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    26 June 2022, Volume 38 Issue 6
    Recent Advances in CRISPR/Cas-based DNA Base Editing
    LAI Xin-tong, WANG Ke-lan, YOU Yu-xin, TAN Jun-jie
    2022, 38(6):  1-12.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0311
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    CRISPR/Cas-based genome editing has revolutionized biological technology,which is achieved by introducing DSB(double strand break)in target DNA sites followed by cellular DNA repair via non-homologous end joining(NHEJ)or homology-directed repair(HDR). NHEJ is the main repair pathway in most of cells,it characterized as repair is simple,efficiency is high and it is extremely easy to have errors,and thus presents randomly and unpredictable insertions and deletions(indels). Point mutations represent the most prevalent form of genetic mutations,which determine the many important agronomic traits in crop plants and cause most of known human genetic diseases. Base editing enables targeted single base substitutions of high efficiency and predicted editing outcomes with neither inducing DSBs in the genomes nor requiring donor templates. Based on its great applicable potentials in the gene therapy,crop breeding as well as basic research,base editing rapidly gains much attentions with numerous applications in agricultural as well as biological fields and considerable progresses having been made to broaden its capabilities. In this review,we summarize the current advances in DNA base editing,focus on improvements in editing efficiency,precision,specificity as well as editing scope,and discuss current limitations and future directions.

    Research Progress in Plant Response to Cd Stress
    LIU Zi-ran, ZHEN Zhen, CHEN Qiang, LI Yue-ying, WANG Ze, PANG Hong-bo
    2022, 38(6):  13-26.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1286
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    Cadmium(Cd)is a heavy metal pollutant with heavy toxicity,ranking the first in soil inorganic pollutants. Cd can be absorbed by plants because of its strong fluidity and solubility. It affects the normal physiological metabolism of plants,reduces crop quality,and endangers human health through the transmission and enrichment of the food chain. Therefore,the response mechanism of plants to Cd stress has become a research hotspot. This paper reviews the damage effects of Cd stress on plant physiological metabolism and molecular level,and summarizes the tolerance mechanism that plants have evolved under Cd stress,aiming to provide a theoretical basis for cultivating low-accumulation crops and super-accumulation plants of remediating Cd contaminated soil.

    Research Progress in Plant Flavanone-3-hydroxylase Gene
    DUAN Yue-tong, WANG Peng-nian, ZHANG Chun-bao, LIN Chun-jing
    2022, 38(6):  27-33.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1253
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    Flavanone 3-hydroxylase(F3H),as a central enzyme for plants to enter the branch of different flavonoid metabolites,can generate dihydroflavonol,a common precursor substance for the synthesis of anthocyanins and flavonols,and it plays a very important regulatory role in the synthesis of flavonoids. From the discovery,structure,function,and expression regulation of the F3H gene,this article reviews the research progress and regulation network of F3H gene in regulating the synthesis of anthocyanins and flavonols,and prospects the future direction. It is aimed to provide a reference for understanding the regulation mechanism of F3H gene on the metabolic synthesis pathway of plant flavonoids,and also help researchers to use F3H with genetic engineering method to obtain the new plant germplasm via directive breeding.

    Endocytosis of AtRGS1 Involved in the Regulation of G-protein-mediated Arabidopsis Development and Stress Responses
    GU Pan, QI Xue-ying, LI Li, ZHANG Xi, SHAN Xiao-yi
    2022, 38(6):  34-42.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0849
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    Heterotrimeric G protein complex, classically consisting of Gα,Gβ,and Gγ subunits, mediates an essential signaling pathway across the PM (plasma membrane) in most eukaryotes. In plants, heterotrimeric G proteins regulate multiple biological processes such as plant growth and development, hormones and sugar signal transduction, and defense processes via RGS(regulator of G-protein signaling protein)protein in PM. The endocytosis of PM-localized proteins is involved in the control of their quantity on the PM in response to environmental stimuli and development signals. In Arabidopsis, several studies recently revealed that various external signals induced the endocytosis of AtRGS1, thus promoted the dissociation of AtRGS1 from Gα. Free Gα-GTP,Gβγ subgroup, and internalized AtRGS1 protein could regulate downstream signal transduction, thus affect the corresponding biological processes. In this review, we give an overview of the molecular cytological mechanism of AtRGS1 endocytosis in G-protein-mediated plant development and stress responses in Arabidopsis, aiming to provide a theoretical reference for deep understanding how RGS affects the plants development process and stress responses, as well as shed a new insight on the regulation of signal transduction by endocytosis of PM proteins.

    Structure of ABC Transporter and Research Progress of It in Bacterial Pathogenicity
    CHEN Fu-nuan, HUANG Yu, CAI Jia, WANG Zhong-liang, JIAN Ji-chang, WANG Bei
    2022, 38(6):  43-52.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1175
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    Adenosine triphosphate binding cassette transporter(ATP-binding cassette transporter,ABC transporter)is known as the most numerous protein family with multiple roles. Functionally,ABC transporter mediate the cross-membrance transmission of multiple substrates by using the energy generated by ATP hydrolysis. ABC transporters exist in most organisms including bacteria,fungi,nematodes,fruit flies,plants,and mammals. Most of ABC transporters were originally discovered by studying the drug resistance(multiple drug resistance and multidrug resistance)of eukaryotes. Recently,there are broad researches on the roles of ABC transporters in bacterial pathogenicity. This paper summarized the structure and mechanism of ABC transporter and the role of it in bacterial pathogenicity,discussed the significance and issues in further studying the mechanism of ABC transporter and control of bacterial diseases. The cell surface or secretory factors associated with ABC transporters is potential targets of antimicrobial therapy or vaccine development,which provides a new idea for the prevention of bacterial diseases.

    Advances in Research on the Effects of Joint Medication on the Drug Resistance of Aeromonas hydrophila
    ZHAO Hai-qing, LI Yun, LIANG Yan-nei, LIU Zhe, REN Ya-lin, LI Jin-juan
    2022, 38(6):  53-65.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1004
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    Aeromonas hydrophila is one of the most serious pathogenic microorganisms threatening the aquaculture industry,and it also has characteristic of zoonotic diseases. Due to the using of antibiotics or antibacterial drugs and exposure to hospitals wastewater,livestock and poultry farming wastewater,the problem of multi-drug resistance of A. hydrophila has become increasingly prominent,which contributes potential risks to the aquiculture safety and human being health. The rational joint medicationcan not only slow down and prevent the emergence of bacterial resistance,but also reduce drug residues. This paper first analyzes the principles followed by the joint medication and the direction to be paid attention to,expounds the causes of drug resistance and the mechanism of the effect of the joint medication on drug resistance,and synthesizes the dynamics progress of the joint medication to block drug resistance in A. hydrophila. Finally,the thoughts and prospects of how to proceed are proposed,aiming to provide prevention and control strategies and solutions for the aquaculture industry to control the drug resistance of A. hydrophila through joint medication.

