Table of Content

26 December 2020, Volume 36 Issue 12

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Study on the Function of Terpene Synthase Gene tps2 and Its Promoter Functional Segment in Zea mays

WANG Dan, LI Sheng-yan, LIU Jin-ping, LANG Zhi-hong

2020, 36(12):  1-11.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0563

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Insect pest is the main reason causing the yield and quality of corn reduced. The tps2（terpene synthase 2）gene is inducible by herbivore and TPS2 catalyzes the production of many terpenoids. To analyze the function of tps2 and its expression regulation mechanism,qPCR was used to detect the expression of tps2 under different hormones and armyworm treatments. The transgenic maize TPS2OE was obtained by Agrobacterium-mediated transformation using a tps2 overexpression vector and it was used to study the selective behavior of Asian corn borer. The functional segment of tps2 promoter was tested by transient expression system of maize protoplast. The results showed that plant hormones（JA,SA,and ET）and armyworm induced high expression of tps2;and the expression of tps2 in TPS2OE and the target terpenoids were significantly higher than that in control B73. The number of corn borer eggs on the leaves of TPS2OE and B73 was insignificant,while the larvae on TPS2OE were fewer,and the damage areas were also smaller. The promoter truncation analysis found that the required functional element of the tps2 promoter was between -183 to -129,and there were repressor binding sites between -227 to -183. In summary,tps2 expression is induced by plant hormones and armyworm,and TPS2OE plants have a certain inhibitory effect on the larvae of Asian corn borer. The required functional element of tps2 promoter is between -183 to -129.

Transcriptome Analysis of Drought-resistant Genes in Hainan Shanlan Upland Rice

ZHAI Nan-xin, CHI Hui, XIA Yue-lin, LIU Cai-yue, PEI Xin-wu, YUAN Qian-hua

2020, 36(12):  12-20.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0422

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Shanlan upland rice is an unique local upland rice in Hainan province,which is rich in drought- resistant gene resources. In this study,a Hainan Shanlan upland rice variety,Baishanuo was used to study the changes of transcriptome level after simulated drought stress. The main results were as follows：2 791 genes were differentially expressed after simulated drought stress,and 184 transcription factors were significantly up-regulated after simulated drought stress. These transcription factors include Znf,AP2/ERF,MYB,NAC,bZIP and so on,among which the number of Znf transcription factors was the largest. Expression analysis showed that some unannotated genes,such as Os01g0910800,their expressions increased with the time of stress,and the multiple of expression was obvious. The transcriptome data and digital gene expression profiling data obtained in this study provide information for the study of drought resistance mechanism of Shanlan upland rice,and lay a foundation for further analysis of the drought resistance mechanism of Shanlan upland rice.

Genetic Relationship Analysis of Sorghum Breeding Materials Based on Simplified Genome Sequencing

ZHANG Yi-zhong, FAN Xin-qi, YANG Hui-yong, ZHANG Xiao-juan, SHAO Qiang, LIANG Du, GUO Qi, LIU Qing-shan, DU Wei-jun

2020, 36(12):  21-33.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0344

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The objective of this study is to explore the genetic structure and relationship of sorghum breeding materials,and to provide theoretical basis for crossbreeding,germplasm innovation and heterosis group construction in the future. Specific-locus amplified fragment sequencing（SLAF-seq）was used to sequence 37 sorghum materials. The sorghum genome（Sorghum_bicolor_v3.1）was used as the reference genome for enzyme digestion prediction and the Nipponbare rice were as the control,and single nucleotide polymorphism（SNP）markers were developed and applied to genetic structure and genetic relationship analysis of breeding materials. Results showed that 106.19 Mb reads length data were obtained by sequencing from the 37 sorghum materials. The reads of the samples varied from 1 461 206 to 4 628 462. The paired-end mapped reads efficiency of the control data was 92.15%,the enzyme digestion efficiency was 89.69%,which indicated that SLAF database was normal. The average quality of sequencing Q30 was 89.60%,and the average GC content of all samples was 45.71%. A total of 226 724 SLAF tags were developed,of which 105 053 were polymorphic SLAF tags. A total 185 451 effective single nucleotide polymorphisms（SNPs）were obtained by sequence analysis. Based on these SNPs,37 sorghum breeding materials were divided into two groups by genetic structure analysis and principal component analysis. The maintenance lines and foreign materials were grouped into one,the restoration lines and Chinese materials were grouped into another one,and the grouping result was basically consistent with the actual genealogy. The genetic distance between materials was estimated based on SNP markers and the distance ranged from 0.0098 to 0.8841. The genetic distance between L2R and L17R was the smallest,and the genetic distance between 3765 white B and L17R was the largest. In this study,the blood relationship of some foreign materials was clarified. It was further found that the sterility lines of Durra and Kafir-caudatum were most distantly related to trend Kaoliang restoration lines,and more attentions should be paid to it in selecting parents for sorghum cross breeding.

