Current Biotechnology

Progress of Slow/controlled Release K Fertilizers in Flue-cured Tobacco
HAN Dan1, LIU Jin2, LI Yanyan3, TU Shuxin4, HUANG Wuxing1, HAO Sanhui1, XU Zicheng1*
2017, 7(4): 261-265. DOI: 10.19586/j.2095-2341.2016.0118
Abstract ( ) PDF (1116KB)( )

Developing the slow/controlled release fertilizers (SRFs) of flue-cured tobacco has great significance in protecting ecological environment and reducing agricultural production costs. In this paper, the internal and external research and application literatures of SRFs were summarized. The SRFs’ concept and characteristics were introduced firstly, and then the effects of SRFs on the growth, K content, quality and economic traits of flue cured tobacco were reviewed, and some problems in current research were pointed out. The SRFs was beneficial to the growth of tobacco, increase of K content in soil and leaves, improvement of quality and enhancing the yield and output value of flue-cured tobacco. But it also had insufficient research in the coated material of SRFs and mechanism, and had poor study about the SRFs mixed with the ordinary fertilizer applications. Therefore，it is necessary to discuss the development of present situation, prospects, problems and countermeasures about the SRFs at home and abroad, so as to provide theoretical basis for industrialization of the SRFs in our country.

Biological Characteristics and Applications of Endothelial Progenitor Cells
LU Tengfei, WU Yangnan, PEI Wenhua, MA Yuehui*, GUAN Weijun*
2017, 7(4): 266-271. DOI: 10.19586/j.2095-2341.2017.0009
Abstract ( ) PDF (1162KB)( )

Endothelial progenitor cells (EPCs) are the precursor cells of the vascular endothelial cells. EPCs is now being more and more introduced into the basic research in medicine, cell biology and other fields about vascular disease with the study of its culture, identification, homing and differentiation. Studies have shown that endothelial progenitor cells can promote wound healing and the recovery of vascular function, becoming a new method to construct cardiovascular tissue engineering. This paper reviewed the biological characteristics of EPCs and its application in animal ischemia models and clinical trial. The paper also analyzed the challenge of EPCs application. Finally, we prospected the research direction of EPCs in the future.

Advance on Detection Method of Insecticide Resistance
MA Yuting, WEI Juan, LI Xianggan*
2017, 7(4): 272-278. DOI: 10.19586/j.2095-2341.2016.0153
Abstract ( ) PDF (2392KB)( )

Under widespread and long-time usage of insecticides, insects develop various degrees of resistance to insecticides, and the number of insect resistance varieties and resistance level to pesticides are increasing year by year accordingly. These situations make the monitor motheds become the focus so that the detection methods have been improved. This paper introduced two groups of methods commonly used for detecting insecticide resistance: Bioassay methods and molecular biology methods. Bioassay methods include artificial diet mixed drug, leaf dipping, topical application, spray and dusting, residual film, soaking method and IRAC No.5. And molecular biology methods include PCR-REN, PASA, RAPD, TaqMan Real-time PCR, KASP and DNA sequencing. By comparing insect characteristics, environmental factors, experimental conditions and objectives, researchers could select appropriate detection methods to quickly obtain insecticide resistance information and timely and effectively adjust the insect control strategy.

Advances in Enzymatic Transformation of Rare Ginsenosides
GUO Feng, LIU Huanhuan, QIU Zhidong, WANG Weinan*
2017, 7(4): 279-283. DOI: 10.19586/j.2095-2341.2017.0017
Abstract ( ) PDF (1122KB)( )

Ginsenosides are one of the main active components of the plants, which belong to the genus Panax of Araliaceae family. Studies have shown that the pharmacological effects of rare saponins, produced by metabolism in vivo, on anti-tumor, anti-inflammatory and anti-aging are stronger than those of the prototype saponins. Therefore, preparation of rare saponins has become a popular topic in the field of ginseng research. Enzymatic transformation of ginsenosides is one of the primary ways for the preparation of rare saponins, in which many breakthroughs have been made recently. In this paper, the results of these studies were reviewed from the perspectives of different glycosidase types as well as their applications in rare saponins biotransformation so that it could lay a solid foundation for the large-scale development and utilization of rare saponins.

Progress on Magnetic Nanoparticles Immobilized Enzymes
CHEN Jing, LENG Juan, YANG Xiai, LIAO Liping, XIAO Aiping*, LIU Liangliang*
2017, 7(4): 284-289. DOI: 10.19586/j.2095-2341.2017.0022
Abstract ( ) PDF (1145KB)( )

Magnetic nanoparticles with magnetism characteristics and the special performance of nanomaterials, are widely applied in many kinds of fields. This paper reviewed progress on the magnetic nanoparticles, including types, features, preparation and surface modification. The paper also summarized the latest development of immobilized enzyme technology of lipase, lactase, amylase and a combination of two or more, which was aimed to provide reference for the study of magnetic nanoparticles immobilized enzymes.

