Biotechnology Bulletin

Effect of Histone Deacetylase Inhibitor（HDACi）on CRISPR Editing Efficiency of Wheat and Transcriptomics Analysis

DAI Wen-shuang, LIU Hui-yun, DU Qing-guo, ZOU Cheng, WANG Ke

2021, 37(1): 2-14. doi:10.13560/j.cnki.biotech.bull.1985.2020-0645

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This study focuses on the relationship between histone deacetylase inhibitors（HDACi）and chromatin status and CRISPR/Cas9 editing efficiency. Different concentrations of nicotinamide（0,2.5,and 5 mmol/L）and sodium butyrate（0,5,and 10 mmol/L）were used to treat wheat seedlings for 7 d and 14 d,and the results showed that sodium butyrate seriously inhibited wheat growth,however,nicotinamide had little effect on it. Moreover,transcriptome sequencing of wheat seedlings treated with nicotinamide revealed some genes which were conducive to promoting the opening of the chromatin state,including 6 methyltransferase synthesis pathway genes. In addition,the TaAGO4a transgenic wheat materials in T₂ generation that had not been edited were treated with nicotinamide,and results showed that a heterozygous mutant plant with mutations in the homologous alleles on 3A and 3B chromosomes was detected in the 5 mmol/L treatment for 14 d,and the editing efficiency increased from 0 to 8.3%;there was no mutant found in other treatments or control group. This study proves that nicotinamide can indeed improve the editing efficiency of wheat genes as a new strategy.

Lipidomics Analysis of Saccharomyces cerevisiae with Tolerance to Phenolic Inhibitors

GU Han-qi, SHAO Ling-zhi, LIU Ran, LIU Xiao-guang, LI Ling, LIU Qian, LI Jie, ZHANG Ya-li

2021, 37(1): 15-23. doi:10.13560/j.cnki.biotech.bull.1985.2020-1338

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The mechanism of the adapted Saccharomyces cerevisiae tolerating to phenolic inhibitors was analyzed by lipidomics from the phospholipid profiles on cell membrane. The lipids of S. cerevisiae wild-type（WT）and adapted strains cultured under the phenolic compound stress were detected using liquid chromatography mass spectrometry（LC-MS）and systematically compared and analyzed by statistical method. The results showed that 565 lipid metabolites were identified,including 185 cell membrane phospholipids. The adapted strain presented the increase in the relative abundance of phosphatidylcholine（PC）,phosphatidylethanolamine（PE）,phosphatidylinositol（PI）,and the phospholipids with long-chain（C32-C36）and diunsaturated fatty acyl chain,while compared with the WT strain. The result of statistical analysis showed that most significant difference in the phospholipids of the WT and the adapted strain were PC and PE with long-chain and unsaturated fatty acyl chain. The mechanism of S. cerevisiae tolerating to phenolics was speculated that the plasma membrane integrity was improved via membrane phospholipids remodeling,i.e.,selectively shielded to phenolic compounds,thus the cells remained high activity.

Advances in Analysis Methods of Mass Spectrometry-based Metabolomics

TIAN He, SHUI Guang-hou

2021, 37(1): 24-32. doi:10.13560/j.cnki.biotech.bull.1985.2020-1383

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Mass spectrometry（MS）-based metabolomics analyses are now widely applied to identify and quantify small-molecular metabolites in biological samples for uncovering endogenous alterations in cells after their exposure to exogenous stimulus. Duo to the complicated components in biological samples and a big span of polarities of metabolites ranged from hydrophilic to hydrophobic metabolites,a series of extraction protocols and the matched liquid chromatography（LC）and MS conditions are required to obtain the quantitative and qualitative information of target components. To some extent,the diversity of structure and physicochemical properties of small molecule metabolites limits the high-throughput acquirement of whole metabolic profiles from biological samples. Present review focuses on the introduction and discussion of extraction methods of varied metabolites,as well as their corresponding LC-MS conditions.

Spatially Resolved Metabolomics：Progress and Challenges

YIN Zhi-bin, HUANG Wen-jie, WU Xin-zhou, YAN Shi-juan

2021, 37(1): 32-51. doi:10.13560/j.cnki.biotech.bull.1985.2020-1374

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Spatially resolved metabolomics is developed by integrating mass spectrometry imaging（MSI）and metabolomics methods for accurate determination of types,contents,and spatial differentiation of endogenous and exogenous metabolites within plant and animal tissues. Benefiting from the unique features of label-free and non-specific detections,as well as high sensitivity,high chemical coverage,and simultaneous detection of elements and molecules,MSI has gained an increasing attention as a powerful tool in spatial and temporal visualization of various metabolites,peptides,and proteins. Herein,we introduce the current research status of metabolomics and MSI techniques,then focus on the emerging applications of spatially resolved metabolomics in plant and animal tissues,as well as at the single cell levels,and finally prospect the current bottlenecks and future developing trends in spatially resolved metabolomics. As an emerging molecular imaging-based omics technique,spatially resolved metabolomics is featured for its label-free and visualized detection of absorption,distribution,metabolism,and excretion of exogenous drugs in animal tissues,and provides insights into the biosynthesis,transport pathways,and accumulation of various metabolites in plant tissues. This technique facilitates further applications in the discovery of targeted drugs,understanding of pathological mechanisms,and a spatial metabolic network closely related to the growth and development of plants and animals.

