Table of Content

26 November 2019, Volume 35 Issue 11

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Cloning and Expression Analysis of St4CL Gene in Solanum tuberosum

XIE Hai-juan, FAN Xi-de, YE Guang-ji, ZHOU Yun, WANG Jian, YANG Yong-zhi

2019, 35(11):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0505

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4-coumarate：CoA ligase（4CL）is one of the most important enzymes in phenylpropane metabolism,thus cloning 4CL from Solanum tuberosum and analyzing its expression will lay foundation for studying its function. A gene named St4CL was cloned from Qingshu 9,bioinformatics for it was conducted,and qRT-PCR was used to detect the expressions of St4CL in the different tissues and varieties. The results demonstrated that the CDS of St4CL gene was 1 710 bp,and it encoded 569 amino acids. The molecular weight of protein was 61.83 kD,and the isoelectric point was 5.53. Bioinformatics analysis showed that St4CL was a stable hydrophobic protein with random coil（42.36%）,α-helix（28.47%）,extended strand（21%）,and β-turn（8.08%）. Subcellular localization prediction showed that St4CL was located on the thylakoid membrane of chloroplast. Phylogenetic analysis revealed that St4CL protein had the highest sequence similarity（about 97%）with and the closest genetic relationship to Sl4CL in Solanum lycopersicum. The promoter of St4CL gene contained many cis-acting elements related to stress response,such as abscisic acid responsive element,gibberellin-responsive element,MYB binding site involved in drought and flavonoid biosynthesis,etc. qRT-PCR analysis showed that St4CL expressed in all tissues,the highest in leaves followed by flowers,and the lowest in roots. The expression of St4CL varied in different varieties,with the highest expression in Qingshu10 and the lowest expression level in Xiazhai 65.

Clone and Expression Profile Analyses of the Gene CaATP9 in Pepper（Capsicum annuum L.）Male Sterility Line

ZHANG Jing-rou, SHAO Gui-fang, WANG Jiao, ZHANG Shui, YANG Ting-yu, DENG Ming-hua

2019, 35(11):  9-15.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0283

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In this research,the pepper cytoplasmic male sterile line 9704A and the homologous maintainer line 9704B were used as experimental materials to study the differences between the primary structure of gene CaATP9 and the editing of RNA,and the real-time quantitative PCR was used to study the spatiotemporal expression of gene CaATP9. The results showed that the primary structure of gene CaATP9 did not differ between the two materials;the gene CaATP9 has five C→T editing sites during transcription,resulting in the conversion of four hydrophilic amino acids（DNA template）into four hydrophobic amino acids and enhancing the stability of the protein;gene CaATP9 is expressed in different tissues of pepper,but the expression level in reproductive tissues is generally higher than that in vegetative tissues;the expression level of gene CaATP9 in the male sterile line differed from that of the homologous maintainer line in the development of flower buds. It is speculated that this difference in expression leads to the disorder of the energy supply of the buds of the cytoplasmic male sterile line,affecting the normal development of microspores and showing infertility.

Cloning and Expression Analysis of the Pear Black Spot Resistant Gene PpEMS1

DUAN Min-jie, YI Hong-wei, WANG Jin, WU Zheng

2019, 35(11):  16-21.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0240

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This study aims to clone the gene related to pear disease resistance and to preliminary analyze the effect of the gene on resistance and defense of pear black spot. Based on homologous gene cloning method,a gene predicted to be the receptor protein kinase EMS1 was cloned from the ‘Huang-guan’ pear,temporarily named as PpEMS1. The gene then was analyzed by bioinformatics,Southern Blot,subcellular localization and quantitative real-time PCR. As result,its CDS was 3 894 bp,containing an open reading frame of 3 891 bp and encoding 1 296 amino acids. Southern blot analysis indicated that this gene existed as a single copy in ‘Huang-guan’ pear,‘Huang-hua’ pear and ‘Cui-yu’ pear,while it existed as multiple copies in ‘Ai-dang’ pear. Subcellular localization demonstrated that the fusion protein was expressed in both cytomembrane and nucleus. The results from the quantitative real-time PCR revealed that the expression of the PpEMS1 gene increased first and then decreased in both ‘Huang-hua’ and ‘Ai-dang’ pears and was induced at 1 d,2 d,3 d,4 d,5 d,6 d,7 d and 8 d after inoculating black spot pathogen. It is inferred that the PpEMS1 gene may be associated with the pear black spot resistance.

Analyse on Monosaccharide Composition of Polysaccharides in Ziziphus jujuba Mill cv. lingwuchangzao Fruit from Zhongning Areas in Ningxia by GC-MS

ZHANG Ying-cai, SU Wei-dong, CHAI Ya-hong

2019, 35(11):  22-29.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0304

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The monosaccharide composition of polysaccharides in Ziziphus jujuba Mill cv.lingwuchangzao fruit from Zhongning areas in Ningxia was studied to provide experimental basis for the development and utilization of different areas Ziziphus jujuba Mill cv.lingwuchangzao. The fruit of Ziziphus jujuba Mill cv.lingwuchangzao from Zhongning areas in different developmental periods was used as experimental material to research the change rules of polysaccharides content and monosaccharide composition by the methods of hot water extraction and gas chromatography-mass spectrometry（GC-MS）. The main research results are followed：the crude polysaccharides obtaining ratio in Ziziphus jujuba Mill cv. lingwuchangzao fruits from Zhongning areas in different developmental periods is respectively 0.43% in the early bulking period,0.527% in the rapid enlargement period,0.80% in the coloring period,and 0.618% in the fruit maturation period. The crude polysaccharide in different developmental periods fruit was fractioned respectively to four fractions（Ju-0,Ju-1,Ju-2,Ju-3）by DEAE-52 cellulose ion-exchange columns,and Ju-2 had the highest contents,this indicated that the main components of polysaccharides in fruit was acid fractions. Finally,the refined polysaccharides in the different developmental periods fruit were analysed by the GC-MS,the results showed that polysaccharides were composed of ten monosaccharides：arabinose,rhamnose,ribose,fucose,xylose,mannose,galactose,glucose,glucuronic acid and galacturonic acid,and did not contain fructose,the contents of arabinose,galactose,ribose,rhamnose,glucuronic acid and galacturonic acid were higher,followed by the fucose and glucose,and the contents of xylose and mannose were the lowest,but the monosaccharide composition and contents are different in different polysaccharides component. Conclusion,The refined polysaccharide contents present ascendant trend with the development of the fruits,the monosaccharide composition and contents of the refined polysaccharide of Ziziphus jujuba Mill cv.lingwuchangzao fruits from Zhongning areas in different developmental periods are different respectively in different polysaccharides component.