    Research Progress in Antibiotic Adjuvant and Antibiotics in Antibacterial Aspects
    ZHU Hao, ZHANG Yan-wei, LIU Run, LIANG Yan, YANG Yi, XU Tian-le, YANG Zhang-ping
    2022, 38(6):  66-73.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0027
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    Antimicrobial resistance has been a global health challenge,which increases the difficulty of controlling and treating life-threatening bacterial infections. Despite ongoing efforts to identify new drugs or alternatives to antibiotics,no new classes of antibiotic or their alternatives have been clinically approved in the last two decades. A combination of antibiotic adjuvants and antibiotics inhibit bacterial resistance determinants or restore antibiotic sensitivity,and offer a sustainable and effective strategy to combat multidrug-resistant bacteria. The current review summarizes the classification and mechanism of antibiotic adjuvants,as well as discusses the challenges and development trends of antibiotic adjuvant and antibiotic combination strategy.

    Rapid Crude Extraction of Genomic DNA from Solanum lycopersicum for PCR
    SHEN Heng, LIU Si-hui, LI yue, LI Jing-tao, LIANG Wen-xing
    2022, 38(6):  74-80.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1123
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    It is necessary to extract plant genomic DNA and perform PCR cloning when conducting the studies of molecular biology on plants. Currently,the CTAB(Cetyltrimethyl Ammonium Bromide)is commonly used to extract plant DNA in laboratories,but the steps are relatively cumbersome and require multiple organic solvents. Specifically,it takes a long time when there are many samples. The DNAs from multi-species were extracted by NaOH,and this study introduces a method of extracting plant genome DNA,which is faster,economical,safer and more efficient. The major steps include the followings:Quickly grinding the plant tissues using liquid nitrogen;quickly extracting DNA by alkaline lysis(0.5 mol/L NaOH);neutralizing the extracted DNA sample with TE buffer;and performing PCR with neutralized DNA as template. Using this simple DNA extraction method and combining PCR detection,the rapid preliminary identification of transgenic S. lycopersicum was completed. Furthermore,it was confirmed that this NaOH method could be used for extracting DNA from the different tissues of S. lycopersicum. The entire extraction process was simple and of short-time. Thereby,DNA extraction for a large number of plant samples could be completed in a short time without using toxic regents such as chloroform and isopropanol etc. The completion time of DNA extraction is about 1/5 of the time while using CTAB method. In addition,the extracted DNA from different tissues are of good integrity and high quality,which meet regular PCR detection,which can be used for gene cloning and for transgenic tomato screening. This method demonstrates potential utilization value and prospects.

    Effects of Analysis Methods on the Analyzed Results of 16S rRNA Gene Amplicon Sequencing in Bacterial Communities
    ZHONG Hui, LIU Ya-jun, WANG Bin-hua, HE Meng-jie, WU Lan
    2022, 38(6):  81-92.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1102
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    Bacterial 16S rRNA gene amplicon sequencing is one of the most widely used method in environmental microbiology. However,there are many ways to determine the minimum taxonomic unit of the sequencing reads,and its effect on the downstream analysis results of microbial diversity needs to be further explored. This study extracted DNA from 5 groups of environmental samples(forest soil,farmland soil,wetland soil,lake sediment,and lake water)for 16S rRNA gene amplicon sequencing,and based on different taxonomic resolutions,5 types of minimum taxonomic unit division method(Sequence clustering OTUs at 97%,98%,99%,100% similarity,and the DADA 2 algorithm to calculate ASVs)were used to compare and analyze the influences of minimum taxonomic unit division methods on microbial community diversity,composition and its correlation with environmental factors. The results showed that higher community α diversity(Chao1 and Shannon)and β diversity(P < 0.05)were obtained by increasing the taxonomic resolution. Compared with the OTU,which was clustering by sequence similarity,the ASV method reduced Chao1 and PD indices to some extent. For community composition,the division method of the taxonomic unit mainly affected the proportion of some low-abundance genera(< 0.2%),but had a smaller effect on the composition of higher taxonomy level(phylum level). In addition,the results of redundant analysis demonstrated that increasing the level of taxonomic resolution would allow the environmental factors to have a higher interpretation of the microbial community,and a concurrently it also affected the order of interpretation degree of community composition by environmental factors. In sum,this study clarified the effect of different division methods of the minimum taxonomy unit on the diversity,composition and association with environmental factors,which provides theoretical guidance for the subsequent study of environmental microbiomics.

    Pathway Engineering Modification of Corynebacterium glutamicum for Shikimic Acid Production
    NIE Li-bin, YI Ling-xin, DENG Yan, SHENG Qi, WU Xiao-yu, ZHANG Bin
    2022, 38(6):  93-102.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1058
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    Shikimic acid,as an intermediate metabolite of aromatic family,is the precursor of oseltamivir phosphate for the synthesis of an anti-avian influenza drug. Currently,the production of shikimic acid mainly depends on the plant extraction method with shortcoming in high cost and long period. Due to the advantages in low production cost and short period,microbial fermentation for the synthesis of shikimic acid has turned into the hot spot of research. In order to construct a recombinant C. glutamicum capable of producing shikimic acid,the metabolic pathways of shikimic acid in C. glutamicum was modified via metabolic engineering at genetic level. The yield of shikimic acid significantly increased by blocking the catabolic pathway,relieving the feedback inhibition,and blocking the competitive metabolic pathway. The final strain C. glutamicum SKA06 produced 7.61 g/L of shikimic acid during 72 h shake flask cultivation,which was 68 times higher than that of the original strain. Moreover,genetic modification based on chromosome engineering overcame the problems of unstable passage and antibiotics addition caused by the introduction of plasmid,which may provide important reference for the development of shikimic acid producing strains.

