipids yield by 3 nitrogen sources at 3.5 mmol/L was higher than that at 5.9 mmol/L and 17.6 mmol/L,and while under 3.5 mmol/L it was the highest 4.18 g/L by urea as nitrogen source,following 4.07 g/L by sodium nitrate,and the lowest 3.42 g/L by ammonium bicarbonate. Nitrogen deficiency increased the proportion of neutral lipids in Eustigmatos sp. The proportion of neutral lipids at 3.5 mmol/L was higher than that at 5.9 mmol/L and 17.6 mmol/L. At 3.5 mmol/L condition,the content of neutral lipids in urea group,ammonium bicarbonate group and sodium nitrate group were 79.0% TL（total lipids）,77.0% TL and 75.7% TL,respectively. The fatty acid composition of Eustigmatos sp. was mainly C14∶0（myristic acid）,C16∶0（palmitic acid）,C16∶1（palmitoleic acid）,C18∶1（oleic acid）,C18∶2（linoleic acid）,C20∶4（arachidonic acid）and C20∶5（EPA）. The content of C16∶1 was 51% in total acid fatty（TFA）. The highest percentage of C20∶5（EPA）was at 17.6 mmol/L,7.18% TFA by sodium nitrate,followed by 6.20% TFA by urea and the lowest 4.82% TFA by ammonium bicarbonate. In conclusion,Eustigmatos sp. presents great development potential.

Tolerance and Adsorption of 2 Photobionts to Heavy Metal Cu and Zn

Jimilamu JIAMALI, Miheriban ABILIMITI, Guhainisha MAIMAITI, Ainiwaer TUMIER

2019, 35(6):  69-75.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0018

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In the bioremediation of heavy metal pollution,the potential of algae absorbing heavy metals has attracted the attentions of many researchers. In order to investigate the difference in adsorption and tolerance of photobiont isolated from lichen body to heavy metals Cu²⁺ and Zn²⁺,2 photobionts（B and P.E）were used as research materials,Evan’s blue staining method,BCO and dithizone spectrophotometric method were to determine the cell viability of photobiont,as well as the Cu²⁺ and Zn²⁺ content in culture medium and algae. The results showed that the cell viability and heavy metal adsorption of the 2 photobionts differed under different concentrations of Cu²⁺ and Zn²⁺. Tolerance to Cu²⁺ stress and adsorption was as following P.E > B. Under Zn²⁺ stress,the tolerance after 9 d of culture was P.E > B;as the culture time prolonged,the cell viability of P.E decreased sharply and was lower than that of B,but the adsorption rate was still at P.E > B. Moreover,the tolerance and adsorption of 2 photobionts to Zn²⁺ stress were significantly higher than Cu²⁺ stress. The study found that the adsorptions of 2 photobiont to Zn²⁺ stress were significantly higher than that to Cu²⁺ stress. The study found that the two algae species of Peltigera lichen,special organisms from the symbiosis of bacteria and algae-,had higher tolerance and adsorption to Cu²⁺ and Zn²⁺ stress than some freely-growing algae.

Construction of An Oxidation Pathway of Xylonic Acid in Bacillus subtilis for Production of Glycolic Acid

LING Xie, JI Ming-hua, DUAN Hai-yan, SHI Ji-ping, SUN Jun-song

2019, 35(6):  76-82.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0038

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It is aimed that a strain of a food-safety bacterium producing glycolic acid can be obtained by over-expressing foreign genes. An over-expression system comprising of xylonate degradation pathway from Escherichia coli was constructed and introduced into Bacillus subtilis（A164S）,glycolic acid was detected as the main product in the broth of recombinant B. subtilis,using high performance liquid chromatography（HPLC）,and the conversion rate of xylonite to glycolic acid reached 42.54%. Meanwhile,Bacillus subtilis,because of its own nonspecific aldolase and glyoxal dehydrogenase,obtained the ability to biologically transform xylic acid under the condition that only xylose dehydratase YjhG was expressed. In conclusion,a strain of B. subtilis producing glycolic acid from xylic acid is successfully constructed and the possible existence of YjhH isozyme is uncovered.

