Current Biotechnology

The CRISPR/Cas Gene Editing Technology and Application in Crop Breeding
ZHENG Hongyan, WANG Lei*
2018, 8(3): 185-190. DOI: 10.19586/j.2095-2341.2018.0059
Abstract ( ) PDF (1053KB)( )

Genome editing is of important value in gene function study and crop improvement, which depends on the DNA double-strand breaks (DSBs) at the genomic target sites created by RNA-guided endonuclease. During DSBs repaired through the error-prone non-homologous end joining (NHEJ) process or the more precise homology-directed repair (HDR) process, small insertions, deletions, or DNA fragment replacement will take place in the targeted gene(s). This review started with introduction of the mechanism and development process of genome editing, and focused on the recent progress of CRISPR/Cas based crop breeding, especially in food and cash crops, which was expected to provide useful reference for crop breeding.

Research Progress of CRISPR/Cas9 in Tumor Therapy
LV Baobei, ZHAO Pengxiang, ZHANG Xin, MA Xuemei, XIE Fei*
2018, 8(3): 191-196. DOI: 10.19586/j.2095-2341.2017.0141
Abstract ( ) PDF (1069KB)( )

It has been rapidly applied to oncology studies since the advent of CRISPR/Cas9. In the study of tumorigenesis mechanism, CRISPR/Cas9 can be used to study the role of single nucleotide mutation, chromosomal aberration and other factors in the tumorigenesis mechanism, as well as the screening of function defective genes in tumor cells. In the study of tumor therapy, CRISPR/Cas9 is mainly used in tumors that induced by viruses, such as nasopharyngeal carcinoma and cervical cancer, which could inhibit the carcinogenesis by gene editing of the corresponding viruses. The use of CRISPR/Cas9 can also accelerate the discovery of new oncology-targeted genes. Despite its rapid development and application, the role of CRISPR/Cas9 in tumor research and therapy was limited by a number of factors, including the efficiency of Cas9 and sgRNA delivery, off-target effects, and safety and cost-effectiveness. This article reviewed the progress of the application of CRISPR/Cas9 in cancer research, which was expect to provide a reference for the study of tumorigenesis, metastasis mechanism and tumor therapy.

Genetic Effect and Breeding Application of Myostatin Gene Mutation in Beef Cattle
WEI Zhuying, BAI Chunling, LI Guangpeng*
2018, 8(3): 197-205. DOI: 10.19586/j.2095-2341.2018.0064
Abstract ( ) PDF (4891KB)( )

The myostatin (MSTN) gene mainly expresses and participates in the regulation of skeletal muscle growth and development. The MSTN gene not only has strong evolutionary conservation, but also has more mutation polymorphisms. In several cattle breeding, kinds of sense mutations at different location of the MSTN have been clarified. All of the sense MSTN mutations resulted in hypertrophic skeletal muscles, which the mutant cattle have the double muscle phenotype, and increase the growth and meat yield. Simultaneously, some of the mutant cattle arise varieties of negative effects. This review focused on the mutation types of MSTN gene, the mutated genetic effects as well as the possible application in beef cattle breeding. The paper would provide data for the research on improvement and breeding of local beef cattle in China.

Progress on Application of Proteomic Technology in Meat Authentication and Meat Quality Analysis
HU Zhengyan, WANG Junlin, WU Pinggu*, WANG Tianjiao, WANG Liyuan, TANG Jun
2018, 8(3): 206-213. DOI: 10.19586/j.2095-2341.2018.0022
Abstract ( ) PDF (1098KB)( )

Meat products are important sources of protein and trace elements for human beings. However, the study of meat authentication and meat quality analysis in meat products has been plagued by methodological limitations. Nowadays the application of proteomic technology has immensely promoted the development of meat authentication technologies, and had a profound influence on the research of the potential molecular mechanisms of meat quality. The present review introduced the definition of proteomics and its main research strategies, and comprehensively discussed the progresses on the application of proteomic technology in meat authentication and meat quality analysis. Furthermore, the research prospect of meat authentication and meat quality analysis was also forecasted, which was expected to provide theoretical basis for the quality control of meat products and the study on the factors influencing meat quality.

Progress on the Function of Heat Shock Transcription Factors in Plant Abiotic Stress Tolerance
LI Li, LIU Shuangqing, YANG Yuanhang, DAI Liangying, LI Wei*
2018, 8(3): 214-220. DOI: 10.19586/j.2095-2341.2018.0002
Abstract ( ) PDF (1221KB)( )

Under environmental stress, plants would produce a series of stress responses, and the heat shock transcription factors play important roles in stress tolerance and other life activities in plants by mediating the transcription and expression of heat shock proteins or other heat-inducible genes. The basic protein domains of plant heat shock transcription factors were introduced, and the functions and mechanism of three kinds of plant heat shock transcription factors were expounded when plant subjected to extreme temperature（high temperature and low temperature）stress, drought stress, high salt stress and reactive oxygen species stress. In addition, the development and application prospects of plant heat shock transcription factors in plant breeding and enhancing plant stress resistance were discussed, which may provide theoretical reference for future study on the biological function and mechanism of heat shock transcription factors during regulating plant resistance to stress.