Analysis of Salt Tolerance of Transgenic BpLTP4 Tobacco

SHI Jing-jing, GUO Yi-ping, YU Ying, ZHOU Mei-qi, WANG Chao

2020, 36(12):  34-41.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0498

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The BpLTP4 gene with CDS of 357 bp was cloned from Betula platyphylla Suk. The BpLTP4 protein was a hydrophobic and had a potential transmembrane region. The tertiary structure of the protein contained a hydrophobic cavity. The protein had 8 Cys residue structures unique specifically to LTP family genes. BpLTP4 was closely related to LTP salt- tolerant genes of other species. qRT-PCR analysis showed that NaCl stress treatment for 48 h highly induced BpLTP4 expression. BpLTP4 overexpressed tobacco transgenic lines were obtained by leaf disc method. After 150 mmol/L NaCl stress treatment of transgenic and wild-type（WT）lines,it was found that the transgenic lines had higher salt tolerance than the WT,and the average germination rate was 5.6 times that of WT. The root length was 1.099 times that of WT. DAB,NBT,Evans blue staining were lighter than WT,POD activity was 0.28 times higher than WT,and SOD activity was 0.12 times higher than WT. In response to salt stress treatment,BpLTP4 improves the salt tolerance of transgenic grass by increasing the protective enzyme activity,removing reactive oxygen species,and maintaining cell integrity.

Mining of Genes Related to Reactive Oxygen Species Scavenging in Response to Salt Stress in Nicotiana alata Based on Transcriptome Sequencing

LU Lin, YANG Shang-yu, LIU Wei-dong, LU Li-ming

2020, 36(12):  42-53.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0241

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In order to identify the genes related to reactive oxygen species（ROS）scavenging involved in salt stress response of Nicotiana tabacum seedlings,tobacco plants were treated with 200 mmol/L NaCl,and the samples of the seedlings were collected at 12 h after the treatment. After total RNA was extracted,high-throughput sequencing technology was carried out to sequence the transcriptome. On the basis of GO and KEGG analysis of the differentially expressed genes,ROS scavenging genes involved were mined,and the results of transcriptome sequencing were verified by qRT-PCR. The results showed that there were 7 239 genes（P < 0.01）with more than two-fold in the expression of tobacco genes after high salt treatment,among which 4 037 genes were up-regulated and 3 162 genes were down-regulated. The functions of these differentially expressed genes were classified into 159 GO items in three categories：biological process,cell components and molecular functions,and were significantly enriched in 12 KEGG metabolic pathways. Meanwhile,the expressions of 45 genes related to ROS scavenging processes changed significantly in tobacco plants under high salt stress,among which 28 genes were up-regulated and 17 genes were down-regulated. The results of this study suggest that the enhanced ROS scavenging ability could be one of the essential mechanisms of N. alata plants in response to salt stress.

Isolation,Identification of a Cellulose-degrading Bacterium with IAA-producing Ability and Optimization of Its Culture Conditions

WU Jing, NIE Cai-e, ZHU Yuan-yuan, HUANG Wei, MA Chao, JIANG Ying, ZHU Lin, GAO Hong-jian

