Optimization of Agrobacterium-Mediated Genetic Transformation System of Armillaria gallica / 中国实验方剂学杂志

ObjectiveTo optimize the existing genetic transformation system of Armillaria gallica to improve the transformation efficiency and lay a foundation for the follow-up research on Armillaria molecular marker-assisted breeding and gene function. MethodThe genetically transformed plasmid pH101-PAgGPD-GFP-TrpC was constructed，transformed into Escherichia coli，amplified， and cultured，and the plasmid was extracted. The extracted plasmid was transformed into four different agrobacteria LBA4404，EHA105，GV3101，and AGL-1，respectively. The transformed agrobacteria were used for impregnating A. gallica，and the agrobacteria with the highest conversion rate were screened out. Then the agrobacterium-mediated genetic transformation system of A. gallica was optimized from the type and concentration of antibiotics，co-culture time，concentration of bacterial solution， and impregnation method. The phenotype profiles of A. gallica under different conditions were observed using Synbiosis ProtoCol 3. ResultThe optimized genetic transformation conditions of A. gallica were as follows： the Agrobacterium strain of EHA105 at absorbance A600 nm=0.6， the co-culture time of 2 d， the infection mode of negative pressure impregnation for 10 min， the primary screening medium of PDA medium containing 400 mg·L-1 cefotaxime sodium and 10 mg·L-1 hygromycin，and the secondary screening medium of PDA medium containing 12 mg·L-1 hygromycin. ConclusionIn this study，the existing genetic transformation system of A. gallica was optimized，and there was a significant difference in the transformation rate before and after optimization （P<0.05）. After optimization，the transformation efficiency of A. gallica was about 4.33%，which was about eight times higher than that before optimization.

Developing biosecurity training program under the background of new engineering education in colleges and universities / 生物工程学报

The global biosecurity situation has become increasingly severe in recent years. The threats from outbreaks of epidemics, misuse and abuse of biotechnology, biological weapons and so on are emerging, which make biosecurity an important part of national security. Meanwhile, as a new interdisciplinary program, the education for biosecurity is still in its infancy. The challenges of domestic and international biosecurity and the quick development of biosecurity industry necessitate the establishment of security disciplines, which expose the deficiencies of talent pool in the interdisciplinary field involving biosecurity in China. Especially under the background of new engineering education, the importance of biosecurity training program has become more obvious than before. On the basis of our teaching experience, we systemically introduced the specific measures to improve the training system of biosecurity in the context of new engineering education, such as establishing an integrated curriculum system, constructing innovative teaching modes, exploring general education reform plans, and perfecting continuous improvement mechanisms.

Optimization of SRAP-PCR System for Valeriana officinalis var. latifolia and Primer Screening / 中国实验方剂学杂志

Objective:To establish the sequence-related amplified polymorphism （SRAP）-polymerase chain reaction （PCR） system for <italic>Valeriana officinalis</italic> var. <italic>latifolia</italic>，so as to lay the theoretical and technical foundations for the breeding of<italic> V. officinalis </italic>var. <italic>latifolia</italic>. Method:Single factor test was applied to investigate the effects of <italic>Taq</italic> Mix dose，Mg²⁺concentration，template DNA concentration，and <italic>Taq </italic>DNA polymerase content on SRAP-PCR amplification of <italic>V. officinalis </italic>var. <italic>latifolia</italic>，based on which the orthogonal experiments were performed to optimize the SRAP-PCR system for <italic>V. officinalis </italic>var. <italic>latifolia</italic>. The effective primers that could be used for genetic diversity studies of <italic>V. officinalis</italic> var. <italic>latifolia </italic>were selected under the optimal reaction condition. Result:The results of the single factor test showed that <italic>Taq </italic>Mix dose within the range of 8-11 μL resulted in better amplification. The addition of a low concentration of Mg²⁺，the medium to low concentrations of template DNA，or the low concentration of <italic>Taq</italic> DNA polymerase enhanced the amplification efficiency or richness. As demonstrated by the orthogonal experiments，the influencing degrees of related factors on SRAP-PCR amplification of <italic>V. officinalis</italic> var. <italic>latifolia </italic>were sorted in a descending order as follows： <italic>Taq</italic> Mix dose><italic>Taq</italic> DNA polymerase content>Mg²⁺ concentration>template DNA concentration. The optimal reaction system for <italic>V. officinalis</italic> var. <italic>latifolia </italic>was determined to consist of 11 μL of <italic>Taq</italic> Mix，30 ng of template DNA，0.025 mmol·L^-1Mg²⁺，1.5 U<italic> </italic>of<italic> Taq </italic>DNA polymerase，5 μmol·L^-1 forward primer，and 5 μmol·L^-1 reverse primer，which was supplemented to 20 μL with ddH₂O. The optimal annealing temperature was 36.8 ℃. A total of 17 pairs of effective primers with high band resolution and polymorphism were selected from 88 primer pairs for SRAP-PCR of <italic>V. officinalis</italic> var. <italic>latifolia</italic>. Conclusion:The established SRAP-PCR system for <italic>V. officinalis</italic> var. <italic>latifolia</italic> is stable， which can be used for genetic diversity studies of <italic>V. officinalis</italic> var. <italic>latifolia</italic>.