Influence of mglB Gene Knockout in Engineered Escherichia coli Producing Ethanol on the Xylose Utilization of Mix Sugar Fermentation

XU Qiong-dan, WANG Yong-ze, WANG Jin-hua, ZHAO Xiao

2019, 35(6):  83-90.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0962

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To investigate the influence of glucose non-PTS transferring gene and its synergistic effect with PTS transferring gene on xylose utilization,a glucose transferring gene mglB was knocked out from engineered Escherichia coli SZ470 and its ptsG mutant SZ470P via RED homologous recombination technique,thus mglB-deleted SZ470M and ptsG/mglB-deleted SZ470P were constructed. The fermentations of mix sugar（3% glucose +2% xylose）by 4 strains were compared,and the transcriptional changes of genes related to xylose transferring and metabolism through all 4 strains were also investigated. The results showed that there were no obvious differences between SZ470M and SZ470 in fermentation. The xylose consumption rate,yield and conversion rate of ethanol by SZ470PM were 0.37 g/L,23.25 g/L and 82.6%,respectively,which increased by 32%,9.8% and 5.8% than those in starting strain SZ470P,respectively. The transcriptional analysis also demonstrated that the transcriptional levels of genes related to xylose transferring and metabolism rose in both SZ470M and SZ470PM rose. In sum,the knockout of both ptsG and mglB synergistically increase the xylose utilization rate,which contributes to the efficient utilization of xylose when using lignocelluloses materials for fermentation.

Identification and Characterization of Bacteria Degrading Polyvinyl Alcohol

LIU Ya-lan, DUAN Meng-jie, LIN Xiao-shan, ZHANG Yi

2019, 35(6):  91-98.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1045

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The aim of this work is to isolate and identify the strains which can effectively degrade polyvinyl alcohol（PVA）from the composting degradation process of PVA foams and further to study the degradation effects and characteristics of these strains on PVA. First,the PVA foam was degraded after 3 years composting,and we used Fourier Transform Infrared Spectroscopy（FTIR）to detect the molecular structure change of the foam. Then we used Finley method to screen the strains with better degradability from the degraded foams. Further we combined morphological characteristics,gram staining experiment and 16s rRNA gene sequence alignment to identify the strains. Finally we employed UV ultraviolet spectrophotometer to measure the degradation rates of PVA by various strains and the OD₆₀₀ of cells in shake flask,and we also compared the degradability of PVA by the screened strains at 2 alcoholysis degrees. The results showed new carboxyl groups and hydroxyl groups appeared in the molecular structure of the PVA foam after 3 years composting degradation;4 strains were screened and identified,including Bacillus sp. DG01,Bacillus sp. DG02,Paenibacillus sp. DG03 and Paenibacillus sp. DG04. The degradation rates by these 4 isolated strains at 3.0 g/L PVA1788 were 67.27%,74.99%,56.74% and 59.70%,respectively,and the degradation rates at 3.0 g/L PVA1799 were 58.27%,54.47%,43.32%,and 46.59%,respectively. The PVA foams presented obvious group changes after the composting;the growth of 4 strains was coupled with the degradation of PVA,and the low alcoholysis degree of PVA was more beneficial to the biodegradation.

Effect of Antimony on the Enzyme Activity of Danio rerio

YUE Xin, YANG Ai-jiang, XU Peng, HU Xia, ZHU Huan-yi, BAO Xin

2019, 35(6):  99-106.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1084

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We aim to study the changes of various enzymes’ activities in organisms under heavy metal antimony stress and to identify the response mechanism of organisms to antimony stress. The changes of different enzyme activities in zebrafish were explored at different concentrations of antimony solution（0,10,20,30,and 40 mg/L）and period（24,48,96,and 144 h）. The results showed that the concentration of alkaline phosphatase in zebrafish was not dependent on the concentration of antimony,while the other 7 enzymes were significantly inhibited. With the increase of antimony stressing time,the activities of adenosine triphosphate and catalase in zebrafish presented a trend of first decreasing and then increasing;the activities of peroxidase,superoxide dismutase,acetylcholinesterase,lactate dehydrogenase,and acid phosphatase showed a “zigzag” trend;while the activities of alkaline phosphatase showed a trend of first increasing and then decreasing. In conclusion,antimony significantly affects the free radical regulation,excitation conduction,energy metabolism,substance transport,body growth and immune defense in zebrafish.