    Whole Genome Identification of Barley NRAMP and Gene Expression Analysis Under Heavy Metal Stress
    LI Yi-han, YU Lang-liu, LI Chun-yan, ZHANG Meng-meng, ZHANG Xiao-qin, FANG Yun-xia, XUE Da-wei
    2022, 38(6):  103-111.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1006
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    Natural resistance associated macrophage protein(NRAMP)is an important type of membrane transporter in plants,it plays an important role in transporting and reusing heavy metal ions. Studying the mechanism of NRAMP family gene expression regulation under Mn2+,Cd2+ and Cu2+ stress would provide a theoretical basis for further revealing the roles of the NRAMP family genes against heavy metal stress. Bioinformatics methods were used to screen and identify NRAMP family gene sequences from the whole genome of Hordeum vulgare L.,analyzes the gene structure,conserved motifs,evolutionary characteristics and expression profiles of its family members. Moreover,qRT-PCR method was applied to detect the expression of the NRAMP gene in barley seedlings under different heavy metal ion stress. The results showed that there were 10 NRAMP family members in the barley genome,which were mainly located on the cell membrane. Phylogenetic analysis demonstrated that the conserved motifs and gene structures of closely related family members were similar,and the genetic relationship between barley and rice NRAMP family members was closer. The results of quantitative analysis showed that the expression levels of HvNramp1 and HvNramp2 were significantly up-regulated under Mn2+ stress. Under Cd2+ stress,all genes’ expression levels were up-regulated except HvNramp8. Under Cu2+ stress,the expression levels of the remaining 6 genes except HvNramp3 were significantly up-regulated. The above results indicate that the NRAMP family genes can respond to the stress of heavy metal ions through positive regulation.

    Identification of AHP Gene Family in Cucumis sativus and Its Expression Analysis Under Abiotic Stress
    ZHOU Guo-yan, YIN Shan-shan, GAO Jia-xin, WU Chun-cheng, YAN Li-ying, XIE Yang
    2022, 38(6):  112-119.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1338
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    Histidine-phosphate transporter(AHP)is one of the important proteins in cytokinin signal transduction pathway,which plays an important role in plant growth and development and resistance to a series of abiotic stresses. Exploring the molecular characteristics and expression pattern of AHP in C. sativus can provide theoretical basis for further exploring the function of AHP gene in C. sativus and improving abiotic stress tolerance of plants. The number,evolutionary relationship,cis-acting elements,gene structure,development and expression patterns of AHP gene family members under abiotic stress in C. sativus were analyzed. The results showed that there were a total of 7 members of AHP gene family in C. sativus,which were unevenly distributed on 6 chromosomes. Phylogenetic analysis indicated that the AHP gene family members of C. sativus were closely related to Benincasa hispida and Citrullus vulgaris Schrad L. Its promoter sequence mainly contained cis-acting elements including light response,low temperature response,drought response,hormone response and defense stress. Transcriptome analysis showed that CsAHP1,CsAHP3 and CsAHP4 were significantly up-regulated or down-regulated under abiotic stress and the expressions of CsAHP1 was the highest on flowering day. Furthermore,real-time fluorescence quantitative PCR(qRT-PCR)results indicated that CsAHP1 and CsAHP3 were significantly up-regulated under drought and high temperature stress,and CsAHP1,CsAHP2 and CsAHP5 were significantly up-regulated under low temperature and low light stress. The AHP gene family of C. sativus might be closely related to plant development and abiotic stress response.

    Promoting Effect of Graphene Oxide on the Root Growth of Arabidopsis thaliana
    GAO Cong, XIAO Chu-jian, LU Shuai, WANG Su-rong, YUAN Hui-hua, CAO Yun-ying
    2022, 38(6):  120-128.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1188
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    This work aims to clarify the promoting effect of graphene oxide(GO)on the growth of Arabidopsis thaliana,so as to provide a theoretical basis for the application of nanomaterials in agricultural production. Seeds of A. thaliana were cultured in 1/2 MS with different concentrations of GO. The primary root length,lateral root number,root activity,production of superoxide anion free radical,superoxide dismutase activity and expression of root growth-related gene were measured. After 20-200 μg/mL GO treatment,the taproot length of A. thaliana increased by 4.6%-43.0%. Compared with the control(without GO),and the difference was significant within 50-200 μg/mL. Treatment with 50 μg/mL GO significantly promoted the formation of lateral roots,and the number of lateral roots increased by 27.1% compared with the control,while treatment with higher or lower than 50 μg/mL was not conducive to the formation of lateral roots. The results showed that 50 μg/mL GO promoted the taproot length and lateral root number of A. thaliana. It was also found that the concentration of 50 μg/mL GO increased the length of meristem and elongation zone of root tip,but had no effect on the root tip diameter and root cap length. The tissue staining of triphenyltetrazole chloride(TTC)and nitrio blue tetrazolium(NBT)indicated that 50 μg/mL GO increased the root activity,superoxide dismutase activity and decreased the production of superoxide anion. Gene expression analysis demonstrated that ADC1 and DAR2 were down-regulated and IQM3 was up-regulated,which promoted the taproot elongation. The expressions of ARF7,ARF19,ERFII-1 and IQM3 were up-regulated, which promoted the increase of the lateral root number. Treatment with 50 μg/mL GO could promote root growth of A. thaliana. The increase of root activity,the decrease of superoxide anion and the up-regulation of root-related gene expression are the main reasons that GO promotes the growth of root system.

    AtSCL4,an Arabidopsis thaliana GRAS Transcription Factor,Negatively Modulates Plants in Response to Osmotic Stress
    XU Hong-yun, ZHANG Ming-yi
    2022, 38(6):  129-135.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0963
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    Exploring the biological function of Arabidopsis thaliana GRAS transcription factor AtSCL4 in response to osmotic stress would lay foundation for studying the function of GRAS protein in abiotic stresses. Wild-type and AtSCL4 mutant were used as experimental materials,and physiological index determination and qRT-PCR methods were applied to study the biological mechanism of AtSCL4 regulating plant osmosis resistance. The study revealed that AtSCL4 was significantly up-regulated by osmotic stress,and mutants of AtSCL4 showed improved osmotic resistance than WT. In response to osmotic stress,AtSCL4 negatively regulated AtMYB61 expression to increase stomatal closure,resulting in reducing water loss. Furthermore,AtSCL4 improved proline and betaine levels by regulating the transcripts of P5SC1 and BADH. In addition,AtSCL4 increased antioxidant enzyme activity by negatively regulating AtSOD1 and PER4 expressions,leading to the decrease of reactive oxygen. AtSCL4 negatively modulated osmotic stress tolerance via the induction of stress-responsive genes and physiological changes.

