Table of Content

26 February 2020, Volume 36 Issue 2

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Advances in Successional Development and Early Establishment of the Chicken Intestinal Microbiota

WANG Jing, DAI Dong, WU Shu-geng, ZHANG Hai-jun, QI Guang-hai

2020, 36(2):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1197

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The gastrointestinal tract houses a large and diverse microbiota,and these microbiota play an important role in growth and health of chickens,contributing to feed digestion and nutrient absorption,development of the intestinal villus and crypt morphology,and improvement of the hosts’ immune systems. Understanding the spatial and temporal variability of gut microbiota and early bacterial colonization will help to develop new microbiota-based intervention strategies in poultry production. This review discusses the composition and successional development of chicken intestinal microbiota,and early microbiota establishment,and summarizes the latest progresses.

Research Advances on Non-starch Polysaccharide in the Regulation of Intestinal Microflora in Pigs

YANG Li-jie, ZENG Xiang-fang, QIAO Shi-yan

2020, 36(2):  9-16.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1224

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As the component of cell wall,fiber is the most widely distributed and abundant renewable resource on the earth. As the most important component of fiber structure besides lignin,non-starch polysaccharide（NSP）plays an important role in the functional regulation of fiber. The mechanism of fiber regulation in intestinal microflora has become a hot topic in the field of microbiology. However,as a complex mixture,the results of studying fiber are not consistent. The difference in fiber source,NSP content,and the incomplete assessment system are the reasons for the diverse results. In this paper,the research of NSP on the regulation of intestinal microflora in pigs in recent years was reviewed,and also the significance and problems of NSP in assessing the balance between dietary fiber and intestinal microflora were discussed,aiming to further expound the dialogue mechanism between nutrients and intestinal microflora and to provide reference for promoting the intestinal health of animals and the rational utilization of fiber.

Recent Advance in the Study of the Regulation of Early Life Gut Microbiota by Probiotics in Livestock

MA Tao, DIAO Qi-yu

2020, 36(2):  17-26.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0842

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Probiotics are defined as live microorganisms that confer a health benefit on the host（human/animals）,including bacteria,fungi（i.e.,yeasts）,etc. Probiotics demonstrate beneficial effects on the animal by improving the growth and immunity,and therefore have been proposed to be a promising alternative to antibiotics. It is suggested that the interaction between probiotics and gut microbiota results in the beneficial effect of probiotics,despite that the mechanisms remain largely unknown. This review summarizes the most recent advances in regulating microbiota in gastrointestinal tract of early age livestock（piglet,chicken,and ruminant）by probiotics based on high-throughput sequencing technology. The paper also highlights the future directions,including how probiotics interact with gut microbiota and affect their functions,how probiotics affect gut microbiota of livestock under different health conditions in early age,and how host factors affect the efficacy of probiotics in terms of regulating gut microbiota of livestock in early age.

Research Progress and Trends on Rumen Microbiota in Dairy Cows

WU Jia-jin, ZHU Sen-lin, ZHOU Mi, SUN Hui-zeng

2020, 36(2):  27-38.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1180

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There is an increasing interest in studying rumen microbiota in dairy cow nutritional trials in recent years,aiming to link its compositional changes to the feed composition,performance traits（i.e. feed efficiency,milk yield,milk fat,etc.）,health related traits（i.e. ruminal acidosis and subacute acidosis）as well as environmental traits（i.e. methane emission）. Additional researches have highlighted the role of microbiota in rumen development in multiple ruminant species. The majority of the findings on the rumen microbiota are based on the sequencing of the marker gene amplicon,which gives the picture of the taxonomic microbial composition and how it shifts under different treatments. Although the emerging metagenomic and metatranscriptomic analyses enabled in-depth exploring of the rumen microbial functions,it also generated more challenges in data analysis and interpretation. Up to date,most of the papers heavily rely on correlation and speculation analysis without clearly clarifying the mechanisms behind. In this paper,we reviewed the progress and limitation of dairy cow rumen microbiota,including the relationship between rumen microbiota and milk production efficiency,methane emission and rumen development,and we also discussed the research trends of rumen microbiota in dairy cows.