Remediation of Salt Damage to Tomato Seedlings from Root Application of Salicylic Acid

SUN De-zhi, YANG Heng-shan, SONG Gui-yun, FAN Fu, HOU Mi-hong, PENG Jing, HAN Xiao-ri

2019, 35(11):  30-38.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0571

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This work is to explore the regulation effect of root application of salicylic acid（SA）eliminating salt stress damage to tomato seedlings,and to provide scientific basis for rational use of SA to solve salt damage in tomato cultivation and breeding salt-tolerant tomato varieties. Under the condition of nutrient solution cultivation,a hydroponic experiment was conducted to explore the effects of different concentrations（50,100,200,400,and 800 μmol/L）exogenous SA on plant growth,chlorophyll content,gas exchange,chlorophyll fluorescence parameters,membrane peroxidation,and antioxidase activity in tomato cultivar（‘Qinfeng Baoguan’）seedlings under stress of 100 mmol/L NaCl. The results showed that the growth inhibition of tomato seedlings under salt stress was effectively alleviated after treated with different concentrations of SA. Meanwhile,the chlorophyll content,net photosynthetic rate（Pn）,stomatal conductance（Gs）,transpiration rate（Tr）,maximum fluorescence of leaves（Fm）,PSII maximum photochemical efficiency（Fv/Fm）,actual photochemical efficiency（Φ_PSII）,and photochemical quenching coefficient（qP）increased to varying degrees,while intercellular CO₂ concentration（Ci）,minimum fluorescence Fo and non-photochemistry quenching coefficient（NPQ）significantly decreased. The amplitude of each index reached the maximum especially when the concentration of SA was 200 μmol/L. When tomato seedlings were put under salt stress,the superoxide dismutase（SOD）,peroxidase（POD）activity,malondialdehyde content,and electrolyte exudation rate of theirs leaves raised remarkably,while their catalase（CAT）activity did not change so obviously. Application of exogenous SA treatment at various concentrations promoted the increase of the three enzyme activities above,along with making the leaf malondialdehyde content and electrolyte exudation rate markedly reduced;moreover,the change was the most significant when treated with 200 μmol/L SA. Studies have demonstrated that exogenous SA could relieve the oxidative damage caused by salt stress mainly by enhancing the photosynthetic capacity of seedling leaves,hence improving the salt tolerance of tomato plants. The treatment with 200 μmol/L SA showed the best results under the experimental conditions,.

Increase of Flavonoids Content in Arabidopsis Heterologous Expressing CiCHI Gene

YANG Fei-yun, BAI Jie, LIU Kun, WANG Rui-gang

2019, 35(11):  39-45.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0669

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Chalcone isomerase（CHI）is the second key enzyme in flavonoids synthesis pathway,and plays an important role in regulating the whole metabolic pathway. Clarifying the function of CiCHI would provide the theoretical evidences for further revealing the anti-stress mechanism of Caragana intermedia. Degenerate primers were designed according to the conserved domain of known CHI genes,and the full length of CiCHI gene sequence was cloned using C. intermedia cDNA as template,then was confirmed by sequence and phylogenetic analysis. The transcript level of CiCHI was induced by ultraviolet（UV）,NaCl and abscisic acid（ABA）treatments as analyzed by quantitative real-time PCR（qRT-PCR）. The expression of CiCHI was the highest in leaves,relatively high in stems,and the lowest in roots. Heterologous expression of CiCHI significantly increased the content of total flavonoids in transgenic Arabidopsis. All these results confirmed that CiCHI was functional in transgenic Arabidopsis.

Cloning of Yak TNFAIP6 Gene and Its Temporal Expression in Ovarian Activity

MA Hong-cheng, XIONG Xian-rong, MU Song-yin, HAI Zhuo, QIN Wen-chang, LI Jian

2019, 35(11):  46-54.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0596

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The aims of this study are to investigate the sequence characteristics of yak（Bos grunniens））TNFAIP6 gene and to analyze its temporal expression differences in different tissues and ovary at different stages of estrous cycle. Total RNA and protein from the heart,lung,spleen,kidney,liver,uterus,small intestine,gastric,muscle,and ovary tissues at different stages of estrous of healthy female yaks were extracted. The coding sequences of TNFAIP6 gene were cloned by RT-PCR and bioinformatics analysis of it was conducted. The expression level of TNFAIP6 gene in different tissues was detected by semi-quantitative PCR. Furthermore,the expression levels of protein and mRNA in yak ovary at different stages of estrous cycle were detected by Western blot and qRT-PCR. The collected data were statistically analyzed. The results showed that the CDS region of yak TNFAIP6 gene was 830 bp,encoding 279 amino acids. The analysis of proteins indicated that TNFAIP6 was hydrophilic and acidic-stable protein,had non-transmembrane domain and signal peptide,including Link and CUB structural domains. The secondary and tertiary structure of it was mainly composed of random coil and α-helix. Through homology alignment,the yak TNFAIP6 had high homology with cattle and wild yak. TNFAIP6 gene of yak from semi-quantitative PCR was widely expressed in various tissues and its expression in ovary,uterus,spleen and muscle was relatively higher than in other tissues. The expression level of TNFAIP6 in the follicular stage was significantly higher than that in the other two stages in ovary（P<0.01）,while there was no significant difference between the corpus luteum stage and red body stage（P>0.05）. The results of Western blot were basically consistent with qRT-PCR. In sum,the TNFAIP6 gene may be involved in the reproductive action of yak ovary,and the result provides basic reference in exploring the action mechanism of TNFAIP6 in the activity of yak ovary.

