Biotechnology Bulletin

Sweet Sorghum—a High Efficient and Quality Forage Crop

ZHANG Dan, WANG Nan, LI Chao, XIE Qi, TANG San-yuan

2019, 35(5): 2-8. doi:10.13560/j.cnki.biotech.bull.1985.2018-0719

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Since the 21st century,the economy of our country has developed rapidly,and the demand for meat products has increased year by year. As a result,the rapid development of livestock farming has been accompanied by the increasing shortage of forage supply,and leads to the high cost of traditional feed. Thus it is important to find an alternative feeding forage. Sweet sorghum,as a high quality forage crop,has high sugar content,large biomass,rich in nutrients,and in additional strong tolerance to stress,can be planted in marginal land. As forage,sweet sorghum can be fed to cattle,sheep,donkeys,fish,rabbits,geese,etc.,and as well as be a portion for pigs. Recently,more and more research on the application of sweet sorghum have been carried out,and the planting area has been expanded year by year in foreign countries and in many places in China,which has a very good prospect of development. It will play an important role in meeting the demand for meat products in our country and promoting the development of livestock farming in the future. This article summarize the advantage and disadvantage of sweet sorghum as forage and point out the future direction about how to breed it for widely usage.

Research Progress on the Stem Juiciness of Sweet Sorghum

LENG Chuan-yuan, HAO Huai-qing, JING Hai-chun

2019, 35(5): 9-14. doi:10.13560/j.cnki.biotech.bull.1985.2019-0035

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Sweet sorghum（Sorghum bicolor）is recognized as a new ideal biofuel crop with the remarkable of stems accumulating high amounts of water and sugars,and the water content in stem has an important influence on the sugar yield of sweet sorghum. Recent significant progress has been achieved in the studies of stem juiciness in sweet sorghum with the development of plant molecular biology and large-scale re-sequencing technology. This paper reviews the progress in the stem juiciness of sweet sorghum and prospects the future research,aiming to fully understand the regulation mechanism of stem juiciness in sweet sorghum and provide a theoretical basis for accurate molecular breeding.

Progress on Genetic Research of Sorghum Grain Weight

HAN Li-jie, CAI Hong-wei

2019, 35(5): 15-27. doi:10.13560/j.cnki.biotech.bull.1985.2019-0105

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Sorghum is the fifth largest cereal crop in the world. Breeding high-yielding varieties of sorghum is of great significance on alleviating food security in the world. Grain weight is an important component of crop yield,increasing grain weight is an effective way in the improvement of sorghum yield. Grain weight is a complex trait controlled by quantitative trait loci（QTL）. At present,some QTLs controlling grain weight in sorghum have been located on the genetic map,which distribute on the all 10 chromosomes of sorghum. In this paper,the genetic characteristics,the influencing factors and the research progress of QTL mapping for sorghum grain weight were summarized,and the detected QTLs were compared and analyzed. The homologous genes of grain weight related genes already cloned in rice and maize were searched in sorghum genome and compared with the mapping intervals of sorghum grain weight QTLs,aiming at providing a basis for marker-assisted breeding and fine mapping or cloning of major QTLs for sorghum grain weight.

Research Advance in Glutinous Sorghum for Making Sauce-flavor Liquor in China

DING Yan-qing, ZHOU Leng-bo, WANG Can, CAO Ning, CHENG Bin, GAO Xu, PENG Qiu, SHAO Ming-bo, ZHANG Li-yi

2019, 35(5): 28-34. doi:10.13560/j.cnki.biotech.bull.1985.2019-0080

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Glutinous sorghum grains are the major raw materials for making sauce-flavor liquors such as Maotai,Xijiu,etc. Guizhou province,the home of famous liquors in our country,is the main cultivation area of glutinous sorghum for making sauce-flavor liquor. However,there are some issues in the research and production of glutinous sorghum for sauce-flavor liquors,for example,the narrow background of genetic resources,the weak research foundation on traits for liquor-making quality,relatively lagging-behind breeding methods,lower yield and serious degradation of main varieties,which resulted in that the sorghum production could not meet the growing demand of liquor-making industry in this region. In this paper,we reviewed the research progress of glutinous sorghum breeding for sauce-flavor liquor,the effect of grain traits on the brewing quality of sauce-flavor liquor,as well as known genes/QTLs for grain quality traits at home and abroad. Finally,we discussed the existing issues and future research directions,aiming to provide a basis for future research of glutinous sorghum for sauce-flavor liquor.

