Current Biotechnology

CRISPR/Cas9-mediated Gene Editing Technology and Potential Applications in RNA Editing
REN Wenlin1, ZHANG Jun2, XU Guixia2, MOU Yani1, LOU Yang3, WU Songqing2*
2017, 7(1): 1-6. DOI: 10.3969/j.issn.20952341.2017.01.01
Abstract ( ) PDF (1132KB)( )

The CRISPR-Cas9 system, from Streptococcus pyogenes, has gained widespread application as a genome editing tool. Previous studies showed that CRISPR-Cas9 system has the potential utility as a universal nucleic acid-recognition technology, that is Cas9 system can also allow RNA identification and editing in live cells. By fusing RCas9 to different protein factors, it may be possible to modulate gene expression, control the localization of RNA, alter the composition of RNAs and enable imaging of specific RNA. In this paper, the mechanism of CRISPR-Cas9 system-mediated immunity was introduced, in particular, we compared RCas9 to previous methods for RNA targeting and discussed potential uses ranging from live imaging of transcriptional dynamics to regenerative therapies and applications in synthetic biology, and finally prospected the application of this new system in life science, which was expected to provide reference for CRISPR-Cas9 system application in RNA editing.

Application of SSR and SNP Molecular Marker in Litchi Breeding
LIU Wei, JIANG Nonghui, YUAN Peiyuan, QIU Yanping, FAN Chao, YANG Xiaoyan, XIANG Xu*
2017, 7(1): 7-12. DOI: 10.3969/j.issn.20952341.2017.01.02
Abstract ( ) PDF (1121KB)( )

Litchi breeding has been mainly resort to seedling selection and phenotype selection for a long term. Considerable difficulties in the initial stage and slow progress of genetic improvement in litchi were mainly due to its unclear genetic background of germplasm, chaos of cultivar name, lack of high efficient modern technology such as early identification of hybrids and functional gene exploration. It is urgent to develop and improve marker-assisted breeding in litchi, so as to overcome the obstacles of traditional breeding and provide new technical support for genetic improvement in litchi breeding. In this paper, we summarized the application of litchi SSR and SNP, two kinds of specific molecular marker, on germplasm genetic relationships analysis, core collection construction, germplasm accurate identification and molecular barcode construction, hybrid identification, as well as genetic map construction. The paper was expected to provide theoretical and practical reference for the breeding application of specific molecular markers in litchi.

Progress on Pharmacological Effects of Flavonoids from Propolis
GHUNIQIGUL Abake, AYNUR Yusup, MAYNUR Dawut, QIMENGUL Turghun, YIMIT Rahman*
2017, 7(1): 13-19. DOI: 10.3969/j.issn.20952341.2017.01.03
Abstract ( ) PDF (1144KB)( )

Flavonoids are secondary metabolites which are widely existed in natural plants. It has a strong effect on health care. Propolis is a natural product, collected by honeybees from various plant sources. This paper summarized the function of flavonoids in propolis, such as antitumor, injury protection, lower blood sugar levels, anti-oxidation and so on, which was aimed to provide reference in pharmacological effect of propolis flavonoids.

Progress on Selecting, Breeding and Fermentation of Trametes versicolor Strains and Polysaccharide Extraction Technology
LI Xiaokang, DAI Jingsha, LI Chengbin, CHEN Ru, QIU Xiaoying*
2017, 7(1): 20-24. DOI: 10.3969/j.issn.20952341.2017.01.04
Abstract ( ) PDF (1108KB)( )

Trametes versicolor is a Chinese traditional medicinal fungus. Polysaccharide krestin, the main active component of T. versicolor, has been widely used in the field of food and medicine. Therefore, its biological functions have been paid more and more attention. However, there are some problems in selection and breeding of superior T. versicolor strains and polysaccharide yield, which restricts the development of T. versicolor industry. In this paper, the selecting and breeding of T. versicolor strains, fermentation and polysaccharide extraction technology were summarized, which was expected to provide a reference for the optimization strategy research of high-yield T. versicolor polysaccharide in the future.