Functional Identification of RstA in Pseudomonas fluorescens Strain 2P24

LI Di-yin, HE Yong-xing, HAN Jian-ting, LI Kun, WANG Zhi-ping, LI Miao-hui

2019, 35(6):  107-113.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0097

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This work is to identify the function of transcriptional regulator RstA from OmpR subfamily and clarify the role of RstA in the regulation of efflux pump EmhABC in Pseudomonas fluorescens strain 2P24. Cofitness data was obtained from Cofitness Browser to explore potential functions of RstA. The rstA and emhABC-deficient mutant strain ΔrstA and ΔemhABC were constructed by homologous recombination and minimum inhibitory concentration（MIC）values of wild-type strain,ΔrstAand ΔemhABC to several antibiotics were detected. Quantitative real-time PCR assay and β-galactosidase assay were performed to detect the transcriptional levels of emhABC in the wild-type strain and ΔrstA. Electrophoretic mobility shift assay（EMSA）of the purified His-RstA protein were employed to access the interaction between RstA and the emhABC promotor. As results,RstA presented similar fitness pattern with EmhABC,which was predicted to be responsible for adapting several antibiotics stress conditions,and the tolerances of ΔrstA and ΔemhABC to multiple antibiotics decreased. Comparing to the wild-type strain,ΔrstA showed 3-fold down-regulation to the transcription and expression of emhABC. Results of EMSA indicated that the recombinant purified protein His-RstA was able to bind to the promoter of emhABC specifically. Transcriptional regulator RstA directly activates the expression of efflux pump EmhABC by binding to the emhABC promotor and contributes to multi-antibiotic resistance in P. fluorescens strain 2P24 .

Immunoregulatory Effect of Polysaccharides from Codonopsis pilosula on the Ana-1 Macrophages in Mice

SHI Bao-zhong, HU Jian-ran, LI Ping, XU Kai

2019, 35(6):  114-118.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0889

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The immunoregulatory effect of polysaccharides from Codonopsis pilosula on the Ana-1 macrophages of mice was investigated. MTT method was used to determine the effects of C. pilosula polysaccharides on Ana-1 cell proliferation. ELISA assay was applied to detect the effects of C. pilosula polysaccharides at different doses on TNF-α and IL-1β secretion in Ana-1 macrophages. C. pilosula polysaccharides at different doses were given to mice by intrapefitoneal injection,then the thymus index,proliferation of lymphoma cells,and the levels of TNF-α and IL-1β in the serum of mice were determined. The in vitro test showed that C. pilosula polysaccharides significantly increased TNF-α and IL-1β secretion of Ana-1 macrophages in a dose-dependent way,but presented no effect on Ana-1 cell proliferation. The in vivo tests indicated that the spleen index,lymphocyte proliferation index,and the levels of TNF-α and IL-1β in the serum of middle-dose and high-dose polysaccharides groups significantly increased in a dose-dependent way,compared to control mice. Therefore,C. pilosula polysaccharides may effectively improve the immune activity of Ana-1 macrophages in mice,i.e.,improve the immune functions of mice.