Research Progress on Interaction Between Rumen Microorganisms and Host and Its Dietary Regulation

WANG Bing, LUO Hai-ling

2020, 36(2):  39-48.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0747

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There is an interaction between rumen microorganisms and hosts. The genetic information of host animal affects rumen microorganisms,while the ruminal microorganism can be regulated by dietary ingredients,nutrient levels,and exogenous additives. In recent years,the analysis of the crosstalk between ruminal microorganism and host through multi-omics technology and the underlying mechanism has become the research hot topic. This review summarizes the research progress of the relationship between rumen microorganism and host and the regulation roles of dietary factors,more specifically,the relationship between ruminal microorganisms and host genome,the relationship between rumen microbes and animal performance,and the effects of diets formula,and addition of probiotics and plant secondary on rumen microorganism,and further prospects the development trend and application of ruminal microorganism research were developed.

Research Progress of Butyrate Regulating Rumen Epithelial Development in Young Ruminants

WANG Lu LIU, Li-xiang, SUN Da-ming, LIU Jun-hua

2020, 36(2):  49-57.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1234

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Rumen,a very important and specific organ in ruminants,is responsible for feed digestion,nutrient absorption and metabolism,thus its developmental state directly affects animal performance and health. The rumen of newborn calves and lambs has not been well developed,thus it is unable for them to fully digest and absorb solid feed. Therefore,the promotion in rumen development of ruminants through nutritional strategies is vital to maintain animal health and improve production performance. Butyrate,the main end product of rumen microbial fermentation of plant feed,is also the important energy source of rumen epithelia and host. The topic of butyrate regulating rumen epithelial development has lost none of its original charm. This paper mainly introduces the development of rumen epithelial morphology and function and the research progress of butyrate regulating rumen epithelial development in young ruminants.

Research Advances on Short-chain Fatty Acids of Metabolites of Gut Microbiota in Aquatic Animals

LIU Yu, DING Qian-wen, RAN Chao, YANG Ya-lin, WANG An-ran, ZHANG Hong-ling, ZHANG Jin-xiong, LI Jie, Rolf Erik OLSEN, Einar RINGØ, ZHANG Zhen, ZHOU Zhi-gang

2020, 36(2):  58-64.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0826

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Short-chain fatty acids（SCFAs）refer to organic fatty acids with less than 6 carbon atoms in the carbon chain. They are mainly produced by anaerobic glycolysis of indigestible carbohydrates in food,mainly including acetate,propionate,butyrate,etc. With the deep research of microbial flora in the digestive tract,microbial metabolites,short-chain fatty acids,have also been reported to have important physiological functions. This paper mainly reviews the role of SCFAs in the nutritional metabolism and immune disease resistance of aquatic animals. Meanwhile,this review outlooks the weakness of researching SCFAs in aquatic animals,aiming to promote the research process and actual application of SCFAs in aquatic animals.

Screening and Identification of Cellulase-producing Strains Isolated from Crayfish Intestine

FENG Guang-zhi, SHI Hui, LIU Bo, WU Yu-ting, WANG Yue-lin, SHI Yu

2020, 36(2):  65-70.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0789

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The intestinal microbes are the main driving force for crayfish to degrade cellulose and hemicellulose. In order to explore the probiotic resources and genetic resources of degrading cellulose in the crayfish intestine,the cellulase-producing bacteria were isolated from crayfish intestine by plate screening method,and the genome of the maximum active strain was analyzed by Illumina MiSeq high-throughput sequencing. Morphological and 16S rRNA gene molecular identification showed that strain 13 was Bacillus velezensis,and strain 33 was Bacillus subtilis. Strain 33 has multiple hydrolase activities and high cellulose-degrading ability. The high-throughput sequencing results indicated that the bacterial genome contained many carbohydrate-related enzyme genes. Crayfish intestines are rich in cellulose-degrading bacteria,and Bacillus bacteria accounts for a certain proportion.

