Table of Content

26 August 2019, Volume 35 Issue 8

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Genome-wide Bioinformatics Identification of PPR Gene Family and Expression Profiles Analysis in Wheat

GUO Cai-juan, GONG Jie, LIU Yong-jie, ZHAO Chang-ping, GAO Shi-qing, WU Hua-wei

2019, 35(8):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0165

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The aim is to screen the PPR（Pentatricopeptide repeats）gene with fertility restoration function for the hybrids of the two-line hybrid wheat,and to further study the function and regulation mechanism of PPR genes. After bioinformatics analysis of wheat genome-wide data,1 351 PPR members were obtained. Then,the chromosome location and distribution of PPR gene family in wheat were analyzed,and the phylogenetic tree was constructed. Total 23 PPR genes related to fertility restoration were obtained by homology align and analysis of them with reported fertility restorer genes in rice. Real-time fluorescence quantitative PCR was performed to screen and identify the expression pattern of 23 fertility restoration genes on young spikes of 2 different restorer lines of high and low,one sterile line,and their F₁ generation in two-line hybrid wheat at meiotic stage. Based on phenotypic analysis of important agronomic traits such as seed setting rate,it was found that the 4 PPR genes showed obvious over-dominance patterns in the high-restorer hybrid combinations,and correspondingly these 4 genes basically presented low-parent dominance expression patterns in low-restorer line hybrid combinations. It is preliminarily confirmed that these 4 PPR genes may be involved in the regulation of fertility restoration of the two-line hybrid wheat.

Screening and Identification of an Antagonistic Bacterium Against Rhizoctonia solani and Analysis of Biocontrol Factor

SONG Ben-chao, ZHAO Dong-mei, YANG Zhi-hui, ZHANG Dai, ZHAO Zhi, ZHU Jie-hua

2019, 35(8):  9-16.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0084

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In order to screen the biocontrol bacteria against Rhizoctonia solani,plate confrontation method was used to screen 9 strains of bacteria with better bacteriostatic effect from potato rhizosphere soil. The strain Z17-2 with the best antibacterial activity was identified as Bacillus amyloliquefaciens based on morphological,physiological and biochemical characteristics and phylogenetic analysis of 16S rDNA（GenBank accession number：MK271286）and gyrB（GenBank accession number：MK295695）sequences. Strain Z17-2 demonstrated antibacterial activity against Rhizoctonia solani,Alternaria solani,Fusarium oxysporum,Fusarium sambucinum,and Streptomyces scabie,while no inhibitory effect on Erwinia carotovora. By measuring the ability of producing lipopeptide,protease,cellulase,pectinase,amylase,iron carrier and phosphorus- and potassium-solubilizing,it was confirmed that strain Z17-2 contained lipopeptide-synthesized gene sfp,fenB,bacD and mycB,and produced protease,iron carrier,cellulase,while no pectinase,and presented no ability of dissolving phosphorus and potassium. The antibacterial ability of the lipopeptide extract in the toxic medium was determined to be 70.38%.

Effects of Continuous Cropping of Lettuce and Rotation of Lettuce-Spinach on Soil Bacterial Community Structure

HONG Jie, KANG Jian-yi, LIU Yi-qian, GAO Xiu-zhi, YI Xin-xin

2019, 35(8):  17-26.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0182

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The objective is to compare the differences in bacterial communities in soil between for the continuous cropping of lettuce and lettuce-spinach rotation. Soil samples were collected from depths of 0-20 cm. High-throughput sequencing technology was used to analyze the bacteria present in soil samples before and after lettuce planting. Sequencing data presented that 46 865 effective sequences were received after quality filter,and 21 692 OTUs were obtained through biological information on similar levels of 97% OTU statistical analysis. Heatmap and community composition analysis showed that bacteria 40 phylum,89 classes,190 orders,371 families,and 693 genera in the samples were detected,and the proportion of Proteobacteria,Firmicutes and Actinomycetes in the continuous process gradually reduced while the change in rotation process was stable. Moreover,the beneficial bacterial genera,including Agromyces,Arthrobacter,Flavobacterium,Lysobacter and Microbacterium,gradually became dominant in the rotation soil samples. The yield of rotating lettuce and catalase activity increased compared with that of continuous cropping. The results of principal component analysis demonstrated that there was significant difference between soil samples in rotation and continuous cropping. After the fifth consecutive planting of lettuce,the yield and catalase activity of lettuce decreased 24.1% and 20.7% respectively,and the obstacles for continuous cropping appeared;while the yield and catalase activity of lettuce in rotation were improved 49.6% and 10.5% respectively,compared with that in continuous cropping. In conclusion,there is the rich diversity of beneficial bacteria in rotating lettuce with spinach,and which increases with the increasing of rotating process,and partially alleviates the problems that occur during lettuce cultivation.

Screening and Identification of a Salt-tolerant Growth-promoting Bacterium and Its Effect on the Growth of Maize Seedlings

SUN Pei, WANG Gang, ZHANG Ya-nan, LI Qian, JI Jing, YANG Dan, YUAN Dong, , WANG Chang, WANG Yu-rong, WANG Ping

2019, 35(8):  27-33.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0145

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A strain of salt-tolerant bacteria was screened and isolated from maize rhizosphere using 10% salt concentration LB medium. The strain was identified by morphological characteristics and 16S rDNA sequence,and its growth-promoting characteristics were analyzed. The effect of the strain on maize seedling growth under salt stress was studied by pot experiment. The results showed that the strain was Kocuria rhizophila Y1 with phosphorus solubility. Under normal conditions,when K. rhizophila Y1 was colonized in the rhizosphere of maize seedlings,the plant height and root length increased by 44.96% and 49.45%,the total chlorophyll content increased by 74.44%,respectively,while the MDA content decreased by 0.65 times. Under salt stress,plant height and root length increased by 1.2 and 1.17 times,total chlorophyll content increased by 56.24%,CAT activity increased by 0.9 times,respectively,while MDA content decreased by 0.34 times. Therefore,K. rhizophila Y1 can be used as a biological fertilizer in saline soil to alleviate the inhibition of salt to the growth of maize seedlings and subsequently to promote the growth of maize seedlings.

