Current Biotechnology

Advance in Research of Centromeres in the Meiotic Process
ZHANG Feng1, LIN Juan2, ZHAO Jun1*
2013, 3(4): 233-237. DOI: 10.3969/j.issn.2095-2341.2013.04.01
Abstract ( ) PDF (1673KB)( )

Centromeres are the important functional elements of chromosomes in eukaryotes, their roles played in mitosis and meiosis processes have been paid more and more attentions. Though the chromosomes vary greatly in DNA sequences, their roles are conserved in all eukaryotic organisms, which make sure the faithful segregation of chromosomes and correct cell division. With the continuous development of molecular biology techniques, we have a deep understanding of the functions of centromeres. Here we review the function of centromeres during the meiotic progress.

Progress on Three Defence Genes eds1, atr/nrc1 and ahl19
QI Xi-liang, CHENG Hong-mei*
2013, 3(4): 238-22. DOI: 10.3969/j.issn.2095-2341.2013.04.02
Abstract ( ) PDF (1432KB)( )

Defence genes play an important role in plant resistant to disease. In this paper we introduce three defence genes' (eds1,atr/nrc1 and ahl19) function in defenses to pathogens and the role in their signal pathway. EDS1 encodes an essential component of disease resistant R genes in salicylic acid dependent resistance pathway TIR-NB-LRR class. AHL19 acts as a positive regulator of plant defense. NRC1 is also required for the HR pathway induced by the R proteins Cf-9, Pto and Rx. It provides theoretical basis to explore the mechanism of plant resistance to disease, especially to the plants which lack the antigen to the disease such as cotton Verticillium Wilt. It is possible to breed new varieties with high resistance to Verticillium Wilt of cotton by using those defence genes.

Research Situation of Mutagenesis Breeding in Millet
ZHANG Wen-xing, ZHAO Jin-feng
2013, 3(4): 243-247. DOI: 10.3969/j.issn.2095-2341.2013.04.03
Abstract ( ) PDF (1164KB)( )

Being oen of modern breeding techniques, mutagensis breeding is an important supplement of select breeding and crossbreeding. Mutation breeding technology is rarely applied in millet. In this paper, we introduced the mutagenic mechanism and biological effects of chemical mutagenesis such as the ethylmethane sulfonate (EMS), diethyl sulfate (DES),etc, and physical mutagenesis likes ion beam implantation, radiation treatment, space induction techniques and so on used in millet. Then we analyzed the current status and problems of mutagenesis techniques in millet research and application, and propose the development direction and key research areas of millet mutagensis breeding in the future.

Distinguishment of Seeds Impurity and SSR Loci Heterozygosis in The Three Line Hybrid Cotton Using Multiplex PCR-SSR Markers
MA Wei-jun, REN Ai-min*, YIN Guo, ZHANG Yu-juan, HAN Qiu-cheng, CUI Ming-hui
2013, 3(4): 248-251. DOI: 10.3969/j.issn.2095-2341.2013.04.04
Abstract ( ) PDF (2956KB)( )

Hybrid seed purity decides the hybrid yield. The conventional identification method lasts long time and can not display the seed purity promptly, so not to meet the production need. Since it is difficult to distinguish the bands pattern differences of hybid lines caused by mixed seeds and SSR loci heterozygous, in order to improve the accuracy of hybrid purity identification, 4 pairs of SSR primers showing stable polymorphism in the parents were selected from 196 pairs of primers, and combined according to the characteristic of PCR banding pattern, the results showed that the multiplex PCR-SSR technology could distinguish hybrid seed impurity and SSR loci heterozygous, so as to provide technical support for rapid and accurate seed purity identification of hybrids.

Expression of Porcine Interferon-alpha in Silkworm, Bombyx mori and its Antiviral Activity Detection
ZHONG Lu-long1, YI Yong-zhu2, DING Nong3, ZHANG Jin-wei3, LIU Ji-xing1*
2013, 3(4): 252-256. DOI: 10.3969/j.issn.2095-2341.2013.04.05
Abstract ( ) PDF (2493KB)( )

Porcine interferon-alpha(PoIFNα) was expressed in silkworm larvae using silkworm-baculovirus expression system. The porcine interferon-alpha gene was artificially synthesized after codon optimization. The optimized target gene was cloned to the baculovirus transfer vector pVL1393 and the recombinant plasmid pVL-PoIFNα was constructed. pVL-PoIFNα was co-transfected with linearized Bm-BacPAK6 DNA into Bm-N cells. The recombinant proteins were successfully detected in hemolymph by Western blot analysis. The recombinant PoIFN-alpha expressed in hemolymph were verified to be of high antiviral activity by inhibiting the cytopathic effect of VSV*GFP in Vero cells, which was about 106 U/mL.

