Table of Content

26 August 2020, Volume 36 Issue 8

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Synthesis and Identification of Distant Hybrids Between Gossypium arboreum and Gossypium gossypioides

WANG Yi-fan, ZHENG Yun, RONG Er-hua, WU Yu-xiang

2020, 36(8):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1249

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Gossypium arboreum as a female parent had distant hybrid with Gossypium gossypioides as male parent. Artificial pollination was used to synthesize hybrid F₁,and the authenticity of the F₁ hybrids was verified,aiming to further doubling it into a new allotetraploid germplasm. Repeated pollination and gibberellin boll keeping measures were used to improve the boll setting rate of hybrid,and the F₁ was identified by morphology and SSR molecular markers. The results showed that the F₁ hybrids had typical post-zygotic reproductive isolation,and the hybrids died in the process of plant culture. Most leaf types of the F₁ hybrid seedlings were amid parents and tended to resemble male parents as a whole. The results of SSR molecular markers demonstrated that the hybrid F₁ not only amplified complementary bands of parents,but also new bands that parents did not have appeared. Statistical analysis revealed that the total genetic components of the hybrid F₁ accounted for 45.91% of the female parents,40.98% of the male parents and 13.11% of new combination bands. The study not only proves the authenticity of hybrids at the molecular level,but also shows that AD genome interaction and genetic recombination occur along with the hybridization process.

Regulation of Lanthanum on Rice Transcriptome Patterns Under Copper Stress

ZHONG Yu-qing, CHEN Jia-jia

2020, 36(8):  8-14.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0110

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This work is to investigate the effects of rare earth lanthanum on rice transcriptome under copper stress,and to identify key genes and functions of lanthanum-regulated responses in rice against copper stress. The optimal copper stress and lanthanum treatment concentrations were screened by antioxidant enzyme activity,transcriptome sequencing and differential expression analysis were performed,and differential expression levels were verified by qRT-PCR. Differential expression analysis of sequencing data revealed that 3 222 genes were significantly up-regulated and 3 798 genes were significantly down-regulated. Four cell wall defense-related genes CHIT13,Laccase,Expansin,and GH3.4 were verified by qRT-PCR. Total 95 Gene Ontology terms and 112 KEGG pathways were obtained by function and pathway enrichment analysis,and they enriched in molecular functions such as kinase and oxygen molecule binding,cell components such as cell wall and plasma membrane,biological processes such as secondary metabolism and lipid metabolism,as well as pathways e.g. phenylpropane biosynthesis,and plant hormone signal transduction. The molecular mechanism is that La increases the tolerance of rice to copper stress by regulating cell wall formation and components.

Cloning and Expression of ClKptA/Tpt1 Gene from Cunninghamia lanceolata（Lamb.）Hook

YANG Shi-quan, PENG Dan, FEI Wen-jie, YANG Feng, QU Gao-yi, TANG Wei-wei, OU Jian-ping, DENG Xiang-wen, ZHOU Bo

2020, 36(8):  15-22.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0177

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Cunninghamia lanceolata（Lamb.）Hook is one of the most important fast-growing timber tree species in southern China. Phosphorus is an essential nutrition in plant growth and development. However,the concentration of phosphorus of soil is relatively low in the subtropical region of China. In order to elucidate how C.lanceolata fully uses phosphorus to promote its growth under phosphorus limitation,the phosphate transferase gene ClKptA/Tpt1 was cloned by RT-PCR from C. lanceolata,and genetic relationship between the protein and KptA/Tpt1 of other species was analyzed. Meanwhile,the tertiary structure of the protein was predicted,and the protein was expressed in Escherichia coli. Furthermore,the expression patterns of ClKptA/Tpt1 in the different parts of C. lanceolata treated with different concentrations of phosphorus were detected. The result showed that the total length of CDS（code sequence）of ClKptA/Tpt1 was 1 143 bp,and it encoded 380 amino acids,which had the closest relationship with the KptA/Tpt1 of Nelumbo nucifera and the furthest relationship with Solanum lycopersicum. The molecular weight of ClKptA/Tpt1 expressed in E. coli was 55.5 kD. The amino acid sequence of ClKptA/Tpt1 did not have a transmembrane structure,and tertiary structure of ClKptA/Tpt1 main constructed by α-heli and β-sheet. ClKptA/Tpt1 expressed in the leaves at the highest and the lowest in stem. The expression level of ClKptA/Tpt1 and lignin content of stem increased with phosphorus content increasingly added in the soil of planted C. lanceolata. These results lay a theoretical foundation for elucidating the molecular mechanism of fully utilizing phosphorus to promote lignin biosynthesis in C. lanceolata.