Differentially Physiological and Biochemical Responses of Grape Cells to Contacted Co-culture Endophytic Fungal Strains

YU Man, CHEN Jing-chao, QU Jin-zhuo, LIU Fang, PAN Xiao-xia, YANG Ming-zhi

2019, 35(8):  34-41.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0211

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Endophytic fungal strains were classified according to the differentially physiological and biochemical responses of grape cells during their co-culturing with endophytic fungi,which may be conducive to screening endophytic fungal resources with varied application values. Grape callus（Vitis vinifera L. cultivar：Gamay）and 47 endophytic fungal strains from 18 genera isolated from grape varieties Rose honey,Cabernet Sauvignon and Xiahei were chosen as experimental materials to construct endophytic fungi-grape cell contacted co-culture system. Further the physiological and biochemical effects of these endophytic fungal strains on grape cellular,growth,anthocyanin contents,phenylalanine ammonia-lyase（PAL）activities and others were analyzed. Ours results indicated that different endophytic fungal strains initiated different degrees of impacts on the co-cultured grape cells in growth,anthocyanin contents and PAL activity. Among all those used endophytic fungal strains,20 conferred less damages to grape cells,6 significantly promoted the co-cultured grape cells in growth,11 significantly promoted the co-cultured grape cellular PAL activity,and 5 promoted the co-cultured grape cells’ anthocyanin contents. Beside the development of candidate endophytic fungal strains,this research provided a methodology for purposely screening candidate endophytic fungal resources.

Screening,Identification and Antifungal Properties of a Bacterium with Antagonistic Activities Against Mycotoxin-producing Aspergillus spp.

LIU Duan-mu, WU Yi, LIU Yun, LIANG Zhi-hong

2019, 35(8):  42-50.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0074

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To obtain the antagonistic strains against Aspergillus ochraceus and Aspergillus flavus and to fully exploit the beneficial microbial resources,efficient antagonistic strains against Aspergillus ochraceus and Aspergillus flavus were isolated and purified from plantation soil and Vigna angularis. Then,the morphology,physiological and biochemical reactions,16S rDNA phylogenetic analysis and specific PCR analysis were applied for the identification of the antagonistic bacteria. Furthermore,the bacteriostatic stability of the fermentation supernatant,etc. were studied. Strain SC-B15,isolated from the plantation soil,had strong antagonistic effects on A. ochraceus and A. flavus growth,and the bacteriostatic rates against A. ochraceus and A. flavus reached 47.30±13.17% and 52.44±2.78%,respectively. SC-B15 was identified as Bacillus amyloliquefaciens. The inhibition zone diameters of the supernatant against A. ochraceus and A. flavus were 15.03±2.66 mm and 13.95±2.62 mm,respectively. Meanwhile,the bacteriostatic substances remained stable at 20-40℃ and pH=6-10,and were insensitive to trypsin and papain. The indicated strains appeared abnormal changes of cavitation,swelling and deformation with microscope. In sum,B. amyloliquefaciens SC-B15,isolated from plantation soil,and its supernatant,efficiently inhibit the A. ochraceus and A. flavus growth and demonstrate certain development application value as antifungal agent in the foodstuff and feedstuff.

Research on the NAMase of Ensifer meliloti 1021 and Regulation Mechanism of 3-Cyanopyridine

GUO Jing-jing, GUO Lei-lei, ZHAO Yun-xiu, DAI Yi-jun

2019, 35(8):  51-58.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0027

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This work aims to clone and express the NAMase of Ensifer meliloti 1021,and to investigate its enzymatic properties and explore the enzymatic mechanisms regulated by 3-CP. PCR was used to amplify the gene nam of E. meliloti 1021 and the constructed recombinant nam-containing plasmid was imported to Escherichia coli Rosetta（DE3）cells for heterologous expression. Ni-NTA affinity chromatography was employed to purify the protein. The effects of temperature,pH,organic solvents and metal ions on NAMase activity were explored,and the immobilization of NAMase was further investigated. The results showed that the full-length of nam was 636 bp,encoded a 22.6 kD protein with PI of 5.5. The optimal pH for NAMase was 7,it retained over 97.1% of its initial activity after incubation at 30-50℃ for 2 h,and the optimal temperature ranged in 30-70℃. Ag²⁺ and isoamyl alcohol demonstrated the highest inhibitory effect on NAMase activity. 3-CP inhibited nicotinic acid production during the transformation of nicotinamide by E. meliloti 1021 and over-expressed NAMase in E. coli Rosetta（DE3）,and this was not a substrate inhibition.

