Table of Content

• 
• Current Biotechnology
  Volume 10 Issue 2 25 March 2020
• Research Progress of miRNA Involved in Regulation of Plant Embryo and Endosperm Development
• XING Lijuan1，LIU Yueping2*，WANG Lei1，XU Miaoyun1*
• 2020, 10(2):  109-116.  DOI: 10.19586/j.2095-2341.2019.0120
• Abstract ( ) PDF (1204KB)( )
Kernel trait is important to yield, it is the final embodiment of grain yield and one of the most complicated traits in breeding. Kernel is mainly composed of embryo and endosperm. Endosperm is the organ where nutrients are accumulated and stored, which mainly provides nutrition for embryo germination and growth. The development, proliferation and enrichment of endosperm cells determine the weight and quality of kernel. Kernel development is a very complicated biological process, involving the temporal and spatial expression of many genes and the regulation of transcription level and post-transcription level. MicroRNAs (miRNAs) are a class of endogenous non-coding small RNA (21～24 nt), which can regulate gene expression in plants at post-transcriptional level through targeted degradation and translation inhibition. miRNA and its target genes form a precise regulatory network to participate in seed development. Based on this, the generation and mechanism of plant miRNAs was summarized, and the research progress of miRNAs regulatory function in plant embryo and endosperm development was also summarized, so as to provide better research direction for further identification of miRNAs related to maize kernel development and analysis of their regulatory function.
• Progress on Photobioreactors for Microalgae Cultivation
• LI Lei, ZHANG Hongbing, LI Wentao, LI Huixuan
• 2020, 10(2):  117-123.  DOI: 10.19586/j.2095-2341.2019.0115
• Abstract ( ) PDF (1748KB)( )
Microalgae, with ability to fix CO2 and purify organic wastewater, has attracted much attention in the fields of environmental protection, food and feed (bait), medicine and bio-energy development, etc. However, large-scale cultivation and its industrialization are still the difficulties to be solved urgently. In this paper, the characteristics and structures of photobioreactors, which are often used for large-scale cultivation of microalgae, were reviewed. The closed microalgae photobioreactor can preferably regulate the cultivation conditions of algae species, and is not easy to be polluted. The purity of algae species is easy to be controlled, but the cultivation scale is small and the production cost is high. However, open-type microalgae photobioreactor cannot control the growth environment of algae species accurately, but it is widely used because of its large production scale, high yield and low production cost. The best method is to integrated the two advantages, first, use the closed decay algae bioreactors for pilot light amplifier, a large number of reproductive effects, and then put into open decay algal light bioreactors for large-scale commercial production. The method is hopeful to be micro algae light bioreactors development direction. This paper was expected to provide reference for micro algae large-scale cultivation.
• The Application of System Biology in the Study of Cognitive Dysfunction
• XU Xiru1, ZHANG Biao1, GU Xiaoqun2
• 2020, 10(2):  124-129.  DOI: 10.19586/j.2095-2341.2020.0005
• Abstract ( ) PDF (1082KB)( )
Cognitive dysfunction can lead to a significant decline in daily life ability and social adaptability of patients, and seriously affect the life quality of patients. According to numerous studies in recent years, the occurrence and development of cognitive dysfunction is related to the disorders of genes, proteins, microorganisms and other internal environmental factors. In this review, based on the research of system biology, we reviewed the studies of cognitive dysfunction from the level of metabonomics, proteomics, genomics and intestinal microbiology conducted by domestic and foreign researchers in recent years, and looked forward to the application of system biology in the investigation of cognitive dysfunction, which was expected to provide reference for related research of cognitive dysfunction.