Progress on Functional Markers for LMW-GS in Wheat
GAO Zhenxian, CAO Qiao, HE Mingqi*, TIAN Guoying, WANG Fei, SHAN Zilong, HAN Ran
2018, 8(3): 221-228. DOI: 10.19586/j.2095-2341.2018.0024
Abstract ( ) PDF (1123KB)( )

Wheat is one of the major grain crops in China. Low molecular weight glutenin subunit (LMW-GS) plays a crucial role in determining the unique baking quality of wheat. In recent years, detecting the type and composition of LMW-GS by molecular markers technique has become one of the hotspots in the study of wheat quality improvement. The latest research progress on the structure of LMW-GS gene and protein, and its classification and functional markers were summarized in this paper, and then the significance of exploitation and utilization as well as the existing problems of the LMW-GS functional markers for the Glu-A3, Glu-B3, Glu-D3 loci in wheat were also discussed. Finally, it was pointed out that the importance of innovation of LMW-GS marker technology and supplement of subunit types to the improvement of wheat quality,which was expected to accelerate the application of LMW-GS functional markers in wheat breeding with high quality.

Progress of BAG-1 Protein and its Regulation Effects on Nerve Systemic Diseases
ZHANG Yating1,2, JIN Xiaoyan2, ZHONG Guohui2, LI Jianwei2, LI Yingxian2*, MA Xuemei1*
2018, 8(3): 229-236. DOI: 10.19586/j.2095-2341.2017.0159
Abstract ( ) PDF (3166KB)( )

The protein encoded by B-cell CLL/lymphoma 2-associated athanogene-1 (BAG-1) is a multifunctional binding protein that contains various domains with different functions, which interacts with Bcl-2, Hsp70/Hsc70, Raf-1, steroid hormone receptors and DNA, plays an essential role in cell apoptosis, signaling transduction, gene transcription, cell proliferation and differentiation. BAG-1 is involved in the development and progression of nervous system diseases, such as Alzheimer disease, Parkinson disease, Huntington disease, stroke, spinal cord injury, and neuropsychiatric disorders. The progress of structure and basic function of BAG-1 and its effects on nervous system diseases was summarized , which was expected to provide reference for related research and therapy of nervous system diseases.

Expression and Application of a Pectate Lyase from Metagenome in Pichia pastoris
DONG Junshuai1, QIN Xing2, ZHANG Zhiwei1*, ZHANG Yuhong2*, ZHANG Wei2
2018, 8(3): 237-245. DOI: 10.19586/j.2095-2341.2018.0041
Abstract ( ) PDF (4725KB)( )

Pectate lyase can be used for the treatment of wastewater containing pectin, pulp bleaching, and bio-refining of cotton and linen textiles. In this study, a pectate lyase gene pela was obtained from pectin-rich soil based on high throughput metagenomic sequencing technology. The pela was ligated into pPIC9 vector and transformed into Pichia pastoris GS115. The PELA activity was 10.8 U/mL after induction with methanol for 10 h in 3 L fermentor. The optimal temperature and pH of recombinant PELA were 45℃ and 9.0, respectively. PELA has good stability at pH 7.5~11.0. The specific activity of the recombinant enzyme was determined to be 244.12 U/mg. Use polygalacturonic acid as substrate, the kinetic parameters Km and Vmax were determined to be 0.26 mg/mL and 488.40 μmol/min·mg, respectively. EDTA and metal ions Cd2+, Zn2+, Mn2+, Cu2+ and Fe3+ showed strongly inhibitory effects on PELA activity, while the enzyme activity was enhanced by 1 mmol/L Ca2+ and 2 mmol/L K+. The recombinant PELA was applied to treat the ramie fiber. The percentage of weight loss was reached 9.2% after 4 h treatment. The whiteness, dispersionramie and softness of ramie fiber were enhanced significantly. The PELA from metagenome has a good potential application for ramie degumming.