Progress of Collagen Peptide Metal Chelates and Related Production Technologies#br#
SUN Shanshan1,2, GAO Yahui1, CHEN Junde2*
2017, 7(4): 290-295. DOI: 10.19586/j.2095-2341.2017.0024
Abstract ( ) PDF (1141KB)( )

Collagen peptide metal chelates are formed by collagen peptides and metal ions through coordinate covalent bonding or adsorption binding. They have been considered as the potential approach to delivering metal mineral elements to consumers with many virtues, such as higher bioavailability, higher safety and higher bioactivity. Chelate mechanism, stability, absorption and utilization, functional activity and production technologies of collagen metal chelates were reviewed and the development prospect were also foreseen in this paper, which was expected to provide reference for related reasearches.

Target Trait Test and Multiple PCR Detection of Insect-resistance and Herbicide Tolerance Transgenic Maize SW-1
ZHANG Xin, DONG Yufeng, WANG Xujing, WANG Zhixing*
2017, 7(4): 296-303. DOI: 10.19586/j.2095-2341.2016.0160
Abstract ( ) PDF (4208KB)( )

Stacked transgenic maize SW-1 containing cry1Ie, cry1Ab/cry2Aj and G10evo-EPSPs, was cultivated by hybridization using transgenic maize IE034 and 12-5 as parents. Agronomic traits test showed that SW-1 had the characteristics of insect-resistance and herbicide tolerance. The multiplex PCR detection system was established by optimizing specific primers, annealing temperature and primer concentration. Multiplex PCR detection system used an endogenous reference gene zSSIIb, the reaction volume was 30 μL, the best annealing temperature was 62℃ and the amount of each primer was 0.3 μL (10 μmol/L ). Using this multiplex PCR detection system, the target gene cry1Ie, cry1Ab/cry2Aj and G10evo-EPSPs can simultaneously be detected, when the mass percentage of SW-1 in the product was more than 0.5%. The results provided a good technical support for the molecular detection of stacked genetically modified maize SW-1. Meanwhile, this study provided scientific data for multiplex PCR detection of other transformation containing cry1Ie、cry1Ab/cry2Aj and G10evo-EPSPs genes.

Cloning and Prokaryotic Expression of the Hemocyanin Subunit TypeⅠ from Calliptanus italicus
WANG Di, ZHANG Zao, WANG Zhangping, YIN Hong*
2017, 7(4): 304-309. DOI: 10.19586/j.2095-2341.2017.0023
Abstract ( ) PDF (3130KB)( )

Hemocyanins are copper-containing (Cu2+) proteins in vivo of insect and play important roles not only in respiratory but also in energy storage, antibiosis and antivirus. To investigate the structure and expression profiles of Calliptanus italicus hemocyanin subunit typeⅠ(CitHc-1), the complete cDNA (2 335 bp) of CitHc-1 was obtained by RACE method. The CitHc-1 contained an open reading frame of 2 079 bp encoding 692 amino acids. The predicted molecular weight is 79.88 kDa. Sequence alignment revealed that the CitHc-1 shared an identity of 91%~94% with hemocyanins from other Acridoidea species. The dendrograms of the arthropod hemocyanin subunit typeⅠinferred by Neighbor-joining (NJ) method showed that, Calliptanus italic hemocyanin subunit typeⅠand Locusta migratoria hemocyanin subunit typeⅠformed a subclade. In order to further study the structure and function of hemocyanin subunit typeⅠfrom Calliptanus italicus, the CitHc-1 prokaryotic expression vector was established successfully and the recombined plasmid was transfected into BL21(DE3). The result of SDS-PAGE showed that the molecular weight was consistent with the theory molecular weight (32 kDa). The results provided a theoretical basis for further analysis on the function of CitHc-1 in C. italicus.