Cloning,Recombinant Expression and Enzymatic Properties of α-Amylase Gene from Laceyella sp.

ZHAO Hai-yan, SONG Chen-bin, LIU Zheng-ya, MA Xing-rong, SHANG Hui-hui, LI An-hua, GUAN Xian-jun, WANG Jian-she

2020, 36(8):  23-33.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1053

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Cloning and heterologous expression of amylase gene in the amylase-producing strain screened from soil were conducted and their enzymatic properties were analyzed. An amylase-producing strain was isolated and numbered MF-8-1,which was from the soil sample near the Anyang Flour Mill by using the solid starch screening medium. Then the amylase gene AmyL was cloned by corresponding primers with homology cloning. Further the recombinant plasmid of pET-22b-AmyL was constructed by ligating Amy and expression vector pET-22b（+）,and transformed into expression host strain Escherichia coli BL21（DE3）. Finally,the recombinant enzyme was isolated and purified and its enzymatic properties were determined. As results,AmyL was expressed successfully in E. coli BL21（DE3）,and the molecular weight of recombinant enzyme AmyL was 55 kD. Recombinant enzyme presented maximum activity at pH 6.0 and 45℃. It had better temperature stability when the temperature was lower than 60℃. The enzyme was highly stable at pH 6.0-10.0. The kinetics studies of recombinant enzyme showed that the specific activity,K_m and V_max of the enzyme were 185.39 U/mg,0.95 mg/mL and 343.12 μmol/（min ·mg）,respectively. AmyL is characterized by stable enzyme activity under medium temperature and alkalinity,thus it has potential application value in detergent,textile,paper making and other industries.

The Characteristics of Phosphate Solubilization of Rock Phosphate by Phosphate-solubilizing Bacterium JL-1

LI Wen, WANG Tao

2020, 36(8):  34-44.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1054

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In this study,rock phosphate was used as the phosphorus source for testing the phosphate-solubilizing ability of Acinetobacter indicus JL-1. The single-factor experiments and response surface test were utilized to determine the optimal conditions for degrading rock phosphate,and the optimal phosphate solubilizing condition was as follows：17 g/L glucose as carbon source,1.2 g/L the compound nitrogen source of ammonium sulfate and yeast powder at a mass ratio of 1∶1,2.0 g/L rock phosphate powder,incubation temperature 28℃,pH 7.0,and inoculum size 1%. Under the optimal condition,the dynamic phosphate-solubilizing curve showed that the maximal solubilizing phosphate concentration was 4.65 μg/mL at 60 h,the phosphate-solubilizing rate was 2.34%,and the number of colonies reached the highest,which was 10.65 log（CFU/mL）,while the pH reached the lowest. The Shapei experiment demonstrated that 72.08% of the available phosphorus was directly released,7.63% was stored in the cells,and 20.29% was assimilated by the strain.

The Mechanism of Dissolving Inorganic Phosphorus by Bacillus megaterium ZS-3 and Its Growth Promotion of Cinnamomum camphora

QIAN Ting, YE Jian-ren

2020, 36(8):  45-52.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0114

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The purpose of this study is to explore the inorganic phosphorus-dissolving characteristics and corresponding phosphorus-dissolving mechanism of Bacillus megaterium ZS-3,and to provide a theoretical basis and technical approach for utilizing and improving the utilization rate of poorly soluble phosphorus resources in soil. NBRIP medium,molybdenum anti-colorimetric method,dilution coating method,and acid-base titration method were applied to determine the amount of phosphorus solubilized,colony growth and titratable acidity of the strain. Scanning electron microscope（SEM）was used to observe the surface morphology of calcium phosphate Ca₃（PO₄）₂. High performance liquid chromatography and p-nitrophenyl phosphate disodium were adapted to measure the types of organic acids,content and activity of phosphatase in the fermentation broth. Selecting 2 different types of soil for pot experiments,the effects of strain ZS-3 on the growth of Cinnamomum camphora and the physicochemical properties of soil were checked. As results,the maximum amount of dissolved phosphorus by strain ZS-3 occurred at 96 h with 188 μg/mL when it was continuously cultured for 168 h. The maximum number of cells reached 2.34×10⁹ CFU/mL at 72 h. The pH dropped the most quickly at early 24 h from initial 8.06 to 5.14. The titratable acidity was slowly increasing. SEM experiment showed that the strain ZS-3 caused the calcium phosphate surface uneven and rough after interacting with it. The activities of acid phosphatase and alkaline phosphatase were low during the dissolution by ZS-3. A large amount of organic acids were generated during the ZS-3 culturing,mainly acetic acid and formic acid,their contents were 484.12 ng/μL and 255.1 ng/μL,respectively. The application of strain ZS-3 promoted the growth of C. camphorain planted in two different types of soil. The soil available phosphorus content increased and there was more significant increase of available phosphorus content in low phosphorus soil. In sum,the strain ZS-3 can efficiently dissolve inorganic phosphorus and promote the growth of C. camphora,and is expected to provide resources for the development of phosphorus-solubilizing microbial fertilizer.