• Progress in Application of Laser Capture Microdissection in Tumor Multi-omics Study
• CHANG Wenjing, ZHAO Yanyan*
• 2020, 10(2):  130-136.  DOI: 10.19586/j.2095-2341.2019.0101
• Abstract ( ) PDF (1120KB)( )
In today’s society, tumor has become an important disease threatening human health due to its high morbidity and mortality. The research and exploration of its pathogenesis and treatment methods are also deepening. With the development of single-cell multi-omics sequencing technology, the cell heterogeneity of tumor tissues has gradually been recognized by researchers. In order to solve this problem, laser capture microdissection (LCM) technology came into being. LCM technology is a sample collection technology that accurately obtains a specific cell population or individual cells from organs or tissues under the direct visualization of a microscope. LCM technology combined with a variety of molecular biological methods can carry out multi-omics studies on heterogeneous tissues, enriching the existing tumor proteomics, genomics and transcriptomic maps. Therefore, LCM technology has become a powerful tool for studying specific expression and molecular mechanisms, and has been widely used in the field of oncology. Based on this situation, the principle, advantages of LCM and its application in tumor multi-omics study were reviewed, and its possible development direction in the future was prospected, so as to provide new ideas for tumor research and treatment.
• Research Progress on PCR Enhancer in Nucleic Acid in vitro Amplification Detection Technology
• XU Lei1，XIAO Guiqing2，SHENG Xiaojing1，QI Zhiqing1*，DIAO Yong1*
• 2020, 10(2):  137-143.  DOI: 10.19586/j.2095-2341.2019.0105
• Abstract ( ) PDF (1130KB)( )
Under the background of the widespread prevalence of diseases caused by various highly pathogenic pathogens, avian influenza viruses, food-borne microorganisms, etc., it is particularly important to use PCR technology for rapid laboratory diagnosis of the first or first wave of cases. At the same time, a variety of PCR-based detection technologies have been developed to diagnose, prevent or predict diseases more quickly, with high throughput and sensitivity. However, in the detection of actual pathogens, the results of low sensitivity and poor accuracy often occur, so the PCR enhancer was added in the PCR amplification system. The PCR enhancer could improves the performance of PCR in terms of yield, specificity, sensitivity, etc. so as to optimize nucleic acid detection, solve the application bottleneck of pathogen detection, and save valuable time for the first pathogen detection. This review introduced the application and mechanism of PCR enhancer in combination with PCR-based nucleic acid in vitro amplification detection technology, in order to provide some reference for the practical application of nucleic acid detection.
• Progress in Application of Toll-like Receptor 3 Agonist in Vaccine Adjuvant
• CAO Xiaoya1, XU Fuzhou1, GUO Jie1, WEN Tong2, SU Xia1, ZHOU Hongzhuan1, YANG Bing1, CUI Yifang1, GUO Fangfang 1*
• 2020, 10(2):  144-151.  DOI: 10.19586/j.2095-2341.2019.0116
• Abstract ( ) PDF (2818KB)( )
Pattern recognition receptors (PRRs) play key roles in the innate immune system and are important host sensors that can recognize pathogen-associated molecular patterns (PAMPs) displayed by invading pathogens. PAMP is an independent immunomodulator with a variety of biochemical components and special structures, and it is gradually considered as the key component of many modern vaccines. Toll-like receptor (TLR) 3 agonist is a synthetic double-stranded RNA (dsRNA) that activates various pathways of host immune defense in a pattern like viral infection. When properly combined with an antigen, TLR3 agonist can be used as a PAMP-adjuvant to regulate and optimize antigen-specific immune response. Based on this, the mechanisms of action of TLR3 and its agonist were mainly discussed, as well as the application progress of TLR3 agonist in vaccine adjuvant, so as to provide new ideas for the research of animal vaccines.