Effect of Cryopreservation Density on Cryopreservation Effect of Peripheral Blood Mononuclear Cells

LIN Ke-jia, LIU Fu-ping, MA Dong-lei, HU Rui, WEI Zong-ke, TAN Yi

2019, 35(6):  119-124.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0646

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The objective of this study is to investigate the effect of cryopreservation density on the cryopreservation of peripheral blood mononuclear cell（PBMC）. Fresh PBMC group（Group F）and 3 PBMC cryopreserved cell density groups：2×10⁷ cells/mL（group A）,4×10⁷ cells/mL（group B）,6×10⁷ cells/mL（group C）were set up. By comparing these data,such as the survival rate of PBMC before and after cryopreservation,as well as the amplification multiples,lymphocyte subsets,and in vitro killing efficiency of multiple cytokines-induced killer（CIK）cells after resuscitation,the cryopreservation effect was validated. Results shows as,the cell viability of group F,group A,group B and group C before and after cryopreservation were（96 + 0.3）%,（95.6 + 0.4）%,（94.7 + 0.2）% and（94.9 + 0.4）%,respectively,i.e.,the cell viability of group B and group C were significantly lower than that of group A（P<0.05）. At 14 days after cell amplification,the cell amplification of group F and group A,group B,and group C were（156.4 + 18.2）times,（160.2 + 28.4）times,（126.1 + 19.8）times,and（110.4 + 11.3）times,respectively,i.e.,that cell amplification of group B and group C was significantly lower than that of group F（P < 0.05）. There was no significant difference in the results of lymphocyte subsets before PBMC freezing and after PBMC resuscitation. Compared with group F,at 14 d after cell amplification of group A,group B,and group C,the lymphocyte subsets and the in vitro killing efficiency of CD3+,CD3+CD4+,CD3+ CD8+,CD3-CD56+ and CD3+CD56+ demonstrated no significant difference（P>0.05）. In conclusion,too high cryopreservation density may affect the cryopreservation effect of PBMC so as to indirectly affect the cell resuscitation. However,the too low cryopreservation density increases the cryopreservation volume so as to greatly raise cost. Therefore,choosing appropriate cell cryopreservation density is an issue that must be considered for a cell library or cell bank.

Effects of Small Molecule Precursors on Bafilomycin A1 Biosynthesis

ZHOU Jian, SUN Fei, FANG Zhi-kai, JIANG Hong

2019, 35(6):  125-130.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1001

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The objective of this work is to explore an effective method of increasing bafilomycin A1 productivity via understanding the effects of small molecule precursors on bafilomycin A1 biosynthesis. The effects of different precursors on the biosynthesis of bafilomycin A1,the optimal concentration and feeding time of the effective precursor were mainly studied. As result,valine was the optimal precursor for bafilomycin A1 biosynthesis. By feeding of 0.2% valine into the fermentation broth at the 36th hour of cultivation,the bafilomycin A1 production reached 285.5 mg/L,which was 78.4% higher than that in the condition without any addition of precursor. In conclusion,feeding precursors significantly influences bafilomycin A1 biosynthesis,and the addition of valine is effective in increasing bafilomycin A1 production.

Microsatellite Polymorphism and Its Correlation Analysis with Body Size Traits of Tan Sheep

LI Biao, ZHANG Rui-ying, WANG Xiao-qi, ZHANG Cun-fang, DUAN Zi-yuan

2019, 35(6):  131-137.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0073

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Tan sheep,a special Mongolia sheep breed in China,is famous with its delicious meat and slow growth rate. In our study,a total of 29 high polymorphic microsatellite markers were used to screen 96 Tan sheep individuals,which were sampled from the basic ewe population in Yanchi Tan sheep reservation farm in the Ningxia Hui Autonomous Region,China. Analysis of genetic diversity,population structure and co-relationship between molecular genotype and measured phenotype（body weight and body size traits）of the 96 samples were carried out,and the following results were obtained：the average（Na）and mean effective（Ne）numbers of alleles,the average expected heterozygosity（He）,the average observed heterozygosity（Ho）,and the average polymorphism information content（PIC）were 9.5,4.5,0.72,0.64 and 0.69,respectively. The sampled population held high genetic diversity,the correlation between MAF33 and body height and chest depth existed,and BMS1788 was associated with rump height and chest circumference. Also,5 microsatellite markers including BL41,BMS835,BOVILS56,MAF33 and BMS500 were significantly correlated with the cannon circumference. These six microsatellite loci may be useful in further marker-assisted breeding for Tan sheep.

