Development of highly efficient electrocompetent cells for electroporation of Geobacillus thermoglucosidasius NCIMB 11955 / 生物工程学报

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×104 CFU/(μg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×106 CFU/(μg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.

Advances in Agrobacterium tumefaciens-mediated transgenic cucumber / 生物工程学报

Cucumber (Cucumis sativus) is an important vegetable crop in the world. Agrobacterium-mediated transgenic technology is an important way to study plant gene functions and improve varieties. In order to further accelerate the transgenic research and breeding process of cucumber, we described the progress and problems of Agrobacterium tumefaciens-mediated transgenic cucumber, from the influencing factors of cucumber regeneration ability, genetic transformation conditions and various additives in the process. We prospected for improving the genetic transformation efficiency and safety selection markers of cucumber, and hoped to provide reference for the research of cucumber resistance breeding and quality improvement.

An Enzymolysis-Assisted Agrobacterium tumefaciens-Mediated Transformation Method for the Yeast-Like Cells of Tremella fuciformis

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

Effect of cryopreservation on the efficiency of exogenous gene, genetic transformation and expression level of Arabidopsis thaliana

Background: Cryopreservation refers to the storage of a living organism at ultra-low-temperature for long-term preservation of plant germplasm. The effect of cryopreservation on the efficiency of exogenous gene genetic transformation and expression level were studied herein. In this work, transgenic Arabidopsis thaliana were successfully conserved in vitro by cryopreservation methods. Results: The effects of osmotic stress due to cryoprotectants during pretreatment and of storage at -196ºC on the stability, the efficiency of genetic transformation and the expression level of exogenous gene were analyzed in Arabidopsis. The results showed that there had not any significant increasing in the efficiency of genetic transformation after cryopreservation, and our observation was not in agreement with earlier reports. Transgenic Arabidopsis lines over-expressing ATOST1 gene were used for the real-time PCR analysis, and the result indicated that the expression of the ATOST1 gene was up-regulated about 2.4-fold in the transgenic seedlings tissues retrieved from cryopreservation than those non-cryopreserved counterparts. Conclusions： Cryopreservation could improve the expression of exogenous gene, however, could not promote the genetic transformation obviously.

Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.

The efficiencies of different transformation methods of E. coli DH5α strain, induced by several cations like Mg2+, Mn2+, Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.

Study on effect of Cassia Seed Budding transforming Organic Vanadium / 国际中医中药杂志

Objective To Explore the effect of Cassia Seed budding transforming organic vanadium. Methods Separate organic vanadium from inorganic vanadium in Cassia Seed sprouts by dislysis bag. The content of total vanadium and inorganic vanadium were determined by using the method of Graphite Atomic Absorption Spectrophotometry. The transformation efficiency of organic vanadium in the Cassia Seed spouts was calculated. Results The seed coat of Cassia Seed could accumulate massive inorganic vanadium existing in environment during the process of its natural growing period,but the transformation efficiency of organic vanadium mainly occurred inside seed coat of Cassia Seed. Condusion Cassia Seed budding is one pathway of the biologic organification of vanadium.

Protective Effect of KangAiZengXiao Decoction on Immunological Deficit Rats With Implanted Tumor / 中国医师杂志

Objective To study the effect mechanism of anticancer synergic (KangAiZengXiao,KAZX) decoction on gastric cancer in immunologic deficit rats with implanted tumor. Methods Forty rats were divided into normal control group(n=10), model group(n=15) and KAZX treating group(n=15). Model group and KAZX group were subcutaneously injected axillary the left upper limb with 0 5ml gastri cancer cells,MGC-803 cell(1?10 7/ml) after irradiated by 60 Co(dose:10Gy). Then the three groups were treated with KAZX decoction or distilled water every day, respectively. After three weeks, the rats were sacrificed,and blood WBC, the weight of spleen, activity of NK cell and transformation efficiency of lymphocyte were also detected.Results The rate of loaded tumor in KAZX group was significantly lower than in model group(20% vs 66 7%,P