Research on the Localization and Function of Atp4 in Schizosaccharomyces pombe

LI Qin, ZHANG Lin-lin, HUANG Ying

2019, 35(8):  59-63.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0111

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In this study,we studied the localization of Atp4 in Saccharomyces pombe and the mechanism of its involving in the mitochondrial function. The technologies utilized in the article included gene knock,fluorescence microscope,mitochondrial extraction,biochemical treatment,Western blotting,and so on. The Δatp4 deletion mutant glycerin medium grew defectively,and it was mitochondrial respiratory-deficient strain. The Atp4 with GFP label in C terminal showed co-localization with mitochondrion by fluorescence microscope observation. Treating Atp4-Flag and vyHL6381 mitochondria by biochemical reagents TritonX-100 and protease K and sodium carbonate revealed that Atp4 located in mitochondrial endometrium. Western blotting assays demonstrated that Atp4 deletion led to the sharp drop of Cob1、Cox1、Cox2 and Atp6 protein and the light drop of Cox and Cox4. Sum of above results,Atp4 plays its function in mitochondrion,and it is essential in maintaining the normal expressions of mitochondrial proteins and function of mitochondrial respiratory chain.

Response of Butyrate-oxidizing Microbial Community to the Co-effects of Antibiotics and Activated Carbon

FENG Gao, ZHANG Yu-chen , GOU Min, CHEN Ya-ting

2019, 35(8):  64-76.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0271

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An anaerobic digestion chemostat using butyrate as the sole carbon source was constructed in this study. 16S rRNA high-throughput sequencing was employed to investigate the dynamic changes of butyrate-degrading microbial community under inhibition of chlortetracycline（CTC）and co-effects of CTC and granular active carbon（GAC）,and to explore the interactions among microorganisms under environmental stress and their responses to sole CTC inhibition and co-effects of CTC and GAC. Our results showed that known syntrophic butyrate degrading bacterium Syntrophomonas（11.6%）and the aceticlastic methanogen Methanosaeta（48.5%）respectively dominated the bacterial and archaeal community in the original chemostat. Addition of 40 mg/L and 50 mg/L CTC led to the inhibitory effects on methane production reduction by 40.4% and 49.3%,respectively. Although Syntrophomonas presented tolerance to CTC,bacteria（e.g.,unclassified Firmicutes and unclassified Comamonadaceae）having positive correlations with it and known acetate-oxidizing bacteria Tepidanaerobacter were significantly inhibited by CTC,which thus affected the degradation of butyrate and caused the accumulation of metabolites,further resulting in the reduction of methane. Methane production reduced by 2.9%,48.5%,and 64.7% when adding GAC along,as well as adding GAC under the 40 mg/L and 50 mg/L CTC,respectively. Co-occurrence network analysis revealed that the addition of GAC significantly enhanced the activity of Geobacter and bacterium that showed positive correlation with it（e.g.,Azonexus）. However,Methanosaeta and Methanoculleus showed significantly negative correlation with most of the bacteria（e.g.,Azonexus）. Thus,the addition of GAC may indirectly affect the activity of Methanosaeta and Methanoculleus.

Isolation of Cold-adapted Cellulose-degrading Bacteria Using Three Different Carbon Sources and Analysis on the Degrading Ability of Consortia

MENG Jian-yu, JI Jin-hua, JIA Li-juan, GUO Hui-qin, TAO Yu, FENG Fu-ying

2019, 35(8):  77-84.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0956

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The cold-adapted cellulose-degrading bacteria were screened from the forest soil of Hulun Buir city using avicel,carboxymethylcellulose（CMC）and D-salicin as carbon source respectively at 4℃. The strains of high enzyme activity were respectively mixed to construct cold-adapted cellulose-degrading consortia and their filter paper degradation abilities were measured. As a result,172 strains of cold-adapted cellulose-degrading bacteria were isolated. Of them,the bacteria isolated with avicel or CMC as carbon source belonged to 4 classes,and the bacteria isolated with D-salicin as carbon source belonged to 6 classes. The first dominant class was γ-Proteobacteria with ratio of 54%,48% and 55%,respectively;the second dominant class was α-Proteobacteria with ratio of 28%,26% and 34% respectively. The first dominant genus was Pseudomonaswith ratio of 35%,26% and 26% respectively,the second dominant genus screened with avicel as carbon source was Rhizobium with ratio of 15%,the second dominant genera screened with CMC as carbon source were Rhizobium and Oerskasty with ratio of 12%,the second dominant genus screened with D-salicin as carbon source was Lelliottia with ratio of 19%. In 27 cold-adapted cellulose-degrading consortia,D13'1'' and D13'2'' presented the highest filter paper enzyme activity of 158.02 U/mL,which was 5-10 times more than that of any single strain in its consortia. D13'1'' and D13'2'' are the optimal flora combination of cold-adapted cellulose-degrading consortia with high application potential.

Enhanced Furfural Tolerance in Zymomonas mobilis by the Overexpression of Antioxidant Genes

WEN Yuan, XIA Juan, QI Liang-hua, LIU Xiao-wei, LIU Chen-guang, BAI Feng-wu

2019, 35(8):  85-94.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0040

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Furfural is a major inhibitor in lignocellulosic hydrolysate,which induces the generation of reactive oxygen species（ROS）. Exploration of relationships between antioxidant genes and furfural tolerance contributes to enhanced tolerance performance of strains. ;By overexpressing endogenous antioxidant genes NADH oxidase（ZMO1885）,NADP⁺ reductase（ZMO1753）,and glutathione reductase（ZMO1211）in Zymomonas mobilis,tolerance performance,ROS,and NADH/NAD⁺ were measured. The results suggested that overexpression of ZM4 ZMO1885 reduced the intracellular ROS level by 52.1%,increased the biomass by 38.9%,and improved furfural tolerance of ZM4/ZMO1885 with no significant interference of cellular NADH/NAD⁺ in the medium containing 2 g/L furfural,compared to the control. However,recombinant strain ZM4/ZMO1753 and ZM4/ZMO1211 did not demonstrate significantly elevated furfural tolerance. In the simulated lignocellulosic hydrolysate,the biomass of ZM4/ZMO1885 was 46.9% higher than that of the control strain,and the sugar consumption rate increased by 110.3% with the increased ethanol productivity by 195.2%. Collectively,the overexpression of antioxidant genes affects furfural tolerance performance,among which ZMO1885 overexpression improves furfural tolerance through promoting ROS elimination and intracellular furfural conversion without redox level perturbation.