The Epidemiological Situation, Prevention and Control of Tuberculosis Complicated with Diabetes Mellitus
JIANG Hui1,2,3, LIU Yuqin1,2,3
2017, 7(1): 25-29. DOI: 10.3969/j.issn.20952341.2017.01.05
Abstract ( ) PDF (1100KB)( )

Tuberculosis and diabetes are common and frequent diseases. Due to the rapid growth of the number of patients with diabetes, the double burden of this cross is affected by the emergence of the TB epidemic. The immune dysfunction caused by diabetes, which can increase the susceptibility to tuberculosis. Patients with severe tuberculosis, accelerate the course of diabetes. This paper reviewed the current situation of the prevalence of tuberculosis combined with diabetes. We also summarized the possible mechanisms of the two disease merger, detection methods, prevention and control measures. Finally, the paper made a forecast of the further prospects on the development of two disease prevention and control.

Functional Analysis of Plastidial Folylpolyglutamate Synthetase in Arabidopsis Under N-limited Conditions
XU Bosi1, MENG Hongyan2, ZHANG Chunyi1*, JIANG Ling1
2017, 7(1): 30-37. DOI: 10.3969/j.issn.2095-2341.2017.01.06
Abstract ( ) PDF (4382KB)( )

Folylpolyglutamate synthetase (FPGS) catalyzes the addition of glutamate residues to the γ-carboxyl of tetrahydrofolate in different positions, to form folylpolyglutamates. AtDFB is the FPGS that located in plastid of Arabidopsis thaliana. In this study the Arabidopsis T-DNA insertion mutant (Atdfb-3) of AtDFB gene was used to explore the function of AtDFB gene by comparing the phenotype of wild type and atdfb-3 under various nitrogen conditions. The results showed that Atdfb-3 mutant displayed shorter primary root length and decreased folate content, compared with the wild type under the nitrogen-limited condition. The phenotype of AtDFB overexpression line under Atdfb-3 background restored to the wild-type level, and the folate content increased under the nitrogen-limited condition. In conclusion, Arabidopsis AtDFB gene plays an important role in folate biosynthesis pathway, and affects the primary root development under the nitrogen-limited condition.

Cloning and Expression Analysis of ZmERF1 in Maize
SHI Qingling1,2, DONG Yongbin1*, ZHOU Qiang1, MA Zhiyan1, QIAO Dahe1, DENG Fei1, LI Yuling1*
2017, 7(1): 38-42. DOI: 10.3969/j.issn.2095-2341.2017.01.07
Abstract ( ) PDF (2850KB)( )

ERFs are downstream component in ethylene signaling and involves in plant responses to ethylene. In this study, an ERF gene, designated as ZmERF1, was isolated from 20 DAP endosperm of inbred Dan232 (Zea mays L.) and its sequence and expression characteristics were analyzed. ZmERF1 contains 811 bp with a 690 bp ORF, and encodes 230 aa with a single conserved AP2 domain. Online protein structure prediction revealed that there are three β-sheets and one α-helix in the AP2 domain. The ZmERF1 localized in the nucleus by online protein prediction. ZmERF1 was closely homologous with sorghum SbERF and rice OsERF. Prokaryotic expression and Western Blot analysis showed that ZmERF1 proteins could be obtained in vitro. The expression level of ZmERF1 proteins in early-stage was lower than that in later-stage of endosperm (36 DAP) in Dan232. ZmERF1 may play a key role in later-stage of endosperm development in maize. The results of this work uncovered characteristics of ERFs in maize, and provided a basic for exploring their biological functions in grains development.