    Isolation and Identification of Bacillus subtilis K-268 and Its Biological Control Effect on Rice Blast
    ZU Xue, ZHOU Hu, ZHU Hua-jun, REN Zuo-hua, LIU Er-ming
    2022, 38(6):  136-146.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1124
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    The use of biocontrol bacteria is an effective measure for green prevention and control of rice blast. In order to discover biocontrol bacteria with high efficiency against Magnaporthe oryzae,an antagonistic bacterium K-268 with the optimal inhibitory effect on M. oryzae from the healthy rice leaves of the susceptible variety K020268 was obtained by using the plate confrontation method and its antimicrobial spectrum was determined. Using morphological observation,physiological and biochemical identification 16S rRNA and gyrA sequence analysis,the identity of the strain was determined. In addition,the physical characteristics of the strain and its control effect against rice leaf blast were preliminarily studied. Results showed that strain K-268 had inhibitory rate of(86.30 ± 0.70)% on the mycelial growth of M. oryzae,and it demonstrated the inhibitory effects on 14 plant pathogenic fungi,such as Rhizoctonia solan,Exserohilum turcicum and Diaporthe citri,etc. Strain K-268 was identified to be Bacillus subtilis,its logarithmic growth period was 14-32 h,the optimal;growth temperature was 30℃,the optimal pH was 6.0-7.0,and had good salt tolerance. The results from in vitro inoculation experiments showed that the disease rate in the prevention group and the treatment group was14.81% and 23.46%,respectively,when the bacterial solution was diluted by 10 times to 6×108 CFU;and the effect of which was not significantly different from that of the 750 times diluted 75% tricyclazole diluent(WP). Results of in vivo spray inoculation showed that the control effects on the rice leaf blast were 59.00% and 60.23%,respectively,when the 10 times diluted fermentation liquid(6×108CFU/mL)were sprayed 24 h before and after inoculation with Magnaporthe oryzae conidia suspension(1×106 /mL),respectively. These effects were close to the 40% rice blast spirit(EC)with 500 times dilution. Therefore,K-268 was confirmed to have certain development and application value in the biological control of rice blast.

    Cloning,Expression of Helianthus annuus HaLACS1 Gene and Identification of Its Functional Complementation in Saccharomyces cerevisiae
    YANG Jia-bao, ZHOU Zhi-ming, ZHANG Zhan, FENG Li, SUN Li
    2022, 38(6):  147-156.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1174
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    This work aims to investigate the functionality of long chain acyl-CoA synthetase(LACS)gene in Helianthus annuus L. in the oil accumulation and responses to abiotic stresses,for laying a foundation in the its oil synthesis and application in the tress responses. RT-PCR was applied to clone the CDS sequence of the HaLACS1,and bioinformatics method was to analyze the characteristics of the HaLACS1. Real-time quantitative PCR was employed to detect the expression patterns of HaLACS1 in different tissues and in responses to NaCl,PEG and ABA treatments. The fusion protein of GFP and HaLACS1 was constructed and transformed into Arabidopsis thaliana protoplasts for subcellular localization analysis. The HaLACS1 gene was transformed into a LACS-deficient yeast strain YB525 to examine the complementation effect and analyze its substrate. The results showed that the length of HaLACS1’s open reading frame was 1 980 bp,encoding 659 amino acids. The phylogenetic tree analysis demonstrated that HaLACS1 was highly similar with the AtLACS1 in A. thaliana and LsLACS1 in Lactuca sativa. The transient expression assays revealed that HaLACS1 was located in endoplasmic reticulum. qRT-PCR result showed that HaLACS1 was ubiquitously expressed in all tissues of H. annuus,but highly expressed in seeds at early developmental stage,followed in the flowers. The transcription levels of HaLACS1 were induced by NaCl,PEG and ABA in H. annuus roots,stems and leaves. Expression of HaLACS1 in the yeast strain YB525 demonstrated that HaLACS1 has LACS enzyme activity. Substrate preference analysis showed that HaLACS1 preferred palmitic acid(C16:0)and oleic acid(C18:1). All these results suggest that HaLACS1 is related to H. annuus in response to abiotic stresses and oil accumulation in the process of seed development.

    Organic Phosphate-solubilizing Bacteria Screening in the Rhizosphere of Paeonia ostii and Study on Their Phosphate-solubilizing Capabilities
    SHEN Jia-jia, HOU Xiao-gai, WANG Er-qiang, WANG Fei, GUO Li-li
    2022, 38(6):  157-165.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0942
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    Paeonia ostii as a new woody oil crop with high oil values,the deficiency of available phosphorus in the soil severely restricts its growth and development,yield and quality. Isolation and screening of highly effective phosphate-solubilizing bacteria(PSB)and playing the role of rhizosphere microorganisms is an effective way to achieve the goal of high yield and high quality of P. ostii and ensure the green development of agriculture. Seedlings of P. ostii ‘Fengdan’ growing for different years were selected as study objects,and phytin was selected as the only phosphorus(P)source in the medium. Then the method of plate dilution was employed to isolate organic phosphate-solubilizing bacteria. And finally the 16S rDNA sequencing technology was used for molecular identification of these strains,and methods of phosphate-solubilizing circle and liquid culture were applied to analyze their phosphate-solubilizing abilities. As results,14 strains of PSB were isolated from the rhizosphere of P. ostii in 1 year,3 and 4 years. Among them,there were 5 strains of Pseudomonas,5 strains of Bacillus,3 strains of Arthrobacter and one strain of Novosphingobium,respectively. The phosphate-solubilizing index and phosphate-solubilizing efficiency of 14 strains ranged in 1.6 to 9.0 and 60% to 800%,among them the phosphate-solubilizing index and phosphate-solubilizing efficiency of strain FD1-15 was the highest. The available P content in liquid culture of PSB ranged in 2.88 mg/L to 5.30 mg/L,and the mobilization ratio ranged in 26.44% to 62.13%. The strain FD3-13 isolated from the rhizosphere of P. ostii with three-year-seedlings presented the strongest phosphate-solubilizing capacity,which belonged to genus of Bacillus. The results provide a theoretical and technical support for improving oil yield and edible quality of oil peony and useful reference for development and application microbial fertilizers.