2020, 36(12):  54-63.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0454

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This study aims to screen strains with the ability of both degrading straw and promoting crop growth from Shajiang black soil for solving problems such as difficult survival status,weak rot-promoting effect and poor crop growth caused by exogenous rot straw. Screening with sodium carboxymethyl cellulose plate and combing with CMC enzyme（endo-1,4-β-D-glucanase）- and IAA（Indole-3-acetic acid）-producing capacity,the potential straw rot- and plant growth-promoting were evaluated. Species identification of strains were conducted via morphological,physiological and biochemical and 16S rDNA sequence analysis,then straw degradation shake flask and corn pot test were used to determine the actual rot- and growth-promoting effect of the strain,and a single factor test was set up to explore the optimal condition for growth and enzyme production of the strain. The screened strain n3 demonstrated the high activity of CMC enzyme（20.60 U/mL）and IAA synthesis（20.15 mg/L）,and was identified as Azospirillum zeae. The results showed that inoculation with n3 increased the rate of straw rot-promoting under liquid fermentation by 54.71%（P <0.01）compared with the control treatment;the root length,average diameter and SPAD value（Soil and plant analyzer development）of maize under soil culture conditions increased by 18.3%,22.0% and 5.24%,respectively（P<0.05）.The optimal nitrogen source and liquid volume of n3 growth,yielding CMC enzyme and IAA were the same as that of yeast powder and 25 mL/250mL,but the optimal pH was 5.0 for CMC enzyme production,and 6.0 for growth and IAA production. Conclusively,the screened A. zeae may be used to produce multi-functional straw-decomposing agent,accelerating the rot-decomposing of straw and promoting the growth of crops in the field,which has broad agricultural application prospect.

The Response of Soil Microbial Community to Repeated Application Clomazone

ZHANG Ying, WU Xiao-hu, LI Xiao-gang, DUAN Ting-ting, XU Jun, DONG Feng-shou, LIU Xin-gang, ZHENG Yong-quan

2020, 36(12):  64-74.  doi:10.13560/j.cnki.biotech.bull.1985.2020-1252

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This work aims to investigate the responses of soil microbial communities and microbes with nitrogen-transforming functions to 2 years consecutive application of clomazone under greenhouse conditions. Soil was sampled after 7,15,30,60,and 90 d of second application and clomazone residue concentration and relevant microbial parameters were determined. The half-lives of clomazone in this experiment were 17.4 d. The results showed that copy numbers of bacteria decreased,whereas bacterial Alpha diversity increased. Copy numbers of fungi（day 15 and day 60）and fungal diversity（day 7 and day 90）significantly decreased. Principal co-ordinates analysis（PCoA）results revealed that the bacterial and fungal community structure significantly changed during the whole culture period. Furthermore,molecular ecological network analysis indicated that the network of clomazone-treated soils contained more nodes,links,higher average degree and average path distance than control soils,i.e.,clomazone application led to remarkable changes in key species of its community network. FAPROTAX functional prediction results showed clomazone mainly reduced denitrification function.

Construction of Prokaryotic Expression Vector of PepA Protein of Vibrio alginolyticus and Identification of Its Acetylation

XU Zhou, FAN Chen-long, DING Yu

2020, 36(12):  75-81.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0444

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The purpose of this study is to construct a prokaryotic expression vector of PepA protein in Vibrio alginolyticus HY9901,to optimize its expression conditions,and to analyze whether or not it is regulated by protein acetylation. Firstly,the pepA gene was cloned via PCR,and an expression vector pET-28a-PepA was constructed and transferred it into Escherichia coli BL21（DE3）.Then,the protein expression and acetylation were analyzed by SDS-PAGE and Western blot. The recombinant expression strain correctly expressed the recombinant protein（60.7 kD）,and its optimal expression conditions was,induced for 5 h at 37℃ by IPTG with a volume fraction of 0.1%. Western blot results showed that PepA protein was acetylated protein,and there was no deacetylation in vitro.

Cloning and Enzymatic Identification of Thermo-tolerant and Endotype Alginate Lyase Gene from Mangrovibacterium sp. SH-52

LI Wei-na, SHEN Dong-ling, ZHANG Yu-xing, LIU Xue-tong, IRBIS Chagan

2020, 36(12):  82-90.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0375

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The objective of this work is to investigate the alginate lyases isolated from anaerobic alginolytic bacteria,to characterize the novel alginate lyase,thus to provide a theoretical basis for the enriching industrial application of alginate lyase. A gene SHA-I encoding an alginate lyase was cloned from an anaerobic bacterium Mangrovibacterium sp. SH-52,sequenced and expressed in Escherichia coli. Its expressed enzyme was purified and the enzymatic characteristics were analyzed. The alginate lyase SHA-I was composed of 362 amino acids with molecular weight of approximately 41 kD. SHA-I belonged to polysaccharide lyase（PL）family 7 and showed 83% amino acid identity with alginate lyase from Lewinella agarilytica. The optimal pH for SHA-I activity was 7.0. SHA-I demonstrated the highest activity at 50℃and remained approximate 60% of the highest activity at 60℃,and 40% at 80℃,indicating that SHA-I was a thermo-tolerant alginate lyase. SHA-I presented substrate specificity to polyG（polymannuronic acid）. It degraded polyG blocks to alginate oligosaccharides as the main products.

