Current Biotechnology

Progress on RNAi-based Transgenic Plants
HUANG Chunmeng1,2, ZHU Pengyu1, WANG Zhi1, WANG Chenguang1, DU Zhixin3, WEI Shuang4, ZHANG Yongjiang1, FU Wei1*
2020, 10(1): 1-9. DOI: 10.19586/j.2095-2341.2019.0106
Abstract ( ) PDF (1690KB)( )

RNA interference (RNAi) refers to the phenomenon of high-efficiency and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is ubiquitous and evolutionarily conserved in eukaryotes. RNAi technology, as a major scientific achievement at the beginning of the 21st century, is currently widely used in disease prevention, gene function research, plant improvement and breeding and other fields. RNAi technology is often combined with transgenic technology for plant improvement and breeding, and agricultural biotechnology products satisfying production needs are created through different vector designs or action pathways. In order to clarify the progress on breeding technology of transgenic plants based on RNAi technology, the research progress of the discovery and mechanism of RNAi phenomenon, the design of transgenic vectors and the delivery methods of small RNA (sRNA) were reviewed. And the research examples and commercialization of transgenic plants based on RNAi technology were also elaborated, with a view to providing reference for related research, thus exerting the maximum application value of RNAi technology and serving the agricultural development in the new era.

Regulatory Status and Challenges of RNAi-based Transgenic Plants in Breeding
HUANG Chunmeng1,2, ZHU Pengyu1, WANG Zhi1, WANG Chenguang1, DU Zhixin3, WEI Shuang4, ZHANG Yongjiang1, FU Wei1*
2020, 10(1): 10-14. DOI: 10.19586/j.2095-2341.2019.0126
Abstract ( ) PDF (1009KB)( )

RNA interference (RNAi) plays a vital role in the growth, development, anti-virus and transposon inactivation of plant, animal and fungi. At present, the RNAi technology has been successfully used to cultivate biotechnological products with excellent characteristics such as disease and insect resistance and quality improvement, which has provided strong support for the green development of agriculture. However, the relevant mechanism of RNAi has not been fully clarified yet, which makes transgenic plants based on RNAi technology face some problems that need to be solved urgently. At the same time, the safe and efficient supervision of the application of RNAi-based transgenic plants also needs to be further improved. Based on this situation, the current regulatory situation of RNAi-based transgenic plants and their breeding challenges were reviewed, and some suggestions for solving the existing problems were put forward, in order to provide new ideas for further application of RNAi technology in agricultural plant improvement and breeding, and provide basis for their regulatory evaluation.

Bio-enzyme Basis of Hydrogen in Biological System
MA Xuemei1, ZHANG Xin1, XIE Fei1, ZHAO Pengxiang1, ZHANG Zhao1, YI Yang1, ZHANG Xiaokang1, MA Shengnan1, LI Qinjian1, LYU Baobei1, LIU Mengyu, YAO Mawulikplimi Adzavon1, SUN Xuejun2, LI Yingxian3
2020, 10(1): 15-22. DOI: 10.19586/j.2095-2341.2020.0002
Abstract ( ) PDF (2985KB)( )

Hydrogen have diverse biological functions. However, the mechanism of hydrogen function has always been debated. It is widely accepted that hydrogen selectively reduces cytotoxic oxygen radicals. However, the evidence of direct reaction between hydrogen and free radicals under physiological conditions is not sufficient, most of which are indirect evidences. It is impossible to distinguish whether hydrogen reacts directly with free radicals or affects the production of free radicals. Hydrogen has antioxidant effect, but our research showed that it was not to eliminate the free radicals after they were generated, but to reduce the generation of free radicals. It was similar to turn off the "switch" at the beginning of the generation of free radicals. Hydrogen could improve the activity of biological enzymes including mitochondrial complex Ⅰ, acetylcholinesterase and HRP, and affected mitochondrial membrane potential and regulate the membrane potential of nerve cells. Some of the oxidoreductases and ion channels of cell membrane are regulated by hydrogen. Taken together, hydrogen has multi-targets with bio-enzyme basis and higher organisms have hydrogenase ativity to metabolize hydrogen. This paper mainly discussed the relationship between hydrogen and free radicals, and the enzyme basis of hydrogen is important for its biological function, which was expected to provide reference for research on the mechanism of how hydrogen function.