Correlation Analysis Between Microsatellite Polymorphism and Production Traits in Black Fur Sheep in Min County

ZHANG Rui, LANG Xia, WU Jian-ping, WANG Cai-lian, LIANG Ting-yu

2019, 35(11):  55-63.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0621

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The genetic diversity of 10 microsatellite loci in the black fur sheep population of Min county and its correlations with production traits were analyzed,which provides a theoretical basis for the protection,utilization and traits improvement of the germplasm resources. A total of 48 black fur sheep of Min county at 12,24 and 36 months old were selected,respectively. Their body weight,body height,body length,chest width,chest depth and chest circumference were measured. Meanwhile,10 pairs of microsatellite primers,PCR and gel electrophoresis technology were used to detect the genetic diversity of the black fur sheep in Min county and to analyze the correlation between various gene types and production traits. A total of 112 alleles were detected at 10 microsatellite loci. The average alleles was 11.2,the average effective alleles was 4.450 8,the average observed heterozygosity was 0.521 8,the average expected heterozygosity was 0.765 7,the average polymorphism information content was 0.728 9;10 loci had various degrees of correlation with body weight,body height,body length,chest width,chest depth and chest circumference. The selected 10 microsatellite loci were polymorphic and could be used to evaluate the genetic resources of black fur sheep in Min county. In addition,the selected loci were correlated with production traits at different degrees,which could be used to guide the trait improvement and breeding of black fur sheep in Min county.

RNA Sequencing Analysis of Cecum Tissues of Jinghai Yellow Chickens Infected by E. tenella

YU Hai-liang, ZOU Wen-bin, WANG Xiao-hui, LIN Yu-xin, DAI Guo-jun, ZHANG Tao, ZHANG Gen-xi, XIE Kai-zhou, WANG Jin-yu, SHI Hui-qiang

2019, 35(11):  64-71.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0257

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This work aims to investigate the differentially expressed genes（DEGs）of cecal tissue of Jinghai yellow chickens infected with E. tenella and the molecular response process and immune response mechanism of the infection. The RNA-seq technique was used to sequence the cecal tissue of E. tenella-infected group and non-infected group on the 7th day of post-infection,and the DEGs were screened for functional and pathway enrichment analysis. The results indicated that there were 2 830 DEGs（P<0.05）between the infected and uninfected groups,of which 1 419 were up-regulated and 1 411 were down-regulated. Ten DEGs were randomly selected for qRT-PCR verification,and the results showed that the fold change of DEGs was highly consistent with the RNA-seq results（r=0.988,P<0.000）,and the coefficient of determination reached 0.975. GO analysis showed that 2 356 DEGs were functionally annotated,and the top 30 significantly enriched GO terms involved mainly cell communication,signal transduction,angiogenesis,and oxidoreductase activity. The KEGG analysis revealed that top significantly enriched signaling pathways included focal adhesions,extracellular matrix-receptor interactions and peroxisome proliferator-activated receptors. Key DEGs in these pathways include ANGPTL4,ACSL5,VEGFC,CD44,and MAKP10,etc.,suggesting that these genes would play an important role in the infection of E. tenella.

On the Detoxification Metabolic Pathways of Boleophthalmus pectinirostris Exposed to Acute Ammonia-nitrogen Stress

YANG Yang, MENG Fan-xing, WANG Ri-xin

2019, 35(11):  72-81.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0313

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In order to explore the ammonia conversion and metabolic mechanism of Boleophthalmus pectinirostris exposed to environmental ammonia nitrogen stress,an acute ammonia nitrogen stress test（8 mmol/L NH₄Cl）in 72 h was conducted in this study. The protein concentration assay was used for detecting enzymes involved in ammonia metabolism,such as glutamine synthetase（GS）activity and blood ammonia levels. Enzyme-linked immunosorbent assay（ELISA）was employed to analyze the expression levels of ammonia metabolic cotransporter,carbonic anhydrase（CA）and sodium hydrogen exchange factor 3（Na⁺/H⁺ exchanger,NHE3）. Meanwhile,quantitative polymerase chain reaction（qPCR）was applied to check the relative expression levels of ammonia metabolism-related genes,such as GS,CA15,NHE,and ammonia transporter Rh type C-1（Rhcg1）. The results showed that under acute ammonia-nitrogen stress,the blood ammonia concentration of B. pectinirostris significantly increased at 12 h and then decreased to the steady level. The hepatic expression of GS gene significantly increased at 12 h and 48 h while its enzyme activity significantly increased at 24 h. The changing tendency of NHE3 protein expression in gill was the same as that of GS activity,while the levels of CA protein significantly increased at 12 h and 48 h,respectively,after stress. The expression levels of ammonia excretion related genes CA15,NHE and Rhcg1 were up-regulated under ammonia-nitrogen stress. The NHE gene was up-regulated earliest（24 h）while CA15and Rhcg1 significantly increased until 48 h,indicating that they participated in ionic ammonia excretion. In brief,under the ammonia-nitrogen stress,B. pectinirostris adopt two main ammonia excretion pathways：1）conversion of toxic ammonia into non-toxic glutamine by hepatic GS enzyme to avoid the excessive accumulation of ammonia in the body;2）facilitating the ammonia excretion through the Rhcg1 and NEH3 synergistically promoted by H⁺ provided by CA enzyme via the protonation of CO₂ in gills.