Functional Analysis of FAD7 Gene in Sweet Sorghum

SONG Yu-shuang， SUI Na

2019, 35(5): 35-41. doi:10.13560/j.cnki.biotech.bull.1985.2018-1102

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Here firstly we isolated and cloned ω-3 fatty acid desaturase（FAD7）gene from sweet sorghum inbred line M81-E. Then we constructed an expression vector to infect Arabidopsis thaliana and obtained the homozygous plants with SbFAD7overexpression. It was preliminarily indicated that the gene played an important role in increasing the cold resistance of plant by measuring and analyzing the contents of Fo,Fv/Fm,electrical conductivity,MDA,superoxide anion radical and hydrogen peroxide content in wild and overexpressed plants under low temperature treatment.

Comparison of Mitochondrial Genome Between A1 Cytoplasmic Male Sterile Line and Maintainer Line of Sorghum bicolor

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

2019, 35(5): 42-47. doi:10.13560/j.cnki.biotech.bull.1985.2019-0089

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Mitochondrial genomic translocation is an important genetic mechanism for cytoplasmic male sterility（CMS）in crops. The main purpose of this study is to explore the translocation region of mitochondrial genomes by comparing the mitochondrial genome between A1 cytoplasmic male sterile line and maintainer line of Sorghum bicolor,and to lay a foundation for cloning related genes of sorghum A1 cytoplasmic male sterility. A1 cytoplasmic male sterile line Tx623A and its maintainer line Tx623B of sorghum were used in this experiment. Their mitochondrial genome sequencing data from Illumina Hiseq（next generation sequencing）and PacBio seq（third generation sequencing）were assembled,and then,the differences of genome structures and genes between the two lines were compared and analyzed. The results showed that the mitochondrial genome sizes of Tx623A and Tx623B were 449 727 bp and 452 772 bp,respectively. The predicted open reading frames（ORFs）were 147 and 145,among which the number of the specific genes for Tx623A were 8 and 6 for Tx623B. Importantly,a 57 kb genome structural variation（SV）region was found after comparing the two mitochondrial genomes,and which may be related to A1 cytoplasmic male sterility. Collectively,our results provide genomic information for cloning related genes of sorghum A1 cytoplasmic male sterility.

Characterization of the Salt Tolerance of Transgenic Maize Line Expressing ABP9

ZHANG Zhao-yang, PANG Jun-ling, HAN Mei, LENG Peng-fei, ZHAO Jun

2019, 35(5): 48-57. doi:10.13560/j.cnki.biotech.bull.1985.2019-0007

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This work is to analyze the salt tolerance of ABP9 transgenic maize,and to study the molecular regulation mechanism of salt tolerance in maize. ABP9 transgenic plants were identified by PCR,Southern blot and quantitative Reverse Transcription-PCR（qRT-PCR）,and their responses at seedling stage to NaCl treatments in Hoagland nutrient solution were molecularly and physiologically characterized with non-transgenic plants as control. The results showed that the ABP9transgene was integrated into the genome in a single copy and expressed at a higher level. Compared with the non-transgenic control,chlorophyll content,Fv/Fm,osmotic adjustment substance,and antioxidant enzyme activities in transgenic maize seedlings under NaCl stress,and the relative water content of leaves during recovery after salt stress all significantly increased,while the aldehyde content and relative conductivity significantly reduced. Several salt stress responsive genes were up-regulated in transgenic plants revealed by transcriptome profiling and qRT-PCR analysis. Our results indicate that enhanced ABP9 expression level contributes to salt tolerance in transgenic maize,providing a valuable germplasm for maize salt tolerance breeding.