Studies on Promoting Ability and Induced Resistance of Biocontrol Bacteria in Rice
ZHANG Xiaofang1, AI Ying2, WEI Lanfang3, SHI Gonglin3,WANG Xing1, LI Fan1, JI Guanghai1*
2017, 7(1): 43-51. DOI: 10.3969/j.issn.2095-2341.2017.01.08
Abstract ( ) PDF (2348KB)( )

Four biocontrol bacteria strains were evaluated for inhibition ability in rice against Xanthomonas oryzae pv. oryzicola(Xooc). Promoting ability of four treatments including seeds soaking, shoots soaking, seedings irrigation and seed pelleting with biocontrol bacteria was studied. At the same time, the induction of three protecting enzymes such as peroxidase, polyphenoloxidase and phenylalnine ammonialyase were studied by application of biocontrol bacteria. The results showed that the four strains had the growth promoting effect and induced resistance of rice seedlings. And the induced resistance and the growth promotion of the strain WY2 was better than other strains. Biocontrol bacteria could enhance the activities of plants protective enzymes as PAL, POD and PPO, and induce plant disease resistance when inoculating Xoc pathogens 24 hours after inoculation of biocontrol bacteria in rice seedlings.

Alteration of Rat Intestinal Pathology and Flora Based on the Chronic Stress Model
WANG Hua, WANG Zhihong*
2017, 7(1): 52-57. DOI: 10.3969/j.issn.2095-2341.2017.01.09
Abstract ( ) PDF (5917KB)( )

To establish the rat chronic stress model using the chronic stress method, and further observe the variation rule of rat intestinal pathology and its flora based on HE staining and ERIC-PCR technique. A total of 10 healthy male SD rats were chosen, and randomly divided into two groups: Control group and model group, 5 rats in each group. Wherein, rats in control group were regular raised, and rats in model group were perform constraint stress for 4 hours everyday using a constraint tube for 30 days, and the rats body weight was recorded before or after modeling, and then performed the behavioral evaluation. In addition, rats were killed, and the intestinal tissues and contents were collected, and then sliced to stain by HE staining, and the genomic DNA was extracted to perform ERIC-PCR assay. Results showed that, compared to control, the body weight, the number of through, upright, grooming significantly decreased, the no moving time of forced swimming significantly prolonged, and sugar water preference significantly decreased. Furthermore, the microvilli of intestinal tract were seriously damaged, and nuclei mild contraction and deeply staining. The intestinal flora obviously changed, and several characteristic bands appeared. Rats chronic stress model was correctly established using chronic constraint stress methods and behavioral evaluation, and provided a significant reference on the study of the chronic stress associated diseases based on pathological assay and intestinal flora expression profile alteration, and exhibited a certain application value.

Isolation and Characterization of a Feather-degrading Bacterium and Cloning of Keratinase Gene
CHEN Xiaotang, LIU Chao, HOU Yanying, ZHANG Xianzhong, LI Yanling, GUO Donghui, SHI Renjiu*
2017, 7(1): 58-64. DOI: 10.3969/j.issn.2095-2341.2017.01.10
Abstract ( ) PDF (2125KB)( )

This study was aimed to isolate and characterize feather-degrading bacteria from soil of discarded feathers. Morphological observation, biochemical analysis and 16S rDNA sequence analysis were used for identifying bacteria, and the enzyme activity and enzyme properties was studied by mensurating protein hydrolysis. The strain was identified as genus Pseudomonas, Stenotrophomonas maltophilia. The optimal pH and temperature for growth were 7.0 and 30℃, optimal inoculum size is 1%, optimal fermentation time is 48 h. The enzyme activity was highest and stable when the feather content was 0.25~0.5 g/50mL. The keratinase gene was cloned which included complete sequence of 2 157 bp and a 1 734 bp open reading frame encoded 580 amino acids from Stenotrophomonas maltophilia. Keratinase of Stenotrophomonas maltophilia has the potential for the application of feather keratin sources for the functional temperature and pH is broad and stable.