Analysis on Codon Usage Bias of aiiA Gene from Bacillus thuringiensis
ZENG Shi-yong, SU Xiao-hui, XIE pan-pan, MAO Hui-min, YANG Mei*
2013, 3(4): 257-263. DOI: 10.3969/j.issn.2095-2341.2013.0 .
Abstract ( ) PDF (4606KB)( )

AiiA, encoded by aiiA gene is one of the homoserine lactone enzyme that can hydrolyze the AHL on the lactone ring of Gram-negative bacteria. Coding sequence of aiiA gene from Bacillus thuringiensis was analyzed by CodonW, CUSP and CHIPS programs for identifying codon bias. Comparing the codon usage of aiiA gene with genomic codon usage of Escherichia coli, Bacillus thuringiensis, Bacillus subtilis and Pichia pastoris, it was indicated that aiiA gene prefers to use the codons ended with A or T; codon bias of aiiA gene from Bacillus thuringiensis was significantly different from those of E.coli, Bacillus subtilis and Pichia pastoris. The result of comparing the usage frequency of aiiA gene in four different hosts showed that there were different degrees of low frenquency regions. The analytic result was very important guideline for designing the aiiA gene to improve the expression of AiiA in those four express systems.

Isolation of a Pullulanase Producing Strain and its Gene Cloning and Enzymatic Properties
WANG Pei-li, CHU Xiao-yu*, WU Ning-feng
2013, 3(4): 264-269. DOI: 10.3969/j.issn.2095-2341.2013.04.07
Abstract ( ) PDF (2183KB)( )

As a starch debranching enzyme, pullulanase can specifically hydrolyze α-1,6 glucosidic linkages in polysaccharide to change amylopectin into amylose. One strain (No.73) with the higher pullulanse activities was selected from the thermal spring in Tengchong, Yunnan Province. The strain was identified as Anoxybacillus sp. by 16S rDNA phylogenetic tree analysis. The full-length sequences of the pullulanase gene from Anoxybacillus sp.73 was obtained by PCR amplification with the degenerate primers designed based on the conservative domains of pullulanase from Bacillaceae. The pullulanase gene pul73 contained 2 121 nucletides, comprising one open reading frame encoding a polypeptide of 706 amino acids. The pul73 gene was cloned and expressed in E. coli BL21 (DE3). The recombinant protein Pul73 was purified by Ni-NTA column and characterized. The optimal pH and temperature of Pul73 were 6.0 and 50℃ respectively. The enzyme showed stability at 65℃ after incubating for 30min. The K_m, V_max and k_cat of the purified Pul73 with pullulan as the substrate were approximately 1.643 mg/mL，1.34 U/mg and 535.8/s, respectively.

Study on CMCase Production of Trichoderma W2 by Solid-state Fermentation
REN Hong-mei, LIU Zuo-jun*, WU Guo-qiang, SHAN Xi-wen
2013, 3(4): 270-276. DOI: 10.3969/j.issn.2095-2341.2013.0 .
Abstract ( ) PDF (2544KB)( )

A strain W2 was separated from the rotted cotton of Hubei province. According to morphology and 18S rDNA sequences analysis, strain W2 belongs to Trichoderma sp. Corn cob was used as substate to optimize solid-state fermentation (SSF) conditions for CMCase production. Four parameters including fermention time, temperature, initial moisture content and pH were optimized and the CMCase activity was acted as response value. The interaction of the respective factors and their influences on CMCase were studied by Box-Benhnken central composite design and response surface methodology, the quadratic equation and response figures were established. The optimal fermentation conditions were obtained based on single-factor tests. Under culture temperature of 25℃, initial moisture content of 63% and pH 6.0, CMCase achieved its high value after 65h fermentation. At last, synchronous fermentation was adopted to produce alcohol, and the alcohol content reached 0.38 mg/mL determined by improved Conway method.

Isolation of Nattokinase Producing Strain and Analysis of its Thermal Stability
ZHAO Zhong-lin1§, LI Shu-ying2§, NIE Ying2, YUAN Chao1, LI Yan3, TANG Xuan-ming2*
2013, 3(4): 277-280. DOI: 10.3969/j.issn.2095-2341.2013.0 .
Abstract ( ) PDF (1176KB)( )

With the Japanese natto strain as control, a natto strain, named BSNK-5, was isolated from Chinese black lobster sauce. The activity of BSNK-5 fermentation broth relative to urokinase activity is 509.64 IU/mL, which increased 83.87% compared to control strain. Production cycle of BSNK-5 fermented natto is shorter, and the natto is golden color with soy sauce flavor. Nattokinase synthesized by BSNK-5 has good thermal stability, which retained about 10% of the activity under 70℃ for 20 min. It is suggested that BSNK5 has great market potential.