Research Progress of Approaches in Single Cell RNA Sequencing Data Analysis

ZHANG Miao, SUN Xiang-rui, XU Chun-ming

2021, 37(1): 52-59. doi:10.13560/j.cnki.biotech.bull.1985.2020-0469

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Single cell RNA sequencing（scRNA-Seq）has been widely used in cell differentiation,tumor microenvironment and etiology of various diseases. Currently improving the accuracy of sequencing results by optimizing data analysis methods has become a research hotspot in the field of sequencing due to the characteristics of low capture rate,high noise,and high variability of scRNA-Seq. Here an overview of the mathematical methods used in the process of data analysis in recent years is summarized,and the advantages and issues in data analysis are discussed,aiming to provide a reference for the development and application of new algorithms,and to gradually improve the reliability of sequencing results.

Research Progress in Single-Cell RNA-Seq of Plant

LI Yi, SUN Chao

2021, 37(1): 60-66. doi:10.13560/j.cnki.biotech.bull.1985.2020-1011

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Single-cell RNA-Seq（scRNA-seq）is a technology to analyze gene expression at single cell level. Currently PCR plate-based and droplet-based protocols are 2 major popular ones used in the studies of plant. Here,we firstly outline the technical principles and data analysis procedure of plant scRNA-seq,and then introduce its research status,focusing on how scRNA-seq can be used to detect distinct plant cell types,to reveal cell evolution trajectories and to construct inter-cell regulatory networks. ScRNA-seq may provide a new perspective on plant research,and help to understand and identify critical cellular biological processes in complex tissues.

Advances in the Studies of Plant Protein Post-translational Modification

LIU Jing, LI Ya-chao, ZHOU Meng-yan, WU Peng-fei, MA Xiang-qing, LI Ming

2021, 37(1): 67-76. doi:10.13560/j.cnki.biotech.bull.1985.2020-0946

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Protein post-translational modification（PTMs）is an important cellular regulatory mechanism. It regulates the activity,structure,positioning and interaction between proteins by covalently binding some chemical small molecular groups on the amino acid side chains of proteins,thereby fine-tuning the dynamic changes of protein’s biological functions. PTMs are one of the fastest and earliest responses of plants to environmental changes. They are the key mechanism of plant proteome diversity and play an important role in plant growth and development and environmental adaptation. This article mainly introduces the research progress of plant PTMs such as phosphorylation,acetylation,succinylation,glycosylation,ubiquitination,crotonylation,S-nitrosylation,2-hydroxyisobutylation,etc. in recent years,aiming to provide references for understanding the key biological functions and research prospects of plant PTMs.

Research Progress of Plant Lysine Acetylproteome Modified in Non-histone Protein

ZHENG Lu, SHEN Ren-fang, LAN Ping

2021, 37(1): 77-89. doi:10.13560/j.cnki.biotech.bull.1985.2020-1080

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Protein lysine acetylation is an important and common protein post-translational modification in plants. The prior researches had focused on acetylation modification of histones in chromosomes and their regulatory mechanisms. Recently,with the development of quantitative acetylproteome,a large number of non-histone proteins with lysine acetylation modification have been discovered,and the universality of its existence in biology and the importance of its function have also been highlighted. Non-histone protein lysine acetylation modification is abundant in different tissues,organs and organelles of plants. It is widely involved in various metabolic processes during the growth and development,and also plays important roles in responses and adaptation to stresses. In this review,we summarize the recent acetylproteome researches in non-histone lysine acetylation modification in plants. Then we describe the distribution characteristics of identified acetylated proteins in different tissues and subcellular organelles. Further we reveal the functions of acetylation modification in growth,development and responses to stresses,crosstalk between lysine acetylation and other protein post-translational modifications. Finally,we prospect and discuss the future research directions.

Microbiome and Its Research Progress of Anaerobic Digestion

LI Ye-qing, JING Zhang-mu, JIANG Hao, XU Quan, ZHOU Hong-jun, FENG Lu

2021, 37(1): 90-101. doi:10.13560/j.cnki.biotech.bull.1985.2020-1217

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A massive amount of organic waste is produced every year in China. The improper disposal of organic waste causes significantly negative impacts on ecology,climate,and human health. Anaerobic digestion has been recognized as a reliable,green and sustainable approach for the treatment of organic waste. However,due to the lack of precise and effective monitoring methods,the microscopic process of an anaerobic digestion is often regarded as a “black box”. With the development of microbiome,researchers have acquired a deeper understanding of the correlation analysis between microbial community and operating parameters,metabolic pathway analysis and other aspects. According to “three stages and four communities” in an anaerobic digestion,typical microbiome are introduced,including 16S rRNA genome,metagenome,metatranscriptome and metaproteome. In addition,the six commonly bioinformatics analysis methods for microbial community are described in detail,such as species composition analysis,α diversity analysis,OTU similarity analysis,and multivariate statistical analysis. Finally,the research progress of microbiology in anaerobic digestion process is systematically reviewed,aiming to provide support for the study of the structure and function of anaerobic digestion,and the development of new anaerobic digestion processes and technologies.