    Characteristics of Endophytic Fungal and Bacterial Community in the Stalks of Sugarcane Cultivars Resistant to Ratoon Stunting Disease
    GAO Xiao-ning, LIU Rui, WU Zi-lin, WU Jia-yun
    2022, 38(6):  166-173.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1197
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    By analyzing the structural characteristics of endophytic fungi and bacteria in the stalks of sugarcane varieties resistant to ratoon stunting disease(RSD),the aim is to understand the roles of endophytic fungi and bacteria in the resistance of sugarcane,so as to provide theoretical basis for ecological control of RSD. Using Illumina MiSeq high-throughput sequencing method,the communities,diversity index,and differences of endophytic bacteria and fungi between susceptible variety YT60 and resistant variety HoCP95988 were compared. The Alpha diversity indexes of endophytic fungi and bacteria in the stalks of susceptible cultivar YT60 was higher than that of resistant cultivar HoCP95988,indicating more species composition. The endophytic bacteria mainly distributed in Proteobacteria,while Ascomycota and Basidiomycota were the dominant phyla of endophytic fungi. The species compositions of 34 genera in 4 phyla Planctomycetes,Chloroflexi,Gemmatimonadetes,and Tenericutes between susceptible and resistant varieties were significantly different. Among them,the relative abundance of different species was higher in susceptible variety YT60. The diversity of endophytic fungal and bacterial communities in the stalks of sugarcane varieties resistant to RSD were relatively rich,and there were significant differences in the composition. Meanwhile,the classification information of different species provided clues to explore the biocontrol microbial resources for control RSD.

    Characteristics of Crambe abyssinica Chloroplast Genome and Its Phylogenetic Relationship in Brassicaceae
    QIAN Fang, GAO Zuo-min, HU Li-juan, WANG Hong-cheng
    2022, 38(6):  174-186.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1078
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    Crambe abyssinica,belonging to Crambe genus in Brassicaceae,has been used as a potentially renewable industrial oil resource because of its high natural erucic acid content in seed. Here,the chloroplast(cp)genome of C. abyssinica was de novo assembled by Illumina Platform,and its phylogenetic relationship was detected based on the cp genome in Brassicaceae. The cp genome of C. abyssinica had a quadripartite structure of 153 754 bp in length,which were annotated a total of 111 unigenes,including 78 protein-coding genes,29 tRNA genes and 4 rRNA genes. Codon usage showed a bias of A/U-ending codons in the cp genome of C. abyssinica. A total of 41 repeat structures and 59 SSRs(simple sequence repeats)were detected in this cp genome. Comparative analysis showed that the genome sequence was conserved and the protein-coding sequence had high similarity. Synonymous and non-synonymous analysis detected strong positive selection exerted on the ndhF and ycf2 gene in the cp genome of C. abyssinica. Moreover,phylogenetic analysis showed that C. abyssinica was closely related to Brassica and formed a sister group with Sinapis species. This suggests that it is possible to transfer favorable genes from C. abyssinica to other Brassicaceae plants by distant hybridization,and also provide important molecular information for elucidating the evolutionary history of C. abyssinica.

    Expressions and of Genes Response to Signal Substances in MAPK Cascade Pathway Genes in Pinus massoniana
    CHEN Hu, YANG Zhang-qi, SUN Shuang, LI Peng, XU Hui-lan
    2022, 38(6):  187-197.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1089
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    MAPK(mitogen-actived protein kinase)cascade pathway plays an important role in response and regulation of biotic,abiotic stresses,and hormone signals et al. In this study,7 genes related to MAPK cascade pathway in Pinus massoniana were identified. We analyzed their physical properties,functional domains,phylogenetic tree,conserved phosphorylation motifs,and gene interactions. We also tested the expression patterns of these genes under hormone and calcium treatment. The results showed that:these genes had close evolutionary relationships with gymnosperms,such as Pinus taeda and Picea asperata. These 7 genes had MAPK domain and typical catalytic amino acid motif. D(I/L/V)K was the common active motif of MAPKs genes of Pinus massoniana. PmMAP4K did not interact directly with other three members of MAPK cascade pathway. Genes in MAPK cascade might regulate the biosynthesis of various hormones by phosphorylation in response to abiotic stress. The results of gene expression showed that all the seven genes responded to MeJA,GA,SA,ABA and Ca2+ treatments,but the expression patterns were different. The results showed that the response of PmMAP4K to Ca2+ treatment was the most obvious followed by PmMAP2K gene,and other genes had no obvious response to Ca2+ treatment only. Four hormones and hormone-plus-Ca2+ treatments increased the expression of genes,respectively. The expression of PmMAPK in MeJA+Ca2+ treatment was higher than that in MeJA treatment alone,while the expression of genes under treatments of each GA,SA,and ABA was higher than that under treatments of each GA,SA,ABA plus Ca2+. This study provides theoretical support for the analysis of the interaction between MAPK cascade pathway genes and signal pathway in respond to abiotic stress.

    Transcriptome Sequencing in the Leaves of Pontederia cordata with Cadmium Exposure and Gene Mining in Phenypropanoid Pathways
    XIN Jian-pan, LI Yan, ZHAO Chu, TIAN Ru-nan
    2022, 38(6):  198-210.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1151
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    To provide theoretical base for the mechanism of phenylpropane metabolic pathway involved in Pontederia cordata against heavy metal exposure,and to analyze the transcriptome sequences in the leaves of P. cordata exposed to cadmium(Cd),the obtained Unigenes were compared and annotated with seven databases including NR,NT,PFAM,KOG,Swiss Prot,and KEGG as well as GO,and the tendency of differentially expressed genes(DEGs)were analyzed,and DEGs involved in phenypropanoid metabolic pathway were mined. Results showed as follows. 1)A total of 221 392 unigenes were obtained,of which 170 175 unigenes were successfully annotated in the public database,and 6 506 unigenes were annotated for 31 secondary metabolic pathways. 2)A total of 168 355 were obtained after comparing the Unigenes from the leaves of P. cordata with dadabase Swiss Prot and NR. And 84 673 CDS sequences were obtained by Esacan software. 3)Trend analysis demonstrated that 20 025 DEGs were clustered into three significant profiles including two down-regulated profiles(cluster 0 covering 2 631 DEGs and cluster 1 covering 3 153 DEGs)and one up-regulated profiles(cluster 5 covering 3 733 DEGs). 4)With Cd2+ the exposure,there were 26 DEGs identified in P. cordata leaves in 6 h and 48 h by further mining the transcriptome data,which encoded 3 crucial enzymes in the common pathway of phenylpropane metabolism,5 crucial enzymes related to lignin biosynthesis,and 7 crucial enzymes responsible for flavonoid biosynthesis. P. cordata is involved in the accumulation and detoxification of Cd in the leaves by regulating the biosynthesis of guaiacyl lignin and up-regulating the gene expression of cinnamate-4-hydroxylase(C4H),anthocyanin synthase(ANS),anthocyanin-3-O -glucosyltransferase(3GT),caffeonyl coenzyme A-O methyltransferase(CCoAOMT),and flavonol 3-methyltransferase(F3OMT). The genes involved in phenylpropane metabolic pathway in the leaves of P. cordata against Cd stress is preliminarily revealed by transcriptome sequencing,which provides a theoretical basis for further research on the function of phenylpropane metabolic pathway in the resistance of the plant to heavy metal stress and related mechanisms.