The Relationship Between Gut Microflora and Sociality of Honeybee

WANG Hong-fang, XU Bao-hua

2020, 36(2):  71-76.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0876

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Honeybees are the typical eusocial insects living in social groups. Compared with solitary insects,they harbor special gut microflora. The relationship between bees’ unique gut microbes and their sociability is a fascinating scientific issue. Guts of honey bee workers are dominated by 9 distinctive bacterial phylotypes. The dominant species and abundances vary among various niche of digestive tract. There are few bacterial phylotypes and low abundance in foregut,but rich phylotypes and high abundances in hindgut,accounting for over 99% of microbes in whole gut. The microflora varies among the bees with different social division. Honeybee gut microbes can regulate their castes differentiation,social labor division,food intake behavior and lifespan by affecting insulin signals,the synthesis of juvenile hormone and vitellogenin,and the antioxidant stress ability. Besides,gut microbes in honeybee also play roles in activating immunity,inhibiting pathogenic bacteria,degrading food,promoting nutrient absorption,detoxifying,and fermenting honey and bee bread. The profile of honeybee gut microbes and the relationship between gut microbes and honeybee sociality are reviewed in this paper.

Research Progress on Intestinal Microbe and Sex Hormone Related Diseases

HUANG Hai-min, LAN Xiu-wan, WU Yao-sheng

2020, 36(2):  77-82.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0955

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Trillions of microbes inhabit the human intestine,forming a complex microecological community. It is clear that intestinal microorganisms provide nutrients and energy to the host and are involved in the occurrence and development of human diseases. With the deepening of relevant researches,growingly evidences indicate that changes in intestinal microorganisms cause variations in sex hormone levels,thus leading to a series of related diseases. In this review,we discuss the relationships between intestinal microorganisms and sex hormone-related diseases such as polycystic ovary syndrome,breast cancer and ovarian cancer,etc.,aiming to provide new ideas for the diagnosis and treatment of human diseases.

Role of Gut Microbiota in Stress-induced Hypertension in Rats

WU Qin, XU Zi-yang, LIU Li-ping, ZHANG Wen-ying, SONG Si-yuan

2020, 36(2):  83-90.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1215

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This work is to observe the variation of gut microbiota in stress-induced hypertension in rats and to investigate the role of gut microbiota in the pathogenesis of stress-induced hypertension in rats. The model of stress-induced hypertension rats was prepared by：Rats were subjected to plantar electrical stimulation and noise twice a day for 2 h at an interval of 4 h for consecutive 14 d. Measurement of blood pressure was conducted by：arterial catheterization in rats,and then blood pressure was recorded by Powerlab system. The model of pseudo germ-free rats was prepared by adding 4 antibiotics of 1 g/L ampicillin,500 mg/L vancomycin,1 g/L neomycin,1 g/L metronidazole file in their drinking water for 4 weeks. The 16S rRNA V3-V4 region sequence of microbe in rat feces was determined by Illumina MiSeq sequencing technique,and the community structure and diversity were compared and analyzed. Chronic stress increased blood pressure,heart rate and serum norepinephrine concentration in rats. α diversity analysis showed that the richness and diversity of gut microbiota decreased in stress rats,and β diversity analysis showed that the species composition of gut microbiota in stress group decreased and the difference became greater. In the pseudo germ-free rats,the gut microbiota species was simple with only a small number of Proteobacteria and the chronic stress did not increase blood pressure,heart rate as well as serum norepinephrine concentration. Stress may cause disorders of gut microbiota in rats and stress-induced hypertension may depend on gut microbiota.