Advances in the Histology Study,Function and Application of Insect Intestinal Flora

HU Zi-yuan, XIA Qiang

2021, 37(1): 102-112. doi:10.13560/j.cnki.biotech.bull.1985.2020-0859

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With the development of insect microecology in recent years,more and more attentions have been paid to the study of histology of intestinal flora. Because there are many kinds,large numbers and various functions of the intestinal flora of insects,it is critical to select the research methods of its histology,which is closely related to whether the research is scientific,efficient and reasonable. The common methods of studying the histology of insect intestinal flora include metagenomics,proteomics,metabonomics,culturomics and combined application of multi-omics. Insect intestinal flora plays a key role in the long-term coevolution with the host,such as nutrition metabolism,growth and development,and protection and defense. With the deepening of its function research,the application research of insect intestinal flora is glowingly extensive. In this paper,we summarize the research in recent years from four aspects：the overview of insect intestinal flora,the selection of omics research methods,the function and application of insect intestinal flora,aiming to provide references for the follow-up study of insect intestinal flora.

Research Advance in the Resistome in the Microbiome of Livestock Animals

MA Tao, LU Wei, LI Song-li, FAN Xia

2021, 37(1): 113-122. doi:10.13560/j.cnki.biotech.bull.1985.2020-1195

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Most antibiotics are currently used in livestock production which provides humans with food such as meat,milk,and eggs;consequently,resistances to antibiotics cause a huge threat to the public health in the world. In order to limit the spread of antibiotic resistance from livestock production to humans,it is essential to understand which specific resistance genes are carried by the microbiota in gastrointestinal tract of livestock animals or products. Resistome refers to the collection of all identified antibiotic resistance genes in any given environmental microbiota,and resistome analysis becomes a hot topic in the research field of livestock production. First,we highlight the most recent advances in the resistome analysis and its affecting factors based on sequencing technique in the microbiota of gastrointestinal tract of livestock（piglet,chicken,and ruminant）,as well as in the milk microbiota. We also suggest future research directions,including the standardization of resistome analysis pipeline,research on the expression of resistome using metatranscriptomics technique,and the resistance genes carried by mobile genetic elements. This review aims to light on the regulation of antibiotic resistance genes in livestock production.

Progress Based on Metabolomics：Plant Polyphenols and Their Gut Health Benefit

HUANG Xiao-dan, CHEN Meng-yu, HUANG Wen-jie, ZHANG Ming-wei, YAN Shi-juan

2021, 37(1): 123-136. doi:10.13560/j.cnki.biotech.bull.1985.2020-1409

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This article provides a general overview of the classification and the sources of plant polyphenols,as well as the recent studies using metabolomics technology in the realms of 1）the identification of novel polyphenols,2）the key factors in polyphenols synthesis pathways and,3）the biological function of plant polyphenols. The metabolism of plant polyphenols in the gut and their functions as prebiotics in regulation of gut microbiota are also discussed. The current studies show that different plant polyphenols play varied roles in regulating gut microbiota,but most of them tend to promote beneficial bacteria,and the health benefits of polyphenols on gut microbiota are in function via interaction with the host. In a word,plant polyphenols as “probiotics” affecting human health relies on the mediation of intestinal microbes. The probiotic function of individual plant polyphenol deserves further investigation,including the factors of host,diet and other confounding factors,as well as expanding studies on clinical trials.

Application and Prospects of Single-Cell Sequencing in Liver Disease

GUO Dong-dong, SUN Fen, HE Xuan-ang, YANG Dong-ye, HUANG Lai-qiang

2021, 37(1): 137-144. doi:10.13560/j.cnki.biotech.bull.1985.2020-0463

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Emerging single-cell sequencing is a potent technique,which can not only reveal mutations on genomics,transcriptomics and epigenetic modification at the molecular level but also be used to identify new cell types and surface markers. It can also help to detect changes in cell genes,transcriptomics or epigenetic modifications when a disease occurs,to understand the interactions among cells as well as to comprehensively understand tumor heterogeneity. Currently,single-cell sequencing has been applied to the studies on many diseases,and achievements on liver diseases including cirrhosis and liver cancer to some accomplishments are reported. Herein,the applications of single-cell sequencing in liver development and liver diseases are reviewed,underlying mechanisms of liver diseases and the issues of this technology are discussed,and the feasible resolutions are proposed,for instance,developing 3D single-cell sequencing technology may help us to deeply understand the mechanisms of liver deseases.