Functional Analysis of Factor Sig2 Responding to High Temperature Stress of Deinococcus radiodurans
TIAN Ye1, WANG Lin1,2, ZHOU Zhengfu1, ZHANG Wei1, CHEN Ming1*
2017, 7(1): 65-71. DOI: 10.3969/j.issn.2095-2341.2017.01.11
Abstract ( ) PDF (2549KB)( )

σ factor, a subunit of RNA polymerase in prokaryotes, can activate transcriptional initiation through a kind of reversible bind with the active catalytic sites of RNA polymerase core enzyme，promote the accurate identification of RNA polymerase to transcriptional initiation site and eventually activate the expression of corresponding genes specifically. With the strong capacity and environmental adaptability to radiation, oxidation and high temperature，Deinococcus radiodurans（D. radiodurans）contains three σ factors，among which Sig2 may function significantly in its strong environmental suitability. In this study，we developed the sig2 deletion mutant strain，named Δsig2, results showed that Δsig2 were more sensitive to heat and had significantly lower survivability than control group D. radiodurans wild type under the condition of high temperature（48℃）stress. We also analyzed the QRT-PCR results and verify that the deletion of sig2 contributed to positive or negative regulation of heat stress related genes in a degree. As a result，we speculated sig2 probably involved in the regulation of heat stress response.

Optimization of Electroporation Conditions for Escherichia coli Strain TG1
JIANG Huabo1, XIA Ziyuan1, ZHANG Qiongge2, WANG Chaoqun2, ZHENG Jiaoyang2, LU Bin1*
2017, 7(1): 72-76. DOI: 10.3969/j.issn.2095-2341.2017.01.12
Abstract ( ) PDF (1567KB)( )

To build up a stable protocol of electroporation transformation method with high efficiency and improve storage capacity of genomie library, we investigated the factors affecting the transformation efficiency, including culture temperature, growth state, voltage, the density and volume of competent cells, the amount of the exogenous gene and the volume of glycerol/mannitol buffer solution, which may contribute to construct highly diverse library. Results showed that when cells growing under 16℃ and harvested in mid-exponential phase (OD600 of 0.5), using glycerol/mannitol density gradient centrifugation techniques and adjusting the cell density up to 1×1011/mL, the electroporation voltage of 14.25 kV/cm, the highest transformation efficiency could reach to 9×109 CFU/μg DNA. The results showed that the optimal conditions for electroporation transformation would provide an important access for high-complexity library construction.

Study on Supercritical Carbon Dioxide Extraction of Total Flavonoids from Mulberry Leaves
SHI Tongyu1, MIAO Weiwei2,3, WANG Zhixiong1,3*
2017, 7(1): 77-84. DOI: 10.3969/j.issn.2095-2341.2017.01.13
Abstract ( ) PDF (4771KB)( )

To explore the optimal plan of extracting total flavone from mulberry leaves through the method of supercritical carbon dioxide, this research utilized supercritical carbon dioxide extraction technology and applies response surface method to examine the influence of extraction pressure, extraction temperature, extraction time and flow velocity of entrainer absolute ethyl alcohol on yield of total flavone in mulberry leaves, and selected the optimal technology that mulberry leaves flavonoids extract using supercritical fluid extraction device. The results indicated that, the optimal extraction condition of response surface method was as follows: pressure in optimal extraction plan was 26 MPa, temperature was 50℃, time was 3 hours, and flow velocity was 2.5 mL/min. The predicted total flavone yield under optimal condition was 6.21%±0.05%.

Determination of Trehalose in Transformation Liquid by High Performance Liquid Chromatography
SONG Jinsong, MA Chunyan, ZHAO Wei*, DUAN Yingying, FANG Jian, CAO Dapeng
2017, 7(1): 85-89. DOI: 10.3969/j.issn.2095-2341.2017.01.14
Abstract ( ) PDF (1506KB)( )

Transformation liquid was obtained from the reaction of crude enyme with maltose as oligomer. The crude enyme was fermented by trehalose synthase producing strain which was selected from soil. The concentration of trehalose in the transformation was determined by HPLC with Sugar-PakTM Ⅰ. Results showed that the method could separate the trehalose and glucose completely, accordance with the result of DNS colorimetric method of 1.5g/100mL, but the HPLC operation process and data processing were simpler and easier to control in trehalose content determination.

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