• Analysis of Intestinal Contents Flora of Hairtail Fish (Trichiurus spp.) from the East China Sea Based on High-throughput Sequencing Technology
• LIU Ming, LIU Hui, LI Na, CHEN Xiao, HU Guangchun, LIU Lanzheng*
• 2020, 10(2):  152-157.  DOI: 10.19586/j.2095-2341.2019.0130
• Abstract ( ) PDF (2512KB)( )
Hairtail fish (Trichiurus spp.) is an important economic fish in the East China Sea. In order to identify its own pathogenic microorganisms, we analyzed the intestinal contents flora of hairtail fish. Intestinal contents flora DNA extraction of hairtail fish captured in the East China Sea were collected, Ion S5TM XL high-throughput sequencing platform and bioinformatics analysis were used to construct the 16S rRNA v3-v4 gene sequencing library of 10 samples. 1 221 OTUs (operational taxonomic units) were obtained from 10 samples, which were classified into 28 phylum and 345 genera. At the phylum level, the abundance of Tenericutes and Proteobacteria is relatively high, which are 51.30% and 29.58% respectively. At the genus level, Mycoplasma and Vibrio account for a relatively high percentage, with relative abundance of 50.95% and 13.79%, respectively. The results showed that Mycoplasma in the intestinal contents flora of hairtail fish is relatively high, and there is a possibility of cross-contamination in the food processing. Therefore, it is necessary to be vigilant in the relevant production process, and certain measures should be taken to control Mycoplasma contamination. The results was excepted to provide reference for the study on the discovery, prevention and control of the new pathogen.Trichiurus spp.; intestinal contents flora; 16S rRNA sequencing; Mycoplasma
• Establishment of Detection Method of Hydrogen Concentration in Aqueous Phase
• ZHANG Zhao, WEI Yanan, YI Yang, MA Shengnan, ZHANG Xiaokang, MA Xuemei, ZHANG Xin*
• 2020, 10(2):  158-163.  DOI: 10.19586/j.2095-2341.2019.0119
• Abstract ( ) PDF (1154KB)( )
In recent years, studies have shown that hydrogen molecules have a wide range of biological effects, and drinking hydrogen-rich water (HRW) is the main intake method. But at present, there is few research on the detection method of hydrogen concentration in aqueous phase. In order to establish a detection method which is suitable for measuring hydrogen concentration in aqueous phase, saturated hydrogen-rich water was prepared by using pure water hydrogen generator. Then, hydrogen concentration in aqueous phase was directly measured by hydrogen microsensor. The results showed that the hydrogen concentration had a good linear relationship with microelectrode signal values in different hydrogen concentration ranges (0～1.620 0 mg·L-1 and 0～0.202 5 mg·L-1), and the method detection limit (MDL) was 4.3×10-3 mg·L-1. At the same time, the hydrogen in the aqueous phase was transferred to the gas phase by headspace method, and the concentration of hydrogen was determined by gas chromatography. The results showed that in different hydrogen concentration ranges (0～1.620 0 mg·L-1 and 0～0.202 5 mg·L-1), hydrogen concentration and the peak area of gas chromatography showed good linear relationship, with MDL of 8.7×10-4 mg·L-1. The results of this study showed that both hydrogen microsensor and gas chromatography method can be used to accurately quantify the hydrogen concentration in the aqueous phase, which means that a detection method using hydrogen microsensor and headspace gas chromatography to determine the hydrogen concentration in the aqueous phase had been successfully established.
• A Novel Strategy for Constructing Chondroitin Sulfate Fluorescent Sensor
• MA Xuan, GAO Siyue, LI Kexin, LIANG Zhihong, YAO Zhiyi*
• 2020, 10(2):  164-169.  DOI: 10.19586/j.2095-2341.2019.0103
• Abstract ( ) PDF (2751KB)( )
Chondroitin sulfate (CS) is a kind of protein viscose polysaccharide extracted from animal tissues, which plays a role in immunomodulation, relieving joint pain, anticoagulant activity and protecting neurons. It is of great significance to develop CS sensor for the coarse screening of the relevant pharmaceutical and health care products in the biomedical industry. In this paper, based on the characteristics of simple preparation, rapid response and strong selectivity of fluorescent sensors, a cationic polymer PDDA was used to induce the self-assembly of the pyrene-based molecule HPTS, resulting in fluorescence quenching to construct the sensor. The addition of the analyte caused the recovery of the fluorescence intensity at 510 nm. We finally established a rapid analysis method for CS in the actual sample. The method showed a good linear relationship at a concentration of 0～5 μmol·L-1. The linear equation was y=1.907 52+0.273 59x (R2=0.998 2), the detection limit was 2.8 μmol·L-1, and the sample recovery was 104%～121%. This sensor features good water solubility, strong specificity and intuitive visualization.