Applications of Single-cell RNA Sequencing in Heart Development,Disease and Medicine

ZHU Qing-yuan, LI Tian-qing

2021, 37(1): 145-154. doi:10.13560/j.cnki.biotech.bull.1985.2020-0396

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Single-cell RNA sequencing（scRNA-seq）can depict the difference in the expression level of the same gene between different cells at the single-cell level,enabling a new understanding of various tissues and organs at the single-cell level. At present,the sequencing research of the heart is transitioning from the bulk RNA-seq to the scRNA-seq,and research articles on the sequencing of mice’s and human’s heart have been published one after another. This article reviews the application of scRNA-seq in heart development,disease and medicine,and discusses scRNA-seq in embryonic heart development,cell heterogeneity of heart,pluripotent stem cells derived cardiovascular cell models,and congenital heart malformation. Finally,we conclude with prospects of scRNA-seq technology for heart development,heart regeneration,heart disease and single-cell precision medicine.

Application and Advances of Multi-omics Analysis in Inflammatory Bowel Disease

ZHANG Li-xing, WANG Li-na, KANG Guang-bo, HUANG He

2021, 37(1): 155-167. doi:10.13560/j.cnki.biotech.bull.1985.2020-1032

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Inflammatory bowel disease（IBD）,comprising ulcerative colitis（UC）and Crohn’s disease（CD）,is an idiopathic inflammatory disease most commonly affecting the ileum,rectum,and colon. It shows highly heterogeneous in terms of clinical manifestation,disease’s progression and therapeutic response. Currently,there is no consensus on the pathogenesis underlying IBD,while the treatment options are quite limited. Overactive inflammatory responses are leading to the damage of intestinal mucosal barrier and disorder of intestinal flora disturbance,which is caused by genetic factors,environmental factors,intestinal micro-ecology and host immunological imbalance. Revealing the complex interaction mechanism in the pathogenesis of IBD,excavating potential therapeutic targets,and developing effective intervention strategies cannot be achieved via mono-omics analysis. Thus,there is a need to apply multi-omics association analysis techniques in further elucidating the pathogenesis of IBD at multiple dimensions. In this review,we retrospect and analyze the application of multi-omics techniques in fields related to IBD research. Moreover,we discuss their potential in the fields of individualized diagnosis and treatment,early diagnosis,and IBD typing,which is aimed to lay a foundation in further investigation of IBD.

Map-based Cloning of Rice Flowering Factor Lfm1 Under Long Days

CHEN Bing, CHEN Guo-xin, ZHANG Zhi-guo

2021, 37(1): 168-173. doi:10.13560/j.cnki.biotech.bull.1985.2020-0613

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Flowering time is one of the most important agronomic traits and determines the regional adaptability and yield in rice. Artificial selection makes rice expand from short-day to long-day and low-latitude to high-latitude,thus rice has gradually evolved a flowering regulation mechanism adapted to long-day. Currently,the mining of rice long-day flowering genes is still very limited although some flowering genes,such as SDG724,RFT1,EHD4,and DTH2 that affect rice long-day trait,have been identified. In this study,a batch of mutant materials with significant differences in flowering period under long-day were obtained by screening the rice mutant library. One of these mutants,lfm1（late-flowering mutant1）,delayed flowering under long-day conditions and was normal under short-day conditions. Through map-based cloning,the Lfm1 gene was initially located near the telomere of chromosome 8. Lfm1 was located near the telomere of chromosome 8. Fine mapping revealed that the Lfm1 gene was located between the molecular markers 8-0.269 and 8-0.283. There were 3 candidated genes in this region of 12 kb. Through sequencing analysis,9 bp deletion at position 2 800 of the sixth exon of the LOC_Os08g01420 was found in lfm1,and lfm1 was an allele of ehd3. It is worth of mentioning that under moderate light conditions（~12 h/12 h）,the mutant lfm1 showed an increase in the number of grains per panicle and a slightly longer growth period,thus it can be used in production potentially. The cloning of Lfm1 gene provides an important gene resource for breeding rice materials adapted to different ecological regions.

Identification and Expression of NAC Transcription Factor FtNAC17 in Tartary Buckwheat

RONG Yu-ping, TANG Bin, LI Peng, ZHANG Jie-qiong, CHEN Qing-fu, ZHU Li-wei, DENG Jiao, HUANG Juan

2021, 37(1): 174-181. doi:10.13560/j.cnki.biotech.bull.1985.2020-0556

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Plant specific NAC transcription factors are involved in plant growth and development and stress response. To study the response of NAC transcription factors in tartary buckwheat to abiotic stress would provide theoretical basis for elucidating gene function. A NAC family’s gene from tartary buckwheat was cloned and then analyzed by bioinformatics. The response of the gene to drought,low temperature,salt,methyl jasmonate,abscisic acid and gibberellin was detected by fluorescence quantitative PCR. The gene encodes 272 amino acids,which is named FtNAC17 and GenBank accession number is MT641452. The analysis of gene structure showed that FtNAC17 gene was composed of two exons and one intron. Multiple alignment of amino acid sequences and phylogenetic analysis revealed that FtNAC17 protein was the closest relative to ANAC002 in Arabidopsis thaliana,belonging to the same subgroup of NAC transcription factor family. FtNAC17 presented varied responses to drought,low temperature and salt abiotic stress.