Effects of Ethanol on Metabolites and Bacterial Community of Rice Straw Cocultured with Rumen Fluid in vitro

LIN Miao, WANG Kuo-peng, CHEN Ying-liang, SUN Wen-jing, FENG Li-mei, HU Zi-xuan

2020, 36(2):  91-99.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1101

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This study aims to study the effect of ethanol addition on rice straw anaerobic fermentation co-cultured with rumen fluid. In vitro fermentation technique by frequent passage and high-throughput sequencing technology were applied to analyze short chain fatty acid（SCFA）yield and bacterial community composition. The results showed that SCFA yield in the rice straw fermentation by in vitro 8^th transferring were significant higher than that by rumen fluid（P<0.01）. Compared with no adding ethanol,ethanol addition significantly increased the proportion of acetate,valerate,and caporate,while decreased propoinate and butyrate proportion（P<0.01）;the total SCFA yield,the proportion of isobutyrate and isovelarate varied insignificantly. The relative abundance of Bacteroidetes phylum decreased,while that of Firmicutes phylum increased by anaerobic fermentation of rice straw（P<0.05）,compared with that by rumen fluid. Furthermore,the relative abundances of Firmicutes and Actinobacteria phyla,Bifidobacterium,unidentified Lachnospiraceae,Succiniclasticum,Desulfovibrio and unidentified Clostridiales genera increased while ethanol was added（P<0.05）. Adding ethanol increased Biomarkers in the rice straw fermentation. The rice straw fermentation and rumen fluid presented close genetic relationship,and adding ethanol significantly changed the bacterial community structure. The proportion of SCFA played an important role in bacterial community of the rice straw fermentation. Our results indicate that in vitro frequent passage and adding ethanol increase the proportion of acetate,valerate,and caporate in rice straw fermentation as ethanol changes the bacterial community of rice straw fermentation.

Transcriptome Sequencing Analysis of Lycium ruthenicum Murr. Under NaCl Stress

MA Yan-jun, DUAN Hui-rong, WEI Jia, RICHARD John Tiika, SHAN Li-shan, MA Rui

2020, 36(2):  100-109.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0492

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This work aims to study the changes of gene expression profiles of Lycium ruthenicum Murr. under salt stress with different concentrations,and to provide a basis for further studying the molecular mechanism of salt resistance of L. ruthenicum. First,the roots and leaves of tissue culture seedlings of L ruthenicum stressed by NaCl at the concentrations of 0（CK）,50 and 250 mmol/L were sampled at stress duration of 0,1 and 12 h,respectively. Then,the sequence analysis was conducted using transcriptome sequencing technique（RNA-Seq）. The results showed that a total of 222.49 Gb raw data were generated by transcriptome sequencing,from which 86 037 Unigenes were obtained. The 46 594 Unigenes,accounting for 54.76% of the total Unigenes,were annotated in the 7 functional databases（GO,KEGG,KOG,NR,Pfam,Swiss-Prot and egg NOG）,while the rest 38 929 Unigenes were not annotated in the known genes databases. These Unigenes were classified into 51 GO categories and 211 metabolic pathways using GO classification and KEGG Pathway cluster analysis. Analysis of differentially expressed genes revealed that the up-regulated genes and down-regulated genes in the leaves and roots of L. ruthenicum increased with the NaCl concentration as well as treatment time increasing. The up-regulated genes（7 514）in leaves were less than down-regulated（9 032）genes,while the up-regulated genes（12 347）in roots were more than down-regulated genes（11 559）. Total 28 325 SSR（Simple Sequence Repeats）loci were discovered in L. ruthenicum under salt stress transcriptome,most of which were single nucleotide SSR and accounted for 70.47% of the total. Comprehensive analysis showed that the responses of L. ruthenicum to salt stress involved many genes and were regulated by several collaborative biological processes. The changes of gene expression level might be the major approach in gene regulation.

Identification and Enzymatic Properties of Strain YB Simultaneously Secreting Highly Efficient Cellulase and Xylanase