Extraction and Optimization of Hydrolysis Conditions of Total Protein from Hippocampus
HU Jiayao, ZHANG Meiyan, WANG Zhenling, WEI Na, GAO Bingmiao*
2017, 7(4): 310-314. DOI: 10.19586/j.2095-2341.2017.0011
Abstract ( ) PDF (2807KB)( )

In order to extract total protein of Hippocampus and optimize conditions for enzymatic hydrolysis, we extracted the total protein of Hippocampus by the method of water extraction of dried Hippocampus. Papain, alkaline protease, neutral protease and trypsin were used respectively under the different pH, time, temperature and E/S conditions to conduct single factor test, and finally the enzymatic hydrolysis conditions were optimized by orthogonal test. The enzymatic hydrolysis condition and protein content under different conditions were determined by SDS-PAGE and BCA, respectively. The optimal enzymolysis conditions were determined according to the degree of proteolysis shown in the charts. The results showed that 1.056 g total protein was extracted from 10 g dried Hippocampus, and its protein concentration was 0.106 g/mL. The best enzyme was alkaline protease. The best enzymatic conditions were pH 9, E/S 5%, hydrolysis temperature 50℃and hydrolysis time 3 h, and the degree of hydrolysis of the Hippocampus protein was 96.9%. The optimal enzymatic hydrolysis conditions have been determined, and the study laid a certain foundation for the study of biological activity of hippocampal polypeptide.

Study on the Antifungal Effect of Bioactive Metabolites Produced by Penicillium bilaiae
ZHANG Nannan1,2, BAI Jing2, ZHANG Liwen2, XU Yuquan2, WANG Huiguo1*, FENG Baomin1
2017, 7(4): 315-319. DOI: 10.19586/j.2095-2341.2017.0020
Abstract ( ) PDF (2462KB)( )

The antagonistic activity of Penicillium bilaiae ACCC 30440 against Fusarium oxysporum and Verticillium dahliae was screened and determined by plate confrontation experiment and growth rate method. The results showed that ACCC 30440 fermentation broth and ethyl acetate extract could inhibit the growth of F. oxysporum and V. dahliae, and the inhibition rate to F. oxysporum and V. dahliae were both more than 40% with 10% fermentation broth, while 20% fermentation broth completely inhibited the growth of these two pathogenic fungi. The half inhibitory concentrations of ACCC 30440 ethyl acetate extract on F. oxysporum and V. dahliae were 518.63 μg/mL and 443.42 μg/mL, respectively. The results indicated that P. bilaiae ACCC 30440 strain could produce bioactive metabolites that inhibited the growth of phytopathogenic fungi.

Research on the Hemostatic Preparation with Corn Microporous Starch
BIAN Wenhai1§, LIU Chengcheng2§, LI Qian2, WANG Chaozhan1*, BIAN Liujiao2
2017, 7(4): 320-326. DOI: 10.19586/j.2095-2341.2016.0126
Abstract ( ) PDF (7710KB)( )

To prepare and evaluate the hemostatic that made of corn microporous starch, a sequential hydrochloric acid and composite enzyme hydrolysis method was utilized for preparation of corn microporous starch. The density and water-absorbing ratio of the microporous starch were determined. The hemostatic effect was evaluated by animal wound hemorrhage model. Results showed that, the density and water-absorbing ratio of the microporous starch were determined to be 3.4×104 /mm2 and 156.98%, respectively. The microorganism and endotoxin had been picked out. The hemostatic time of the microporous starch to the rabbit ear and liver trauma were within 140 s and 80 s, and the hemostasis effect was superior to Yunnan Baiyao. The microporous starch had good biocompatibility and met the safety criterion of biomaterial. It has potential to be a promising biomaterial for hemostasis.

Study on the Function of Ganoderma lucidum Semen Ziziphi Spinosae Capsules on Sleep Improvement
FENG Weiwei1, WU Meiyuan2, DENG Chunmeng1, WANG Yaqian1, MAO Guanghua1, XU Caiquan3, WU Xiangyang1
2017, 7(4): 327-330. DOI: 10.19586/j.2095-2341.2017.0012
Abstract ( ) PDF (1114KB)( )

The function of Ganoderma lucidum Semen Ziziphi Spinosae Capsules (GLSZSC) on sleep improvement was studied. ICR mice were randomly divided into normal control and GLSZSC (225 mg/kg, 450 mg/kg and 1 350 mg/kg) groups and each group with 12 mice. After giving GLSZSC via gavage for 30 days, the effect on sleep improvement was studied by the experiments of sleep-induced directly, prolonging sleep time induced by pentobarbital sodium and affecting mice barbital sodium, respectively. Compared with normal control group, the GLSZSC did not have directly sleep-induced function, whereas the GLSZSC (450 mg/kg and 1 350 mg/kg) could significantly prolong the sleep time induced by pentobarbital sodium (P<0.01 and P<005). The GLSZSC (1 350 mg/ kg) decreased the number of mice in sleep latency induced by barbital sodium (P<0.01) and increased the number of mice in sleep under the threshold hypnogenesis dosage of barbital sodium (P<0.05) obviously. The research showed that GLSZSC had certain effect on improving sleep of mice.