Bioinformatics Analysis and Subcellular Localization of Human HSPA8 Gene

WU Xiao-min, ZHAO Jia-fu, NI Kai, ZHU Xiao-feng, XU Hou-qiang

2019, 35(11):  82-88.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0472

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This work is to investigate the bioinformatics analysis and subcellular localization of human HSPA8 gene. The HSPA8 gene CDS region was amplified by RT-PCR method,and its physicochemical properties,secondary structure and tertiary structure were analyzed by bioinformatics tools and its phylogenetic tree was constructed. The recombinant eukaryotic expression vector pEGFP-C1-HSPA8 was constructed by restriction enzyme digestion and ligation,and transfected into HEK-293T cells. The subcellular localization was observed by fluorescence colocalization. The result showed that the CDS region of human HSPA8 gene was successfully cloned. The results of bioinformatics analysis showed that the human HSPA8 gene CDS region was 1 941 bp in length and the molecular formula was C₃₁₁₁H₄₉₉₈N₈₆₀O₉₉₄S₁₇. The relative molecular weight was 70.89 kD,encoding a total of 646 amino acids. The secondary structure prediction showed HSPA8 protein consisted of the α-helix（41.64%）and random coil（32.20%）dominantly. HSPA8 nucleotide homology and genetic phylogenetic tree analysis showed that humans and chimpanzees were closely related. Fluorescence colocalization results showed that HSPA8 protein was evenly distributed throughout the cell. The bioinformatics analysis and subcellular localization of HSPA8 protein laid the foundation for further exploring the intracellular function of HSPA8 gene.

Construction and Activity Analysis of the PLIN1 Gene CRISPR/Cas9 Vector

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

2019, 35(11):  89-95.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0357

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The objectives of this work are to design and validate the sgRNA being capable of targeted cleaving the PLIN1 gene and to lay a foundation for the establishment of a highly efficient PLIN1 gene-cleaved animal model. Three sgRNAs with favorable evaluation results were designed using predictive software. After adding T7 promoter,the corresponding sgRNA was transcribed in vitro to construct the digestive system and to verify the cleavage activity in vitro. The corresponding gene knockout plasmid vector was constructed,and the plasmid was transferred into 3T3-L1 preadipocytes by electroporation and induced to differentiate. The expression of PLIN1 mRNA in cells was detected by RT-PCR. The expression of PLIN1 protein in cells was detected by Western blot. Three sgRNAs of PLIN1 were successfully transcribed in vitro and the restriction enzyme system was constructed. The cleavage efficiency of sgRNA-PLIN1-1 was 61.8%±9.0%,and that of sgRNA-PLIN1-2 was 64.1%±9.6%. The cleavage efficiency of sgRNA-PLIN1-3 was 34.1%±7.2%,and that of sgRNA-PLIN1-3 group was significantly lower than that of sgRNA-PLIN1-1 group and sgRNA-PLIN1-2 group（P<0.05）. The plasmid vector of three sgRNAs was successfully constructed and electroporated into 3T3-L1 preadipocytes,and the transfection efficiency was about 30%. The expression of PLIN1 mRNA in each positive interference group（P<0.01）significantly decreased at the 4th day after differentiation. Compared with sgRNA-PLIN1-3 group,there was a significant difference in the expression level of PLIN1 mRNA in sgRNA-PLIN1-1 group and sgRNA-PLIN1-2 group. The expression of PLIN1 protein in each positive interference group significantly decreased（P<0.01）,and there was no significant difference in the expression of PLIN1 protein between the positive interference groups. In conclusion,all three sgRNAs may cleave the exon 2 of the PLIN1 gene in a target manner,and the cleavage activity of the gRNA-PLIN1-1 group and the sgRNA-PLIN1-2 group is slightly higher than that of the sgRNA-PLIN1-3 group. In vitro active cleavage experiments may well predict intracellular cleavage and are an effective means of screening highly active sgRNA.

Eukaryotic Expression of Extracellular Domain of Pseudorabies Virus gE Protein and Preparation of Monoclonal Antibodies

LANG Qiao-li, WU Meng, HUANG Nan, HE Qi-lin, GE Liang-peng, YANG Xi

2019, 35(11):  96-103.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0310

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This work aims to construct eukaryotic expression vector of the extracellular domain of gE gene for generating stable soluble gE protein in HEK293F cells,and to obtain specific monoclonal antibody of gE by hybridoma screening. Bioinformatics was used to analyze the gE gene sequence,and the expression vectors pcDNA3.4-gE-6×His and pcDNA3.4-gE-mFc were constructed and then transfected into HEK293F cells via transient transfer method for the expression and further purification. Western blot and SDS-PAGE were used to identify and compare the fusion protein gE-6×Hia and gE-mFc. Pseudorabies inactivated whole virus was used to immunize the mice,hybridoma fusion cells were obtained by electrofusion,positive hybridoma cell lines stably and specifically binding to gE protein were screened by gE-mFc protein and IFA. As results,the pcDNA3.4-gE-6×His and pcDNA3.4-gE-mFc expression vectors were successfully constructed and the gE-6×His and gE-mouse Fc soluble proteins were obtained after expression and purification. The results of SDS-PAGE and Western blot showed that gE-mFc had a higher expression level and better stability than gE-6×His,and the purity reached 85% after only one purification. Further,9 stably and specifically hybridoma positive cell lines were screened using gE-mFc protein. In sum,this is the first time that stable and soluble gE protein is expressed by mammalian cell expression system,and by which gE specific monoclonal hybridoma cell lines are screened,laying a fine material foundation for the development of pseudorabies diagnostic reagents.