Directed Domestication of Copper Tolerance for Enhancing Low-grade Chalcopyrite Bioleaching by Acidithiobacillus caldus

CUI Ya-quan, FENG Shou-shuai, HUANG Xing, CHEN Jin-cai, YANG Hai-lin

2019, 35(8):  95-102.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0103

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In the late stage of low-grade chalcopyrite bioleaching,the accumulated Cu²⁺ would inhibit the cell growth of bioleaching microorganisms and affect leaching efficiency,therefore,it is crucial to domesticate the copper tolerance of bioleaching microorganisms. After 6 months copper tolerance domestication of Acidithiobacillus caldus,the key parameters of the original strain and the domesticated strain in the pure culture and bioleaching process under different copper stress（0,1 and 3 g/L Cu²⁺）were analyzed. The highest specific growth rate（μ_max）in pure culture under 3 g/L Cu²⁺ stress increased from 0.21 d^-1（13 d）before domestication to 0.54 d^-1（10 d）. The efficiencies of copper leaching by the domesticated strain in 0,1 and 3 g/L Cu²⁺ leaching system increased by 17.64%,70.93% and 306.09% respectively,compared to the original one. The morphological observation of the leached slag by scanning electron microscopy（SEM）indicated that the degree of corrosion reduced along with the increased copper stress. Under the same stress conditions,the slag from domesticated leaching system presented more potential adsorption sites and obvious corrosion marks. Fourier transform infrared spectroscopy（FTIR）analysis of key functional groups showed that there were more sulfur-containing groups in the slags of domesticated leaching system. X-ray diffraction（XRD）analysis of slag composition implied that these derivatives in iron and sulfur elements such as Fe₃O₄ and FeS in the domesticated bioleaching system were rich. In sum,the domesticated A. caldus has strong resistance to copper stress and maintains a more active biochemical leaching effect in leaching system,which is expected to play a potential advantage in the similar industrial bioleaching process.

Effects of Phosphorus Concentrations on Growth and Metabolism of Seawater Spirulina platensis

CHEN Hao, YANG Bing-jie, LI Tao, WU Hua-lian, WU Hou-bo, XIANG Wen-zhou

2019, 35(8):  103-110.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0181

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Phosphorus plays an important role in the growth and metabolism of microalgae. In order to explore the feasibility of regulating the accumulation of major metabolites in seawater Spirulina by adjusting phosphorus concentration,we analyzed the physiological and biochemical effects of different phosphorus concentrations on seawater Spirulina platensis by using K₂HPO₃·3H₂O as phosphorus resource and setting 5 phosphorus concentrations 0.005-0.04 g/L based on seawater Zarrouk medium. The result showed that biomass production and protein content of seawater S. platensis were enhanced along with the increase of phosphorus concentration,and the maximum were 0.48±0.02 g/L and 59.23±0.61%DW,respectively,at the concentration of 0.04 g/L. The total polysaccharide content decreased significantly and then tended to be stable with the increasing of phosphorus concentration,and the maximum was 25.96±1.61%DW at phosphate concentration of 0.005 g/L. The content of C-phycocyanin,allophycocyanin and total phycobiliproteins increased first and then decreased along with the increasing of phosphorus concentration,and the maximum reached at the phosphate concentration of 0.03 g/L by 14.56±0.99%DW,4.21±0.19%DW and 18.77±0.39%DW respectively. The content of chlorophyll a and carotenoids increased along with the increasing of phosphate concentration,with the maximum value of 1.01±0.01%DW and 0.35±0.02%DW,respectively. The variation of phosphorus concentration generally demonstrated no significant effect on total lipids content and fatty acids composition（P>0.05）. The results showed that low phosphorus concentration was beneficial to the accumulation of polysaccharides;while high phosphorus concentration was beneficial to the synthesis of protein and phycocyanin,chlorophyll and carotenoid,thus the synthesis and accumulation of the main high-value products of seawater Spirulina could be effectively adjusted by changing phosphorus concentration.

Expression and Binding Activity Analysis of Chitin Deacetylase SeCDA7 from Spodoptera exigua Larvae

ZHANG Wei, BI Yang, ZHAO Dan, ZONG Zhao-li, GUO Wei

2019, 35(8):  111-117.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0137

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Chitin deacetylase（CDA）is an important component of insect chitin metabolism enzymes,thus recognized as the key target in pest control. RT-PCR method was used to clone a gene secda encoding CDA in Spodoptera exigua midgut. The secda was 1 431 bp in length,containing an ORF 1 134bp（GenBank accession number：MG604929）. After the signal peptide removed,the secreted SeCDA7 protein was predicted to have a molecular weight 43.156 kD. Domain analysis indicated that SeCDA7 had a polysaccharide acetyltransferase catalytic region belonging to the first class V CDA protein. Then,the prokaryotic and eukaryotic recombinant expression vectors were constructed,and Sf9 insect cells were infected using the Escherichia coli and Bac-to-Bac insect baculovirus expression system,and SeCDA7 was expressed successfully in the insect cells. The purification results of SeCDA7 protein and analysis of chitin binding activity showed that SeCDA7 protein had chitin binding activity. Fluorescence quantitative PCR results demonstrated that secda7 gene mainly expressed in the midgut. In sum,the heterogeneous expression of the chitin deacetylase gene secda7 in S. exigua is achieved and the SeCDA7 is identified to have chitin binding activity,which provides a theoretical basis for further investigation on the physiological function of the chitin deacetylase of S. exigua.