Screening and Identification of Marine Electricity-producing and Lipase-producing Bacteria and Preliminary Study on Its Culture Conditions

HUANG Yang-tian, LU Yu-biao, HUANG Yi-tie, MENG Fan-long, XU Kai-wen, LI Peng

2020, 36(12):  91-97.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0833

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The objective of this work is to study the characteristics and electricity-producing of lipase-producing bacteria in the sea. The strains with lipase-producing feature were screened from the intertidal marine mud in Zhoushan sea area. Olive oil was used as the sole carbon source for plate screening,NaOH titration was used to determine the lipase activity,and the effect of fermentation condition on the lipase activity was preliminarily studied. To determine the taxonomic status of strains,16S rRNA gene analysis was used for identification,phylogenetic tree was constructed,and the effects of various culture conditions on the enzyme production activity were preliminarily studied through single-factor experiment. The microbial electric performance was preliminarily explored. The results showed that two strains with lipase-producing activity were selected from the intertidal mud,labeled S11 and S22,respectively. Molecular identification results revealed that S11 and Pseudomonas putidawa WAB1947,S22 and Bacillus substills LSRB were the closest in phylogenetic status respectively. The results of single-factor experiment demonstrated that the corresponding enzyme activity of the two strains was the best when the culture time was 48 h,the initial pH value was 7.0,and the urea content was 0.35 g/L. Microbial fuel cell（MFC）was constructed for voltage acquisition,and the results showed that the maximum output voltage of S11 and S22 was 0.055 V and 0.031 V,respectively. In conclusion,strain S11 and S22 is Pseudomonas putida and Bacillus substills,respectively,and they present certain electricity-producing capacity.

Eukaryotic Expression,Purification and Activity Identification of Rat His-Akt1 Recombinant Protein

MENG Li, DU Cai-ping

2020, 36(12):  98-103.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0497

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To obtain rat His-Akt1 recombinant protein with high activity,His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed firstly,and then transfected into HEK293 cells. Next,the His-Akt1 was purified by Ni column affinity chromatography（Ni-NTA）,and its activity was then identified. His-Akt1 cDNAs were amplified from the full-length recombinant Akt1-pcDNA3.1 by PCR and cloned into eukaryotic expression vector pcDNA3.1. The HEK293 cells transfected with His-Akt1-pcDNA3.1 plasmid were collected and sent to Ni-NTA. His-Akt1 purity was identified by SDS-PAGE,Coomassie blue staining and Western blot. Dialytically purified His-Akt1 finally was subjected to Western blot analysis to detect Ser473 and Thr308 phosphorylation levels. His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed successfully and efficiently expressed in HEK293 cells. The Coomassie blue staining and Western blot results showed that high-purity His-Akt1 recombinant protein was obtained by purifying 2 mg overexpressing His-Akt1 protein samples with Ni-NTA and eluting with 100 mmol/L imidazole. In addition,the Ser473 and Thr308 phosphorylation（i.e. activation）of the His-Akt1 after dialysis presented both high levels. The recombinant His-Akt1-pcDNA3.1 eukaryotic expression vector is constructed successfully,and the protein highly expressed,and the purified His-Akt1 recombinant protein demonstrates high enzymatic activity.

In Vitro Fermentation of Monosodium Glutamate with Human Gut Microbes

LIU Shu-jun, CHEN Miao, WANG Feng-zhong, BAO Yu-ming, XIN Feng-jiao, WEN Bo-ting

2020, 36(12):  104-112.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0600

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This study aims to investigate the effects of monosodium glutamate on the composition and functions of human gut microbes. In vitro fermentation of gut microbes using sodium glutamate as substrate was conducted,and the content of short-chain fatty acids（SCFA）and γ-aminobutyric acid（GABA）in the fermentation broth was determined. The results showed that the total SCFA yield in the fermentation with monosodium glutamate was significantly higher than the control without substrate（P<0.01）,and the content of butyric acid increased by 26 times. The conversion rate of monosodium glutamate reached 72.36% after in vitro fermentation. The results from in vitro fermentation with monosodium glutamate showed that the relative abundance of Bacteroidete and Proteobateria phyla in vitro fermentation with monosodium glutamate increased（P<0.05）if compared with the original feces,while that of Firmicutes and Actinobacteria phyla decreased（P<0.01）. Escherichia-Shigella and Bacteroides genera increased（P<0.01）while monosodium glutamate was added. Our results indicate that in vitro fermentation with monosodium glutamate increase the content of SCFAs in fermentation broths,especially the butyric acid,and affect the composition and function of human gut microbes;moreover,monosodium glutamate may convert into GABA via in vitro fermentation with gut microbes.