In Vitro Antioxidative and Anti-proliferative Activities of Extractions from Six Common Edible Mushrooms

CHEN Zi-han, LIU Jin-juan

2019, 35(11):  104-108.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0407

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In this study,the antioxidant and anti-proliferative activities of the extracts from Hypsizygus marmoreus,Lentinus edodes,Agrocybe cylindracea,Pleurotus ostreatus,Flammulina velutipes and Volvaria volvacea were evaluated. The antioxidative activities were determined by DPPH radical-scavenging,OH·radical scavenging,and total antioxidant capacity and reducing power. The anti-proliferative activities were detected by Alamar blue assay. Total phenolics and flavonoids contents of 6 edible fungi ranged from 155.76 to 283.54 mg/L and from 5.19 to 49.79 mg/L,respectively;the highest was in V. volvacea,A. cylindracea and L. edodes,while that in P.s ostreatus and H. marmoreus was the lowest. The total antioxidant capacity and reducing power of A. cylindracea and L. edodes（22.94 U/Land 20.10 U/L,0.26 and 0.233,respectively）were better than the others. The ability of scavenging DPPH· ,·OH by V. volvacea was the highest（48.8% and 75.12%,respectively）,while there was no differences among the capacities of scavenging O₂^-·. The extractions from 6 common edible mushrooms demonstrated the anti-proliferation activity against HepG2,SGC-7901,NCI-H460,MDA-MB-231 and LO2 cells. A. cylindracea showed the highest activity for HepG2 and NCI-H460（IC₅₀=4.62±2.13,4.96±1.84 mL/L）,and V. volvacea showed the highest activity for SGC-7901and MDA-MB-231（IC₅₀=5.01±1.03,6.95±1.03 mL/L）. All extractions showed low cytotoxic effect on LO2 cell. These data suggest that these 6 edible fungi appear significant antioxidative and antitumor activities in experiments in vitro,among them,P. ostreatus,L. edodes and F. velutipes have the strongest antioxidant and antitumor proliferation activities.

Comparative Study on Endophytic Fungi Diversity of Kobresia humilis in Floccularia luteovirens

GUO Jing, XIE Zhan-ling, LUO Tao, XUE Zhi-feng, GUO Jian-juan, LI Fa-xiong, ZHANG Xiu-juan

2019, 35(11):  109-117.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0385

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In order to study the endophytic fungi diversity of Kobresia humilis in the habitat of Floccularia luteovirens,the diversity of endophytic fungi in K. humilis was studied by traditional isolation culture method and macrogenomic method. The results showed that：66 endophytic fungi were isolated from K. humilis and identified by traditional method,which belonged to Ascomycota and were classified into 7 orders,namely Pleosporales,Xylariales,Eurotiales,Helotiales and Hypocreales,Mucorales,Glomerellales. The dominant species were Stagonospora bicolor;the diversity index,richness index and evenness index of roots were 1.63,1.56 and 0.82,respectively. F. luteovirens was not obtained by isolation. 587 endophytic fungi were detected by macrogenomic method. Ascomycota accounted for 77.21%,Basidiomycota 16.21%,and the remaining 6.58% were unclassified fungi,the dominant population was Fusarium sp. The diversity index,richness index and evenness index of roots were 3.13,39.08 and 0.48,respectively. The F. luteovirens was detected and its relative abundance was 11.39. Comparing the two methods,there were abundant endophytic fungi resources in K. humilis in the habitat of F. luteovirens. F. luteovirens was one of the dominant endophytic fungi populations. This study proved for the first time that F. luteovirens was an endophytic fungus of Kobresia genus,it is a new breakthrough in studying the nutritional types of floccularia luteovirens and its interaction with plants.

Molecular Identification and Drug Resistance Analysis of Plesiomonas shigellode Isolated from Fish

DOU Peng-peng, WANG Li, ZHANG Hua, ZHENG Yao

2019, 35(11):  118-123.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0349

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To identify a strain（L-1）isolated from a sick Pelteobagrus fulvidraco and analyze its drug resistance,the morphology,physiological and biochemical analysis,16S rDNA gene and 23S rRNA gene amplification,and phylogenetic tree construction were carried out,and its drug resistance was determined by drug susceptibility test and drug-resistant genes test. The results showed that the strain L-1 was Gram-negative bacterium in short rob. The reactions to gelatin liquefaction,aesculin and arabinose and so on were negative,while that to arginine,tryptophan and glucose and so on were positive. The length of sequence fragment by 16S rDNA gene amplification was 1 461 bp. BLAST alignment showed that the similarity between the splicing sequence of L-1 and Plesiomonas shigellode in NCBI was 99%. In the phylogenetic tree of the strain L-1,the strain L-1 was the closest to P. shigellode. Only the target band with a size of 280 bp was amplified from the L-1 via 23S rRNA,while all other strains did not. Thus L-1 was comprehensively determined to be P. shigellode. Antimicrobial susceptibility test showed that L-1 was sensitive to cefoperazone,centamicin,etc.,and was moderate to tetracycline,and was resistant to benzocillin,ampicillin and other drugs. Drug-resistant gene Sul2,TEM,ant（3''）-Ⅰa and aac（6'）-Ⅰb in the strain L-1 were detected,while Sul1,Sul3,aph（3'）-Ⅱa and aac（3）-Ⅱa were not.