Correlation Between FSHR and ESR1 and Breeding Traits in Leizhou Black Duck

ZOU Kun, CUI Hong-yan, XUE Yuan, ZHANG Shao-wei, LU Li-li, ZHAO Zhi-hui, SU Ying

2019, 35(8):  118-126.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0094

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The aim of this study is to explore effect of the laying-related gene FSHR and ESR1 on Leizhou black duck. Quantitative PCR was employed to investigate the expression pattern of FSHR and ESR1 in hypothalamus-hypophysis-ovary axis of 0-120 d Leizhou black duck,and PCR-SSCP was used for analyzing the correlation between FSHR and ESR1 with breeding traits. The results showed that the overall expression trend of FSHR increased stably at 0-90 d and decreased slightly at 90-120 d in hypothalamus and ovary,but there was no significant difference（P>0.05）. The overall expression trend of ESR1 increased in 0-120 d but there was fluctuation at 90 d in ovary and difference was not significant（P>0.05）. The SNP for FSHR（g.856937G>A,g.922947A>G）and ESR1（g.190744A>G）were significantly associated with laying performance（P<0.05）,but were not correlated with egg quality（P>0.05）. The results suggest FSHR and ESR1 play an important role in early gonad development in Leizhou black duck,and three identified SNPs may be used as genotype maker for egg laying.

Effect of Adding Hawthorn and Astragalus Mixtures on the Plasma Metabolome of Perinatal Dairy Cows

ZHANG Meng, LIU Guo-lin, LI Xiang-long, CHEN Yong-hong, BAI Ling-rong, LUO Fang, LI Ya-chao, TAO Jin-zhong

2019, 35(8):  127-137.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0300

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This work aims to study the effects of adding hawthorn and astragalus on the plasma metabolites of prenatal dairy cows. Twenty healthy and high-yielding dairy cows with similar age,parity and body condition were selected,and then randomly divided into 2 groups,i.e.,control group A（n=10）：fed basal diet;test group B（n=10）：supplemented with compound Chinese herbal hawthorn and astragalus（150 g per cow）. Feeding for consecutive 14 days while the perinatal circle started,collecting 10 ml blood from the front tail vein before morning feeding,high performance liquid chromatography-mass spectrometry（HPLC-MS）combined with principal component analysis（PCA）and orthogonal partial least squares-Discriminant analysis（OPLS-DA）were used for pattern recognition of plasma metabolic profiles in both groups. Plasma differential metabolites between 2 groups were screened based on the VIP>1,P<0.05,and FC>1.3 or FC<0.77. The ROC curve was employed to further determine the recognition ability of these differential metabolites to plasma classification in 2 groups. The results showed that there was significant difference in the metabolism profile of D-glutamine and D-glutamic acid,linoleic acid,taurine and hypotaurine,alanine,aspartic acid and glutamic acid,ammonia,leucine and isoleucine between the test group and the control group. Nine significant up-regulated differential metabolites were identified by ROC curve：creatinine,5,2'-O-methylcytidine,myristoleic acid,L-leucine,nicotinate,3-hydroxybenzoic acid,hypoxanthine,γ-glutamyl proline and hippuric acid. In summary,the addition of hawthorn and astragalus to prenatal feed affects the amino acid metabolism,fat metabolism and glucose metabolism of dairy cows.

Galactose Feeding in CHO Cell Culture Process：the Impacts on the Cell Growth,Metabolism and Glycosylation of Fc Fusion Protein

XIAO Zheng, FAN Li, TAN Wen-song

2019, 35(8):  138-145.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0138

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This work aims to get insights into the effects of feeding galactose on the CHO cell growth,metabolism and product expression during fed-batch culture process. Through substituting glucose with equimolar galactose in the feed medium of CHO cell culture process,the impacts of feeding galactose with varied ratio on cell growth,metabolism and product characteristics of Fc fusion protein were investigated. Results showed that the data from shake flask indicated more than 60% galactose substitution adversely affected cell growth,and the pH of late culture process increased significantly. Moreover,with the increase of galactose substitution proportion in the feed medium,metabolic by-product lactate concentration rapidly decreased,while accumulation of ammonia increased significantly;in addition,the concentrations of glutamic acid and alanine also showed a gradient increase. Within low substitution percentage（0%-40%）of galactose,the titer and sialic acid content increased with the increase of substitution proportion;however,within a higher proportion（60%-100%）,they both decreased with the replacement ratio increasing. Through stably controlling the culture pH in the bioreactor,by replacing the 40% of total glucose with galactose,the titer and total sialic acid content of Fc fusion protein were enhanced by 43% and 37% respectively. In sum,substitution of glucose with galactose in CHO fed-batch process may greatly enhance product expression and sialic acid content,and is conducive to establish the high-yield and high-quality CHO culture process.