Relationship analysis for Ludisia discolor germplasm resources based on SCoT molecular marker / 中草药

Objective: To make a distinction between Ludisia discolor and its relatives genus in molecular level, SCoT markers were employed to assess the genetic relationship and construct the DNA fingerprint. Methods: Orthogonal design method were carried out to optimize the suitable SCoT-PCR reaction system based on five factors. The optimum annealing temperature and SCoT primers were also screened. The 12 germplasm resources were used as materials, the screened primers were selected to analyze the genetic relationship of 12 materials. POPGENE was used to calculate the genetic diversity, NTSYS was performed to analyze cluster, and DNA map was constructed. Results: The optimized SCoT-PCR reaction system was constructed and a total of 12 rich bands were screened out as the primers of SCoT molecular marker with polymorphism ratio of 98.98%. According to Nei's genetic similarity coefficient, a total of 12 materials were divided into three cluster when coefficient was 0.45. Goodyera schlechtendaliana was in category I with seven L. discolor lines, indicating that these samples had close relationship. In category II, there were three samples came from Anoectochilus roxburghii. Moreover, a green L. discolor sample was alone clustered into category III. The DNA fingerprint map by the SC8 primer could identify the 12 materials. Conclusion: There are rich genetic diversities in 12 samples of L. discolor and its relatives genus, and the construction of DNA fingerprint map provides a theoretical basis for the identification of L. discolor and its relatives genus, which were tested in this study.

Establish and optimization of inter-simple sequence repeat PCR reaction system of Gnaphalium affine / 药学实践杂志

Objective To provide the experimental basis for the subsequent genetic diversity research through establishing and optimizing the inter-simple sequence repeat PCR (ISSR-PCR) reaction system of Gnaphalium affine. Methods The single-factor experimental method and full experimental method were used to optimize the ISSR-PCR reaction system of Gnaphalium affine. Under the optimal system, after screening primers and corresponding annealing temperatures, the systematic feasibility was verified. Results The optimal ISSR-PCR reaction system was consisted of 10 μl Premix Taq DNA polymerase, 0.3 μmol/L primer, 10 ng DNA template, and sterilized water added to 20 μl. Finally, 10 primers were screened from 100 universal primers, and verification results indicated the system had high stability, good reproducibility, and the selected primers had good polymorphism. Conclusion The ISSR-PCR amplification system of Gnaphalium affine was established for the first time and the primers with appropriate annealing temperatures were filtered out, which provided a reference for the subsequent genetic diversity research of Gnaphalium affine.

Establishment and application of a drug screening model targeting somatostatin receptor 2 / 中国药理学通报

Aim To establish a cell model to detect the activity of somatostatin (SST) by targeting somatostatin receptor 2 (SSTR2), and to provide a simple and stable evaluation method for the drug screening of SSTR2 agonists and somatostatin analogues (SSTA). Methods The target gene of SSTR2 was integrated into the pEGFP-N3 vector, and the recombinant plasmid was constructed and transfected into HEK293 cells. After G418 screening, positive clone was selected and the stable cell lines were obtained by expanding culture. The stable cell lines were identified by fluorescence cell imaging, Western blot and qPCR. A calcium flow detection system was established to optimize cell number, fluorescence dye concentration and incubation time. Finally, the screening model was used to detect the different batches of the marketed somatostatin preparation Stilamin. Results SSTR2 stable cell lines were successfully constructed, and the receptors were mainly distributed on the cell membrane. The optimal conditions for calcium flow detection were determined as follows; 30 000 cells/Well, Fluo-4/AM indicator concentration was 3 p,mol • L -1 ~5 u,mol • L-1 , incubation time was 45 min. Under this condition, EC50 value of Stilamin in different batches was stable. Conclusions SSTR2 overexpressed stable cell lines are successfully constructed and calcium flow detection method is optimized to provide a simple and stable model for the screening of somatostatin receptor agonists.

SCoT-PCR Primer Selection and Reaction System Optimization of DNA of Marchantia convoluta in Guangxi / 中国药房