Regulation of DDX20 Gene on the Migration of Gastric Carcinoma Cell
LIU Jun1,2, ZHOU Chunyan2, JIN Li2, CHEN Gang2, XIAO Qian2,3*, ZHU Huanzhang1*
2017, 7(4): 331-337. DOI: 10.19586/j.2095-2341.2017.0018
Abstract ( ) PDF (8649KB)( )

To study the regulation of DDX20 gene on the migration of gastric carcinoma cell based on the cell wound scratch assay, the DDX20 gene expression level assay of gastric mucosa cell GES, gastric carcinoma cell MGC803, SGC7901 and MKN74 were further detected by real-time PCR to confirm the high and low expression cell line of DDX20 gene. We designed and packaged DDX20 gene, and interfered Lentivirus. Then we screened the stable transfection cell lines by the assay of real-time quantitive polymerase chain reaction (RT-qPCR) and Western blot, and finally perform the cell wound scratch assay and gathered images based on the stable transfection cell lines. Results showed that, compared with the gastric mucosa cell GES, the expression level of DDX20 gene in the gastric carcinoma cell of MGC803 was the highest, and second in the gastric carcinoma cell of MKN74, and the minimum in the gastric carcinoma cell of SGC7901. After transfection of the DDX20 gene overexpression Lentivirus, the expression level of DDX20 gene and protein was significantly increased. After transfection of DDX20 gene intervention Lentivirus, the expression level of DDX20 gene and protein was significantly decreased, especially in the sh-RNA-02 intervention Lentivirus. In addition, overexpressed DDX20 gene could significantly accelerate the migration of gastric carcinoma cell, inhibited down-regulated the expression of DDX20 gene could inhibit the migration of gastric carcinoma cell. DDX20 gene is a tumor metastasis associated gene, and the migration of gastric carcinoma could be decreased when the expression of DDX20 was inhibited, and the results provided a potential treatment target in clinic, and exhibited a certain application value of DDX20 in clinic.

Effects of pyk2 Knockout on the Proliferation, Migration and Invasion of Human Glioma Cell Line by CRISPR/Cas9
WANG Chaojia1, WANG Hui1, HU Juntao1, HU Shengli1, ZHANG Yuqiang1, CHENG Yusi1, LEI Shangguo2*
2017, 7(4): 338-344. DOI: 10.19586/j.2095-2341.2017.0010
Abstract ( ) PDF (5811KB)( )

Two pairs of CRISPR/Cas9 recombinant plasmids which targets pyk2 genes were constructed to investigate the effects of Pyk2 (proline rich tyrosine kinase 2) on the proliferation and invasion of human glioma cell line U251. The plasmids were transfected into human glioma U251 cells after they were identificatied, and the wild type U251 cells were set as the control group. Positive monoclonal cells were selected by enzyme digestion and Western blot analysis, then the mutation sites were revealed by sequencing. Compared with the control group, the proliferation ability of the experimental group (the positive monoclonal cells) was decreased by the RTCA method (P<0.05). The invasion and migration ability of the cells in experimental group were significantly inhibited by Transwell chamber method and Scratch test, and the differences were statistically significant (P<005). The matrix metalloproteinase 9 (MMP9), which was used to verify the invasion ability, was significantly decreased in the experimental group than in the control group by Western blot method. Through the above methods, we successfully obtained the stable cell lines with target gene knockout, and confirmed that knockout of pyk2 gene could significantly inhibit the proliferation, migration and invasion of human glioma cells.

Optimizing of Supercritical Carbon Dioxide Extraction of Flavonoids Ingredients from Mulberry Leaf by Thogonal Experiment Method
XIE Yulong1, SHI Tongyu1, MIAO Weiwei2,3, WANG Zhixiong1,2*
2017, 7(4): 345-349. DOI: 10.19586/j.2095-2341.2016.0127
Abstract ( ) PDF (1784KB)( )

The intent of this study is to explore the best solutions for the supercritical CO2 extraction of flavonoids from mulberry leaves. This study used supercritical carbon dioxide extraction technology, detected the effects of extraction pressure, extraction temperature, extraction time, entrainer flow on the yield of total flavonoids in mulberry leaves through orthogonal experiment method, to optimize the best process conditions. Results showed that: orthogonal experiment for the optimum extraction conditions were as follows: extraction pressure was 25 MPa, extraction temperature was 40 ℃, extraction time was 3 h, entrainer anhydrous ethanol velocity was 2.5 mL/min. The expected flavonoids yield rate was 6.19%±0.26%, the repeatability was good. Flavonoids extraction rate by the supercritical carbon dioxide extraction from mulberry leaves was high. This study provided theoretical and experimental basis for further development of mulberry leaf.

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