Soluble Expression and Purification of hTNFR1 in Escherichia coli
HAN Ping1, GAI Yuanming2, ZHU Beiwei1*, ZHANG Dawei2*
2018, 8(3): 246-253. DOI: 10.19586/j.2095-2341.2018.0021
Abstract ( ) PDF (7242KB)( )

The human tumor necrosis factor type 1 receptor (hTNFR1) gene was cloned into pET-22b expression vector, this study successfully constructed the recombinant plasmid pETH1 and transformed it into Escherichia coli BL21 (DE3) expression strain for shake flask fermentation, realizing heterologous expression of hTNFR1 in E. coli. However, all the proteins were in the forms of inclusion bodies. To improve the soluble expression of the target protein existed in the sedimentation in E. coli, we used two strategies of fusion tagging and molecular chaperone for protein folding. Results showed that, the fusion of NusA to the N-terminus of hTNFR1 increased the solubility; based on NusA-hTNFR1, seven molecular chaperones was overexpressed, among which the molecular chaperone tig was screened for it showed significant promotion on the soluble expression of hTNFR1 in E. coli; we purified protein by Ni-NTA affinity chromatography, then, the tig of NusA in the N-terminus of hTNFR1 was removed by enzymatic hydrolysis with TEV protease. After identification by Western blot, the hTNFR1 protein with high purity was obtained. The results laid a good foundation for further study on the physiological activity of hTNFR1 as well as the treatment of diseases.

Truncated Protein Expression, Purification and Polyclonal Antibody Preparation of PrsA1 Gene from Listeria monocytogenes
SUN Jingjuan1, QIU Jingxuan1, ZENG Haijuan1, DING Chengchao1, WANG Guangbin2, LI Jie1, WANG Shujuan1, LIU Qing1*
2018, 8(3): 254-260. DOI: 10.19586/j.2095-2341.2017.0163
Abstract ( ) PDF (4366KB)( )

Listeria monocytogenes is a common food-borne pathogen that causes listeriosis and poses a tremendous threat to food safety. prsA1 is conservative and specific. Signal peptides, transmembrane regions and spatial antigen epitopes of PrsA1 were predicted in this study by SignalP 4.1 Server program, TMHMM Server V.2.0 program and SEPPA 2.0 program, respectively. The results showed that the N terminus of PrsA1 contained a signal peptide and a transmembrane region, and that the protein had good epitopes. Therefore, it could be used as a detection target. On this basis, non-transmembrane region sequence of prsA1, Δ84prsA1, was acquired through PCR. The recombinant plasmid pET30a-Δ84prsA1 was constructed and transferred into Escherichia coli to induce expression of Δ28PrsA1. Δ28PrsA1 was purified by Ni-IDA-Sefinose Column. The titer of polyclonal antibody prepared by purified Δ28PrsA1 was determined by indirect ELISA, and the titer was as high as 1∶128 000. Western blotting analysis showed that the polyclonal antibody could recognize PrsA1 protein extracted from Listeria monocytogenes. In this study, bioinformatics methods was used to screen the detection target and analyze the epitopes, and polyclonal antibody was prepared successfully, which provided a practical basis for the screening of detection target and immunological detection of Listeria monocytogenes.

Study of the Correlation Between MbBV Infection and Cyclophilin A in Hi5 Cells
HU Yan, XU Cuixian, ZHANG Pan, YU Dan, ZHANG Binyue, LI Ming*
2018, 8(3): 261-268. DOI: 10.19586/j.2095-2341.2017.0143
Abstract ( ) PDF (2707KB)( )

The Microplitis bicoloratus bracovirus (MbBV) is one type of polydnavirus, which is mainly found in parasitic wasps (Hymenoptera: Braconidae) and plays a vital role in parasitism. Cyclophilin A (CypA) is a peptidyl-prolyl cis-trans isomerase involved in several different cellular events including the immune response. During the process of insect cells infected by MbBV, whether there was any relationship between MbBV and the level of CypA was mainly investigated. The results showed that the MbBV gene can be detected in Hi5 insect cells after 24 h of infection with MbBV, during which the transcriptional level and protein expression level of cypa were both upregulated. When CypA was silenced or suppressed in MbBV-treated Hi5 cells, vank86, a gene encoding one member of MbBV, was remarkably down-regulated via analysis of qRT-PCR. And overexpression of CypA in Hi5 cells leaded to an upregulation of the vank86. These results indicated that the level of CypA in insect cells might be correlated to the MbBV infection.

Effect of LSD1 Gene Knockout on K562 Cell Cycle
REN Sirui, WANG Bingrui, GUO Qing, FENG Tiantian, WANG Ding, GAO Jie*, SHI Lihong*
2018, 8(3): 269-273. DOI: 10.19586/j.2095-2341.2018.0031
Abstract ( ) PDF (1519KB)( )

The aim of the study is to uncover the mechanism of reduced proliferation of K562 cells resulting from LSD1 depletion. Making use of the LSD1-/- K562 cell lines, cellular apoptosis was measured by Annexin V/PI double staining with flow cytometry. Cell cycle was examined using PI staining with flow cytometry. We want to explore whether the apoptosis level of K562 cells is altered and whether cell cycle is affected after knockout of LSD1 gene. The results showed that there was no induction of apoptosis in K562 cells after LSD1 depletion; the cell cycle of K562 cells arrested at the G0/G1 phase, cells decline in the DNA duplication phase. The results indicated that LSD1 depletion inhibits K562 cells proliferation mainly via impairment of cell cycle but not apoptosis.

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