Isolation of an Acetoin-producing Strain Resistant to High Concentration Glucose and Identification of Metabolites

HUO Ming-yue, ZHANG Ge, SU Yu-long, LI Xing, ZHANG Hai-bo, ZHENG Rong, LIU Hao-bao

2020, 36(8):  53-60.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0119

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In order to obtain an acetoin-producing strain that is resistant to high concentrations of glucose,a new strain was screened from tobacco field soil using high-sugar medium and acetoin coloring solution;then the screened strain was sequenced and identified by whole genome technology and 16S rDNA sequence alignment analysis,and tolerance of the strain was tested at different concentrations of glucose in the medium. Finally,the GC-MS（gas chromatography-mass spectrometry）was used to qualitatively analyze the fermentation products of the strain and the capacity of acetoin-producing was analyzed by preliminary fermentation for 72 h. The results showed that the strain obtained was Staphylococcus aureus and named as PX03. In the glucose tolerance test,the strain PX03 still grew in a medium containing 500 g/L glucose,with an OD₆₀₀ of 65;grew the best at a concentration of 300 g/L glucose,with an OD₆₀₀ of 100. GC-MS qualitative analysis revealed that the strain produced a large amount of aroma components,including acetoin（22.51%）,2,3-butanediol（21.29%）,acetic acid（13.97%）,3,4-dihydroxy-3,4-dimethyl-2,5-hexanedione（8.52%）,pyranone（5.24%）and 3-methyl-bbutanoic acid（4.47%）. The preliminary fermentation results demonstrated that the yield of acetoin of this strain reached 41 g/L.

Isolation and Identification of Lignin-degrading Bacteria from the Gut of Termite and Their Degradation Characteristics

LI Feng, HUANG Shu-shi

2020, 36(8):  61-68.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0128

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The objectives of this work are to enrich and isolate lignin-degrading bacteria from the gut of termite,and to study their degradation characteristics. A lignin-degrading bacterium was first isolated from the gut of a termite species,Microtermes pakistanicus,and then identified by 16S rRNA gene sequence analysis for their phylogeny property. Further,with a variety of evaluation methods,such as FTIR and TGA,the degradation characteristics of lignin for the strain were analyzed. As results,a strain MP-132 was identified as Raoultella ornithinolytica. The degradation efficiency was up to 53.2% after 7 d culture when alkaline lignin was used as sole carbon source. Laccase and lignin peroxidase peroxidase reached maximum activities 32 U/L and 105.6 U/L,respectively. Moreover,strain MP-13 led the azure B and aniline blue decolorized. The results from the FTIR and TGA analysis showed that strain MP-132 uniquely presented an attractive capability to deconstruct lignin components. In conclusion,this work systematically reveals the degradation characteristics of strain MP-132,which may provide fine germplasm resources for the biodegradation of lignin.

Enhanced Furfural Tolerance in Saccharomyces cerevisiae by the Overexpression of GLN1 Gene

WU Yu, WANG Jin-hua, ZHAO Xiao

2020, 36(8):  69-78.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0033

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Furfural is the main inhibitor of straw hydrolysate,and it can inhibit the growth and fermentation performance of Saccharomyces cerevisiae. The glutamine synthetase gene GLN1 of S. cerevisiae was inserted into the vector pESC-URA,and then introduced into the original yeast BY4741 to construct the recombinant yeast YEA9,and its furfural tolerance and fermentation performance were measured. The results showed that at a concentration of 1.55 g / L furfural,the biomass of YEA9 increased by 3.7 times,the sugar consumption rate increased by 8.6 times,the final ethanol concentration increased by 9.2 times,and the sugar alcohol conversion rate increased by 10.6%. Glutathione content increased by 22.4%,NADH oxidase activity increased by 5.1%,NAD ⁺ / NADH values did not change significantly,and the level of reactive oxygen species was lower than that in BY4741. In the simulated hydrolysate,the biomass of YEA9 increased by 1.75 times,the sugar consumption rate increased by 6.1 times,the final ethanol content increased by 5.3 times,and the sugar alcohol conversion rate increased by 0.5%. In summary,overexpression of the GLN1 gene can increase the enzyme activity of glutamine synthetase in S. cerevisiae,the content of glutamine and glutathione,and the enzyme activity of NADH oxidase,while reduce the accumulation of ROS,and ultimately enhance the furfural stress tolerance and ethanol fermentation efficiency.