Application Effect of a Novel Plant Immune Inducer ZNC in Tobacco

GUO Mei-yan, LIU Bao-you, LI Yang, ZHANG Xiao-ying, CEHN Yu-guo, DING Xin-hua

2021, 37(1): 182-188. doi:10.13560/j.cnki.biotech.bull.1985.2020-0673

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Immune inducer ZhiNengCong（ZNC）,an ethanol extract of endophyte Paecilomyces variotii,has function of promoting plant growth and enhancing disease resistance. Our study aims to evaluate the induced disease resistance and growth and development effect of ZNC on tobacco while ZNC were sprayed at the different stage of tobacco. The results showed that the 3 concentrations of ZNC showed fine control effect on viral diseases,and reduced the occurrence of rhizome diseases and later leaf spot diseases. The control effect of 500 ng/mL ZNC on tobacco virus and brown spot was the best,reaching 57.07%,indicating that this inducer had a favorable inducement effect on the main tobacco diseases. Compared with CK,the yield increased 6.00% and 5.41% respectively in 50 ng/mL and 500 ng/mL treatments,and the content of total sugar,reducing sugar,total nitrogen and K₂O also increased in 500 ng/mL,i.e.,increasing yield and improving quality occurred at certain level. More experiment demonstrated that 1 000 ng/mL ZNC treatment showed anti-disease effect,however,it presented significantly inhibitory effect on tobacco growth. This showed that ZNC had a fine effect on tobacco growth and disease resistance within a certain dose range,and further experiments are needed to determine it.

Genetic Diversity Analysis of 73 Vitis amurensis and Its Hybrids Offsprings Based on SSR Molecular Markers

WANG Yan-li, YANG Yi-ming, FAN Shu-tian, ZHAO Ying, XU Pei-lei, LU Wen-peng, LI Chang-yu

2021, 37(1): 189-197. doi:10.13560/j.cnki.biotech.bull.1985.2020-0416

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The genetic diversity and genetic relationship of Vitis amurensis between Russia and Northeast China were analyzed with the SSR molecular marker technology, aiming to provide references for utilization and innovation of grape germplasm resources and molecular marker-assisted breeding of species. In the study, 11 pairs of SSR primers with fine polymorphism were selected and used to analyze the genetic diversity and genetic relationship of 10 Vitis amurensis from Northeast China and 63 Russia-introduced grapes. Total 75 alleles were detected in 73 grape resources by 11 pairs of primers, the number of observed alleles ranged from 3（VVIN31）to 10（VVS2）with the average of 6.818 2. The number of effective alleles（Ne）ranged from 1.305 0（VVIN31）to 5.280（VVS2）with the average of 3.519 6. The Shannon’s polymorphism information index（I）ranged from 0.467 8（VVIN31）to 1.883 0（VVS2）with the average of 1.373 6. The polymorphism information content ranged from 0.233 7（VVIN31）to 0.809 8（VVS2）with the mean value of 0.644 0, but the sites VVIN31 and VMCA12 only had low moderate polymorphism. Nei’s genetic diversity index ranged from 0.809 8（VVS2）to 0.233 7（VVIN31）, with an average value 0.644 4, indicating that the genetic diversity of each site was quite different and the alleles were not evenly distributed in the population. The observed heterozygosity ranged from 0.166 7 to 0.931 5 with the average value of 0.464 2, the expected heterozygosity ranged from 0.815 4 to 0.235 3, with an average value of 0.648 9. The heterozygosity of different sites varied greatly, and the average observed heterozygosity was lower than the expected heterozygosity, indicating that there was a certain inbreeding rate in the population, the heterozygotes was lack, and there were many homozygotes. The average coefficient of genetic differentiation was 0.1183 and the average of gene flow Nm was 1.864 1, showing that medium genetic differentiation of germplasm and large gene flow. The result of cluster analysis demonstrated that the relationship between Chinese grape resources and Russian wild grape resources was close, but that with Russian selective grape resources was far. The genetic diversity of selected varieties in Russia was higher than that of Chinese grape varieties and wild grape resources in Russia. Elven pairs of SSR primers have rich polymorphism information, 73 grape resources generate moderate genetic differentiation and abundant gene flow, and the genetic diversity of Russian grape resources is rich, which can be used for breeding new grape varieties.