    Influence on Expression of Jasmonic Acid Signaling Pathway Gene in Tetranychus urticae Fed on Mite-resistant and Mite-susceptible Cassava Cultivars
    HAN Zhi-ling, CHEN Qing, LIANG Xiao, WU Chun-ling, LIU Ying, WU Mu-feng, XU Xue-lian
    2022, 38(6):  211-220.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0993
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    This study aims to clarify how the mite density and feeding time affect gene expressions of jasmonic acid signaling pathway when Tetranychus urticae fed on mite-resistant and mite-susceptible cassava cultivars,which can not only provide a theoretical basis for elucidating the important role of jasmonic acid signal pathway in cassava resistance to insect,but also can offer references for identifying and evaluating mite resistant germplasm resources. Here,mites with different densities(15,25,35,45,50 and 55 mites/leaf)were inoculated on the leaves of mite-resistant cassava cultivar C1115 and mite-susceptible cassava cultivar Mianbao,respectively. And then the expressions of jasmonic acid signaling pathway genes(DAD1,LOX2,OPR3 and JAR1)were analyzed at 1,2,4 and 8 d post-infestation,respectively. The results showed that the expressions of LOX2,OPR3 and JAR1 in 2 d post-infestation either mite-resistant cassava C1115 and mite-susceptible cassava Mianbao significantly increased compared with those before infested,in addition,the expressions of those three genes were significantly higher in C1115 than Mianbao(P<0.05),while the expressions of DAD1 showed great fluctuation as the mite density and infestation time increased. This study revealed that under the conditions of relative high mite density and short mite infestation period,the expressions of jasmonic acid signaling pathway genes like LOX2,OPR3 and JAR1 were significantly higher in mite-resistant cassava cultivars compared with the susceptible ones. Those genes might probably correlate with cassava resistance to mite,which needs further investigation.

    Effects of Soil Types on Bacterial Community Diversity on the Rhizosphere Soil of Arachis hypogaea and Yield
    XU Yang, ZHANG Guan-chu, DING Hong, QIN Fei-fei, ZHANG Zhi-meng, DAI Liang-xiang
    2022, 38(6):  221-234.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0912
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    The objectives of this work are to understand the relationship between bacterial community diversity of the rhizosphere soil and the yield of peanut(Arachis hypogaea)in different soil types,and to define the regional dominance of peanut production characteristics in various soil types. Using 6 soil types from the main peanut-producing areas,pot experiments were conducted to study the bacterial community structure and changing characteristics on the rhizosphere soil and non-rhizosphere soil through high-throughput sequencing technology. As results,bacterial community diversity and richness of the six soil samples showed that the bacterial richness and diversity of Shaoyang red soil of Hunan province were lower,while those of Luanxian brown soil of Hebei province were relatively higher. Actinobacteria,Proteobacteria,Chloroflexi,Acidobacteria,and Gemmatimonadetes were the common dominant phyla,whereas significant differences of the bacterial abundance were found in different soil types,among which Chlorobacteria was the most abundant in Shaoyang red soil,while Actinobacteria and Proteobacteria were enriched in the other soil types. Moreover,bacterial abundances in the rhizosphere and non-rhizosphere soils were different,and the abundances of all bacteria(including Actinobacteria,Proteobacteria,Chlorobacteria,Acidobacteria,Gemmatimonadetes,and Firmicutes)in Shaoyang red soil of Hunan province varied more than that in other areas. Redundancy analysis showed that soil physical and chemical factors of different soil types such as soil acidity(pH),organic matter(ORM),available nitrogen(N),available phosphorus(P),and available potassium(K)had different influences and positive and negative correlation with bacterial community abundance and species abundance. There was a significantly positive correlation between the bacterial community and soil factors in Jiaozuo tide brown soil of Henan province,and a significantly negative correlation between the bacterial community and soil factors in Shaoyang red soil of Hunan province. Statistical analysis demonstrated that the pod yields of Gongzhuling black soil in Jilin province,of Shihezi brown calcium soil in Xinjiang province,and of Yinan brown soil in Shandong provice were higher,while the pod yield of Shaoyang red soil in Hunan province was lower. The abundance of dominant Proteobacteria in Gongzhuling black soil of Jilin province,Shihezi brown calcium soil of Xinjiang province,and Yinan brown soil of Shandong was higher than that in Shaoyang red soil of Hunan province,and Sphingomonas was also higher in Yinan brown soil of Shandong province and Gongzhuling black soil of Jilin province. It was inferred that Proteobacteria and Sphingomonas were beneficial to peanut growth and the increase of pod yield. In conclusion,although the bacterial composition of peanut rhizosphere in different soil types was similar to some extent,the differences were obvious,and the abundance of some bacteria showed unique characteristics. Proteobacteria and Sphingomonas may be beneficial phylum and genus contributed to the increase of peanut pod yield,respectively. The peanut pod yield in Shaoyang red soil of Hunan province was the lowest,and the soil endowment advantage of peanut production was lower.