Study on the Cholesterol-lowering and Antioxidant Abilities of a Tri-lactobacillus In Vitro

HUANG Yuan-xia, PENG Chuan-hai, DING Ning, QIU Zhong-ping, LI Xing, ZOU Mei-hui

2020, 36(12):  113-120.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0399

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In order to obtain a tri-lactobacillus that is easy to colonize in vivo and has strong ability of lowering cholesterol and anti-oxidation,three strains of Lactobacillus casei S1,Enterococcus faecium S4 and Lactobacillus harbinensis S6 were used to form tri-lactobacillus. The artificial gastrointestinal tolerance,cholesterol-lowering ability,yield of γ-aminobutyric acid（GABA）,and antioxidant activity of tri-lactobacillus were investigated. The results showed that the survival rate of tri-lactobacillus was 47.88% and the number of viable bacteria was 1.25×10⁹CFU/mL after 24 h of treatment with artificial gastric intestinal fluid. The total cholesterol removal rate,specific activity of bile salt hydrolase and GABA yield were 73.43%,151.40 U/mg and 1.387 mg/mL,respectively;and the reducing power,superoxide radical clearance rate and hydroxyl radical clearance were 1.52,95.68% and 89.93%,respectively. It was significantly higher than the control group（P < 0.01）. The tolerance of tri-lactobacillus to artificial gastric and intestinal fluid is strong,and it has strong ability of lowering cholesterol and anti-oxidation,thus it can be further developed as functional microecological preparation or health care product.

Progress of the Structural and Functional Analysis of Plant Transcription Factor TIFY Protein Family

YANG Rui-jia, ZHANG Zhong-bao, WU Zhong-yi

2020, 36(12):  121-128.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0307

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The TIFY protein is a kind of plant-specific transcription factor in many plants. Depending on its conservative domain,it can be classified into four subfamilies：TIFY,JAZ,ZML,and PPD. Here,we reviewed the research progress on the structure,distribution,and biological function of TIFY protein in regulating the growth and development of plants,responding to various stresses and different hormone signals,aiming to lay a theoretical foundation for in-depth research and utilization of TIFY protein family.

Progress of Plant Cuticular Wax Synthesis and Its Regulatory Factor WIN/SHN

LI Xiao-pei, WANG Si-ning, SHI Jing-jing, GAO Zhi-min

2020, 36(12):  129-136.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0572

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Plant cuticular wax is a hydrophobic protective layer by which plants directly contact with the external environment. Because it can regulate the water in plants to deal with abiotic stresses such as drought,salt and cold,it plays an important role in plant growth and development. Among different species,the structure and composition of the plant cuticular wax vary largely,but all have similar wax synthesis pathways,and remarkable research results on these pathways has achieved. Many transcription factors（TFs）are involved in the wax biosynthesis of plants. WIN/SHN,which belongs to the AP2 TF family,regulates the expressions of wax synthesis-related genes by binding to their promoters,thereby affecting the waxy synthesis. Based on the synthesis of plant cuticular wax,this article focuses on the review of WIN/SHN TFs,including their structural characteristics,expression patterns,transcriptional regulation and the effects on physiology and phenotype,as well as their responses to stresses. Furthermore,the complexity of wax synthesis is also discussed. In view of the important value of wax for life science and agricultural production practice,it is of great significance to carry out in-depth research on the mechanism of wax synthesis,regulation,transport and deposition regulated by the genetic and environmental factors including WIN/SHN TFs.