Enhancement of Anti-tumor Effect of a Ribosome-inactivating Protein by Cell Penetrating Peptides and Saponin

LIU Yang, CAO Xue-wei, LU Mei-ya, WANG Fu-jun, ZHAO Jian

2019, 35(8):  146-154.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0198

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A ribosome-inactivated protein named as Burkholderia lethal factor 1（BLF1）was fused with the cell penetrating peptide（CPP）HBP and co-administered with esculentoside A（EsA）to enhance the antitumor effect of the recombinant protein BLF1-HBP. Both the recombinant protein BLF1 and BLF1-HBP were expressed in Escherichia coliBL21（DE3）and then purifed with NI-NTA. The inhibitory effects of the two recombinant proteins were tested and compared in tumor cell line HepG2,MCF-7,A549 and HeLa with the MTT assay. The transmembrane efficiency of the recombinant proteins was observed by laser confocal microscopy and the anti-tumor effect was analyzed with flow cytometry. The result showed that CPP HBP effectively increased the inhibitory effect of BLF1 in all 4 tested tumor cell lines,and the most significant inhibitory effect was observed in HeLa cells,which was increased by 47.5 times. When BLF1-HBP was co-administered with EsA,the antitumor activity of BLF1-HBP was further improved and the IC₅₀ value in MCF-7 cells decreased from 6 840 nmol/L to 0.57 nmol/L. Laser confocal observation revealed that EsA effectively promoted the translocation efficiency of BLF1 recombinant protein. Flow cytometry analysis showed that EsA greatly enhanced the apoptosis effect of tumor cells induced by BLF1-HBP. Therefore,the fusion of BLF1 and HBP and co-administration with EsA may significantly increase the antitumor effect of BLF1,and this enhancement is due to the induction of more tumor cell apoptosis.

Effects of Inflammation and Tumorigenesis on the Imbalance of Hematopoietic Progenitor Cells

LI Xiao-yu, LIU Lin, XING Bing, TANG Jing, LIU Ya-ping, ZHOU Zu-ping, PU Shi-ming

2019, 35(8):  155-161.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1008

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This article mainly investigates the effects of inflammation and tumors on hematopoietic progenitor cells（HPCs）in the hematopoietic-immune system of mice,and explores the correlation between hematopoietic-immune lines in inflammation and tumor status. AOM（Azoxymethane）and DSS（Dextran sulfate sodium salt）were used to induce the mouse model of inflammatory colon cancer,and flow cytometry was used to the abundance and cell cycle of hematopoietic progenitor cells in normal,inflammatory and tumor mice. In the process of inflammation and tumor progression,the percentage of multi-potent progenitors（MPPs）in the bone marrow of mice significantly increased,and common myeloid progenitors（CMPs）and megakaryocytes/megakaryocyte/erythrocyte progenitors（MEPs）and granulocyte/macrophage progenitors（GMPs）demonstrated similar changes,whereas common lymphoid progenitors（CLPs）decreased significantly. Moreover,in the inflammatory and tumor states,the division ability of myeloid progenitor cell was significantly stronger than the normal physiological state. In addition,the tumor peripheral blood CD4⁺/CD8⁺ T cell ratio decreased significantly in the tumor state. In the process of inflammation and tumor progression,HSPCs were biased toward myeloid differentiation. In the tumor state,the cellular immune function of mice was unbalanced;in addition,long-term inflammatory damage may cause imbalance of the immune system and eventually lead to tumor formation.

Biosynthesis of Ganglioside Oligosaccharide Fluoride in Escherichia coli

LIU Xin-ping, TAN Yu-meng, ZHANG Xue, FENG Yan, YANG Guang-yu

2019, 35(8):  162-169.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0229

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Gangliosides are glycosphingolipid molecules that present in all vertebrate cells and show important physiological functions and pharmaceutical activities. Synthetic biology provides an opportunity to construct biological synthetic system for the efficient production of gangliosides,in which the synthesis of oligosaccharide fluoride intermediates is a critical step. Herein,the genes of CMP-NeuAc synthase（neuA）from Neisseria meningitidis,α-2,3-sialyltransferase（cst）,β-1,4-GalNAc transferase（cgtA）,β-1,3-galactosyltransferase（cgtB）and UDP-GlcNAc C4 epimerase（gne）from Campylobacter jejuni were introduced into Escherichia coli JM107. Then the biosynthesis pathways of GM3,GM2,and GM1 oligosaccharide fluoride were successfully constructed in E. coli by using lactose fluoride and sialic acid as substrates. A highly sensitive detection method was developed based on pre-column-derivatization-HPLC. The engineered strains were optimized by permeases overexpression and promoter replacement,and the yields of GM3,GM2,and GM1 oligosaccharide fluoride reached 81.5 mg/L,18.1 mg/L,and 12.3 mg/L respectively in shaking flasks fermented,which laid a promising foundation for further transformation by metabolic engineering.

Biological Functions of Protein Fatty Acylation in Plant Cells

YU Ming-xiang, SONG Shui-shan

2019, 35(8):  170-177.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0095

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Protein fatty acylation modification is an important form of protein translation modification,and plays an important role in cell signal transduction,growth and metabolism. N-myristoylation and S-acylation are the two major forms of fatty acylation. Long-chain fatty acids are covalently bound to proteins,which alter the structure of the protein and affect a range of physiological functions of the cell. In recent years,compared with the functional studies of protein fatty acylation in fungi and animal cells,the study of plant protein lipid acylation and its biological functions are relatively backward,and the two are not identical,causing researchers broad interests. Extensive studies have found that the plant protein N-myristoylation and S-acylation modification process requires the corresponding soybean acyltransferase and S-acyltransferase to catalyze,respectively. Through the study of two transferase-deficient mutants,these two acyltransferases were found to be involved in plant seed germination,flowering length and phenotypic normalization. N-myristoylation and S-acylated proteins were inserted into the membrane at the corresponding positions by hydrophobic acyl bonds for membrane anchoring;participating in the regulation of plant growth,signal transduction and immune response processes. This paper reviewed recent advances in the biological functions of N-myristoylation and S-acylation in plant cells and discusses the role of lipid-modification of plant G-protein coupled receptors（GPCRs）in the sensing of bacterial signaling molecules N-acylhomoserine lactones（AHLs）,providing theoretical guidance for the use of genetic intervention techniques to improve crop production,quality and resistance.