Effects of Nano-TiO₂ and Tetracycline Exposure on Drug-resistant Escherichia coli

ZHANG Yong-li, YUAN Wei, YANG Qing-xiang

2019, 35(11):  124-131.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0528

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The drug-resistant Escherichia coli（M13）and sensitive quality control strain（E. coli ATCC25922）were used to study the effects of nano-TiO₂ and tetracycline on the growth of E. coli. The results showed that the addition of nano-TiO₂ further inhibited the growth of sensitive bacteria compared with the addition of the gradient concentration of tetracycline alone,while the drug-resistant bacteria did not change much. The antibacterial rate of nano-TiO₂ alone or co-exposure to the drug-resistant bacteria was below 40%,while that to the sensitive bacteria ranged in 80% and 100%. The drug-resistant strain M13 was resistant to antibiotics and also to nano-TiO₂.There are some commonalities between microbial resistance mechanisms against antibiotics and other environmental virulence factors,but the mechanism of microbial resistance under co-exposure conditions remains to be further studied.

Identification,Fermentation and Biocontrol Efficiency of Trichoderma harzianum SKD-ZX-1

LU Hong-sheng ZHANG Xue GAO Yu-ting SUN Pei-ming QIU Meng-meng

2019, 35(11):  132-140.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0586

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Trichoderma harzianum is an important biocontrol bacterium and has an antagonistic effect on pathogenic bacteria of vegetable diseases. In order to determine the biocontrol effect of the isolated T. harzianum SKD-ZX-1,the fermentation broth of the strain was determined and analyzed by GC-MS,and the effect of this strain on the bacterial flora in tomato root rot soil was analyzed by metagenomic sequencing. Further the antagonistic experiment of the strain against Alternaria spp. and Alternaria solani were carried out by plate-to-sputum culture method. The results showed that the fermentation liquid of T. harzianum SKD-ZX-1 contained multiple antibacterial ingredients,namely diisobutyl phthalate,dibutyl phthalate,and di（2-ethylhexyl）phthalate,and increased the types of bacterial flora in the soil. Moreover,Enterobacter increased by 46% in the experimental group compared with the control group,while the genus Pseudomonas and Clostridium decreased by 5.28% and 36.1%,respectively. The inhibition rates of T. harzianum against the two pathogens was 100%,and the inhibition coefficient reached Ⅰ. The present study demonstrates that T. harzianum SKD-ZX-1 has significant inhibitory effect on Alternaria alternata and Alternaria solani,and its fermentation broth has certain antibacterial effect.

Research Progress of microRNA in Plant Development

HUANG Jun-jun, LIU Wen-wen, GUO Ya-ru, JIANG Tian-hui, REN Qing, WANG Hua-hua, LIANG Wei-hong

2019, 35(11):  141-149.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0526

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microRNA（miRNA）is a type of endogenous non-coding small RNA that is widely present in plants and is about 20-25 nucleotides long. By degrading the target gene mRNA and inhibiting its translation,the expression of the targeted gene is controlled at the post-transcriptional level and thus a variety of biological functions,including plant growth and development,reproduction and stress response,were regulated. Previous studies have shown that miRNA and its target genes are not only a key regulator in plant timing transformation,but also play an important regulatory role in shoot tip development,leaf morphogenesis,floral organ development and flowering time. This review focuses on the latest research progress of miRNAs in regulating plant growth and development and developmental plasticity,and explores and prospects the need-to-be clarified issues in the studies of plant miRNAs,aiming to provide a reference for deeply analyzing miRNAs in regulating plant tissues and organ patterns,and the role of them in plant morphological diversity and molecular regulatory networks.

Research Progress on Transcription Factors Regulating Plant Seed Development

WU Yu, SHEN Yong-bao, SHI Feng-hou

2019, 35(11):  150-159.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0043

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Plant seeds are the unique reproductive organs to gymnosperms and angiosperms,also the main food source for human being survival. The process of seed development,including morphological development and seed maturation,is governed by a complex network of transcription factors,which regulate and supervise the entire process. Transcription factors include 4 functional domains：DNA binding region,oligomerization site region,nuclear localization signal region and transcription regulatory region,which regulate gene expression by interaction between the domains and the cis-element. According to current research,the WOX family regulates germ development,the HAP3 family regulates embryo morphogenesis and cell differentiation,the MADS-box family regulates the development of embryo and ovule,the NAC family regulates the growth and development of ovule integument,the bHLH family regulates the size and the morphology of seed coat cells,and the MYB family is a positive regulator of seed coat development. Members of the HAP family,LEC1,and members of the AFL subfamily of the B3 superfamily,LEC2,FUSCA3 and ABI3,interact to form a regulatory network that regulates the mature development of the seed. The Dof family of the zinc finger superfamily regulates the development of endosperm,the synthesis of storage proteins and the change of fat content in seeds,the bZIP family regulates the expression of storage genes,the AP2/EREBP family regulates the accumulation of seed volume and weight,protein and oil,and the WRKY family,IKU,MINI3,SHB1 gene and KLU gene regulate the size of a seed. This paper reviews the structure and types of transcription factors in plant seed development,analyzes the order and function of the genes,dissects the molecular regulation mechanism of seed development,and prospects the research direction and prospects,aiming at providing theoretical basis and new ideas for improving seed quality.

Genetic Engineering Progress and Breeding Tactics on Blue Flowers

LI Chong-hui, YIN Jun-mei

2019, 35(11):  160-168.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0450

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Blue flowers possess highly ornamental value,but its coloration mechanism is very complicated. A great number of progresses have been achieved in the study of the pigmentation mechanism and genetic engineering in blue flower. Here the chemical,physiological,and molecular mechanisms of blue flower color are summarized. Many breeding practice for blue flowers are reviewed,including overexpression of key enzyme genes on anthocyanin biosynthetic pathway,expressing inhibition of entogenous competitive enzyme genes,introduction of regulatory genes for vacuoles pH regulation,and overexpression of metal ions transporter genes. Several tactics of genetic engineering for blue flowers are summarized based on these breeding practices,aiming to provide the reference for the genetic engineering breeding of blue flowers.