YANG Bin, LI Xiao-bo, ZHOU Lin, OU Pei-yu, JIN Xiao-bao

2020, 36(2):  110-118.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0497

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The objective of this work is to screen and identify fungi that can degrade lignocellulose and to study its enzyme production characteristics. The Congo red plate coating method was used to screen the fungi with ability of degrading lignocellulose. Then the ITS-rDNA sequence analysis was combined to identify the enzyme production conditions,and the DEAE Sepharose Fast Flow anion exchange and Chromatography and Sephadex G-100 gel chromatography were employed to separate and purify the crude enzyme solution precipitated by ammonium sulfate,and finally the enzymatic properties were studied. A strain YB degrading lignocellulose was screened and identified as Trichoderma virens. The maximum viability of cellulase and xylanase during fermentation was 313.53±26.78 U/mL and 18120.87±500.37 U/mL,respectively. The cellulase（CMC enzyme）Ib,IV and xylanase Ia were isolated and purified,and their molecular weights were 58.5 kD,22.8 kD and 44.5 kD,respectively,as determined by SDS-PAGE. The optimal enzymatic reaction conditions for the three enzymes were：50℃,pH5.0. Among them,xylanase effectively degraded corncob xylan into xylose and various xylooligosaccharides. In conclusion,the strain Trichoderma virens YB can secrete lignocellulose efficiently degrading enzyme and thus has potential for application in xylanase and xylooligosaccharide production.

Endophytic Fungi Diversity in the Needles of Pinus densiflora with Sphaeropsis sapinea

XIE Xian, LIANG Jun, ZHANG Ming, HU Rui-rui, CHENG Yuan, ZHANG Xing-yao

2020, 36(2):  119-125.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0836

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The objective of this work is to study the interaction between shoot blight of pine and endophytic fungi in needles at the microbial level and to analyze the community structure and diversity of endophytic fungi for providing basic data in the prevention of shoot blight of pine. High-throughput sequencing technology was used to determine the diversity and community structure of endophytic fungi in different level of blight of P. densiflora. Results demonstrated that the richness of endophytic fungi in the P. densiflora needles showed an upward trend,and the diversity index tended of decreasing first and then increasing. Ascomycota and Basidiomycota were phyla of the highest relative abundance in the asymptomatic needles,and the dominant species of endophytic fungi were Lapidomyces and Selenophoma. The disease caused the relative abundance of dominant species to decrease. In conclusion,the determination of fungi in the needles of P. densiflora clarified the diversity and community structure of endophytic fungi in the needles of P. densiflora with different levels of S. sapinea.

Bacterial Community Characteristics of Cultured Seawater with Microecological Preparations in Different Seasons

WANG Yong-yan, ZHAO Bing-he, LIANG Guang-yu, LI Yun, XU Yang-cang

2020, 36(2):  126-133.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0817

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Bacterium,as one of important factor to maintain aquaculture water stability,is often used to evaluate the quality of aquaculture water. Microecological preparations are widely applied in aquaculture water to remove harmful substances. This work aims to study the changes of bacterial community structure and bacteria diversity after adding microecological preparations into cultured seawater during different seasons,and to explore the effects of environmental factors of water quality on bacterial community. The high-throughput sequencing technology was used to amplify the bacterial 16S rRNA V3 and V4 regions,and the distribution and diversity of bacterial community in cultured seawater were analyzed after adding completely commercial microecological preparations in the winter,spring and summer. Redundancy analysis（RDA）was employed to analyze the relationship between water quality and bacterial communities in different seasons. The diversity was rich,the Proteobacteria and Bacteroides were main dominant bacteria shared in three seasons. Of them the Tenacibaculum（38.28%）,Zobellella（30.78%）and Halomonas（13.55%）were dominant in the winter,the Glycocaulis（34.00%）,Halomonas（11.36%）and Planktosalinus（9.67%）were dominant in the spring,and the Pseudofulvimonas（28.94%）,Zobellella（17.51%）,Planktosalinus（15.86%）,Marinobacter（12.30%）were dominant in the summer,the bacterial genera varied obviously in different seasons. RDA analysis showed that environmental factors at different stage affected bacterial community in various ways,and temperature,salinity,pH,and dissolved oxygen presented significant correlations with abundance of 10 dominant genera in 3 seasons;the dominant genera were positively correlated with salinity and dissolved oxygen in winter and spring,and positively correlated with temperature and pH in summer. In conclusion,microecological preparations demonstrate the great advantages in purifying aquaculture waters,each in whole may play each own regulation ability though the structure and diversity of bacterial community are changed in different seasons. This study provides new ideas for further treating cultured seawater and applying new microecological preparations.