Effects of Plasma Membrane Localization of Arabidopsis thaliana CBL9 on the Growth of Pollen Tube Tip

ZHOU Li-ming, FANG Wei

2019, 35(5): 58-63. doi:10.13560/j.cnki.biotech.bull.1985.2018-1093

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This work is aimed to understand the role of Arabidopsis thaliana CBL9（calcineurin B-like protein 9）in the growth of pollen tube tip. Based on the transient expression system,the CBL9 with YFP-tag were transiently expressed in tobacco pollen grains by gene gun,and the overexpression phenotypes and subcellular localization patterns of CBL9-YFP were observed. At the conservative sites for N-terminal acylation of CBL9,a mutant version of CBL9,CBL9G2A was constructed via overlap-PCR-mediated site mutagenesis,and the changes in their phenotypes and localization were compared and studied. The results demonstrated that CBL9-YFP was localized to plasma membrane（PM）of growing pollen tubes and granular organelles. CBL9 overexpression induced a severe loss of growth polarity,leading to a short tube with the swelling tip. CBL9G2A was evenly distributed in cytosol of pollen tubes,and its overexpression did not cause the depolarization growth of pollen tubes. These results suggested that N-terminal acylation site of CBL9 is an important factor in correct targeting to PM,which is required for its function in polarized growth of pollen tubes.

Effects of T1084d and T1084A Point Mutations in the NtTkr Tail of Nicotiana tabacum on Coiled-helix Structure and Interaction with Target Proteins

XU Lin-na, HU Meng-ke, TONG Wen-yan, LI Fen

2019, 35(5): 64-69. doi:10.13560/j.cnki.biotech.bull.1985.2018-0957

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As a novel member of kinesin family,NtTkr is predominantly expressed in the vigorous division tissues of tobacco,and in it there are 3 coiled-coils（CC1,CC2 and CC3）. It is known that the point mutation and T deletion of T1084 in the conventional CC3 motif of the tail may affect the distribution of NtTkr,and which is inferred to be affected by the protein substrate binding with it. In order to determine the effects of deletion and point mutations of T1084 on the interaction with candidate proteins,T1084 deletion（T1084d）and T1084A point mutations were introduced into the tobacco NtTkr tail by overlap extension PCR,cloned into pGBKT7 to construct bait expression vector,and transferred to AH109 and mated to Y187 with pGADT7-H2A/Sulfer/NT3 for two-hybrid analysis. The results showed that T1084A was similar to T,but bluing earlier and the color was darker than T;while T1084d did not perform as T did. The primary structure and Coiled-coil software analysis showed that T1084 was located in the ‘a’ position of CC3 and was a polar hydrophilic amino acid. Substituting it with non-polar hydrophobic alanine enhanced the hydrophobicity of heptapeptide repeat domain（HR domain）,and resulting in the prolongation of repeated sequence of heptapeptide,while the deletion of T1084 resulted in its shortening. The results indicate that increasing the hydrophilicity of HR domain and the length of heptapeptide repeat sequence may enhance the interaction of NtKrp with the candidate protein,while shortening of coiled coil may result in a decrease of their interaction.

Inheritance Study on Fruiting Body Color of Pleurotus djamor（Rumph. ex Fr.）Boedijn

ZHANG Ze-hua, GAO Wei, WU Xiang-li, HUANG Chen-yang, CHANG Ming-chang, ZHANG Jin-xia

2019, 35(5): 70-75. doi:10.13560/j.cnki.biotech.bull.1985.2019-0127

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The color of fruiting body is one of the most important commercial traits of edible mushrooms and an important objective for breeding superior mushroom cultivars. In this study,a pink strain CCMSSC00450 and a taupe strain CCMSSC04445 of Pleurotus djamor（Rumph. ex Fr.）Boedijn were used as dikaryotic parental lines,and the constitutive monokaryons of two strains i.e.,PDm52 and PDm17 for strain CCMSSC00450,PDm99 and PDm14 for CCMSSC04445,were obtained by protoplast monokaryonization. After the cross-mating of 4 constitutive monokaryons,4 hybrid dikaryons were generated and cultivated for phenotyping. The 4 hybrid strains showed different color,i.e.,pink for PDm52×PDm99,pinkish gray for PDm52×PDm14,taupe for PDm17×PDm14,and white for PDm17×PDm99. A total 207 single spore isolates of the pink hybrid（PDm52×PDm99）were randomly selected as the segregating population of the color of pink fruiting body and PDm17 as the tester line,the test-cross population was constructed. Fruiting results showed that the color of the test-cross population was obviously segregated in a continuous distribution pattern varying from dark to light with pink,light pink,white,etc. The results revealed that the pink of the fruiting body was not completely dominant with respect to the white,and the white was recessive. Color of fruiting body of P. djamor is a quantitative trait controlled by multiple genes with high heritability. Meanwhile,a significant correlation was detected between the colors of the cap and gill.