• Synthesis of Quercetin Glycoside Catalyzed by Microbial Glycosyltransferase and Evaluation of Their Anti-inflammatory Activity
• WANG Cheng, DOU Wenfang*, HE Lili
• 2020, 10(2):  170-175.  DOI: 10.19586/j.2095-2341.2019.0112
• Abstract ( ) PDF (1501KB)( )
Glycosylation of quercetin was carried out using microbial-derived glycosyltransferases GT-1, GT-2 and BTGT-1. It was found that the three enzymes can catalyze the production of quercetin-3-O-glucose (isoquercitrin) with quercetin and UDP-glucose as substrate. The product formation rates of GT-1,GT-2 and BTGT-1 catalytic reactions were 5.33%, 15.18% and 63.82%, respectively. The water-soluble and anti-inflammatory activity of quercetin, quercetin-3-O-glucose and quercetin-N-acetyl-D-glucosamine in vitro were tested. It was found that the water solubility of isoquercitrin and quercetin-N-acetyl-D-glucosamine was 13.8 times and 15.4 times higher than that of quercetin, respectively, clearly suggesting that glycosylation can greatly improve water solubility of quercetin. The toxic effect of quercetin glycoside on RAW264.7 cells was lower than that of quercetin at the same concentration, but the three compounds showed significant effects on the release of NO, IL-1β and IL-6 in LPS-induced RAW264.7 cells in a dose-dependent manner, which futher displayed that they have a certain anti-inflammation potential.
• miR-148a Inhibits Glycolysis and Cell Proliferation of Breast Cancer Cells Through Targeted Regulation of Hexokinase 2 Gene
• ZHANG Haitao, ZHANG Yuxin*
• 2020, 10(2):  176-184.  DOI: 10.19586/j.2095-2341.2019.0109
• Abstract ( ) PDF (3932KB)( )
In order to investigate the effects of miR-148a and hexokinase 2 (HK2) gene on glycolytic metabolic pathway of human breast cancer cells and their possible mechanisms, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of miR-148a in various breast cancer cell lines, and then breast cancer cell lines with relatively low expression of miR-148a were selected as research objects. Furthermore, the effect of miR-148a expression on glucose uptake, lactic acid production and cell proliferation index of breast cancer cells was observed to explore the effect of miR-148a on glucose metabolism ability of breast cancer cells. Subsequently, the targeted relationship between miR-148a and HK2 genes was predicted through the online database of TargetScan, and then verified through the double luciferase reporting experiment, Western blot and gene recovery experiment, so as to further clarify the mechanism of miR-148a and HK2 in the glycolytic metabolic pathway of breast cancer cells. Through qRT-PCR, the expression of miR-148a was found to be decreased in various breast cancer cell lines, especially in MDA-MB231 (P<0.000 1). Overexpression of miR-148a significantly decreased glucose uptake, lactic acid production and cell proliferation of MDA-MB231 cells (P<0.01). While inhibition of miR-148a expression significantly increased glucose uptake, lactic acid production and cell proliferation index of MDA-MB231 cells (P<0.01). Predicted by TargetScan online database, miR-148a had partial binding sites with the 3′ untranslated region (3′-UTR) of HK2 gene; and the double luciferase report experiment found that miR-148a was bound to the wild-type HK2 3′-UTR luciferase report vector and not to the mutant HK2 3'-UTR. Western blot analysis showed that overexpression of miR-148a significantly decreased the expression of HK2 protein in MDA-MB231 cells (P<0.000 1), while inhibition of miR-148a promoted the expression of HK2 protein significantly (P<0.05). Gene recovery experiment showed that overexpression of HK2 gene significantly increased glucose uptake, lactic acid production and cell proliferation index of MDA-MB231 breast cancer cells (P<0.01). The overexpressed miR-148a vector and the overexpressed HK2 vector were co-transfected into MDA-MB231 cells, and miR-148a reversed the increasing of glucose uptake and lactic acid production caused by HK2, and inhibited cell proliferation. Therefore, studies suggested that miR-148a could inhibit glycolytic metabolism and cell proliferation of breast cancer cells MDA-MB231 by targeted inhibition of HK2 gene expression.