Light-responding Gene StRSM 1 Mediated by Chlorophyll-binding Protein CP24 Regulates Chlorophyll Accumulation

SA Shi-juan, WU Han-yu, ZHANG Xiao-ping, ZHENG Rui, YAO Xin-ling

2021, 37(1): 198-204. doi:10.13560/j.cnki.biotech.bull.1985.2020-0502

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Mutation on RSM（RADIALIS-like SANT/MYB）,a member of the single MYB transcription factor family inverts darkness-induced chlorophyll reduction,and the reason for it is unknown yet. To reveal how RSM regulates chlorophyll accumulation,StRSM 1-overexpressed positive transformant line of Nicotiana tabacum and Solanum tuberosum were generated,and chlorophyll accumulation and other physiologic phenotypes were assayed. The result showed that over-expression of RSM1 led to light green leaf and a less chlorophyll accumulation,and inhibition of RSM 1 expression increased chlorophyll accumulation. Expression assay of genes encoding chlorophyll metabolism and binding demonstrated that over-expression of StRSM 1 increased the expression of chlorophyll-binding protein CP24 under darkness and changed expression pattern of CP24. The above findings indicate that light-responding transcription factor StRSM 1 negatively regulates chlorophyll accumulation. CP24 involves in the regulation. The result will play an important role in further clarifying how RSM 1responds to light and deeply understanding the light response involved in RSM1.

Total Carbohydrates and β-glucans Accumulation of Rhodosorus sp. SCSIO-45730

DAI Lu-mei, LI Tao, WU Hua-lian, WU Hou-bo, XIANG Wen-zhou

2021, 37(1): 205-214. doi:10.13560/j.cnki.biotech.bull.1985.2020-0639

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Rhodosorus sp. has the potential to be used in medicine,healthcare and bioenergy due to its capacity of accumulating high carbohydrates content. In order to evaluate its industrial potential,the growth,total carbohydrates and β-glucans accumulation of this strain in 4 different culture media with different salinities were investigated. Results showed that the modified ASW medium was more beneficial for the biomass and total carbohydrates accumulation in Rhodosorus sp. SCSIO-45730 compared to other media（f/2,Conway and Erdschreiber）. The salinity adaptability of this alga was studied using ASW medium as the basic medium. Results indicated that this alga grew normally in the salinity of 18‰-58‰. The cell size gradually increased and the algal cell aggregated with the increase of salinity. The highest biomass,total carbohydrates content and productivity,and β-glucans productivity of Rhodosorus sp. SCSIO-45730 were obtained at the salinity of 38‰,which was 13.70 g/L,48.52% DW,412.58 mg/L·d and 119.70 mg/L·d,respectively,while the β-glucans content decreased with the increase of salinity. The total carbohydrates and β-glucans volumetric yield of this alga in outdoor flat-plate photobioreactor were 0.73 g/L and 0.23 g/L,respectively. In conclusion,Rhodosorus sp. SCSIO-45730 is capable of adaptation to a wide range of salinity and has high potential for total carbohydrates and β-glucans production. It is considered as an optimal feedstock for the production of bioethanol and β-glucans.

Effects of Surfactants on Deltamethrin Degradation by Acinetobacter junii LH-1-1

WU Min, TANG Jie, HU Qiong, LEI Dan, ZHANG Qing

2021, 37(1): 215-222. doi:10.13560/j.cnki.biotech.bull.1985.2020-0674

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It is aimed to investigate the effects of surfactants on the degradation of deltamethrin（DM）by strain. The previous screened Acinetobacter junii strain LH-1-1,which has a high degradability on DM,was selected as target strain. The critical micelle concentration（CMC）of three different types of surfactants（CTAB,AES,and Tween-20）werefirst determined by fluorescence spectrometry,and then the effects of the CMCs of CTAB,AES,and Tween-20 on the strain’s growth,and solubility and degradation of DM by the strain were investigated. The CMC of CTAB,AES,and Tween-20 was 0.793 mmol/L,0.547 mmol/L,and 0.031 mmol/L,respectively. The results showed that the strain growth was not significantly affected by using CTAB,while inhibited by AES,and Tween-20 had a certain promotion on the strain’s growth. The solubility of DM was significantly improved by using CTAB,AES and Tween-20. The degradation rate of DM was significantly enhanced by A. junii LH-1-1 in the presence of CTAB. When the concentration of CTAB was 2 CMC,the degradation rate of DM was 98.20% within 72 h,which was 24.31% higher than the positive control group（without the addition of surfactants）. The cationic surfactant CTAB may significantly improve the degradation of DM by A. junii LH-1-1.

Effect and Influencing Factors of Immobilized Microspheres on Degradation of Phthalate Esters in Soil