Algae-bacteria Symbiosis Increases Biomass and Oil Production of Chlorella emersonii

ZHAGN Jing-jie, DUAN Lu-lu, CHENG Wei-lan, JI Chun-li, CUI Hong-li, LI Run-zhi

2019, 35(5): 76-84. doi:10.13560/j.cnki.biotech.bull.1985.2018-1098

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The large-scale cultivation of microalgae is often accompanied by bacteria and their infection. Limited information is available for the effect of bacteria in phycosphere on microalgae growth and the mechanism of algae-bacterial symbiosis. In order to establish a beneficial algae-bacterial symbiotic system and increase the biomass production of microalgae,Chlorella emersonii was selected as the test material,and microflora in phycosphere was isolated and then identified by 16S rDNA sequencing. The dominant growth-promoting bacteria were screened by co-culture of algae and bacteria（1∶1）. Various algae-bacterial co-culture systems at different ratios were artificially constructed to analyze the effects of dominant growth-promoting bacteria on microalgae growth and biomass yield. The results showed that 6 bacteria were isolated from the phycosphere of C. emersonii strain SXND-25,belonging to the 4 genera Pantoea,Pseudomonas,Enterococcus gallinarum and Escherichia coli. Of them,Pseudomonas and Pantoea were the dominant growth-promoting bacteria. Compared with other co-culture systems at different ratios of algae and bacteria,a symbiotic system of C. emersonii and Pantoea（1∶5）showed outstanding growth-promoting effects. The biomass of C. emersonii increased to 5.86 g/L on the 8th day of cultivation,the oil content was 26.88% in algal cells,with 1.575 g/L total oil production and 554-564 mg/L monounsaturated fatty acid（MUFA）. Another excellent combination was 1∶1 co-culture of C. emersonii and Pseudomonas. The biomass of C. emersonii on the 8th day was 4.12 g/L,the oil content of algal cells was 29.50%,and the total oil production reached 1.215 g/L,but low MUFA content（168-175 mg/L）. The present study demonstrated that adding the appropriate amount of growth-promoting bacteria in the cultivation of C. emersonii simultaneously increased algal biomass and oil production,providing solid scientific reference for investigating the algal-bacterial interaction effect and the application of beneficial algal-bacterial symbiosis system in commercial production of microalgae.

Preparation of Recombinant Human SOD1/3 Transgenic Goat and Detection of Expressed Products

ZHOU Min-ya, LU Rui, ZHANG Ting, YUAN Ting-ting, LU Yao-yao, YAN Kun-ning, YUAN Yu-guo, CHENG Yong

2019, 35(5): 85-92. doi:10.13560/j.cnki.biotech.bull.1985.2018-0877

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In this study,we employed human SOD1（hSOD1）and SOD3（hSOD3）as encoding sequence along with goat beta-casein/CMV chimeric promoter to construct mammary gland-specific expression vectors rhSOD1 and rhSOD3,which then co-transfected goat fetal fibroblast cells. PCR and sequence analysis of the amplified products were applied to screen and obtain the SOD1/3 cloned lines,and somatic cell nuclear transfer（SCNT）was used to prepare bitransgenic goat. Whether or not successful integration of foreign genes in newborn goats was verified by PCR and sequence analysis of amplification product,and the expressed product was analyzed by Western blotting,ELISA and in vitro activity test. The results were as such：6 SOD1/3 transgenic goat fetal fibroblast cell lines were obtained,one original bitransgenic goat（♀）was prepared,rhSOD1 and rhSOD3 were detected in the milk from the transgenic goats,and their concentrations were 88.81± 8.36 mg/L and 267.82± 12.67 mg/L,respectively. The biological activity of recombinant human SOD in the goat milk was 1 432±1 57 U/mL. This study implies that two expression vectors and single marker gene can simultaneously transfect goat fetal fibroblast cells to obtain the transgenic cell line with double gene integration,the SOD1 and SOD3 functional genes may co-express in the mammary gland,and the expressed products show fine biological activity.