• Study on Reducing the Concentration of Dimethyl Sulfoxide in Hepatocyte Cryoprotectant by Response Surface Methodology
• QIU Guanyu, LI Weijie*, LIU Baolin
• 2020, 10(2):  185-189.  DOI: 10.19586/j.2095-2341.2019.0107
• Abstract ( ) PDF (4476KB)( )
In order to effectively reduce the concentration of dimethyl sulfoxide (Me2SO, DMSO), reduce its toxicity as a cryoprotectant for cryopreservation of cells, and improve cell survival rate, this experiment targeted hepatocytes and adopts response surface method for cryoprotection. The formulation was optimized based on response surface methodology. The results showed that the optimal ratio of cryoprotectant was DMSO concentration 3%, glycerol concentration 6%, trehalose concentration 0.1%. Under this condition, hepatocyte rewarming survival rate was 84.35%, which could effectively reduce the concentration of DMSO. The result reduced the toxic damage that caused to the liver cells and laid the foundation for subsequent research.
• Effect of Two Treatments of Essential Oil Fumigation on the Storage Quality of Flammulina velutipes
• LIU Hong, YUN Jianmin*, MAO Yongqiang, HE Tiantian，BI Yang
• 2020, 10(2):  190-197.  DOI: 10.19586/j.2095-2341.2019.0128
• Abstract ( ) PDF (1237KB)( )
To develop an efficient green preservative for Flammulina velutipes, the fresh Flammulina velutipes was used as the test material, and Zanthoxylum essential oil and clove essential oil were used as the fumigant, screening tests for the types and concentrations of essential oils for the preservation of F. velutipes were conducted at room temperature (25±1)℃ and low temperature (4±1)℃ conditions. A series of index were measured, including sensory evaluation and some physiological and biochemical index. Results showed that, at room temperature (25±1)℃, the fresh-keeping effect of 0.1 mL·kg-1 Zanthoxylum essential oil and 0.5 mL·kg-1 clove essential oil was more significant (P<0.05). The sensory scores of the 0.1 mL·kg-1 Zanthoxylum essential oil and 0.5 mL·kg-1 clove essential oil treatment group were higher than those of the control by 23.4% and 27.8%, respectively. And both of them can significantly inhibit browning, reduce spoilage and deterioration. In the low temperature (4 ± 1)℃ storage test, it was found that both of 0.1 mL·kg-1 Zanthoxylum essential oil and 0.5 mL·kg-1 clove essential oil can effectively inhibit the increase of respiratory intensity and PPO activity (P<0.05). Among them, the respiratory intensity reduced by 28.3% and 39.6%, respectively. After 15 days of storage, the PPO activity of the essential oil treatment group was reduced by 8.2% and 16.6% respectively compared with the control; the essential oil treatment effectively reduced the production of MDA, maintained a higher level of total phenol content, and reduced the degree of rotten browning. On the 15th day, the MDA content of the control was 1.75 μmol·g-1, while the MDA content of the Zanthoxylum oil and clove essential oil treatment groups were 0.15 and 0.40 μmol·g-1 lower than the control, respectively. The MDA content of clove essential oil treatment group was significantly lower than those of Zanthoxylum essential oil treatment group and control group (P <0.05). This research demonstrated that 0.1 mL·kg-1 Zanthoxylum essential oil and 0.5 mL·kg-1 clove essential oil fumigation have significant effect on the storage and preservation of F. velutipes. Among them, 0.5 mL·kg-1 clove essential oil had the most obvious preservation effect, during the 15 days storage period, F. velutipes still maintains good quality, but the control group had been slightly browned and some began to rot. The results provided theoretical basis for the application of Zanthoxylum essential oil and clove essential oil to Flammulina velutipes storage and preservation after harvest.