The Research Progress of β-Fructosidase Inhibitors

ZHOU Huai-ye, ZHOU Bi-yao, Su Tao

2020, 36(12):  137-145.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0258

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C/VIF（Cell wall/Vacuolar Inhibitor of β-Fructosidase）in higher plants is a small protein family（14-23 kD）with subcellular localizations to the apoplast or vacuoles,which post-translationally inhibits the hydrolytic activity of acid invertases. C/VIF can affect the seeds and fruits development and maturation through fine-tuning of the homeostasis of sucrose metabolism or cell osmotic pressure,and participate in the regulation of adapted abiotic stresses. It is also found to be involved in sugar immunization and defense response to pathogen infection. The gene expression and enzymatic activity of C/VIFs are subject to the transcriptional control by hormones（e.g.,ABA,Auxin,and CK）and environmental cues（e.g.,cold,drought,and wounding）. Here,the molecular and biochemical features,the profiling of spatiotemporal expression patterns,and novel findings of physiological significance and the underlying modulatory mechanisms of C/VIFs are reviewed. Given that the functional roles of C/VIFs remain to be unraveled in woods,this paper provides the basis and lays a theoretical foundation for the subsequent filling of these research gaps.

Application of Metagenomics in Plant Diseases Research

WANG Pan-pan, YANG Ye, LIU Di-qiu, CUI Xiu-ming, LIU Yuan

2020, 36(12):  146-154.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0207

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With the continuous development of sequencing technology,metagenomics has become a current research hotspot and is one of the important approaches of studying plant diseases. Plant disease affects plant normal growth and development,even leads to the death of a plant. Most plant diseases result from pathogenic microorganisms or viruses. In contrast to traditional research methods on microbes,metagenomics can be used to investigate species composition and functional genes in samples by high-throughput sequencing and subsequent bioinformatics analysis,i.e. studying the species diversity. This paper summarizes and compares the analytical strategies of current metagenomics,and its applications in the study of plant rhizosphere microorganisms and plant viruses,and also provides an outlook on future trends,aiming at providing methodological reference in the study of plant diseases.

Research Progress on Regulatory Genes of Important Agronomic Traits and Breeding Utilization in Rice

ZHANG Hai-miao, LI Yang, LIU Hai-feng, KONG Ling-guang, DING Xin-hua

2020, 36(12):  155-169.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0537

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Rice is one of the three main grain crops in China,and it is also one of the guaranteed varieties to ensure the absolute safety of rations in China,thus it has high production value. With the rapid development of technologies such as molecular markers and transgenic technology,more and more researchers have focused on cloning and identifying genes for high yield,high resistance,high nutritional efficiency and other excellent agronomic traits in rice. On this basis,molecular breeding technology is used to improve the target traits in order to obtain rice varieties with excellent comprehensive traits. Compared with traditional hybridization techniques,the application of molecular breeding in rice can break the reproductive isolation between species and shorten the breeding time,therefore make the improvement of rice traits more precise and diversified. This article reviews the recent research progress on the regulation genes related to high nutrient utilization efficiency,hormone,biotic stress,abiotic stress,yield and quality in rice,and prospects on the future direction of rice research and development,aiming to provide a beneficial reference for the scientific research of rice in China.

Advances in the Study of Genes Related to Pollen Exine Sporopollen Synthesis and Transport in Rice

WANG Lian-hong, ZHANG Qiu-yun, LIU Jia-lin, SHEN Ya-qi, OUYANG Lin-juan, HE Hao-hua, HU Li-fang

2020, 36(12):  170-177.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0421

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The pollen exine is the guarantee for the normal pollen development,and the main component is sporpollen,which can establish the structural support for the microspore cytoplasm to protect it from harsh conditions. The normal development of the pollen exine is a complex process of multi-gene expression regulation,there are a large number of genes involved in the development of pollen exine,especially the metabolism of sporopollenin,have been isolated and identified in rice. This paper summarized 22 genes related to rice sporopollen synthesis and transport,as well as their functions,molecular mechanisms and regulatory networks,which is aimed to provide a theoretical basis for further research on the molecular mechanism of rice male reproductive development.

Review on Vegetative Propagation Techniques of Torreya Arn.

WANG Xiao-he, GU Xi-rong, LI Jie, CUI Yao

2020, 36(12):  178-187.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0155

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There are 7 species of genus Torreya in existence,with large barriers of sexual reproduction and scarce wild germplasm resources. Most of them are key protected plants that are gradually endangered to endangered. Asexual reproduction is an essential prerequisite for expanding the population size,protecting,developing and using this genus. The current techniques used are mainly grafting,tissue culture and cuttings. Among them,the success rate of grafting is affected by the affinity and vigor of scion and rootstock,grafting method and grafting period,etc.;tissue culture effect are closely related to plant genotype,explant type and material extraction time,basic medium type,hormone type and concentration,exogenous additives and culture environment;the rooting rate of cuttings is closely related to cutting type,cutting period,cutting substrate,the type,concentration and treatment time of exogenous hormones,and management after cutting. In the future,it is necessary to continue to strengthen the research on asexual reproduction technology,and to explore its mechanism,thus to fundamentally solve the problem of asexual reproduction of the genus Torreya,and finally to comprehensively promote the application of asexual reproduction technology in the expansion of the population of genus Torreya.