Research Progress on Neuroinflammation-related Cells in Traumatic Brain Injury
ZHAO Qinghui, LUO Qiuli, JU Fangdi, HE Jin, YAO Tingting, MA Shengnan, XUN Zhiming, ZHAO Pengxiang, XIE Fei^*
2020, 10(1): 23-29. DOI: 10.19586/j.2095-2341.2019.0078
Abstract ( ) PDF (1061KB)( )

Traumatic brain injury (TBI), also known as craniocerebral injury or head injury, refers specifically to brain tissue damage caused by trauma. However, from mild to severe TBI, there are few treatments to improve the prognosis of TBI patients. Neuritis can cause acute secondary injury after traumatic brain injury and related to chronic neurodegenerative diseases. Therefore, it is particularly important to understand the cellular system involved in the neuroinflammatory response after TBI. This paper mainly focused on the cells involved in the inflammatory response in TBI (such as microglia, astrocytes, oligodendrocytes, neutrophile granulocyte, lymphocyte, etc.). This article reviewed the recent advances in the initiation and interaction of granulocytes and lymphocytes, which was excepted to provide a new strategy for clinical research.

Fungal Laccases and Their Mediator Systems: Sources, Mechanisms and Applications
XU Xin¹, ZHANG Guoqing^1,2*, HU Boyang¹, LIU Zilu¹, SUN Yue¹
2020, 10(1): 30-39. DOI: 10.19586/j.2095-2341.2019.0055
Abstract ( ) PDF (3112KB)( )

Laccases are multicopper oxidases which are widely distributed in plants, fungi, bacteria and insects. They can catalyze aromatic and non-aromatic compounds with final reduction of molecular oxygen to water as by-product. Some small molecular mediators can enhance the substrate range, efficiency and stability of laccases. Laccase mediator systems (LMS), constituted by laccases and their mediators, can more effectively catalyze non-phenolic, polycyclic aromatic hydrocarbons, and other refractory compounds. They have become one of the research hotspots in recent years, and manifest great application prospects in the fields of pulping and bleaching, dye decolorization, and environmental detoxification, etc. This paper reviewed the recent research advancement on sources and functions of laccases, structure and mechanisms of fungal laccases, mediators and their mechanisms, and applications of LMS, which was expected to provide reference for application research of laccases.

Progress on Nitrogen Removal Related Enzymes and Their Coding Genes in Heterotrophic Nitrifying and Aerobic Denitrifying Bacteria
ZHANG Mingxia, LI Anzhang, CHEN Meng, XU Shuaishuai, ZHU Honghui^*
2020, 10(1): 40-45. DOI: 10.19586/j.2095-2341.2019.0092
Abstract ( ) PDF (1393KB)( )

The nitrogen (N) pollution in water has become a serious problem for environment in China, and great effort has been taken to look for an efficient and economic method for N removal. In recent years, researchers have isolated many heterotrophic nitrifying and aerobic denitrifying bacteria from different environments. These functional bacteria showed tremendous potential for application by the reason of their rapid growth, simultaneously nitrifying and denitrifying under aerobic conditions and other organic pollutant removal. At present, the N removal pathway and mechanism of heterotrophic nitrification-aerobic denitrifying bacteria are mainly studied by assaying the intermediate or end product of N cycle, detecting the activity of related enzymes, annotating functional genes in N cycle and referring to the N cycle pathway of autotrophic nitrifying bacteria and anoxic denitrifying bacteria. The complete N conversion pathway and N metabolism mechanism should be further studied and determined. The progress on the related enzymes and their coding genes involved in N removal pathways of heterotrophic nitrification-aerobic denitrifying bacteria were summarized in order to provide reference for theoretical research of heterotrophic nitrifying-aerobic denitrifying bacteria and their application in wastewater denitrogenation.