Research Progress and Prospect of microRNA in Medicinal Plants

YAN Wu-ping, WU You-gen, YU Jing ,YANG Dong-mei, ZHANG Jun-feng

2019, 35(8):  178-185.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0098

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MicroRNAs（miRNAs）are endogenous ~22 nt non-encoding single-strand RNAs and may play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. The study of plant miRNA has rapidly become a research hotspot in the field of plant molecular biology since the plant miRNA was first reported in 2002. This paper reviews the biogenesis and mechanism of plant miRNAs,focusing on research progress of miRNA in medicinal plants. Moreover,the paper prospects the future research direction in this field,aiming to provide references for future research of miRNA in medicinal plants.

Strategies and Advances in Functional Genomics of Aspergillus oryzae

WU Qin-qin, SUN Min, CHEN Yu, FU Ya-qin, ZENG Bin, HE Bin

2019, 35(8):  186-192.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0226

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Aspergillus oryzae,a filamentous fungus,is an important strain in fermentation industry and is often used in the production of soy sauce,Douchi,secondary metabolites and enzyme-like products. In recent years,with the decoding of whole genome sequence of A. oryzae,the functional genomics of A. oryzae has become a hot topic. Due to the immaturity of A. oryzae transformation system and the existence of multinucleation,the research progress of functional genome on A. oryzae is slow. This paper first reviews the genetic transformation strategies of A. oryzae,as well as compares their advantages and disadvantages. Then the paper introduces the research progress of functional genomics of A. oryzae in recent years,mainly including protein secretion,conidia formation and secondary metabolite synthesis of A. oryzae. Finally,the application ofA. oryzae in the production of enzymes and secondary metabolites is prospected.

Advances and Prospects of Synthetic Biology in Lactic Acid Bacteria

LIU Yang-er, GUO Ming-zhang, DU Ruo-xi, HE Xiao-yun, HUANG Kun-lun, XU Wen-tao

2019, 35(8):  193-204.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0086

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As the traditional food-grade microorganism,lactic acid bacteria have been applied in food industry,health care,and clinical medicine for a long time. With the need for special functions of lactic acid bacteria increasing,the traditional screening strain method gradually becomes the bottleneck of hindering the development of lactic acid industry due to its complex technology,long cycle,and low success rate. Fortunately,synthetic biology provides new opportunities for developing novel functional lactic acid bacteria strains by modifying cells to meet the designer’s demands with the method of introducing a gene network of specific functions into the genome. Firstly,this paper summarizes the characteristics of lactic acid bacteria and explores the advantages of lactic acid bacteria as a synthetic biological chassis. Then,this paper reviews the current situation of lactic acid bacteria synthesis biology in component design,vector selection,transformation methods and gene editing techniques. Finally,this paper summarizes the application of engineered lactic acid bacteria in disease diagnosis and treatment,food quality improvement and bioenergy,and discusses the technological breakthroughs promoting the further applications,which would be guidance for the development of synthetic biology in lactic acid bacteria.

Research Progress on the Regulation of LncRNA in the Development of Mammalian Hair Follicle

CHANG Yong-fang, BAO Peng-jia, CHU Min, WU Xiao-yun, LIANG Chun-nian, YAN Ping

2019, 35(8):  205-212.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0187

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Long non-coding RNA is a kind of eukaryotic transcripts that are more than 200nt in length and do not encode proteins by themselves. It is a key regulator of protein-coding gene expression,which is widely distributed in the genome of animals and plants,regulating gene expression at the transcriptional and post-transcriptional levels,and as an important regulator participates biological processes in life development cycles,cell differentiation and diseases. Although,several lncRNAs relevant to skin biology have been reported,the detailed functions and mechanisms of lncRNAs in hair follicle development and hair fibers are rare. The combination of new generation sequencing technology and microarray technology provides more accurate and rapid way for the identification of lncRNAs. It has been showed that some lncRNAs affect the cells proliferation and differentiation of skin hair follicle and the formation of dermal papilla,and their target genes involve in the regulation of in the periodic growth of hair follicles. In this review,the regulatory effect of lncRNA on hair follicle growth and development is summarized in order to provide a theoretical basis for the further study of the role and regulatory mechanism of lncRNA related to mammalian hair follicle growth and development.

Current Status and Challenges of Epigenetic Drug Research and Development

JIANG Rui, LÜ Ke-nao, PAN Xue-feng, CUI Xin-xia, SHEN Shi-gang, DING Liang

2019, 35(8):  213-225.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0099

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Epigenetic modifications do not affect DNA sequence,but do affect the expression of DNA genetic information by affecting the chromatin activity through DNA methylation/demethylation,reversible modifications of various histones,non-coding RNA molecules etc. Clinical drugs based on epigenetic mechanisms are intended to correct the expressions of the disease-associated genes by artificially interfering with the epigenetic modifications of the chromatins in the disease conditions,so as to achieve the preventions and treatments of diseases. To promote the research and development（R&D）of epigenetic drugs and its biotechnological industrialization in China,this paper systematically summarized the R&D current status of the epigenetic drugs,including DNA methyltransferase inhibitors,histone modifying enzyme inhibitors,siRNA,etc.,and also deeply discussed the critical issues that may need to be resolved in R&D and industrialization of epigenetic drugs.