Removal of Bisphenol A in Wastewater by Immobilized Laccase

SUN Kai, CHEN Zheng-jie, WANG Deng-yang, SHU Ru-yu, WU Ji, WEI Fan

2020, 36(12):  188-198.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0377

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Bisphenol A（BPA）is an essential synthetic chemical material,which is generally used as a plasticizer in industrial and residential applications to produce polycarbonate plastics and epoxy resins. BPA may accumulate in wildlife and human body,which leads to imbalance and disorder of the endocrine system of the organism,and even induces cancer risk. Adsorption,Fenton oxidation,electrochemistry,photocatalytic degradation,and biological film filtering are capable of effectively removing BPA in wastewater. However,it is difficult for these conventional technologies to eliminate trace amounts of BPA,and even easily causes secondary damages to the ecological environment. Fortunately,laccase-catalyzed radical oxidation reactions have the advantages of mild reaction conditions,high catalytic performance,controllable operation,cost-effectiveness,and environmental friendliness,and thus it has extremely high strategic value in repairing BPA polluted wastewater. Currently,the application of immobilization technology is not only expected to achieve the continuous removal of BPA by laccase,but reinforces laccase-mediated oxidative degradation of BPA. The main sources of BPA and its harms to organisms are reviewed,and particularly,the immobilized laccase-catalyzed transformation efficiency and mechanism of BPA are systematically summarized,further the ecotoxicity of BPA intermediate products is also clarified in laccase-mediated reaction processes. These findings are aimed at laying a theoretical basis and technical support for the large-scale application of immobilized laccase in wastewater treatment.

Research Progresses on the Synthetic Biology of Terpenes in Saccharomyces cerevisiae

LI Jia-xiu, CAI Qian-ru, WU Jie-qun

2020, 36(12):  199-207.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0565

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Terpenoids are widely used in the pharmaceutical,food,and cosmetic industries due to their unique and diverse biological activities. Saccharomyces cerevisiae,the simplest eukaryotic cell,is the preferred cell factory for complex terpenoids biosynthesis with high suitability of enzymes from eukaryotic sources. In recent years,construction and optimization of terpenoid metabolic pathways in S. cerevisiae have received much attention in synthetic biological researches. Here we review the synthetic biological strategies of high-value terpenoids from four aspects：optimizing isoprene precursor pathway enzymes,reforming central carbon metabolism,balancing competitive pathways,and expressing heterologous cytochrome P450 enzymes,aiming at providing references for the further researches of terpenoids biosynthesis in S. cerevisiae.

Structure and Catalytic Mechanism of Bacterial Urease Protein

HE Yue, ZHAO Sheng-guo, ZHANG Xiao-yin, ZHENG Nan, WANG Jia-qi

2020, 36(12):  208-217.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0459

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Bacterial urease catalyzes the efficient decomposition of urea to produce ammonia;however,high activity of urease has negative influence on human healthy,agriculture,environment,and so on. It is of great scientific significance to research characteristics of bacterial urease and study how to inhibit urease activity. With the rapid development of molecular and structural biology,more and more structures of urease have been analyzed,and the regulation mechanism of urease activity has been improved constantly,which mark a new stage of urease protein research. This review summarizes the crystal structure and active center characteristics of bacterial urease,catalytic mechanism,mechanism of urease inhibitors,in order to provide some references to urease regulation research.

Green Biotransformation of Protein-derived Biomass

LIANG Xin-xin, TANG Dan, HUO Yi-xin

2020, 36(12):  216-228.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0365

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The large-scale application of bioenergy contributes to environmental protection and sustainable energy supply. At present,the recycling of carbon in the development of biofuel technology has emerged,but the recycling of nitrogen has not been given sufficient attention. Considering the wide variety and huge amount of protein-rich wastes,the conversion of the carbon backbone of amino acids in proteins into high value-added compounds such as biofuels,bulk chemicals and pharmaceutical intermediates is becoming a growing concern. From the perspective of the biological recycling of ammonia,this paper details an efficient,low-cost and sustainable production system of converting protein-rich biomass waste produced by photosynthesis into high-value compounds. In addition,the paper also summarizes the challenges and feasible strategies in further optimizing the biotransformation technology,which provides a reference for the low pollution and efficient production of high value-added compounds in the next step.