Cloning of σ-GST Gene in Anodonta woodiana and Its Expression Analysis Under Pentachlorophenol Stress

XIA Xi-chao, LIU Qing-chun, HUA Chun-xiu, ZHANG Ke, LI Bing-jie, SONG Guo-ying, XUE Shi-peng, YU Rui-xue, WANG Zhong-xiao, ZHANG Qing-yuan, LIU Li

2019, 35(5): 93-101. doi:10.13560/j.cnki.biotech.bull.1985.2018-0871

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Glutathione S-transferases（GSTs）play a prominent role in protecting cells against oxidative stress. Our previous studies reveal that the pentachlorophenol（PCP）causes significant oxidative stress and acute toxic effects on Anodonta woodiana,but its chronic toxicity remains unclear. In order to investigate the chronic effect of PCP,A. woodiana were randomly grouped into PCP-treated group with 13.9 μg/L concentrations of PCP and control group with the same volume of dimethyl sulfoxide. Meanwhile,one GST sequences were cloned from A. woodiana and named as σ-GST,and the effect of PCP on the σ-GST was analyzed. The full-length cDNA of σ-GST consisted of 132 bp 5' untranslated region（UTR）,80 bp 3' UTR and 609 bp open reading frame of an encoded polypeptide by 203 amino acids. σ-GST had the tag sequence of GST and conserved amino acids,and had high homology with σ-like GST sequence of other species. The σ-GST was widely expressed in different tissues such as foot,mantle,adductor muscle,heart,hepatopancreas,hemocytes and gill. PCP treatment resulted in a significant increase of σ-GST expression in the hepatopancreas,gill and hemocytes. In the hepatopancreas,σ-GST mRNA level of PCP-treated group increased more than 28.73% at day 1,then 70.37%（P<0.05）at day 3,reached 6.64 times（P<0.01）at day 15 in contrast to the control group. In the gill,σ-GST mRNA expression showed a up-regulation by 1.14 times（P<0.05）in the PCP-treated group during whole experiment,compared with that of control group. These results indicate that up-regulation of σ-GST expression in A. woodiana is conducive to its resistance to the stress from PCP treatment during experiment.

Optimization of Shake Flask-fermentation Condition for Bacillus amyloliquefaciens M1 Strain and Its Denitrification Performance

LI Li, YAN Yue-gen, WU Hua-ming

2019, 35(5): 102-108. doi:10.13560/j.cnki.biotech.bull.1985.2018-0920

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This work aims to increase the total spores of Bacillus amyloliquefaciens M1 in fermentation,and investigate the anaerobic denitrification effect in wastewater. First,single factor experiments were used to investigate the effects of species and concentrations of various carbon and nitrogen sources,initial pH,temperature,shaking speed,medium volume etc. on the spore yield of M1.Then orthogonal experiments were carried out to optimize 4 significant factors of nitrogen source concentration,inoculation amount,liquid loading amount and temperature. Results showed that the optimal composition of the medium were as such：5 g/L carbon source（soluble starch）,10 g/L nitrogen source[yeast powder and tryptone mix（2∶1,V/V）],1 g/L NaCl. The optimal cultural condition was as：initial pH6.5,temperature 34℃,shaking speed 180 r/min,medium capacity 30%（75 mL/250mL）,and inoculation rate 7%. Under the optimal fermentation condition,the total spores yield of B. amyloliquefaciens M1 was up to 6.8×10⁸ CFU/mL,2.3 times higher than that cultured in LB medium（2.08×10⁸ CFU/mL）. Compared with the control test,test with M1 addition showed 15%-34% higher denitrification.