Expression and Characterization of Aquatic Neutral Phytase Gene from Penicillium sp. C1 in Pichia pastoris

LI Ya-nan, YU Li-hong, CHEN Xin-mei, YANG Hao-meng, HUANG Huo-qing

2020, 36(2):  134-141.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0704

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A new phytase gene was cloned from the genome DNA of Penicillium sp. C1 strain by degenerate primers and TAIL-PCR,and named as phyC1. The whole length of structural gene was 1 477 bp,including a 58 bp intron at the 5&apos; terminus. The gene encoded 472 amino acids and a termination codon,and the first 23 amino acids were signal peptides. The gene encoding the mature protein of phyC1 was efficiently expressed in Pichia pastoris GS115,and then the recombinant enzyme PhyC1 was purified to electrophoretic purity by ammonium sulfate precipitation,anion exchange column and Sephedex chromatography. The enzymatic properties showed that the optimal pH of recombinant PhyC1 was 6.5,and high stability at pH 5.0-10.0. The optimal temperature of recombinant PhyC1 was 50℃. Compared with the commercial phytases,PhyC1 showed two significant properties,such as high activity under low temperature,especially below 20℃,and over 90% relative enzymatic activity remained under neutral pH 7.0. Therefore,PhyC1 has potential application prospect in feed industry,especially in aquatic industry.

Phenotypic Characteristics Combined with Genomic Analysis to Identify Different Colony Morphology Bacillus velezensis ACCC 19742

GUO He-bao, WANG Xing, HE Shan-wen, ZHANG Xiao-xia

2020, 36(2):  142-148.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0750

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During the period of regular transfer and preservation of bacteria in Agricultural Culture Collection of China（ACCC）,it was found that Bacillus amyloliquefaciens ACCC 19742 showed two different colonies on the same medium,named these two different colonies as 19742-1 and 19742-2. The morphology,physiology,biochemistry and genome analysis were used to identify ACCC 19742,and to further determine the taxonomic status of the strain. Firstly,the strains were isolated and purified. Secondly,16S rRNA and gyrB genes were amplified and sequenced. Phylogenetic tree was constructed by MEGA 7.0 software. Physiological and biochemical characteristics of the strains were analyzed by API 20NE,BIOLOG and fatty acid. The ANI and DDH values of the strains were analyzed by whole genome analysis. Only the gluconate assimilation reaction in 2 strains was different according to API 20NE detection,the main fatty acid components were consistent,only the percentage was slightly different. Based on the phylogenetic analysis,16S rRNA gene similarity of the two strains was 100%,while the gyrB gene similarity was 99.4%. The ANI value of the two strains was 99.95% and the DDH value was 99.62%. Combining with genetic and phenotypic characteristics,it was confirmed that the two strains were derived from different morphological variants of the same strain,not caused by pollution. Meanwhile,ANI and DDH values of 19742 and Bacillus velezensis NRRL_B 41580^T were the highest（97% and 77%,respectively）. Evolutionary analysis of 16S rRNA and gyrB also indicated that the strain belonged to the taxonomic status Bacillus velezensis,but not B. amyloliquefaciens. This provides a certain reference value for the preservation of the strain.

Research Progress on Regulation of miRNA in the Heat Stress Response of Plants

LI Ze-qing, LIU Cai-xian, XING Wen, WEN Ya-feng

2020, 36(2):  149-157.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0857

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microRNA（miRNA）is a non-coding RNA comprised of 20-24 nucleotide. It can degrade or inhibit translation of the target gene after transcription through sequence complementation,and regulates gene expression at the post-transcriptional level. miRNAs serve as important regulatory factors involved in plant growth,development and stress response,and it is widely distributed in plant genomes. At present,a number of studies have shown that high temperature not only induces changes in the expression of plant coding genes,but also significantly changes the expression of some non-coding RNAs including miRNAs,which act as an important non-coding RNA that participates in the heat stress response of plants. In this review,we summarize the synthetic pathway,effect mechanism and main functions of miRNA,with a focus on the regulatory mechanism of plant miRNA in heat stress response. It provides new insight for the researches and application of miRNA in resistance to high temperature stress in plants.