Influencing Factors and Formation Mechanism of CaCO₃ Precipitation Induced by Microbial Carbonic Anhydrase

YUAN Liang

2020, 36(8):  79-68.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1218

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Microbial carbonic anhydrase can accelerate the CO₂ hydration,which is of great significance for the study of global carbon cycle. A bacterium producing extracellular carbonic anhydrase was selected to study the effects of temperature,pH value and Ca²⁺ concentration on bacterial growth and carbonic anhydrase activity in this article. The results showed that the activity of carbonic anhydrase at 25℃ was the highest,which was beneficial to the CaCO₃ precipitation. The precipitation of CaCO₃ was the most in the alkaline environment with the initial pH 8.5. When the Ca²⁺ concentration was 50 mmol/L,the growth and reproduction of the bacterium were the best,too low Ca²⁺ concentration affected the generation of CaCO₃,while too high Ca²⁺ concentration seriously affected the growth of the bacterium and reduced the activity of the bacterium. Finally,the mechanism of CaCO₃ precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase accelerated the hydration of CO₂ into HCO₃^-,and HCO₃^- reacted with OH^-and Ca²⁺ to form CaCO₃ precipitation in alkaline environment and in the presence of calcium source.

Isolation,Screening and Identification of Desulfurization Bacteria from Kitchen Waste

WANG Xiao-li, ZHANG Wen-wen, WU Qing, CAO Yun-e

2020, 36(8):  87-95.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0825

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The objective of this work is to obtain microorganisms that can reduce hydrogen sulfide emission in the kitchen waste compost process. Kitchen waste compost was enriched,screened and identified to preliminarily determine the genera of bacteria,and the optimal culture conditions of desulfurization bacterium for deodorization were determined via single factor experiment. Results showed that the two desulfurization strains were identified as KW6 and KSW8,respectively. KW6 belonged to Bacillus and KSW8 belonged to Rhizobium. The remove rate of hydrogen sulfide by KW6 was the highest,when the culture temperature was 30℃,the pH value was 7.5 and the inoculation was 12%,which was 83.5%,65.3% and 73.8%,respectively. The removal rate of hydrogen sulfide by KSW8 was the highest,when the culture temperature was 35℃,the pH value was 6.5 and the inoculation was 20%,which was 85.8%,75.8% and 72.4%,respectively. Comparing with CK,KW6 and KSW8 reduced the releasing amount of hydrogen sulfide by 28.83% and 50.08%,respectively. The optimal deodorization culture conditions for KW6 were temperature 30℃,pH 7.5 and inoculation 12%.The optimal deodorization culture conditions for KSW8 were 35℃,pH6.5 and inoculation 20%.

Bacterial Communities in a Middle School Campus Assessed by High-throughput Sequencing

HUANG Ting, FANG Yuan, FENG Zhou, SHEN He, NIE Yong, ZHENG Xin, WANG Jia-quan, XU Zi-mu

2020, 36(8):  96-103.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0134

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The elementary and middle school campus is a special living environment with the characteristics of large population density,active microbial transmission and communication. The microbial community structure and transmission in campus is a key factor impacting the microbial formation and development of adolescents’ healthy. Taking a middle school campus as the research object,the microbial community structure in different places on the campus was explored,and an attempt was made to clarify the campus microbial community and its characteristic distribution. Microbial samples were collected from where human body may easily be in touch to,such as the armrests in the classrooms and gym stadium,the doors and handrails in laboratories,and handrails in canteen. The V4 regions of bacterial 16S rRNA genes for the samples were sequenced at high-throughput using Illumina HiSeq platform. The results showed that buildings of different usages exhibited distinct microbial communities. There were significant differences in their relative abundances though the microorganisms on the surface of the objects in each building mainly belonged to Proteobacteria,Firmicutes and Actinobacteria at the phylum level and Enhydrobacter,Streptococcus and Staphylococcus at the genus level. For example,Acinetobacter and Aerococcus in the canteen samples showed higher abundances but were absent in buildings at the teaching area. The microbial diversity on the armrest surface of the gym stairs was significantly higher than that of other facilities. Through a preliminary analysis of the composition of bacteria in different locations on the campus,we found that the microbial community in the different buildings of the school campus was determined by their usages,and speculated that the distribution of these characteristics should be closely related to different types of teaching and life activities. This study provides a reference for further studying the impact of the campus environment on adolescents’ physical health.

Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Deltacoronavirus（PDCoV）N Protein

CHEN Rui, FU Jia-yu, LIU Hao-yu, LI Cheng, ZHAO Yu-jia, HU Jing-fei, QU Huan, CAO San-jie, WEN Xin-tian, WEN Yi-ping, ZHAO Qin, WU Rui, HUANG Xiao-bo

2020, 36(8):  104-110.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1196

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This work aims to express and purify the porcine deltacoronavirus（PDCoV）N protein and to prepare its polyclonal antibody. The PDCoV N gene was amplified by RT-PCR and the recombinant prokaryotic expression vector pET28-N was transformed into Escherichia coli Transetta（DE3）,from which the fusion protein was identified by SDS-PAGE. The purified N protein was used to inoculate rabbit and the polyclonal antibody was prepared. The specificity of the serum antibody was determined by Western blot and the serum antibody titer was detected by indirect ELISA. The diagnostic application potential of the polyclonal antibody was evaluated by using indirect immunofluorescence assay（IFA）,immunofluorescence staining（IF）and flow cytometry（FCM）. Recombinant N protein was soluble with a molecular weight of 44 kD. The titer of the antibody was about 1∶204 800. IFA and FCM tests confirmed that the antibody specifically bound to PDCoV and had no cross-reactivity with PEDV and TGEV. IF tests showed that the polyclonal antibody could be used to detect PDCoV in small intestine tissue.

Lipid Droplet Formation in the Pre-adipocytes of Leptin-overexpressed Pig

HU Bin-yue, HU Yang, CHENG Wen-min, ZHAO Su-mei, ZHAO Hong-Ye, WEI Hong-Jiang

2020, 36(8):  111-119.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0072

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This work aims to investigate the effect of endogenous leptin overexpression on lipid droplet formation in pig adipocytes and to provide a theoretical basis for studying the molecular mechanism of Leptin regulating lipid metabolism. The subcutaneous adipocytes of leptin-overexpressed and wild-type pig were isolated and cultured under aseptic condition. Then these cells were induced to be differentiated to produce lipid droplets（LDs）. The area of a lipid droplet and the content of the lipid were observed by microscope after Oil Red O and Bodipy staining. Then the expression levels of lipid droplet formation-related genes were determined by Q-PCR. The cells differentiated to form LDs after 4 d of induction. The results from the Oil Red O and Bodipy staining showed that the number of lipid droplets and lipid contents in the leptin-overexpressed porcine adipocytes were significantly lower than those in the wild type（P<0.05）. Furthermore,the expression levels of lipogenesis-related genes PPAR γ,SCAP,SREPB1 and PLIN2 in the leptin-overexpressed porcine adipocytes were significantly lower compared to the wild type（P<0.01）. Leptin overexpression inhibited the formation of LDs by down-regulating PPARγ,SREPB1,SCAP and PLIN2 genes expression in porcine pre-adipocytes.

Isolation,Identification and Antibiotic Sensitivity Analysis of Bacterial Pathogen from Litopenaeus vannamei with Black Gill Disease

LIN Jia-ming, GE Hui, LIN Ke-bing, YANG Zhang-wu, ZHOU Chen, WU Jian-shao, WANG Guo-dong, ZHANG Zhe, YANG Qiu-hua, WANG Yi-lei

2020, 36(8):  120-128.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1158

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In order to identify the pathogen of black gill disease in Litopenaeus vannamei overwintering parent shrimp and analyze its drug resistance,a dominant strain was isolated from the focus in the gill of a dying diseased shrimp via plate method,and then analyzed by combining the morphology of the strain and its ITS-rDNA gene sequence. The results showed that the dominant strain had 99% sequence homology with Fusarium solani,and the constructed phylogenetic tree clustered as one branch with F. solani;therefore the isolated dominant strain was determined as F. solani. The artificial infection was conducted based on Koch’s rule,and the results confirmed that the pathogen had strong pathogenicity and disease shrimps had black gill. The shrimps infected with concentrations of 5.0×10⁵ CFU/mL（25 µL/shrimp）for 7 d died 53.33%. Agar dilution method and M38-A2 scheme of NCCLS were used for drug sensitivity test with selected 11 common drugs. After preliminary screening,it was found that the strain was sensitive to clotrimazole,econazole,terbinafine,nystatin,natamycin and ketoconazole. The MIC of econazole was 1 μg/mL,that of terbinafine（TBF）was 4 μg/mL,that of clotrimazole and natamycin was 8 μg/mL,and that of miconazole and itraconazole was 16 μg/mL. In conclusion,the pathogenicity,drug resistance and taxonomic status of the dominant strain were determined in this study,which may provide a reference for the prevention and control of black gill disease of overwintering parent shrimp.