Degradation of Ethyl Formate by Aeromonas salmonicida subsp.salmonicida

ZHANG Shan-shan, YAO Xiao-long, WANG Ke, YOU Ya

2019, 35(5): 109-117. doi:10.13560/j.cnki.biotech.bull.1985.2019-0178

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Ethyl formate is widely present in wastewater from the chemical industry and is very harmful to both organisms and the environment. Microbial degradation is one of the important approaches for the treatment of ethyl formate. In this study,a strain of ETH-3 capable of degrading ethyl formate was screened from activated sludge by enrichment culture method and plate scribing method. Then,the strain was identified as Aeromonas salmonicida subsp. salmonicida by morphological observation,physiological and biochemical tests and 16S rDNA identification. The optimal growth conditions of strain ETH-3 and its ability to degrade ethyl formate were determined by shake flask test. The results showed that the optimal growth temperature of the strain was 25-30℃,and the optimal growth pH was about 8. The strain had strong ability to degrade ethyl formate. At 30℃,the degradation of 0-10 500 mg/L ethyl formate was completely degraded within 36 h. Analysis of metabolites by GC-MS revealed that ethyl formate was first metabolized by microorganisms to formic acid and ethanol,and then formic acid and ethanol were further degraded by microorganisms into CO₂ and H₂O.

Effect of Mannitol Phosphorylase Gene Knockout on D-mannitol Synthesis

ZHA0 Ya-tong, HE Guang-ming, WENG Ru-ru, SHI Ai-qin, LU Fu-ping, LI Yu

2019, 35(5): 118-124. doi:10.13560/j.cnki.biotech.bull.1985.2018-1039

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In order to explore the effect of mannitol decomposition and utilization pathways on mannitol synthesis,the cmtA,cmtB and mtlA genes in Escherichia. coli PTS system were knocked out by CRISPR/Cas9 on the basis of recombinant strain R1（K-12/pTrc99a-mdh）,which blocked the decomposition pathway of mannitol in E. coli K-12,and the recombinant strain R₃（K-12/∆cmtA∆cmtBmdh⁺）and R₅（K-12/∆cmtA∆cmtB∆mtlAmdh⁺）were obtained. Compared with the original strain R₁,the growth rate of R₃ did not decrease,but that of R₅ decreased significantly. Moreover,R₅ did not grow on the medium with mannitol as the sole carbon source,indicating that the strain could no longer use mannitol as the carbon source after all the three genes cmtA,cmtB and mtlA were knocked out. Finally,the recombinant strain R₅ was constructed,the activity of MDH enzyme was 258 U/mL,and a small amount of mannitol was detected by high performance liquid chromatography,which laid a foundation for further exploring the regulation mechanism of mannitol synthesis in E. coli.

Effect of Signal Peptide-like Sequence in DrwH on Its Antioxidant Function

GUO Lei-zhou, HAN Jia-hui, TANG Yin, LI Jiang, HUANG Cheng, DAI Qi-lin, WANG Jin, PING Shu-zhen, JIANG Shi-jie

2019, 35(5): 125-132. doi:10.13560/j.cnki.biotech.bull.1985.2018-1050

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DrwH,a hydrophobic LEA5C protein of Deinococcus radiodurans,is involved in the process of cell tolerance to oxidative and salinity stresses. Previous studies suggest the presence of a signal peptide-like sequence of 19 amino acids in the N-terminal of DrwH protein. In order to explore the effect of the signal peptide-like sequence on the antioxidant function of DrwH,DrwH protein was truncated to different fragments and then recombinant Escherichia coli strains containing the different fragments were constructed,and their growths were analyzed and compared between normal culture and oxidative stress. The results showed that overexpression of the fusion protein containing the signal peptide-like sequence not only inhibited the growth of E. coli,but also caused DrwH protein to lose its antioxidant capacity. qRT-PCR results demonstrated that,after induction by IPTG,the full-length protein DrwH containing signal peptide-like sequence and the WHy-domain were accumulated at the transcriptional level,and the proteins exerted its biological function under excessive accumulation. This indicates that the signal peptide-like sequence presents an important influence on the expression of the full-length protein DrwH and its antioxidant function.