• Optimization of Spray Drying Technology for Hemp Seed Oil Microcapsules by Response Surface Methodology
• SHI Jiangchuan1,2, SHI Jia2*, YU Mingxiao2, ZHAO Weixue2, XU Hao2, XU Lei2
• 2020, 10(2):  198-204.  DOI: 10.19586/j.2095-2341.2020.0009
• Abstract ( ) PDF (8273KB)( )
In order to prepare hemp seed oil microcapsules with high embedding rate and good stability and expand its application scope in field of food, using hemp seed oil as core material, mono-and di-fatty acid glyceride as emulsifier, sodium caseinate as wall material, solid corn syrup as filler agent, sodium citrate as buffer salt, Sodium ascorbyl palmitate as antioxidant, and hemp seed oil microcapsules with 60% oil-loading rate were prepared by spray drying method. Taking microcapsule embedding rate as response value, on the basis of single factor experiment, dry matter concentration, inlet air temperature and outlet air temperature were used as experimental factors, and Box-Behnken response surface analysis method was used for optimization. Then the surface morphology of hemp seed oil microcapsules was observed by scanning electron microscopy to determine the embedding effect. The oxidation stability of hemp seed oil microcapsules was tested by oil oxidation analyzer. The optimum embedding conditions were determined as follows: dry matter concentration 42%, air inlet temperature 168 ℃, air outlet temperature 74 ℃. Under these conditions, the embedding rate of hemp seed oil microcapsules could reach 92.15%. Scanning electron microscopy showed that the surface of hemp seed oil microcapsules was smooth and crack-free, indicating that the embedding effect of hemp seed oil microcapsules was ideal. The oxidation induction period of the test group (hemp seed oil microcapsule) was longer than that of the control group (hemp seed oil) measured by the oil oxidation analyzer, which could reach more than 30 h, indicating that the stability of the oil can be improved to a greater extent by microencapsulating the hemp seed oil. The research results provided theoretical support for the development and application of hemp seed oil in food industry.
• Study on Extraction and Purification Technology of Fucoxanthin from Copper Algae
• LI Hongyan1,2, WANG Ying1,2*, LIU Tianhong1, JIANG Xiaodong1,2, LI Xiao1,2, SUN Yuanqin1, JI Lei1, TANG Huanhuan1
• 2020, 10(2):  205-213.  DOI: 10.19586/j.2095-2341.2020.0001
• Abstract ( ) PDF (1739KB)( )
In order to systematically study on the extraction and purification of fucoxanthin and further provide research basis for the industrial production of high-purity fucoxanthin, an organic solvent suitable for extracting fucoxanthin from fresh copper algae (Sargassum horneri) was selected, and the optimum technological parameters including extraction solvent concentration, extraction temperature, extraction time, solid-liquid ratio were determined through single factor and orthogonal experiments. Subsequently, silica gel column chromatography was used for purification, and the optimum silica gel column bed height, sample loading amount and elution flow rate were determined through single factor experiments. Finally, the fucoxanthin purified by silica gel column chromatography was further purified by preparative liquid chromatography. The results showed that the optimum process conditions for organic solvent extraction were as follows: methanol concentration 90%, extraction temperature 50 ℃, extraction time 1 h, and solid-liquid ratio 1∶10. Under these conditions, the extraction rate of fucoxanthin reached (0.258 9±0.003 6) mg·g-1 fresh weight (FW) ［(1.078 8±0.015 0) mg·g-1 dry weight (DW)］. The optimum technological conditions for silica gel column chromatography were as follows: the height of silica gel column bed was 10 cm, the loading amount was 6 g, and the elution flow rate was 10 mL·min-1. Under these conditions, the yield of fucoxanthin was 0.176 5 mg·g-1 FW (0.735 3 mg·g-1 DW), and the purity was 87.01%±0.88%. After further purified by preparative liquid chromatography, the yield of fucoxanthin was 0.127 1 mg·g-1 FW (0.529 4 mg·g-1 DW) and the purity was 99.27%±0.22%. The research process was simple and the yield of fucoxanthin was high, which provided an experimental basis for the preparation of high purity fucoxanthin.