Research Advance on Pollen-wall Development in Male Sterility

ZHANG Jiang-jiang, CHANG Li, ZHAO Li-ning, LI De-fang

2019, 35(6):  138-146.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0814

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Pollen wall is a key component of a pollen and plays an important role in pollen development and fertilization. Many courses related with the development of the pollen wall are identified in the study of male sterility,for instance,the degradation of tapetum and callose,the formation and degradation of the primexine as well as the development of a pollen intine,present the special relationship with the male sterility. In this paper we summarize the development of pollen wall based on the the reports of male sterility,aiming at discovering the general pattern in the development of pollen wall as well as providing a theoretical support in studying functional and dissecting mechanism of pollen wall in male sterility.

Mechanisms of Stress and Mitigation of Heavy Metals on Seed Germination of Plants

LI Gui-ling, WANG Qi, WANG Jin-shui, JIA Feng

2019, 35(6):  147-155.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0938

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Heavy metals are non-essential elements to plant growth and have strong biotictoxicity. They are easily absorbed and accumulated by plants，but difficultly degraded，and cause damages to human health by good chain. This paper reviewed the effects of heavy metals on the morphological，physiological and biochemical，and molecular indexes during seed germination，including seed germination，the growth of plant seedlings，the activities of hydrolases such as amylase and esterase activities，antioxidant enzyme activities，soluble sugar and proline content，genotoxicity，and protein gene expression of the plants. In addition，the paper summarized the methods and treatment measures for alleviating toxicities from heavy metals，discussed the existing problems in the current research in this field，and highlighted the research direction for the treatment of plant heavy metal toxicity. It provides reference for the research and treatment of heavy metal pollution

Roles of African Swine Fever Virus Structural Proteins in Viral Infection

OU Yun-wen, LIU Li-jun, DAI Jun-fei, MA Bing, ZHANG Yong-guang, ZHANG Jie

2019, 35(6):  156-163.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0968

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African swine fever（ASF）is an acute,lethal,and hemorrhagic porcine infectious disease caused by African swine fever virus（ASFV）which has been caused serious economic losses to the pig industry. ASFV is a double-stranded DNA virus. The viral genome contains between 150 and 167 open reading frames（ORFs）,and encodes for more 200 proteins,around 50 of them are structural proteins. The structural proteins service as the major composition of viral particles,and play important roles in the assembly of virions,viral adsorption,entry and replication. This paper reviews the roles of ASFV structural proteins in viral infection,and provides guidelines for further studies in the structural proteins of ASFV.

Current Status and Progress on Prokaryotic Transcriptome Study

LIU Meng-yuan, LENG Yan, ZHAI Li-xiang, HE LI-fang, LI Shi-weng

2019, 35(6):  164-171.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0070

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The breakthrough of transcriptional technology in studying prokaryotes transcriptome has shown its unique advantage in revealing the molecular mechanism of prokaryotes’ life process. The transcriptome study of prokaryotes began with pathogenic bacteria,and recently the transcriptome analysis of the degradation mechanism of pollutants by prokaryotes has become a research focus. The metabolic mechanism,mechanism of action and transcription-related genes of degrading bacteria were further explored through multi-histological integration analysis. In this paper,the status and progress of the study on prokaryotic transcriptome are reviewed,i.e.,the degradation bacteria and their transcriptome characteristics in response to heavy metals,aromatic compounds,petroleum hydrocarbons and other organic pollutants are introduced. The transcriptome study of pathogenic bacteria is also focused,including human,animal and plant pathogenic bacteria species,nature,and transcriptome characteristics. The future development and application of transcriptome research in prokaryotes are also prospected. It aims to lay an important theoretical foundation for prokaryotes in environmental pollution control as well as prevention and control of human and animal and plant diseases caused by pathogenic bacteria.