ZHANG Xiao-hong, TAO Hong, WANG Ya-juan, LI Yi-chun, ZHANG Rui

2021, 37(1): 223-233. doi:10.13560/j.cnki.biotech.bull.1985.2020-0675

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This work aims to study the effects and influencing factors of immobilized microspheres on the degradation of phthalate esters（PAEs）in the remediation of phthalate esters-contaminated soil. The method of embedding was used to immobilize the microbacteria extracted from the research group’s previous work with sodium alginate as the carrier. The effects of the immobilized microspheres and the free bacteria on the degradation of PAEs in the soil were compared. In addition,the influences of the pH,temperature,heavy metals and inorganic salts on degrading PAEs were evaluated. The results showed that：（1）Under the same conditions of soil environment,the degradation effect of the immobilized microspheres on DMP（Dimethyl ortho-phthalate）,DnBP（Di-n-butyl ortho-phthalate）and DEHP（Bis（2-ethylhexyl）ortho-phthalate）was higher than that of the free bacteria. DMP was completely degraded in 7 d,DnBP was completely degraded within 10 d,and the degradation rate of DEHP was 63.73% in 20 d. However,the free bacteria completely degraded DMP in 15 d,and completely degraded DnBP in 20 d. DEHP degradation rate was 48.77% in 20 d.（2）At different pH values,the degradation rate of the immobilized microspheres to DMP,DnBP and DEHP was higher than that of the free bacteria. When pH9,the degradation rate of the immobilized microspheres to DMP,DnBP and DEHP peaked at 96.81%,89.39% and 58.35%,respectively.（3）At different temperatures,the degradation rate of the immobilized microspheres to DMP,DnBP and DEHP was also higher than that of the free bacteria. When the temperature was 30℃,the degradation efficiency of the immobilized microspheres to DMP,DnBP and DEHP was the highest in 96.27%,89.19 and 59.01%,respectively.（4）The degradation rate of the free bacteria to DMP,DnBP and DEHP was significantly reduced by heavy metals;while the degradation rate of the immobilized microspheres to DMP and DnBP was only reduced by 16.35% and 9.95%. In addition,the degradation rate to DEHP increased by 2.49%. These indicated that heavy metals had a strong inhibitory effect on the free bacteria,but had little impact on the degradation effect of the immobilized microspheres.（5）Under saline-alkali conditions,neutral salt greatly reduced the ability of the free bacteria and the immobilized microspheres to degrade DMP,DnBP and DEHP. However,alkaline salt and mixed salt had little impact on the degrading bacteria,and enhanced the ability of the immobilized microspheres to degrade DnBP and DEHP. The immobilized microspheres have obviously better degradation effect compared with the free bacteria,and it had better ability to adapt to the outside environment and to resist the pollution of heavy metals and inorganic salts.

Research Advances of Fruit Anthocyanin Accumulation in Rosaceae Plants

LU Wen-ying, ZHAO Lei, LI Tian-qi, CUI He-yun, LIAO Ping-an

2021, 37(1): 234-245. doi:10.13560/j.cnki.biotech.bull.1985.2020-0717

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Rosaceae is an important member of the plant kingdom. Besides the ornamental plants such as roses,it also contains edible fruit trees such as apples,pears and peaches. The fruits of Rosaceae are often rich in anthocyanin and have various colors,which are very popular among consumers. In recent years,fruit scientists have conducted more in-depth studies on the accumulation of anthocyanins in fruits,mainly exploring the molecular mechanisms of environmental factors or hormone signals involved in the accumulation of anthocyanins. This paper reviews the molecular regulation mechanism of Rosaceae fruit coloring,thinks and summarizes how to increase the anthocyanin accumulation of the fruit,and provides theoretical support for improving the commerciality and intrinsic quality of Rosaceae fruit.

Research Progress on High-density Genetic Linkage Map Construction of Important Ornamental Plants：a Review

GUO Li-li, LI Yu-ying, GUO Da-long, HOU Xiao-gai

2021, 37(1): 246-254. doi:10.13560/j.cnki.biotech.bull.1985.2020-1008

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Genetic linkage map is a linear arrangement of the relative positions of the sites in the chromosome or genome based on the recombination frequency between genetic markers. The construction of high-density genetic map may allow the integration of physical map and genetic map feasible and plays an important role in promoting gene map cloning. The utilization of genetic map can effectively improve breeding efficiency and varieties. The precision of the high genetic map of important flowers have not yet reached the level of fine mapping;moreover,few studies on the high-density genetic map construction of important flowers such as Lilium brownii,Crataegus pinnatifida,Tulipa gesneriana and Helianthus annuus have been conducted,which limits the molecular breeding research progress of flower plant. Here the construction process and drawing method of genetic map are summarized. Then the research progress of genetic map construction of important flowers such as Paeonia suffruticosa,Prunus mume,Rosa rugosa,Dendranthema morifolium,Cymbidium,Nelumbo,Osmanthus are also reviewed. Furthermore the existing major issues in the construction of high density genetic map of important flower plants are discussed. At last,the development direction of the genetic map construction of important flower plants is prospected. This review might shed a light on providing a reference for studying gene mapping,auxiliary genome assembly,comparative genomics,gene cloning and molecular marker assisted breeding of flowers plants.