    Screening of β-lactamase CTX-M-14 Chinese Medicine Inhibitor and Enzyme Inhibition of Rutin
    ZHAO Zi-yu, WANG Chun-guang, LV Jian-cun, LI Ji-kai, ZHANG Tie
    2022, 38(6):  235-244.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1105
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    The production of β-lactamases is a common mechanism for bacteria to develop drug resistance,and its main representatives are extended-spectrum β-lactamases,of which CTX-M-14 is showing an epidemic trend worldwide. In order to find an inhibitor to CTX-M-14 for restoring the susceptibility of bacteria to β-lactam antibiotics,in this experiment firstly a prokaryotic expression vector of CTX-M-14 was constructed,and CTX-M-14 was used as the target site for screening with the help of virtual screening tool Autodock Vina,and finally a monomeric inhibitor of Chinese medicine,rutin,with good binding effect was obtained. Its relevant potency by DS Visualizer was analyzed,the combined inhibitory effect of rutin and antibiotics was verified by co-inhibition test,and the enzyme inhibiting effect of rutin and clavulanic acid were compared by enzyme kinetic test. The results showed that rutin formed multiple hydrogen bonds and van der Waals forces at the binding site of CTX-M-14,and its binding force was 9.9 kcal/mol. Rutin and cefotaxime sodium showed synergistic effect on Escherichia coli E320(FICI=0.236);0.312 5 mg/mL of rutin and cefotaxime sodium showed synergistic effect CTX-M-14 protein(FICI≤0.375);1.25 mg/mL of rutin and cefotaxime sodium had no effect on BL-21 introduced into empty plasmid pET-28a(+)(FICI=1.5);both rutin and clavulanic acid were competitive inhibitors,but clavulanic acid had better enzyme inhibitory effect than rutin. This experiment demonstrates that rutin may competitively inhibit the CTX-M-14 activity and has a potential as β-lactamase inhibitor.

    Effects of CBM41 N-terminal Truncation on the Enzymological Properties of the Pullulanase from Bacillus subtilis 168
    FU Qiao, LIN Qi-lan, XUE Qiang, XIONG Hai-rong, WANG Ya-wei
    2022, 38(6):  245-251.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0927
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    Different N-terminal truncated variants were constructed using N-terminal truncation to modify the protein structure of Bacillus subtilis 168 pullulanase,the effects of truncated mutation on enzymatic properties were studied. Three variants,M1(ΔN2),M2(ΔN4)and M3(ΔN6),by deleting the first 2,4 and 6 residues from the N-terminus respectively,were cloned and expressed in Escherichia coli BL21 cells by genetic engineering method. The optimal temperature of three variants was 40-45℃,and the optimal pH was 6.0,which was consistent with the wild-type pullulanase(WT). Tm values of WT,M1,M2 and M3 were 48.57,50.03,48.43 and 49.50℃,respectively. The specific activity of M1,M2 and M3 were all higher than those of wild-type,increasing by 1.18,1.60 and 2.44 times,respectively. Km values of WT,M1,M2,and M3 were 23.89,29.01,17.29 and 19.08 mg/mL,respectively. These results indicate that variants with the improved properties can be obtained by N-terminal truncation of pullulanase,which provides new methods and ideas for improving the thermal stability,specific activity and substrate binding capacity of this enzyme.

    Medium Optimization for Laccase Production by Acrophialophora sp. Z45 and Its Decolorization of Dyes
    JIA Chen-bo, SU Yi-huang, MA Xiu-mei, WANG Chun-li, XU Chun-yan
    2022, 38(6):  252-260.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1221
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    The strain Acrophialophora sp. Z45 is a laccase-producing fungus,which was discovered in the previous study of our laboratory. In this study,based on the size of guaiacol-oxidation zone on different medium,the factors influencing the laccase production by strain Z45 were studied. The orthogonal experiments were designed to optimize the basic medium for laccase production and the difference in laccase activity before and after medium optimization was then compared. Finally,the ability of strain Z45 to decolorize eight synthetic dyes was evaluated based on the correlation between laccase and dye decolorization. The results based on the single factor experiment and orthogonal test showed that the optimized medium for laccase production was as follows:sucrose as carbon source,sodium nitrate as nitrogen source,the carbon to nitrogen ratio was 45∶1,with a pH value of 5.0. The laccase activity of strain Z45 was significantly improved after medium optimization. Strain Z45 had certain decolorization ability to bromophenol blue,neutral red,methylene blue,methyl blue and crystalline violet,among which the decolorization ability to triphenylmethane dye methyl blue was the strongest. The crude enzyme solution of strain Z45 decolorized methyl blue rapidly. On the solid medium with lower concentrations of methyl blue,the time for complete decolorization of methyl blue was not affected by the dye concentration,but at higher concentrations of methyl blue,the time for complete decolorization was gradually extended with the increase of dye concentration.

    Effects of Temperature on the Growth,Total Lipid and Eicosapentaenoic Acid Synthesis of Eustigmatos sp.
    XU Jin, LI Tao, LI Chu-lin, ZHU Shun-ni, WANG Zhong-ming, XIANG Wen-zhou
    2022, 38(6):  261-271.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1293
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    Eustigmatos sp. has received great attention due to its high content of eicosapentaenoic acid(EPA). Temperature is an important factor affecting the synthesis of polyunsaturated fatty acids. In this study,Eustigmatos sp. JHsu-01 was used as the experimental strain and cultured in the 2 conditions,high temperature(30℃)and low temperature(15℃). The growth,lipid accumulation,fatty acid composition and the expressions of key genes involved in lipid synthesis were measured and the effects of temperature on the EPA synthesis patterns were investigated. The results showed that the low-temperature enhanced the synthesis of membrane lipids and EPA in Eustigmatos sp.,and the highest EPA content was 2.78% DW. Glycolipids were the main carrier of EPA,but temperature changed the distribution of EPA between glycolipids and neutral lipids. The results of transcriptome demonstrated that under low temperature conditions,the expression of key genes in de novo fatty acid synthesis,triacylglycerols(GPAT,plsC,PLPP and DGAT),glycolipids(MGD and DGD),sulfolipids(SQD1 and SQD2)and ω-3 synthesis pathways(Δ5 Des,Δ6 Des and Δ15 Des)were up-regulated. In conclusion,low temperature is an important culture condition to promote the synthesis of EPA in Eustigmatos,and it is also an optimal cultivation approach for obtaining high-content glycolipid EPA. The research results may provide a theoretical and technical guidance to increase the production of EPA.