Progress on Oligosaccharide Oxidase
TONG Lige, LUO Huiying, MENG Kun^*
2020, 10(1): 46-52. DOI: 10.19586/j.2095-2341.2019.0091
Abstract ( ) PDF (3091KB)( )

Oligosaccharide oxidase is a new type of oxidases, which belongs to the auxiliary activity family 7 (AA7). It can catalyze various oligosaccharides into corresponding aldehyde acids and generate hydrogen peroxide in the reaction process. According to the substrates, the reported oligosaccharide oxidases are classed into gluco-oligosaccharide oxidase (GOOX), xylo-oligosaccharide oxidase (XOOX) and chito-oligosaccharide oxidase (COOX), etc. Oligosaccharide oxidase has a wide range of applications, and can be used in multiple fields such as food, medicine, feed, bio-fuel, and so on. However, few studies were carried out on oligosaccharide oxidase to present at home and abroad. Based on this situation, several aspects including enzymatic property, molecular structure, catalytic mechanism, molecular improvement and industrial application about oligosaccharide oxidase were reviewed in order to provide some insights into the research and application of oligosaccharide oxidase.

Research Progress on Butyric Acid Production by Biotechnology
WANG Jieming1, SHI Yali1,2*, LIU Anli1, LIU Zhanying1,2, LAN Hui1, HAO Jianxia1, YAO Zhipeng1, ZHANG Qingcui1
2020, 10(1): 53-59. DOI: 10.19586/j.2095-2341.2019.0058
Abstract ( ) PDF (1034KB)( )

Butyric acid is an important industrial chemicals, which has been used widely in food additives and pharmaceutical industry. Nowadays, butyric acid production in industry scale is dominated by chemical synthesis with starting materials derived from crude oil. Compared with organic chemical synthesis, the advantage of microbial fermentation include wide material sources, low energy consumption in fermentation, environmentally friendly, sustainable production with sustainable adding material. The production of butyric acid through microbial fermentation has generated a favorable business climate. Five issues of butyric acid production with bacteria through fermentation were presented in this review, including the characters of butyric acid, the characters of the butyrate-producing strains, the metabolic pathway and regulation of the butyrate-producing strains, the process of producing butyric acid by fermentation, the utilization of butyrate-producing bacterias and their products. These were good indicatiors for the genetic-engineering of the butyrate-producing bacterias and the optimization of the production process.

Application of Duplex Droplet Digital PCR for Detection of Genetically Modified Soybean
LIU Xiao1, ZHU Pengyu2, JING Xiaoyan2, LI Zhihong1, ZHANG Yongjiang2, LI Xiang3, FU Wei1,2*
2020, 10(1): 60-66. DOI: 10.19586/j.2095-2341.2019.0077
Abstract ( ) PDF (4834KB)( )

In this study, a duplex PCR detection method for three genetically modified soybean of MON87705, MON87769 and DP356043 was established by droplet digital PCR (ddPCR) platform. Taking advantage of specificity and quantitative range of duplex digital PCR, the primer probe combination and experimental procedure was optimized, exogenous genes and reference gene were detected. The experiment result showed that the primer probe combination used in the experiment only produced fluorescent signal for the target soybean line in the digital PCR method. It can be used in the detection of genetically modified soybean for its specificity. The genetically modified ingredients content was basically the same as reference parameters. The quantitative detection limit was 0.5%, and the qualitative detection limit was 0.05%, which could meet the needs of the detection of low-purity sample. The study also determined the gene copy number of transgenic soybean, which indicated that the dual digital PCR system established in this study can accurately and stably meet the actual detection needs, and has a good development prospect in practical application.