Study on Expression,Purification and Activity of Recombinant Human Osteopontin in Mammalian Cells

ZHOU Mei-qi, XIAO Xin-yi, YANG Zhuo-yi, BAI Si-yi, CHEN Hui, YUAN Yun-sheng

2020, 36(8):  129-135.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0053

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This work aims to transfect human embryonic kidney 293E（HEK293）,to secretly express and purify his-tagged recombinant human osteopontin（rhOPN）using mammalian expression vector with transient transfection technology,as well as to study how the rhOPN promotes the cell proliferation of colorectal cancer and non-small cell lung cancer（NSCLC）. The expression vector pcDNA3.1-OPN for OPN fusion 6×his-tagged was constructed and synthesized,and was transiently transfected into HEK293E cells with a polyetherimide（PEI）. The rhOPN was purified by a nickel affinity chromatography column. The mobility and purity of the purified rhOPN in gel was analyzed by SDS-PAGE electrophoresis and Western blot. In addition,the binding capacity between rhOPN and anti-OPN antibody was accessed by ELISA,and the effects of the rhOPN on the proliferation of colorectal cancer and NSCLC cells were analyzed with CCK-8（Cell Counting Kit-8）. The results demonstrated that rhOPN was successfully expressed by transiently transfecting pcDNA3.1-OPN into HEK293E cells and 95% purity was achieved. The rhOPN was well recognized by anti-OPN antibodies,and it significantly promoted the proliferation of HT29 cells in vitro at a concentration of 36 μg/mL. It also remarkably promoted the proliferation of NSCLC H1299 and HCC827 cells in vitro at a concentration of 20 μg/mL. In sum,the rhOPN is successfully expressed in HEK293E cells using mammalian cell transient transfection technology and purified to be in high purity. It is found that the rhOPN can significantly promote the proliferation of human colorectal cancer cells and NSCLC cells and has fine biological activity.

Research Progress of Remorin Protein in Plants

HU Xiao-qian, ZHANG Ying-yi, LI Xin, YAN Hai-fang

2020, 36(8):  136-143.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1185

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The remorin protein is a plant-specific oligo filamentous family protein that is localized in the membrane domain along with the plasma membrane/lipid. The remorin protein has a conserved coiled-coild motif on the C-terminal structure,which can be used to identify potential remorin family proteins,and high sequence variability in N-terminal regions of a remorin leads to its functional diversity. Remorin proteins play a crucial role in plant growth and development,signal transduction and stress response. In this paper,the research on remorin protein in plant organogenesis,crop yield,signal transduction,stress resistance and disease resistance will be reviewed. The mechanism of remorin protein involving in plant development and stress resistance may provide new insights,thus which is conducive to the cultivation of excellent crops with high yield,quality and high stress resistance.

Research Progress on the HAK Function of Plant High Affinity Potassium Ion Transporter

SU Wen, LIU Jing, WANG Bing, LI Wei, DAI Liang-ying

2020, 36(8):  144-152.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0152

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Potassium（K）is an important nutrient element for plant growth and development,and known as the “stress-resistant element” and “quality element”. In low-potassium environments,plants mainly use high-affinity transporters to absorb and transport potassium ions. The KUP/HAK/KT family,the largest high-affinity potassium transport family in the plant,plays a key role in the affinity transport of potassium ions. Here we descripted the basic situation and classification of the KUP/ HAK/KT family of plants,the phylogenetic analysis of the high-affinity potassium ion transporter HAK,the functional studies of the HAK transporter in increasing plant potassium absorption,affecting plant growth and development,and enhancing plant capabilities of resisting biological stress and abiotic stress. Finally,we prospected the to-be-solved issues related to the potassium ion transporter HAK. The deep understanding of the mechanism of plant HAK potassium transporters is of great practical significance in improving the efficiency of potassium fertilizer using,increasing crop yield and quality,and promoting agricultural development.

Crosstalk Between Hydrogen Sulfide and Nitric Oxide Signaling in Plants

SUN Yu-ying, QIU Xue-mei, YE Xin-yu, LI Zhong-guang

2020, 36(8):  153-161.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0084

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During the processes of plant growth,development,and response to environmental stress,which is involved in the crosstalk among signaling molecules,such as calcium（Ca²⁺）,reactive oxygen species（ROS）,hydrogen sulfide（H₂S）,and nitric oxide（NO）. Recently,H₂S and NO are considered to be important second messengers,which take part in multiple physiological processes,including seed germination,plant growth and development as well as response and adaptation to environmental stress. In these physiological processes,the interplay between H₂S and NO can be found. In the light of current research advance on H₂S and NO,this review discussed the anabolism and catabolism of H₂S and NO in plants,as well as their homeostasis in plant cells. In addition,H₂S interaction with NO in plants,by reacting each other,acting on common target molecules,regulating metabolic enzymes each other,and controlling other signaling pathways,was also summarized.