    Establishment of Porcine Fetal Fibroblasts with OXTR-knockout Using CRISPR/Cas9
    LIU Jing-jing, LIU Xiao-rui, LI Lin, WANG Ying, YANG Hai-yuan, DAI Yi-fan
    2022, 38(6):  272-278.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1432
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    This work is aimed to construct OXTR(oxytocin receptor)- knocked porcine fetal fibroblasts(PFFs)via CRISPR/Cas9,which could be used as donor cells to generate OXTR disrupted Bama miniature pig models. Firstly,the pig OXTR and its human counterpart were analyzed by bioinformatics. Next,two sgRNAs targeting the exon region of pig OXTR were designed using online tools(http://www.rgenome.net/cas-offinder/)and ligated into the pX330 plasmid. Finally,the gene targeting plasmids were co-transfected with a neomycin-expression plasmid into early passage of primary PFFs. G418 screening was used to obtain the resistant single-cell colonies,and the Sanger sequencing was used to determine their genotypes. Bioinformatics analysis showed that the OXTR genes of humans and pigs had a close evolutionary distance. The identity and similarity of the amino acid sequence of human and pig OXTR were 91% and 93%,respectively. The RMSD value of the three-dimensional structure was 0.009. The Cas9/sgRNA targeting vectors of gene OXTR were constructed successfully. After the transfection of PFFs cells,drug screening was used to obtain OXTR knockout monoclonal cells,and they were sequenced,as well as the gene mutant type was confirmed. In conclusion,the human and porcine OXTR are evolutionarily conserved and highly homologous. The constructed Cas9/sgRNA expression vector may allow the OXTR gene editing achieved. The OXTR-knocked monoclonal cells were successfully obtained,which paves the early foundation for the generation of OXTR-knocked Bama pig model.

    Cloning of Bos grunniens TGF-β1 Gene and Its Expression in Major Organs of Female Reproductive System
    WANG Nan, ZHANG Rui, PAN Yang-yang, HE Hong-hong, WANG Jing-lei, CUI Yan, YU Si-jiu
    2022, 38(6):  279-290.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1152
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    Transforming growth factor β1(TGF-β1)is a multifunctional growth and differentiation factor,which regulates many physiological and pathological processes of the body,and has important biological functions and broad application prospects. In this study,we collected ovarian,oviductal and uterine tissues from yaks during estrus and pregnancy,cloned the yak TGF-β1 gene,and performed the quantitative analysis of yak TGF-β1 at the gene and protein levels using quantitative real-time fluorescence PCR(qRT-PCR),immunohistochemistry(IHC),and protein immunoblotting(Western blot). The results showed that TGF-β1 gene(GenBank No.MZ004937)was highly conserved,and the nucleotide sequence differed from the TGF-β1 nucleotide sequence of Bos taurus,and the encoded amino acid was mutated from alanine to glycine. It was most closely related to Bos taurus,Bos indicus×Bos taurus,and most distantly related to Canine. The encoded protein was a stably hydrophilic transmembrane protein. TGF-β1 was expressed in ovary,oviduct and uterus tissues of yak during follicular phase,luteal phase and gestation phase. In ovary,the expression of TGF-β1 during gestation phase was significantly higher than that during follicular phase and luteal phase(P<0.05). In oviduct,the expression of TGF-β1 gene in gestation phase was significantly higher than that in follicular and luteal phases(P<0.01). In utero,the expression in follicular phase and gestation phase was significantly higher than that in luteal phase(P<0.05). Immunohistochemistry(IHC)results indicated that TGF-β1 was mainly expressed in ovarian reproductive epithelium,follicular membrane,granular layer of follicle,luteal cells,tubal mucosal epithelial cells,uterine gland(UG)and endometrium cells. The results will provide basic data for further exploring the molecular mechanism of TGF-β1 in reproductive physiology of yaks,and provide theoretical basis for exploring the adaptability of plateau mammals to alpine environment.

    Effects of Lentivirus-mediated Occludin Overexpression on BVDV Infection in BALB/c Mice
    WANG Wan-shun, FU Qiang, WEI Yu-rong, HU Xin-yan, CHEN Jun-zhen, LI Ze-yu, SHI Hui-jun
    2022, 38(6):  291-298.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1156
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    This work aims to study whether the overexpression of tight junction protein(occludin,OCLN)affects the replication of bovine viral diarrhea virus(BVDV)in BALB/c mice. After lentivirus expression vector pLVML-Myc-bOCLN-linker-GFP-IRES-Puro was constructed,it was co-transfected into HEK-293T cells together with the helper plasmids pSPAX2 and pMD2.G. At 48 h post-transfection,OCLN-GFP lentivirus suspension was collected and its titer was measured. Meanwhile,the lentivirus generated with pLVML-Myc-Mcs-linker-GFP-IRES-Puro vector(GFP)was served as negative control. BALB/c mice aged 4-5 weeks were randomly divided into five groups:A(0 d),B(4 d),C(8 d),D(10 d)and E(15 d),with 6 mice in each group. Then 5× 107 IU OCLN-GFP and GFP lentivirus suspension were injected twice continuously at an interval of 48 h. At 96 h after the second injection,BALB/c mice were challenged with 1.68 × 105 TCID50 BVDV. At 0,4,8,10 and 15 d after the challenge,mice were dissected and tissues including liver,spleen,lung,kidney and small intestine were collected. The overexpression of OCLN was detected by real-time fluorescent quantitative PCR(qRT-PCR)and Western blot,and the viral loads of BVDV in different tissues were detected by qRT-PCR. Histopathological sections were made to observe the pathological changes of each tissue. As results,OCLN-GFP and GFP lentivirus were successfully generated. After 96 h of lentivirus infection in BALB/c mice,the expressions of OCLN mRNA and protein increased significantly after OCLN-GFP lentivirus infection. Compared with the control group infected with GFP lentivirus,the BVDV viral load in tissues and organs of OCLN-GFP lentivirus infection experimental group increased significantly from 4 d after challenge. Meanwhile,compared with the control group,at 4 d after BVDV challenge,the lung and liver organs of the experimental group appeared obvious lesions at the earliest time;after 8 d of BVDV challenge,the pathological changes of liver,spleen and lung were more serious than those in the control group. In conclusion,the results shows that OCLN overexpression maysignificantly increase the replication of BVDV in BALB/c mice,and the pathological changes caused by BVDV infection are more serious,which provides an important basis for clarifying the molecular mechanism of OCLN mediated BVDV replication.