Isolation of Bacteria in the Saline Alkaline Soils of Akesu in Xinjiang
LI Liang-qiu1, ZHEN He-yun1, CAI Zhuo-ping1, YAO Qing2, ZHU Hong-hui1*
2013, 3(4): 281-287. DOI: 10.3969/j.issn.2095-2341.2013.04.10
Abstract ( ) PDF (2018KB)( )

In order to understand the diversity of microbial resources in culture media from the saline soil of Tarim Basin in Xinjiang, we isolated and cultured 103 strains of the bacteria from the saline alkaline soils of Akesu in Xinjiang. 16S rDNA sequence phylogenetic analysis showed that these strains belong to the genus Bacillus and other 31 genera. The dominant strains were Bacillus, Streptomyces, Micrococcus, Kocuria and Paracoccus，which occupied 36.27%, 10.8%, 6.9%, 6.9% and 3.9% of the total strains. 16S rDNA of five strains had less than 97% sequence similarity to those published strains, suggesting that they might be new bacterial taxa. The results showed that there were abundant micriobial resources in the saline alkaline soils of Aksu in Xinjiang, providing an important base for the development of microbial resources in saline alkaline soils.

Isolation and Identification of a Salt-tolerance Myxobacteria
ZHANG Xian-jiao, XIE Xiao-lin, CHEN Mei-biao, DUAN Jin-mei, LI Yan-xuan, ZHU Hong-hui*
2013, 3(4): 288-292. DOI: 10.3969/j.issn.2095-2341.2013.04.11
Abstract ( ) PDF (3264KB)( )

Base on conventional isolational method, a salttolerance myxobacteria was isolated from the saline-alkaline soils of Akesu in Xinjiang. According to the morphologies of swarms, fruiting bodies,vegetative cells, myxospores, physiological and biochemical characteristic and the sequence of 16S rDNA, the strain was identified to Corallococcus macrosporus. Analysis of salt-tolerance showed the strain 5x1 could survive in NaCl concentration of 2%.

Development of a Detctection Method for Mycoplasma in Manufacture of Recombinant Therapeutic Proteins
LIU Su-xia§, LIU Xiao-zhi§, YANG Li-xia, ZHOU Wei, CHANG Liang, ZHOU Xing-jun*, GAO Jian*
2013, 3(4): 293-296. DOI: 10.3969/j.issn.2095-2341.2013.0 .
Abstract ( ) PDF (1766KB)( )

For the detection of mycoplasma contamination in Chinese hamster ovary cells (CHO) cell culture, a rapid, accurate, and stable PCR detection method was established. A housekeeping gene encoded glyceraldehyde 3-phosphate dehydrogenase was used as a reference gene, and the internal reference primers and primers for mycoplasma detection were placed in a PCR system simultaneously. The concentration ratio of two kinds of primers was optimized as 5 μmol/L reference gene primers and 10 μmol/L mycoplasma detection primers. PCR method had consistent results with DNA staining method, with less false negative result and short detction time, showing that it could meet the demand for mycoplasma contamination detection in the production process.

A Rapid Detecting Method for Enterobacter sakazakii based on F₀F₁-ATPase Molecular Motor Biosensor
ZHANG Jie1, WANG Yu2, LU Lin1, LIU Yan-hua1, ZHOU qi1, SUN Zi-yan1, GU De-zhou1, SHANG Shi-jin1, ZHANG Hui-yuan1, WANG Pei-rong3, CHEN Guang-quan1, YUE Jia-chang3
2013, 3(4): 297-300. DOI: 10.3969/j.issn.2095-2341.2013.0 .
Abstract ( ) PDF (1391KB)( )

A rapid detecting method for Enterobacter sakazakii based on F₀F₁-ATPase molecular motor biosensor was cons tructed. Specific ITS probe were connected with F₀F₁-ATPase's ε subunit by using avidin-biotin system, and then biosensors were combined with the test samples and negative sample, respectively. To compare the catalytic ATP synthesis, Enterobacter sakazakii DNA in the samples were tested. The result showed that the optimum conditions for detection were the concentration of chro ITS of 0.019 mg/mL and Enterobacter sakazakii DNA concentration of 40 ng/mL. The result was consistent to that of the traditional detection methods and PCR detection method, showing this is in good agreement.

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