Research Progress on Endophytic Fungi Improving Plant Resistance to Salt Stress

LI E, HU Hua-ran, LI Jiao-nan, DU Guang-hui, LIU Fei-hu

2019, 35(11):  169-178.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0499

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Soil salinization is becoming increasingly serious,and it has become a major constraint in plant production. Therefore,research on soil salinization has become a hotspot in recent years. Salt stress mainly causes difficulties in plant uptake of soil water and nutrients. Endophytic fungi may promote plant uptake of soil water and nutrients under salt stress,alleviate salt-induced damages,thereby maintaining plant growth,population structure and biomass. In this paper,we summarized the discovered endophytic fungi and their effects on plant resistance to salt stress,discussed the research prospects of endophytic fungi and current issues,aiming to provide reference and basis for discovering and utilizing of microbial resources to enhance plants resistance to salt stress.

Research Progress on Chitin Deacetylase

ZHANG Yan, GUAN Fei-fei, WU Ning-feng, TIAN Jian

2019, 35(11):  179-186.  doi:10.13560/J.cnki.biotech.bull.1985.2019-0449

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Chitin is the second largest polysaccharide to cellulose in the natural world. Chitosan is a de-acetylated derivative from chitin and has been widely used due to its fine solubility. First,a various deacetylase protein sequences from different sources were collected and aligned,then bioinformatics methods were conducted to carry out in-depth excavation and analysis of their catalytically active central domains,and the similarities and differences of biological sources,catalytic mechanisms and reaction conditions of various deacetylases with different catalytic domains were elucidated. The results showed that the studied deacetylases were mostly derived from fungi and insects,mostly belonged to the CE4 family,and had the catalytic activity center of NodB. It was easier for them to react with acetylated oligosaccharides with a polymerization degree >3,while difficult to catalyze poorly soluble polysaccharides. Most of these enzymes had the maximum enzyme activity at pH 8.0 and 40-70℃,and different divalent metal ions had different impacts on different enzymes. Finally,this paper proposed a new direction for rapid and specific screening of new enzymes,analysis of enzymatic hydrolysis mechanisms and molecular engineering from marine macrogenomic libraries,providing a new insight for researchers in the field to develop highly efficient and highly specific deacetylases.

Research Progress on the Nuclear Reprogramming After Somatic Cells Nuclear Transfer in Mammalian

WU Xiao, ZHUANG Zhan-wei, MA Xiao-li, HUANG Si-xiu, LI Zi-cong, XU Zheng

2019, 35(11):  187-194.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0168

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Somatic cell nuclear transfer technology in mammalian has been developed for more than 20 years,however,the abnormal nuclear reprogramming during embryonic development is critical barrier to the application of this technology. At present,the key method to improve the efficiency of cloning is to overcome the error of reprogramming by adjusting the epigenetic modification in the reprogramming process,thereby improving the development efficiency of reconstructed embryos. Here,we summarize the abnormal of donor nuclear reprogramming in the development of early embryos after nuclear transfer,and discuss the research status in correcting these epigenetic modifications. Meanwhile,we prospect the possible reprogramming events and emerging technologies that influences the development of nuclear transfer embryos.

Research Advances on Type I Interferons Used for Understanding Etiology of Canine Diseases and Treatment of Them

HU Wen, FENG Xun, ZHANG Bao-bao, YAN Zhi-wei, XU Yu-sheng, LIU Yong-sheng

2019, 35(11):  195-200.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0851

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Type I interferons（IFNs）are a group of cytokines with immunological activity. Type I IFNs can function as antiviral role,inhibit cell proliferation,and regulate immunity and anti-tumor effect by the activation and regulation of adaptive and innate immune responses. To date,type I IFNs are mainly used for the therapy of human renal cancer,melanoma and chronic hepatitis B and so on. Some members of type I IFNs have been approved for the treatment of canine parvovirus,feline leukemia virus and feline immunodeficiency virus infections. However,type I IFNs have still not been used for the treatment of canine diseases. This paper reviews the latest advances on the immunologic competence of type I IFNs and IFNs-activated down-stream immune signal pathways,aiming to offer valuable references for researchers in canine diseases.

Summary of Genomics Mining Technology and Its Research Progress in Fungi

XU Jie ,HUANG Jian-zhong, LI Li

2019, 35(11):  201-207.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0170

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Microbial secondary metabolites are important sources of drugs. When the traditional activity-oriented screening mechanism is stagnant，how to adopt new screening methods to reduce the repeated detection of known compounds and to quickly obtain new compounds with novel structures and special biological activities is the key to drug development. Genomic mining technology has become a research hotspot at home and abroad because it may control the directed expression of genes encoding secondary metabolites，combine scientific theories with industrial production and guide them，effectively reduce the development cost and cycle，and contribute to the final market application. In this paper，the research direction of genome mining technology and the technical means of activating silenced gene expression are summarized，the important status of fungi in genome mining and the research achievements in recent years are also described，and the broad prospect of fungi in the field of medicine is highlighted.

Advances in Superovulation of the Cow

WANG Yang-yang, SUI He-ming, ZHAO Shan-jiang, XU Hui-tao, HAO Hai-sheng, PANG Yun-wei, ZHAO Xue-ming, ZHU Hua-bin

2019, 35(11):  208-216.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0417

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Superovulation with exogenous gonadotropin is still the main way to produce transferable embryos in vivo for bovine embryo transfer industry. Although many researches have been carried out to study and refine this technique,but the efficiency of donor cow`s superovulationdid not increase during the last seven decades,because the superovulation result was influenced by multitudinous and intricate factors. This article reviewed the new advances in superovulation of donor cows,aiming to find useful information for increasing efficiency of bovine superovulation and transferable embryos.