Preparation and Identification of Polyclonal Antibody Against the Cap Protein of Porcine Circovirus Type 2

OU Yun-wen, DAI Jun-fei, MA Bing, ZHANG Jie, DENG Hui-xiang, LI Xiao, ZHANG Xin-ming

2019, 35(8):  226-231.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0205

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This experiment was aimed to prepare polyclonal antibodies of Cap protein of porcine circovirus type 2（PCV2）. The gene was amplified from the DNA of PCV2 CAU0673 strain by PCR,whose product was approximately 702 bp. The recombined plasmid pET30a-PCV2-Cap was expressed in E.coli BL21（DE3）and was induced by IPTG. The recombinant Cap protein was purified by Ni-NTA,and used as an immunogen for immunization of New Zealand white rabbits by subcutaneous injection in several sites on back to prepare polyclonal antibodies. The specificity of the serum antibody was determined by western blot,IFA,and the serum antibody titer was detected by indirect ELISA. PCR,digestion and sequencing were used to identify positive plasmid. The results showed that the recombined plasmid pET30a-PCV2-Cap was successfully constructed. The results of SDA-PAGE showed that the recombined protein was successfully expressed in E.coli BL21;（DE3）with a relative molecular weight of 34 kD,mainly existed in a form of inclusion body. The results of western blot showed that this recombined Cap protein was specifically reacted with PCV2 positive pig serum. The prepared rabbit antisera was specifically reacted with the recombined Cap protein and whole virus antigen,and the serum antibody titer was 1∶12 800.

Research of Progress of Strategy and Application of Metabolic Pathway Modification in Escherichia coli

LI Ran, HUANG Yu-qing, JIA Zhen-hua

2019, 35(8):  232-237.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0131

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The metabolic pathway of Escherichia coli can be modified by genetic engineering,which can be used for the synthesis of biofuels,chiral drugs,and their derivatives. E. coli has become a common host microorganism for genetic modification due to its strong stability and easy operation. Using metabolomics and synthetic biology,E. coli can be efficiently used as a target for biocatalytic production. This paper reviews the strategies of metabolic pathway modification of pyruvate,acetyl coenzyme A,mevalonate and shikimic acid in E. coli,and the application of metabolic pathway modification of E. coli in the synthesis of biofuels and block compounds,which provides a holistic idea and method for researchers to study the synthesis of target compounds by E. coli.

Breeding of a New Strain of High-yield Surfactin by Bacillus Interspecies Protoplast Fusion

PAN Xu-yao, WEI Tao, TAN Zhu-hao, LIN Long-zhen, GUO Li-qiong, LIN Jun-fang

2019, 35(8):  238-245.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0235

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This work aims to obtain a new strain of high-yield Surfactin via Bacillus natto and Bacillus siamansi interspecies protoplast fusion. The protoplasts of B. natto and B. siamansi were prepared using B. natto and B. siamansi as parent strains,and the protoplasts were fused by PEG-mediated parental inactivation labeling. A new strain producing high yield Surfactin was obtained after the potential fusions identified by PCR,high-throughput screening by CPC-BTB method,HPLC rescreening and stability verification of fusion strains. The results showed as such：the optimal enzymatic hydrolysis concentration of protoplasts of B. natto and B. siamansi were 0.25 mg/mL and 0.2 mg/mL,respectively;the optimal enzymatic hydrolysis time was 25 min and 20 min,respectively;and protoplast yield were 83.1% and 84.3%,respectively. The conditions of protoplast fusion were as follows：B. natto was inactivated by UV for 80 min,B. siamansi was inactivated at 85℃ for 40 min,and the fusion system was 50% PEG6000 at 38℃ for 15 min. A stable fusion strain F8 was obtained,and it produced a 33.3% increase in Surfactin production compared to sole B. natto and a 60% increase by sole B. siamansi.

Preparation of Anti-lung Cancer Peptides by Enzymatic Hydrolysis of Protein from Enteromorpha clathrata

DUAN Xun-wei, XIAO Gui-qing, WANG Li-xing, WU Wen-lin, DAI Cong-jie, DONG Le

2019, 35(8):  246-252.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0338

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In order to exploit abundant proteins in the green algae Enteromorpha clathrata,protein from E. clathrata was hydrolyzed by the papain enzyme,the optimal hydrolysis process conditions were solid/liquid ratio of 1∶25,enzyme dose of 1 250 U/g protein,hydrolysis temperature of 45.7℃,hydrolysis pH of 7.25 and hydrolysis duration of 120 min. Under these conditions,polypeptides of different molecular weight range were obtained by ultrafiltration,and their anti-lung cancer effects were evaluated preliminarily at the cellular level of HUVEC（human umbilical vein endothelial cell）,A549,H446 and H460. According to the results,the oxidation resistance,proliferation and tubule formation of HUVEC treated with 2-6 kD polypeptide from E. clathrata were inhibited,and the proliferation,mobility,cell cycle of all 3 tested lung cancer cells H446,H460 and A549 were inhibited at varied level,and the cells apoptosis were induced. The 2-6 kD proteins demonstrated significantly better inhibitory effect on H446 and H460 than that on A549. In conclusion,this study proves the feasibility of gaining natural anti-tumor small-molecule peptides by enzymolysis protein from E. clathrata,and provides a novel way to explore the high value utilization of protein resources from E. clathrata.

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2019, 35(8):  253-253. 

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2019, 35(8):  254-254. 

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