Research Progress of RNA Interference Technology in the Field of Entomology

XU Xue-liang, WANG Fen-shan, LIU Zi-rong, FAN Lin-juan, JI Xiang-yun, JIANG Jie-xian, YAO Ying-juan

2021, 37(1): 255-261. doi:10.13560/j.cnki.biotech.bull.1985.2020-0653

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RNA interference（RNAi）is a physiological phenomenon of post-transcriptional degradation of endogenous genes in organism,and it is widely present in organisms. RNAi is mainly induced by short/small RNA to hinder the translation or transcription of the target gene,and which cause the target messenger RNA to be silence. RNAi has the advantages of high efficiency and specificity,thus it is widely used in insect gene function research and has shown great potential for development of novel pest management strategies. This review mainly expounds the silencing mechanism of RNAi,several ways of transferring double-stranded RNA into insects,and the latest research progress of RNAi technology in insects of different orders. Finally,the paper briefly summarizes the shortcomings of RNAi technology,and prospects the application of RNAi technology in pest control,with a view to providing theoretical support for the widespread application of this technology in agricultural pest control.

Research Progress on Metabolic Causes for Hypoxic Stress in Mammalian Animals

PENG Wen-chao, LIU Jian-xin, WANG Di-ming

2021, 37(1): 262-271. doi:10.13560/j.cnki.biotech.bull.1985.2020-0551

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Oxygen is the material basis for maintaining the metabolic homeostasis of mammals. If the oxygen supply is insufficient during the metabolic process,it can cause hypoxic stress. Environmental hypoxia,metabolic hypoxia,and oxygen-carrier dysfunction are known as the key causes of hypoxic stress. The impact of hypoxia on the body metabolism and tissue function of mammals were mainly conducted in lung,liver,digestive tract,muscle,and mammary gland,etc. It can maintain its metabolic homeostasis if a mammal in a hypoxic state has developed a metabolic pattern adapted to hypoxia. On the contrary,if the animal cannot maintain metabolic homeostasis under hypoxic conditions,it will cause oxidative stress or even pathological changes. At present,the research on hypoxic stress in livestock mainly focuses on the metabolic adaptation mechanism of plateau animals;however,the metabolic rate,oxygen consumption and free radical levels of lactating animals are relatively high,but the role of oxygen in the formation of metabolic stress in lactating animals and impact on lactation performance are still under being explored. The current review summarized the causes and consequences of hypoxic stress,aimed to discuss the biological basis of mammalian hypoxic stress,and to provide a theoretical basis for maintaining the health status of lactating animals from the perspective of hypoxic stress.

Rapid Detection of Plant Bacterial Wilt by Loop-mediated Isothermal Amplification

LI Xin-shen, HUANG Xiao-mei, WU Shu-xiu, HUANG Rui-rong, WEI Lin-gen, HUA Ju-ling

2021, 37(1): 272-281. doi:10.13560/j.cnki.biotech.bull.1985.2020-0389

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The purpose of the study is to establish a rapid detection method for Ralstonia solanacearum,which is beneficial to the early diagnosis of plant bacterial wilt in the field. The LAMP（loop-mediated isothermal amplification）method for amplification of cytochrome C gene was established by optimization of reaction conditions. The minimum detection limit with the LAMP method reached 1 pg. The detection could be completed within 1 h without using complicated instruments and the results were visible by human eye. Using this method,R. solanacearum was detected in the stem extracts of manually inoculated eggplant,tomato,peanut,sesame and Amaranthus lividus and potato tuber tissue fluid of potato,especially,it was appropriate for the detection of sesame,peanut,tomato,potato and sweet potato plants suspected of being in disease in the field,and the detection rate by LAMP was much higher than that by PCR method. The LAMP method is a fast,specific,sensitive,and easy-to-use for the field detection of R. solanacearum bacterial wilt,which is suitable for promotion at the grassroots level.

Study on the Identification of Potato Ploidy Using Flow Cytometry Based on Liquid Nitrogen Grinding Method

ZHENG Ying-zhuan, LÜ Yan, YANG Dong-xu, LI Guo-wei, WANG Hong-yang, LI Can-hui

2021, 37(1): 282-288. doi:10.13560/j.cnki.biotech.bull.1985.2020-0478

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The ploidy identification of potato chromosomes is an important part in the evaluation of potato germplasm resources. Flow cytometry can quickly and accurately determine the nuclear DNA content of cells,which is widely used to identify plant’s chromosome ploidy. A high-throughput flow cytometry system that is suitable for potato ploidy identification is established for providing evidences for potato breeding. Twenty pieces of potato cooperative 88,progeny of an induced parthenogenesis,were used as materials,a liquid nitrogen grinding method was used to prepare leaf cell nuclear suspension,which was then compared with the leaf cell nuclear suspension by traditional blade shredding method,and chromosome ploidy of the known tetraploid potato cooperative 88 and diploid potato IVP101 was determined. It was found that there was no significant difference between the ploidy identification results by the two methods,but the liquid nitrogen grinding method was simple and less time-consuming. The flow cytometry based on the liquid nitrogen grinding method quickly and accurately identified potato ploidy. In addition,fluorescent staining time（from 15 min to 12 h）of the nucleus suspension prepared by the liquid nitrogen grinding method did not affect the ploidy determination results,which is convenient for researchers to flexibly choose the staining time in actual operation.

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2021, 37(1): 289-292.

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