Development of SSR Molecular Markers with Sorghum Polymorphism Using Re-sequencing

WANG Ping, WANG Chun-yu, ZHANG Li-xia, CONG Ling, ZHU Zhen-xing, LU Xiao-chun

2019, 35(11):  217-223.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0692

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SSR marker is widely applied in molecule-assisted breeding due to its low cost and easy operation. Currently,sorghum SSR marker is based on sequencing,and although the genome sequence of American sorghum BTX623 is completed,the efficiency is low in screening polymorphism markers in applications. In this study,26 sorghum materials composed of sterile lines and restorer lines were re-sequenced and analyzed by bioinformatics,and 24 441 SSR markers containing at least 2 polymorphic types in 26 materials were developed. These SSR markers generated 2-7 genotypes in 26 sorghum varieties,including 16 694 markers for 2 genotypes and only 77 markers for 7 genotypes. In addition,6 673 polymorphic SSRs at the single-copy gene were screened. Fifty pairs of SSRs on the 10 evenly-covered sorghum chromosomes were randomly selected as bridge markers,and Liaoning sorghum hybrid Liaonuo 3 was used for test. Among them,49 pairs of primers were amplified,the success rate of which achieved 98%. Subsequent test with 2 sorghum varieties and 74 mini-core germplasm showed that 18 pairs of SSR markers among 50 pairs of SSRs presented polymorphism between the 2 varieties. One selected SSR marker generated 8 genotypes in 74 mini-core germplasm. This study demonstrates that re-sequencing sterile lines and restorer lines may effectively develop SSR markers for Chinese sorghum breeding.

Development of SNP Marker Based on the Rehmannia glutinosa Transcriptome Database and Construction of DNA Fingerprint in Rehmannia

GUO Meng-meng, ZHOU Yan-qing, DUAN Hong-ying, YANG Ke, SHAO Lu-ying

2019, 35(11):  224-230.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0409

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Rehmannia glutinosa possesses well-known medicinal properties and nutritional values. Accurately identifying varieties is essential for germplasm management and breeding. Using the SNP molecular markers to identify R. glutinosa germplasm and to construct fingerprints provides a new method for R. glutinosa molecular marker breeding. Rehmannia transcriptome data was applied and compared with three rehmannia transcriptome data in the SRA database to find candidate SNPs,and PCR technology amplification and sequence analysis were used to study the mutations of candidate SNP of 28 Rehmannia cultivars. Total 102 075 Unigenes were obtained from the transcriptome data of R. glutinosa,including 35 339 SNP loci with a frequency of 0.34. A 40 candidate SNPs were randomly selected,and 39 pairs of primers were designed. Seven pairs of good SNP primers were selected by PCR and sequence analysis,including 8 SNP loci with fine polymorphism. The fingerprint of R. glutinosa was constructed using these 8 SNP loci,which allowed to distinguish 17 different rehmannia germplasm,thus it can be used for the identification of interspecific and intraspecific species of R. glutinosa.

Preparation of miR-155 Knockout Cells Mediated by CRISPR/Cas9 Technology

LI Cong-cong, ZHANG Yong-hui, ZHAO Wan-xia, WU Jiao, HAN Hao-yuan, LI Meng-yun, NIU Hui, SONG Su-fang, LI Wan-tao

2019, 35(11):  231-239.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0609

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To study the function of miR-155 in porcine alveolar macrophage cell line（3D4/21）,miR-155 knockout cells were constructed using CRISPR/Cas9 technology. Firstly,4 prime series specific to sgRNAs for targeting miR-155 were designed using sgRNA-cas9 program. Then,sgRNA expression vectors were constructed and co-transfected with pEGFP-C1-cas9 vector into 3D4/21 cells. Next,the cells expressing green fluorescent protein（GFP）were enriched and selected using the FACS-sorting strategy. Finally,T7E I cleavage assay,Sanger sequencing,real-time quantitative PCR（RT-qPCR）and Western blot were used to detect the efficiency of the knockout. We found that miR-155’s genomic sequence was edited using 4 different sgRNAs,while the highest efficiency of knockout reached by sgRNA39,up to 42% after T7EI enzyme digestion and FACS-sorting. Sanger sequencing demonstrated that 31 nucleotides of sgRNA39 cell line were knocked out. The RT-qPCR result showed that the expression levels of miR-155-5p/3p significantly decreased. The Western blot result indicated that the protein expression level of target gene SHIP1 in miR-155 significantly increased. To sum up,the successful establishment of the miR-155 knockout cell line provides cell model for further studying the regulation function of miR-155 in macrophage cells.

Knockout of NODAL Gene in MDA-MB-231 Cell Line by CRISPR/Cas9

LI Nian-feng, FANG Tian-xing, NI Yu-fang, ZENG Fan-cai

2019, 35(11):  240-250.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0426

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In order to study the function of growth differentiation factor NODAL（nodal growth differentiation factor）,the cell line with NODAL gene knockout needs to be established. Two different CRISPR/Cas9 expression vectors targeting different sites and one targeting vector for homologous recombination were constructed and then transfected into triple negative breast cancer cell line MDA-MB-231 by electrotransfection. The genotypes after purinomycin resistance screening and single-cell clones selection were identified by PCR and sequencing,and protein expression was detected by Western blot. Eight single-cell clones identified as positive by allelic homologous recombination assay were re-identified by non-homologous recombination and large fragment deletion analyses. Only one single-cell clone had a frame-shift mutation in the target site. A large fragment deletion occurred at the target sites of the 4 single-cell clones,and the wild-type alleles were detected at the target sites of the 4 single-cell clones. Western blot showed that NODAL expressions in only 2 single-cell clones were not detected,and NODAL expressions were decreased in 3 single-cell clones,and no significant changes in NODAL expressions were found in other 3 single-cell clones. Thus,the single-cell clones with NODAL gene knockout are obtained based on genotype identification and Western blot,which lays a foundation for further research on the molecular mechanism of NODAL in breast cancer cells.