Research Progress on Proteins of PEDV Antagonizing Host Innate Immune Responses

CHENG Wen-yu, BAI Yun, JIA Huai-jie, QIANG Tao-yan, ZHAO Hong-yuan, ZHANG Bo-yi, GUO Xiao-hui

2020, 36(12):  229-238.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0436

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Porcine epidemic diarrhea virus is one of major causative agents of diarrhea in pigs that causes vomiting,watery diarrhea,and severe dehydration in swine,and with topped 100% mortality in neonatal piglets,leading to enormous economic losses within the swine industry worldwide. The encoded 21 proteins of PEDV are not only involved in virus life cycle,but also play critical role in antagonizing host innate immune responses. Presently,ten viral proteins have been confirmed to have abilities in inhibition interferon responses,i.e.,host immune system was escaped through different mechanisms. In order to deeply understand the functions of these proteins in viral infection and mechanisms in regulation host immune responses,the recent progress on the PEDV proteins involved in host immune regulation has been reviewed,aiming to provide theoretical basis for PEDV vaccine development and disease control.

Research Progress on Innate Immunity-Related Coding Genes in the Regulation of Cow Mastitis

HU Qi-chao, LUORENG Zhuo-ma, WEI Da-wei, YANG Jian, JIA Li, WANG Xing-ping, MA Yun

2020, 36(12):  239-246.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0429

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Mastitis is one of the most common diseases in dairy cattle,especially in high-yield individuals,which has caused huge economic losses to the modern dairy industry. In order to clarify the molecular mechanism of dairy cow mastitis and carry out anti-mastitis molecular breeding,scholars have carried out a lot of research works on the regulatory mechanism of innate immunity on dairy cow mastitis. It is found that coding genes such as cell pattern recognition receptors,signal pathway,inflammatory cytokines,inflammatory chemokines,host defensins and antimicrobial peptides play an important role in breast innate immune response. This article summarizes the research progress on classification,expression pattern,molecular regulation mechanism of innate immunity-related coding genes as well as the mining of mastitis-related SNPs in bovine mammary gland,which will provide reference for systematic understanding and further study of the molecular regulation mechanism of dairy cow mastitis.

Sample Preparation and Data Analysis Method for Soybean Seed Proteome

MU Yong-ying, WANG Dao-ping, CHEN Ming, QIU Li-juan, PAN Ying-hong

2020, 36(12):  247-255.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0555

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This study constructs a method for effective sample preparation and analysis of soybean seed proteomes via liquid mass spectrometry,aiming to improve the coverage of soybean proteome analysis. The mature seeds of soybean variety Zhonghuang 35 were used as materials to compare the protein extraction and enzyme digestion methods,liquid phase separation gradients and databases. The results showed that the identified proteins were the most when soybean seed proteome was extracted using modified urea thiourea buffer and digested with Lys-C/trypsin sequentially,then subjected on 90min nano liquid chromatographic separation and analyzed based on UniProt database. Finally,this method was applied to the preparation and analysis of mature seeds of soybean variety Tokachi-Nagaha,and a total of 2 244 non-redundant proteins were identified from three independent samples,in which about 61% could be repeatedly identified. Thus this method is reliable and stabile,and it is suitable for the proteome sample preparation of soybean seed and data analysis.

Short Tandem Target Mimic and Its Application in Analyzing Plant miRNA Functions

ZHENG Wen-qing, ZHANG Qian, DU Liang

2020, 36(12):  256-264.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0257

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MicroRNA（miRNA）,a class of endogenous non-coding RNAs in about 21-24 nucleotides length,regulates gene expression by affecting the stability of target mRNA or by regulating the process of translation. With the development of modern molecular biology technology,more and more studies demonstrated that miRNAs play an important role in the growth and development of plants. However,in the miRNA function research,more effective and widely applicable methods are needed. Here,a specific miRNA targeting method is introduced,namely Short Tandem Target Mimic（STTM）. The basic characteristics,function mechanism of STTM,comparison with other similar technologies,and its application in the study of miRNA function in plants are described,which may provide a technical reference for the study of plant miRNA function in the future.