Research Progress on Molecular Mechanisms of Nitrate-regulated Plant Flowering and Yield

GONG Wei, YU Jian-yuan, ZHANG Xi, SHAN Xiao-yi

2020, 36(8):  162-172.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0141

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Developing early-maturation high-yield varieties is one of the most desirable goals for crop breeding. Nitrogen（N）,the essential nutrient for plant development and growth,plays a key role in the regulation of flowering and yield productivity. Nitrate（NO₃^-）,the predominant N form in the soil,functions as a nutrient and signal to participate in the regulation of plant flowering and yield via various means such as transport,metabolism and signal transduction. In this review,we focus on the molecular mechanisms underlying nitrate-regulated early-maturity and high-yield in model plant Arabidopsis thaliana and Oryza sativa as well as in staple crops. Through this review,we aim to provide a theoretical reference for rational utilization of nitrogen fertilizer,improvement of nitrogen utilization efficiency and cultivation of new varieties of early-maturing and high-yielding crops.

Research Progress of miR172 in Plant Development and Regulation

XU Zi-han, HU Feng-rong

2020, 36(8):  173-184.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0181

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microRNAs（miRNAs）are a class of non-coding small molecular RNA with a length of 19-25 nt regulating the growth and development of plants,signal transduction and stress response at the post-transcriptional level,which has become the hotspot in biology. miR172,a conserved miRNA family in plants,plays an important role in the development of various organs in plants,such as flowering induction,flower organ morphic formation,fruit maturation,and leaf and root growth,through the regulation of APETALA2 genes. In view of this,this paper reviews the development of miR172 in vegetative organs,reproductive organs and other organs of plants in the past 20 years,as well as its functions in the transition of development process,and discusses the effects of other factors on miR172,so as to provide references for the in-depth analysis of the action mechanism and molecular regulatory network of miR172 and its target genes.

Research Progress of Thermophilic Lignocellulase in Cellulose Ethanol Production

LIU Deng, LIU Jun-hong

2020, 36(8):  185-193.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1267

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Focusing on the application of thermophilic lignocellulase in the production of cellulosic ethanol,the technical bottlenecks that limit the commercialization of cellulosic ethanol are summarized. Then the characteristics of thermophilic lignocellulase are briefly introduced,and the research advances on thermophilic lignocellulase in screening,modification,immobilization,heterologous expression,metabolic regulation and synergy are emphasized. Further,the role of thermophilic lignocellulase in the production of cellulosic ethanol is discussed. Finally,the problems while using thermophilic lignocellulase in the production of cellulosic ethanol are presented and its application is prospected.

Mutual Regulation of microRNAs and Epigenetics in Human Cancers

TANG De-ping, YAO Hui-hui, TANG Jin-zhou, MAO Ai-hong

2020, 36(8):  194-200.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0960

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Along with the crucial role of genetic abnormalities,epigenetic aberration is emerging as one key relevant player in human cancers. Nowadays,accumulating evidences suggest that microRNAs（miRNAs）,as an important regulator in epigenetics,enter the epigenetic field. miRNAs and epigenetic machinery are the interactional players in the regulation of gene expression. Indeed,compelling evidence has indicated that epigenetic modifications,such as DNA methylation or histone acetylation,can affect miRNAs expression,and to be potentially responsible for the aberrant miRNA observed in cancer,in turn,miRNAs also can control the epigenetic machinery through directly targeting its enzymatic components. This review will describe the mutual regulation of miRNAs and epigenetics,and particularly focus on illuminating how epigenetics can affect the miRNAs expression,as well as how miRNAs can control epigenetics in human cancer. Finally,the review will discuss the translational clinical implications of the epigenetics/miRNA mutual regulation.

The Interaction Law Between Small Molecular Substances and Aptamers

PENG Yuan-yuan, XIAO Xing-ning, ZHU Long-jiao, TAO Xiao-qi, XU Wen-tao

2020, 36(8):  201-209.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1106

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After nearly 30 years of development,nucleic acid aptamers have gradually become one of the hotspots in many subject areas such as biology,medicine,materials,and physics. The trace detection of small molecular substances is also of great significance in various fields,thus it is necessary to understand the action mode and law between small molecules and aptamers. In this paper,based on the classification of small molecular substances,the action modes between small molecular substances and aptamers are summarized,the existing methods of studying interactions between small molecules and nucleic acids are expounded,and the types of interaction between different target molecules and aptamers are discussed. The results indicate that the aptamer segment tends to "wrap" the target small molecule,allowing the target to "set" in the nucleic acid structure. Small molecule compounds often utilize hydrogen bonding to bind aptamers,and there are hydrophobic and electrostatic interactions between the two. The metal target molecule binds to the negatively charged moiety of the pyrimidine group of the aptamer. This paper classifies the interaction modes and binding sites between small molecules and aptamers,aiming to play a reference and guide for the development of small molecule aptamers.