Research Advances in Biosynthesis of Polyhydroxyalkanoates（PHAs）by Halophilic Bacteria

ZHANG Meng-ying, LI Ya-hui, ZHAN Yuan-long, LIU Chang-li

2019, 35(6):  172-177.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0843

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The accumulated polyhydroxyalkanoates（PHAs）in microorganisms are degradable bio-plastics,and replacing the petrochemical plastics by the green and environmental PHAs synthesized in microorganism reduces the pollution of white waste. PHAs biosynthesis from halophilic bacteria eliminates tedious sterilization and the harsh conditions of sterile cultures,thus ensuring more economical benefits and competitiveness than other microorganisms. Based on the research direction and progress of PHAs biosynthesis by halophilic bacteria at home and abroad,this paper classifies the PHAs synthesis by halophilic bacteria,as well as summarizes and analyzes the factors influencing PHAs synthesis by halophilic bacteria;meanwhile,the paper prospects the development of PHAs synthesis by halophilic bacteria.

Role of m6A RNA Methylation in Tumorigenesis and Development

LONG Wen-lin, GUO Hui, SHENG Jie, SONG Ru-hui, XU Yao

2019, 35(6):  178-186.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1082

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m6A methylation,which was first discovered in 1974,is a kind of methylation modification on RNA molecule. In recent years,it has become a research hotspot in life science. Modifications of m6A are dynamic and reversible in mammalian cells,which have been proposed as another type of epigenetic regulation similar to DNA and histone modifications. This RNA chemical marker is produced by the protein of m6A“Writers”and can be reversed by m6A“Erasers”（demethylase）. In addition,“Readers”can recognize the mRNA of m6A,and accordingly regulate the expression of downstream genes. m6A RNA methylation is involved in all stages in the life cycle of RNA,from RNA processing,nuclear export,translation modulation to RNA degradation,which suggests its potential of influencing a plurality of aspects of RNA metabolism. All of the recent studies reveal that m6A modification is a complicated regulation network in different tissues,cell lines,and space-time models,and it is be closely associated with tumor initiation and progression. This review focuses on the molecular regulatory mechanism of m6A,its physiological significance and the research progress in several kinds of human tumors,aiming at providing new insight for early clinical diagnosis and targeted therapy of cancer.

Research Advances on Afucosylated Trasuzumab

LEI Sha-sha, ZHU Hong-yu, ZHANG Guo-hua, XU Ming-bo, YANG Zhong-fan, YAO Wen-bing

2019, 35(6):  187-195.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1049

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The breast cancer,among the most common malignant cancers in women,seriously endangers women’s life and health,due to its high mortality rate. HER2-positive breast cancer results in the highest incidence and mortality. The trastuzumab presents significant clinical efficacy in the treatment of HER2-positive breast cancer. However,as the consequence of drug resistance,the abnormal expression of HER2 and high medication cost,the trastuzumab encounters huge limitations in the real-world clinical use. It is found that the knockdown of core fucose in the Fc region area of the antibody significantly improves the ADCC effect of trastuzumab and its clinical efficacy. This manuscript mainly reviewed the means to knockdown the core fucose of trastuzumab and the associated clinical advantages of afucosylated trastuzumab,together with the other Fc region engineering methods to improve the ADCC effect of monoclonal antibody.

Research Progress on the Development and Application of Dextranase

CHANG Guo-wei, HUANG Zeng-wei, LI Zhi-de, LIANG Da-feng

2019, 35(6):  196-204.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1021

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Dextranase is a kind of enzyme that can specifically catalyzes the hydrolysis of α-1,6-glucosidic bonds on dextran. Dextran,the substrate of dextranase,is a glucan produced by the fermentation of sucrose via some microorganisms. Dextran is widely used in medicine,food,materials and other fields,but it brings the adverse effects on oral health,sugar production,etc. Studies on dextran,dextran hydrolysates and other polysaccharides are getting increasingly advanced and dextranase plays an increasingly important role;but the overall development level of dextranase preparation is still not high and its application is still limited. This review summarizes the development and researches on the construction of dextranase-producing strains,fermentation and purification of dextranase,dextranase immobilization,characterization of enzymatic properties,enzyme activity enhancement,etc.,as well as the research progresses on applying dextranase in sugar production,food,medicine and materials,including the research results in our team. Then the paper discusses the issues in the development and application and prospects the future research and development directions.