Research Advances in Salt and Alkali Tolerance Improvement Technology at the Seed Germination Stage

ZHU Jian-feng, YANG Xiu-yan, WU Hai-wen, ZHANG Hua-xin

2020, 36(2):  158-168.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0727

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Seed germination is the process of embryogrowth and development from relative static to physiologically active state,which is the most susceptible to the external environment,among which saline-alkali stress is one of the most serious ecological constraints. The saline and alkali land is widely distributed all over China,which is about 1×10⁸ ha and accounting for 10% of the total land area of China. Inhibition of seed germination under saline-alkali stress has become the primary obstacle to plant natural regeneration and agroforestry production in the saline and alkali land in China. At present,the known techniques to improve salt and alkali tolerance of plant at germination stage mainly include physical methods,chemical regulations,biological approaches and breeding techniques. Physical methods include seed priming and seed treating with low temperature,ultrasound,microwave,electric field and so on. Chemical regulation refers to regulation of inorganic and organic exogenous substances. Biological approaches include symbiosis of plant-promoting bacteria and fungi. Breeding techniques include salt tolerance identification,screening and genetically modified techniques. This paper systematically reviewed the research progress of methods and techniques for improving salt and alkali tolerance of plant seeds during germination period,then briefly described the characteristics and shortcomings of these technologies,and finally proposed the suggestions for further work. It is aimed to provide reference for solving the problem of seed germination hindrance in the saline-alkali land,achieving low-cost and large-scale bioremediation of the saline-alkali land and agroforestry production in saline-alkali area.

Research Advances on Functions of SUMO E3 Ligase in Plant Abiotic Stress Adaptation

WANG Yong-ping, REN Wei, WANG Run-juan, SHAO Kun-zhong, GAO Hui-juan, ZHANG Jin-lin

2020, 36(2):  169-177.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0744

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As one of the most important post-translational modifications,sumoylation is widely found in eukaryotes and plays essential roles in the regulation of plant growth,development and stress responses. SUMO E3 ligase has the function of recognizing and selecting substrate,thus directly promotes the binding of SUMO protein and target proteins. At present,a variety of SUMO E3 ligases have been identified in plants. Here,we reviewed the roles of SUMO E3 ligases in plant adaptation to abiotic stresses,including drought,high salinity,high/low temperature,nutrient deficiency and heavy metal toxicity and prospected the future research direction in this field,aiming at providing references for future research on sumoylation in plants.

Research Advance on the Application of Additives in Algae

YANG Shu-ping, ZHANG Lin, XU Ji-lin

2020, 36(2):  178-187.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0523

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Rich in various nutrients and active substances,algae have important economic value and application prospects. Nowadays,open runway pool is often used in algal cultivation,the cost of which is low,but easily affected by environmental conditions. Inappropriate environmental conditions（temperature,acidity,heavy metal,UV,salinity and light stress,etc.）could cause the increase of production cost and the decrease of production output and quality,thus this would severely hinder the development of algal farming and related industries. Additives can not only effectively promote the growth of algae and also alleviate the damage of adverse environmental conditions. In this paper,the application of additives in algae growth and stress resistance in recent years is summarized and the related mechanisms of various additives are sorted and clarified. The results showed that the application of additives in algae was mostly limited to the measurement of growth and physiological indexes,such as the cell density,the photosystem activity,the content of osmotic regulation substance and lipid content,the activity of catalase and nitrate reductase,etc. Only a few studies have extended the expressions of defense genes by molecular technology,trying to further explore the molecular mechanism of algae stress resistance. This paper is expected to provide theoretical basis for researchers to further clarify the signal transduction mechanism of common additives in algae and improve the yield and quality of algal products caused by abiotic stress,which is of great practical significance.