Study on Preservation of Armillaria luteo-virens from Tibet Plateau
2020, 10(1): 67-73. DOI: 10.19586/j.2095-2341.2019.0052
Abstract ( ) PDF (6161KB)( )

In order to prolong the preservation period of Armillaria luteo-virens, acidic electrolyzed water (AEW), short-wave ultraviolet (UVC) and ozone (O₃) were used to carry out single and compound preservation treatment on the harvested Armillaria luteo-virens. The changes of appearance quality of Armillaria luteo-virens after being packaged in preservation bags and refrigerated at low temperature were observed, and the browning degree, hardness, weight loss rate and the activities of polyphenoloxidase (PPO), phenylalanine ammonia-lyase (PAL) were also detected. The results showed that both UVC and O₃ treatments could effectively prolong the preservation period of Armillaria luteo-virens, and cold storage at 4 ℃ could prolong the preservation period of Armillaria luteo-virens from 10 d to 42 d. However, AEW was not suitable for preservation of Armillaria luteo-virens with less water content, due to the introduction of external moisture accelerating its decay. The research results have theoretical guiding significance for the preservation of Armillaria luteo-virens.

The Expression Level of Protein and mRNA of Gene Piwil4, Mael, Ddx4 and its Relation with Hepatoma Detection in the Rat Hepatoma Model
GULIMIRE Yimit, ISKANDER Bakili, WANG Yanjiao^*
2020, 10(1): 74-79. DOI: 10.19586/j.2095-2341.2019.0096
Abstract ( ) PDF (4349KB)( )

The expression level of PIWI and piRNA closed related to cancer tape. The expression levels of Piwil2 genes in the liver tissues is significantly higher than paracancerous tissue in liver tissue. PIWI/piRNA pathway genes including the Piwil4, Mael and Ddx4 are candidate genes, expressed in many kinds of cancer tissues. But the study on PIWI/piRNA pathway genes in hepatoma tissues has not been reported. This study was aimed to explore the roles and possible mechanisms of Piwil4, Mael and Ddx4 genes in the occurrence and progress of hepatocellular carcinoma. We established rat hepatoma model and the serum was extracted to detect the tumor markers by ELISA. The transcription and protein expression levels of Piwil4, Mael and Ddx4 genes in the liver tissues of the control and model groups were detected by qRT-PCR, Western-blot and immunohistochemistry. The results showed that the content of tumor markers in serum of rats with hepatoma increased significantly. And the results indicated that the PIWI/piRNA pathway gene including the Piwil4, Mael and Ddx4 were active in rat hepatoma model. Research showed that surveillance of PIWI expression in rat hepatoma model may be a prognostic markers for detection and treatment of hepatoma.

Effect of gacS Gene Deletion in Gac System on Toxicity of Pseudomonas donghuensis HYS
YOU Jia, GUI Zhe, XIE Zhixiong^*
2020, 10(1): 80-89. DOI: 10.19586/j.2095-2341.2019.0053
Abstract ( ) PDF (5969KB)( )

Bacterial pathogenic factors are an important part of bacterial toxicity. The Gac system is a global regulatory system of bacteria that regulates the production of various virulence factors in bacteria. In previous studies, a gram-negative bacterium, siderophore high-yielding Pseudomonas donghuensis (HYS), was isolated from the waters of East Lake in Wuhan for the first time. In order to explore the effect of Gac system on HYS toxicity, firstly, the position of GacS protein from Gac system in HYS was located through amino acid sequence BlastP alignment. Through bioinformatics analysis, Gac system was determined to exist in HYS. Then gacS fragments derived from P. donghuensis HYS and P. aeruginosa PAO1 were respectively supplemented into gacS gene knockout HYS strain ΔgacS through gene cloning, and the obtained strains were fed to Caenorhabditis elegans, and the toxicity of the strains were estimate by comparing the survival number and days of the nematodes. The results showed that the toxicity of ΔgacS strain was obviously weakened. However, the toxicity of the two complementary strains to nematodes recovered to different degrees, which indicated that Gac system in HYS participated in toxicity regulation pathway, and gacS fragment of P. aeruginosa PAO1 could also play a certain toxicity regulation role in HYS. In order to further explore whether the attenuated characteristics of ΔgacS strain were related to the loss of virulence factors in HYS, the activity of extracellular protease of the strain was detected by defatted milk plate, and the exercise capacity of the strain was detected by swimming motility plate, swarming motility plate and twitching motility plate. Meanwhile, metabolites such as lipopolysaccharide and hydrocyanic acid were also detected. The results showed that the protease secretion of ΔgacS strain completely disappeared compared with HYS, and the three kinds of exercise abilities of ΔgacS were significantly decreased compared with HYS (P<0.05). In addition, the yield of extracellular lipopolysaccharide of ΔgacS decreased to different degrees compared with that of wild type HYS. However, the phenotypes of these virulence factors could all be recovered to the wild type level in the complementary strains ΔgacS/pBBR2-gacS_HYS, and the complementation of homologous gacS gene from PAO1 could even complement the function of gacS in regulating toxic substances to a certain extent. Studies have shown that the potential animal pathogenic bacteria, P. donghuesis HYS, was positively regulating and producing toxic factors through Gac system, thus exerting lethal effect on Caenorhabditis elegans, which provided theoretical basis for organisms to resist HYS infection in the future.