Development of Maintenance Medium Using for Influenza Virus Production and Analysis of Cellular Metabolic Characteristics

XIE Pan, YE Qian, LIU Xu-ping, TAN Wen-song

2019, 35(5): 133-139. doi:10.13560/j.cnki.biotech.bull.1985.2019-0056

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This study developed a maintenance medium supporting the influenza virus production to high yield based on MDCK cell culture and using Simplex Lattice design,and studied the cellular metabolic characteristics during the virus infection under the maintenance medium,meanwhile the cell growth medium was as a control group. The maintenance medium was composed of A and F medium in a ratio of 0.586∶0.432 by the statistical analysis,in which the maximum virus titer was 2^14.062 HAU/100 μL in the 2 L bioreactor,which was 59.55% higher than that of the control group. When the initial concentrations of glucose were the same,the cells in the maintenance medium group enhanced the glucose uptake and the glycolysis pathway,the consumption rate of serine,arginine and isoleucine that were conducive to virus proliferation significantly increased,and the production rate of alanine inhibiting virus proliferation greatly decreased,compared with the control group. Therefore,this work emphasized the importance of maintenance medium development;meanwhile the results provide a methodological reference and theoretical guidance for the development and optimization of other viral vaccine media.

Identification and Functional Analysis of Enhancers-regulated miRNA Feed-forward Loops in Hepatocellular Carcinoma

CHANG Yun-jian, KANG Ran, XUE Xuan, WANG Shao-chang, ZHAO Qing-wen, GUO Zhi-yun

2019, 35(5): 140-145. doi:10.13560/j.cnki.biotech.bull.1985.2018-0846

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Previous studies demonstrate that,as an important network motif,feed-forward loops（FFLs）play an important role in the development of tumor diseases. Three types of elements,namely miRNAs,enhancers,and transcription factors,are widely reported to be closely related to tumors. However,the characteristics of the FFLs and their function in hepatocellular carcinoma remain unclear. Here 5055 liver cancer-specific enhancers were identified based on the DNase high-sensitivity site of HepG2 cells and the ChIP-seq data of modification information of 3 three types of histone H3K4me1,H3K27ac and H3K4me3. In addition,2070 TF-enhancer-miRNA FFLs were identified based on hypergeometric test and component regulation relationship. The analysis result of miRNA target genes in FFLs by GO and KEGG functional enrichment showed that target genes of miRNAs in the FFLs were significantly enriched in tumor or hepatocellular carcinoma-related signaling pathways and biological processes. Among them,hsa-miR-574 was involved in 99 FFLs and highly expressed in HepG2 cells,and significantly enriched in the known cAMP signaling pathway in inhibiting the proliferation of hepatoma cells. These results preliminarily explored the characteristics and functions of the enhancer-miRNA-based FFLs in hepatocellular carcinoma,and it is expected to provide a theoretical and data basis for liver tumor marker recognition based on network motif.

Roles of Dof Transcription Factors in the Regulation of Plant

GUO Yan-xiu, CHEN Jing, WANG Yan-fang, SUN Chun-yu, WANG Yi, ZHANG Mei-ping

2019, 35(5): 146-156. doi:10.13560/j.cnki.biotech.bull.1985.2018-1062

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As a plant-specific transcription-factor,DNA binding with one finger（Dof）protein was firstly discovered in corn in 1993. Dof has only one conserved zinc finger domain containing 52 amino acids. In general,the zinc finger domain contains 4 conserved Cys residues and one Zn²⁺,and these two components are both indispensable for Dof protein. Cysteine mutation or the presence of a divalent ion chelating agent will affect the formation of the zinc finger structure,and then the Dof protein cannot bind to the target DNA,resulting in the loss of its activity. The Cys residue and Zn²⁺ form a CX2CX21CX2C motif by covalent binding of other amino acid residues,and this motif may specifically recognize AAAG（or CTTT）sequences in promoters,thus activating or inhibiting the expression of target genes. Besides,the C-terminal amino acid sequence of the Dof protein was volatile,which led to the evolutionarily diverse and functional diversity of Dof transcription factor. To date,the Dof transcription factor was reported to play an important role in many aspects of plant growth and development,such as seed germination,development of vascular,leaf polarity,development of flower and pollen,photoperiod reaction,metabolism of carbon and nitrogen,regulation of secondary metabolites,and stress responses. This review focuses on the structure,phylogenetic relationship,and motif composition and regulation metabolism of Dof transcription factors,aiming at providing a theoretical basis for the further study of Dof transcription factors.