The Relationship Between Lung Microbiota and Respiratory Diseases

LI Zong-jie, DI Di, LI Bei-bei

2020, 36(2):  188-192.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0905

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The respiratory tract mucosal immune system has good defensive and protective effects against invaded pathogens and strong eliminating ability,therefore the lungs of healthy organisms are considered sterile for a long time. With the development of the culture-independent sequencing technology,studies on the composition structure and immune regulating functions of lung microbiota are much focused. Early life development and maturation of respiratory microbiota are shaped by birth mode,diet structure,living environment and antibiotic expose,and the formation and development of lung microbiota in early life might affect the occurrence and development of respiratory diseases throughout the life cycle. The interactions among lung microbiota,host immune system,intestinal microbiota and respiratory viruses can regulate the immune homeostasis and inflammatory infection of the lung. Therefore,early life intervention of lung microbiota communities can be explored as an effective strategy and novel target to prevent and control respiratory diseases.

Application of RNA Interference Technology in Antiviral and Antiparasitic Research of Aquatic Animals

SONG Hua-li, SUN Xiao-ying, KONG Xiang-hui, LI Li, PEI Chao

2020, 36(2):  193-205.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0908

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RNA interference（RNAi）is a post-transcriptional gene silencing mechanism mediated by double-stranded RNA which inhibits the expression of target genes,and is widely found in eukaryotes. With the development of aquaculture in recent years,diseases of aquatic animals often break out,bringing huge economic losses to farmers. Currently,the mechanism of aquatic animal diseases caused by viruses and parasites still needs to be further studied. The emergence of RNA interference technology provides a powerful tool for studying the pathogenic mechanism of aquatic animal diseases. In this paper,the discovery,mechanism and application of RNA interference in the antiviral and antiparasitic research of aquatic animals are reviewed,and the future research and application of RNAi technology in the prevention and treatment of aquatic animal disease are prospected,providing reference for the control of diseases in aquatic animals.

Construction of Single-walled Carbon Nanotube-loaded Koi Herpes Virus ORF149 Nucleic Acid Vaccine

HU Feng, WANG Qing, LI Ying-ying, ZENG Wei-wei, WANG Gao-xue, ZHU Bin, WANG Ying-ying, YIN Ji-yuan

2020, 36(2):  206-213.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0688

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In order to develop a new type of highly effective vaccine against Koi Herpesvirus（KHV）disease,a single-walled carbon nanotube（SWCNT）was used as a transport vector to construct a KHV ORF149 nucleic acid vaccine. The recombinant plasmid pcDNA3.1（+）-ORF149 was firstly constructed,and its expression was confirmed by transient transfection and immunoblot analysis. Then,the recombinant plasmid was conjugated to SWCNTs by 1,3-dipolar cycloaddition reaction. Scanning electron microscopy and nucleic acid electrophoresis was conducted to identify whether the conjugation was successful. The results showed that specific signals were detected with constructed recombinant plasmid in both immunoblot analysis and indirect immunofluorescence assay after transfection. In the nucleic acid agarose gel electrophoresis,the electrophoretic band of the constructed recombinant vaccine disappeared. Under the observation of scanning electron microscopy,the morphologies of the conjugated SWCNTs with the recombinant plasmid and blank SWCNTs were significantly different.

Improvement of SLiCE Seamless Cloning Method Based on Annealing Buffer

YANG Dong-cheng, CAI Song, ZHAO Xiao, WANG Jin-hua, WANG Yong-ze

2020, 36(2):  214-222.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0468

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The objective of this work is to establish a method to fulfill the Seamless Ligation Cloning Extract（SLiCE）under the condition of low efficiency competent cells. The step of denaturation and renaturation was introduced into SLiCE reaction to improve the efficiency of recombination. Results showed that denaturation and renaturation of DNA fragments using annealing buffer after SLiCE reaction resulted in about 7 times more clones than the control,and the recombination efficiency of SLiCE achieved as high as 100% by using colony PCR verification. Under this condition,the recombinant clones were obtained by transforming the SLiCE products into competent cells with 1.7×10⁶ CFU/μg transformation efficiency,and the recombination efficiency was still up to 100%. In sum,positive clones could be obtained using competent cells prepared by the routine CaCl₂ method if above optimized SLiCE method with denaturation and renaturation steps were performed. This indicates that the seamless cloning efficiency and practicability of SLiCE may be improved via the optimized method.