Fluorescent Detection of Ag⁺ Using Graphene and Functional Nucleic Acid

WU Xue-qi, ZHANG Li-pei, CAO Yang, WEN Xiao-gang, ZHOU Xiao-hong

2020, 36(8):  210-216.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1248

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Based on the principle of mismatched binding Ag⁺ of nucleic acid aptamer,using the different binding forces of graphene to single-stranded DNA and double-stranded DNA,a new fluorescent sensor with C-Ag⁺-C and graphene binding was designed and developed. A high sensitivity,fast and convenient fluorescence detection method was developed to achieve the Ag⁺ detection in turn on form. Under the optimization of detection conditions,the turn on fluorescent aptasensor offered a linear detection range of 150-400 nmol/L with a detection limit of 23 nmol/L for Ag⁺. The recovery rate of the method was 96.6% for Ag⁺-labeled detection in the experimental samples of drinking water,showing the ability to be used for actual sample analysis.

Application in the Detection of Fungal Toxins by Nucleic Acid Aptamer Lateral Flow Chromatography Analysis Technique

ZHAO Ying, WANG Nan, LU An-xiang, FENG Xiao-yuan, GUO Xiao-jun, LUAN Yun-xia

2020, 36(8):  217-227.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0830

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Lateral flow chromatography analysis technique（LFAs）characterized as simple,rapid,economic,and efficient one and can quickly presents the test results under the laboratory condition. New chemical antibody,nucleic acid aptamer body becomes a novel identifying component because of its in vitro screening,easy synthesis and modification,small batch difference and so on. Aptamer lateral flow chromatography analysis technique is the novel and fast detection technique of combing lateral flow tomography technology with aptamer. The LFAs is mainly used for qualitative,semi-quantitative and quantitative detection of contaminants in food safety,environment and clinical analysis. This paper reviews the LFA susing the aptamer combined with markers such as fluorescent,nano-gold and magnetic beads,as well as their applications in the rapid detection of fungal toxins through optics,colorimetry,surface-enhanced raman scattering,which aims at providing a reference in designing aptamer lateral flow chromatography analysis biological sensing technology.

Application Prospects of Stem Cell Technology in the Protection of Indigenous Porcine Germplasm Resources

SUN Jia-dong, SUN Xiao-feng, LI Lan, SHEN Wei, CHENG Shun-feng

2020, 36(8):  228-234.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0014

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Our country is one where a large amount of pigs is breeding,owing 1/3 of the global porcine germplasm resources. However,in recent years,the breeding volume of indigenous pig has decreased sharply,and some breeds are almost extinct. The genetic resource diversity of indigenous pig breeds in China has been seriously damaged,and high-quality genes have been lost continuously. How to effectively protect and conserve these excellent germplasm resources is an urgent and critical issue. At present,the protection of porcine germplasm resources includes in situ conservation,cryopreservation of semen and embryos,DNA library conservation and cryopreservation of somatic cells;however on which there are some limitations. Stem cells are a type of cell population that can self-renew and have the potential to differentiate. It can not only differentiate into different types of cells,but also maintain the stability of its own population through division. Healthy offspring have been created successfully by stem cells in pigs and mice. Recently,a new wave of germplasm resource conservation led by stem cell research is emerging. Stem cell technology may become a new means to protect indigenous porcine germplasm resources.

Efficient Editing of Mouse Galt Gene Based on CRISPR/cas9 System

YUE Peng-peng, GUO Jun-fan, YU Hong-hao, FU Can, WANG Xiao-yan, GAO Jin-tao

2020, 36(8):  235-342.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0237

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GALT gene mutation is the main cause of human type I galactosemia. This paper intends to target the mouse Galt gene through the CRISPR/cas9 system for simulating human GALT gene mutation,so as to lay the foundation for the establishment of an accurate animal model of type I galactosemia. We first analyzed pathogenic mutation sites of the GALT gene of type I galactosemia in China,and designed the sgRNA-directed sequence of the specifically targeted mouse Galt gene that can mimic the pathogenic mutation. Then sgRNA expression plasmid was then constructed and co-transfected with the cas9 expression plasmid into mouse 3T3 cells. The positive transfected cells were selected by puromycin and blasticidin,and the genomic DNA of the positive cells was extracted and the DNA fragment of the target site was amplified by PCR. The gene editing was identified and the editing efficiency was analyzed by TA clone sequencing. The results showed that the three sgRNA-directed sequences designed in this efficiently edited the mouse Galt gene through the CRISPR/Cas9 system,and the editing efficiency was 100%.