Research Progress on Electrical Signal Molecules in Electrochemical Functional Nucleic Acids Biosensors

XIE Yin-xia, WANG Wei-ran, CHENG Nan, XU Wen-tao

2019, 35(5): 157-169. doi:10.13560/j.cnki.biotech.bull.1985.2018-0600

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Electrical signal molecules refer to a class of molecules that have an electrochemical activity as signal-switching role in functional nucleic acids electrochemical biosensors,and can interact with nucleic acids or can be Labeled on nucleic acid strands. Electrical signal molecules are essential part of functional nucleic acids electrochemical biosensors,and crucial for the detection sensitivity and application popularity of functional nucleic acids electrochemical biosensors. In this article we briefly introduced 5 major categories of electrical signal molecules,i.e.,dye class,organometallic complexes,nanomaterials,catalase-like,and organic small molecule. Then we demonstrated their applications in functional nucleic acids electrochemical biosensors,mainly from 3 aspects：the methods of generating electrical signals,practical applications,and the advantages and disadvantages of each electrical signal. In the end,we Look on the future discovery or design of new electrical signal molecules. It’s expected to be useful for the subsequent study of electrical signals.

Construction of a RPA Detection Method for Transgenic Soybean and Its Application

DOU Wen, LI You, LU Xin-yu, QIAN Xue-mei, SHEN Dan-yu

2019, 35(5): 170-175. doi:10.13560/j.cnki.biotech.bull.1985.2018-0936

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The goal of this work is to develop a fast and easy-operating RPA detection method for the GM soybean to monitor the illegal planting and spreading of the GM soybeans in China. Five pairs of RPA primers were initially designed based on the conserved sequences of CaMV-35S promoter and the NOS terminator in GM soybeans. PCR reactions were further performed to screen the primers and a set of RPA primers with the high sensitivity and specificity,Nos163,was selected for constructing RPA test strips. Reaction temperatures and times of RPA were also optimized. No transgenic component was detected from bean sprouts and fresh green soybean in market,as well as soy samples planted in field in Nanjing area using our RPA detection technique. All evidences suggest that RPA detection method is more sensitive than PCR,and can be performed under normal temperature without depending on the expensive equipment. The test results can be easily visualized,therefore which makes it an effective approach for the rapid detection of GM soybean.

Enzymatic Utilization of Solid Waste from Formalin Tanning as Resources Using Microbial Enzyme

ZHENG Xiang, QIN Meng, GAO Pei-ru, QIN Yan-mei, MA Qing-he

2019, 35(5): 176-180. doi:10.13560/j.cnki.biotech.bull.1985.2018-0880

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In order to establish a technology of cleaning and utilizing solid waste as resource in tannery industry,enzymatic hydrolysis of formaldehyde tanning solid waste was carried out by JW enzyme preparation,and the industrial and agricultural applications of enzyme hydrolysate was evaluated. The results indicated that the skin pieces in solid waste were completely hydrolyzed by JW-4 enzyme. The length of animal fiber recovered after the enzymatic hydrolysis of the skin pieces was better than that by scrap knives,and their strength and elongation of fracture were equivalent to that of the original hair fiber. The hydrolysate contained a lot of small peptides and amino acids,in which the concentration of amino acid was 5.23%,the content of freeformaldehyde was 37.34 mg/kg,and the content of heavy metal was lower than the standard of amino acid fertilizer. The wheat rooted 1d in advance after wheat seeds cultivated with the hydrolysate,and plant fresh weight after 6 d cultivation was heavier 13% than the control. Treating leather and solid waste with clean products not only causes no secondary pollution,but also the solid waste could be utilized as resources.

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2019, 35(5): 181-181.

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