Construction of TIG-Fc Eukaryotic Expression Vector and its Effect on the Function of Natural Killer Cells
LI Guohua¹, GAO Jinwei², ZHOU Xisheng³, WANG Xia¹, CHENG Weihua^4*
2020, 10(1): 90-96. DOI: 10.19586/j.2095-2341.2019.0074
Abstract ( ) PDF (2778KB)( )

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is a novel immunosuppressive receptor, which plays an important role in the immune regulatory network of the body. In order to further explore the immunomodulatory function of TIGIT in natural killer (NK) cells, a eukaryotic expression vector for fusion protein composed by TIGIT extracellular domain (TIGIT amino acids 1~135, to retain the functional domain IgV region, this extracellular segment is simply called TIG) and Fc segment of human immunoglobulin G3 (IgG3) was constructed, and the expression of TIG-Fc fusion protein and its effect on NK-92 cell function were preliminarily studied. Using molecular biology method, the gene carrying human TIG and Fc segment of IgG3 were fused, and then inserted into eukaryotic expression vector pcDNA3.1(-) to construct recombinant plasmid pcDNA3.1(-)-TIG-Fc. Subsequently, the recombinant plasmid was transfected into human embryonic kidney (HEK) 293T cells, and the expression of TIG-Fc fusion protein in 293T cells was detected by flow cytometry and Western blot. Then the recombinant plasmid was transfected into NK-92 cells, and the effect of TIG-Fc fusion protein on NK-92 cell proliferation was detected by WST-1, and the effect of TIG-Fc fusion protein on IFN-γ secretion level of NK-92 cells was detected by ELISA. The results showed that the eukaryotic expression vector for human TIG and human IgG3 Fc fusion expression was successfully constructed, and the expression of TIG-Fc fusion protein could significantly inhibit the proliferation of NK-92 cells and their secretion level of IFN-γ (P<0.05), which laid an important foundation for the functional research of TIGIT in the later stage.

Analysis of Lilium Plants Research Trend Based on Bibliometrics
LANG Lixin, DOU Xiaoying, WANG Huan, KONG Ying, BAI Jinrong^*
2020, 10(1): 96-107. DOI: 10.19586/j.2095-2341.2019.0003
Abstract ( ) PDF (4327KB)( )

CiteSpace software was used to analyze articles of the Lilium plants published from 1998 to 2018 in the core database of Web of Science (WoS for short), and conducted bibliometrics statistics and analysis from the perspectives of countries, institutions, authors, journals, and cited articles, so as to understand its research overview and cutting-edge dynamics. The results showed that from 1998 to 2018, a total of 1 058 articles on Lilium plants were published. After 2006, the number of articles published in China ranked the first and fluctuated. Chinese Academy of Sciences ranked the first among the research institutions. Top 2 core authors were from Wageningen University in the Netherlands. The journal with greatest influential and the most number of published articles was Scientia Horticulturae. 80% of research subjects in Lilium belonged to basic science and technical science in Top 10 subject areas. The research focused on the morphology, direction of cell and molecular biology. In recent 5 years, the research contented that highly cited article was related to the verification of the function of active substances in Lilium, indicating that the active ingredients and their efficacy in medicine and health has been the new trend of research.

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