Development of Dipstick for the Rapid Detection of Three Important Monilinia Species on Fruits

LIN Hui-jiao, YANG Hua-wei, GU Heng-sen, JIANG Xiang, ZHANG Hai-lei, LIU Yu-chen, ZHOU Er-xun

2019, 35(6):  205-212.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1006

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A rapid detection dipstick for fruit quarantine in port was studied and developed using three important brown rot pathogens（Monilinia fructicola,Monilinia laxa and Monilinia fructigena）on fruits. Using nitrocellulose membrane as the solid phase carrier,the translation elongation factor 1-alpha（EF-1α）of above 3 Monilinia species as detecting targets,and PCR amplification products of biotin marker as detecting markers,DNA-DNA hybridization was applied in the rapid detection of 3 Monilinia species with the developed dipstick. The specificity and sensitivity of the Monilinia dipstick（abbr. as MON dipstick below）were evaluated by using DNA of brown rot strains and the constructed recombinant plasmids of EF-1α as positive standard samples,respectively. The results showed that the MON dipstick specifically detected 3 Monilinia species;the whole detection process（including DNA extraction and amplification process）was completed in 2 h,and the lowest detection limit reached 10 fg/µL. In sum,the dipstick developed in this study is of high specificity,sensitivity and stability,and the detection result is visible by human eyes,thus it does not rely on expensive equipment and can be expanded and used in local quarantine laboratory.

Construction of Highly Efficient and Rigorous Targetron System in Escherichia coli

CHEN Xiang-hao, LIU Fang, WANG Cai-xia, CHEN Zheng-hong, HONG Wei, CAI Meng-di, ZHANG Zheng-rong, QI Ting-na, LIAO Yong-hui, GU Jun-ying, CUI Gu-zhen

2019, 35(6):  213-220.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0883

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The objective is to construct a highly efficient and rigorous Targetron gene targeting system in Escherichia coli. Firstly,the T7-lac operon from pET28a and the group II intron from pSY6 were assembled to construct an IPTG-inducible Targetron plasmid system in E. coli. Then,taking the lacZ gene as an example,the efficiency and stringency of the IPTG-inducible Targetron targeting system were verified by analyzing the insertion efficiency of the group II introns before and after induction. Finally,a highly efficient and rigorous inducible Targetron gene targeting system in E. coli was obtained after optimizing the IPTG concentration and induction time. As results,group II introns were unable to be inserted into 2 target sites at all in the absence of IPTG induction,the targeting efficiency was 0. When induced for 45 min after adding 0.5 mmol/L IPTG,the targeting efficiency at lacZ-635s site increased to 90.8±5.5%,and the targeting efficiency at lacZ-1063a site increased to 92.6±2.4%. In conclusion,the IPTG-inducible Targetron gene targeting system in E. coli is successfully established,laying a foundation for the research and application of the group II intron.

Construction of ATP Synthetic Strain and Its Application in the Production of S- adenosylmethionine

JIANG Lin-lin, WU Lei, XU Hai-xia, HUANG Jian-li, ZHANG Yong-jin, XU Qi, YANG Yong

2019, 35(6):  221-226.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0977

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In order to achieve ATP production as well as reduce the cost of producing S-adenosylmethionine,a recombinant Escherichia coli strain expressing adenosine kinase,adenylate kinase and acetate kinase simultaneously was constructed for ATP synthesis. The conditions of transforming ATP were optimized,and the optimized reaction system was as：adenosine 30 mmol/L,acetylphosphate dilithium 135 mmol/L,magnesium sulfate 5 mmol/L,borax 50 mmol/L,E. coli strain 2 g/L（wet weight）,initial pH 7.5,the reaction temperature 35℃,and reacting for 3 h,the conversion rate was over 99%. Based on the above result,the reaction system was enlarged in 5 L. After reaction ended,50 g/L（wet weight）recombinant E. coli strain expressing adenosylmethionine synthase,65 mmol/L D,L-methionine and 15 mmol/L magnesium sulfate were added to the reaction system,the concentration of S-adenosylmethionine was up to 8.7 g/L and the conversion rate reached 72.5% after 18 h transformation.