OBJECTIVE:To establish PCR reaction system of DNA of Marchantia convoluta in Guangxi marked with SCoT polymorphism marker technique by screening primer and optimizing reaction condition. METHODS:Modified CTAB method was used to extract DNA of M. convolute from Guangxi;gel electrophoresis and UV spectrophotometry were used to investigate purity and concentration of DNA. Using sample DNA as template,PCR amplification of 36 SCoT primers was conducted,and suitable primers were screened after electrophoresis,staining and imaging of products. The orthogonal experiment of 5 factors and 4 levels was conducted by 5 main factors as DNA concentration,Mg2 + concentration,dNTP concentration,primer concentration,Taq DNA polymerase concentration. The condition of SCoT-PCR reaction system was optimized. RESULTS:Extracted sample DNA bands were neat without RNA contamination,degradation or dispersion of fluorescence;sample well was clear. UV absorbance ratio ranged 1.7-2.0 at 260 nm and 280 nm;purity and concentration of DNA were both suitable for follow-up test. PCR results of 36 primers showed that product band of No. 4 primer was neat without diffuse fluorescence but with best luminance,so No. 4 primer was used for PCR reaction. The optimal SCoT-PCR reaction system contained 30.00 μg/mL DNA,2.00 mmol/L Mg2+,0.20 mmol/L dNTP, 0.40 μmol/L primer,0.50 U/mL Taq DNA polymerase(total reaction volume of 20 μL). CONCLUSIONS:Suitable SCoT-PCR primer of DNA is screened,and reaction system is optimized. It provides technologic basis for variety identification and genetic relationship analysis of M. convoluta in Guangxi.

To optimize dual molecular beacon detection system for rapid identification of Mycobacterium tuberculosis / 国际检验医学杂志

Objective To optimize the experimental system of dual molecular beacon to rapidly detect My-cobacterium tuberculosis and its resistant strains.Methods Fluorescence quantitative PCR was carried out by selecting different magnesium ion concentration,annealing temperature and primer concentration respectively. Finally,the optimum reaction conditions were obtained.Results In order to ensure the efficiency of amplifica-tion and no non-specific amplification,the final selection of the best conditions were as follows,the concentra-tion of Mg2+was 3.0 mmol/L,annealing temperature was 60 ℃,and the concentration of primers was 0.3 mmol/L.Conclusion The optimal condition of dual molecular beacon experiment was established,which en-sured that the detection of Mycobacterium tuberculosis by molecular beacon quantitative PCR had the advanta-ges,such as simple operation,rapid speed,high sensitivity(the minimum detection limit was 1 CFU/mL)and specificity(only Mycobacterium tuberculosis complex including drug-resistant strains could be detected),good reproducibility(coefficient of variation was < 5%)and other advantages.The study provides the necessary conditions for the dual molecular beacon detection of Mycobacterium tuberculosis.

Study on early screening of high saponin ginseng lines / 中国中药杂志

The study aims at screening the specific bands by PCR, quickly and accurately evaluating the quality of ginseng seeding, accelerating the process of ginseng breeding. Based on the correlation of genetic differences and saponin content between individuals, a pair of specific primer GC1 was screened by PCR. According to the experiment by L16 (45) orthogonal test, a PCR system most suitable for GC1 was established, which came out total 25 μL reaction system containing DNA 2.60 mg•L⁻¹, Mg²⁺ 1.44 mmol•L⁻¹, dNTP 0.19 mmol•L⁻¹, primer 0.32 μmol•L⁻¹ and Taq enzyme concentration 0.076 U•μL⁻¹. By comparing the saponin content and the GC1 PCR electrophoretogram of samples, the ginseng, with 1 200 bp specific band by PCR of GC1, the contents of 9 monosodium saponins and their additions were higher than others, which provided a reliable method for accelerating the process of ginseng breeding. The sequence was sequenced and 99% homologous to glycerol-3-phosphate dehydrogenase.

Suggestions for improving public appointment registration platforms in Beijing area / 中华医学图书情报杂志

After the advantages and disadvantages of appointment registration systems in different large hospitals of Beijing were compared, the problems in their appointment registration systems were analyzed and suggestions were put forward for establishing the public appointment registration systems with powerful systematization and compati-bility which are integrated into the EMR system of Beijing.

Establishment and optimization of RAMP-PCR reaction system for DNA of Eupolyphaga sinensis / 中草药

Objective: To provide the basis for the genetic diversity research of Eupolyphaga sinensis by establishing and optimizing the random amplified microsatellite polymorphism RAMP-PCR reaction system and amplification procedure for genomic DNA. Methods: Phenol chloroform extraction of genomic DNA was performed on female E. sinensis. Based on RAMP Primer (ISSR807 + RAPD A5), the orthogonal test was adopted to optimize the RAMP-PCR amplification system on E. sinensis in five factors (Mg2+, dNTPs, primer, Taq DNA polymerase, and template DNA) at four levels, and the suitable anneal temperature of the primer was selected. Results: The optimal Primer (ISSR807+RAPD A5) for RAMP-PCR system (25 μL reaction volume) of E. sinensis was: 10 × PCR buffer (2.5 μL), Mg2+ (1.0 mmol/L), dNTPs (2.0 mmol/L), primer (0.5 μmol/L), Taq DNA polymerase (1 U), and template DNA (1.5 ng/μL); The optimized anneal temperature was 46.8°C. Conclusion: The established and optimized RAMP-PCR reaction system is stable and repeatable, which could provide the basis for the analysis of germplasm resources and genetic diversity of E. sinensis.