Construction of quantitative real-time PCR detection system of transgenic tomato line Zeneca B,Da,F / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the plasmid reference molecules for detection of transgenic tomato line Zeneca B,Da,F using quantitative real-time PCR(qPCR).</p><p><b>METHODS</b>Three plasmid reference molecules pEasy-T3-APX, pEasy-T3-16A and pEasy-T3-16S were cloned based on reverse genetics, which contain the target fragments of tomato endogenous reference gene apx (ERG-apx), gene-specific sequence of pg(GS-pg) and construct-specific sequence of vectors pJR16S/pJR16A (CS-16S/CS-16A) of Zeneca B,Da,F, respectively. Primers and Taqman probes were designed by Beacon Designer 7.5.The specificity, sensitivity, reproducibility and the limit of detection(LOD) of the qualitative and quantitative PCR system based on the plasmid reference molecules were evaluated. PicoGreen was used to measure the DNA concentration of the plasmid reference molecules. Two sets of samples containing 1% or 0.1% (w/w) pEasy-T3-16A or pEasy-T3-16S mixed with pEasy-T3- APX as background DNA were prepared for evaluating the efficacy of the qPCR system.</p><p><b>RESULTS</b>The target fragments for qPCR detection were anchored, ERG-apx 108 bp, GS-pg 108 bp , CS-16S 109 bp and CS-16A 102 bp. The three plasmid reference molecules were confirmed at the expected sizes by restriction enzyme digestion. The qPCR results showed that the RSD of reproducibility were 0.2% to1.5%, LOD was 25 copies, R2 values for these standard curves were 0.994 ~0.998 and amplification efficiencies were 93.3%~102.4%.The bias between the test and true values of two sets of mixed samples ranged from -9.3% to 14.7% after adjusting by conversion factors(Cf).</p><p><b>CONCLUSION</b>The plasmid reference molecules and qPCR system for qualitative and quantitative detection of transgenic tomato line Zeneca B,Da,F have been established successfully.</p>

Optimization of tomato genetic transformation, kanamycin-resistant screening and seed selection / 南方医科大学学报

<p><b>OBJECTIVE</b>To optimize the process of tomato genetic transformation, screening and seed selection using multiepitope antigenic gene (MAG) and truncated major surface antigen 1 (tSAG1) of Toxoplasma gondii as the target insert genes.</p><p><b>METHODS</b>Tomato high-frequency regeneration system was optimized with different choices of media and explants. The genetic transformation procedure was optimized using different tomato cultivars, explants, culture temperatures, media and acetosyringone (AS) supplements. Three concentrations of kanamycin were utilized for resistant selection of the transgenic candidate roots. The selected lines were trained, transplanted to soil and grown in a greenhouse till maturity. Sterile seeding using kanamycin-incorporated medium was conducted for screening transgenic tomato generations.</p><p><b>RESULTS AND CONCLUSION</b>Cotyledons were better than hypocotyls as the regeneration explants. The regeneration rate of cotyledons reached 98% (59/60) using the optimized regeneration medium ZB3. The culture medium and temperature were the key factors for tomato transgenic shoot induction. The number of transgenic buds increased significantly at the appropriate temperature condition (23-/+1 degrees celsius;), and AS of 100 micromol/L in the medium before inoculation also significantly raised transformation rate. The budding rate of Zhongshu No.5 cotyledons was 35% (28/81) using the medium ZB2 under (23-/+1) degrees celsius;. Kanamycin at 80 mg/L was optimal for transgenic plantlet rooting selection with the rooting rate of 48% (31/65). 117 transgenic lines were obtained. Non-transgenic tomato plant growth, especially the root and elongation, was inhibited obviously with kanamycin at 100 mg/L or above, and the roots became purple and lacked lateral roots. The transgenic tomato seeds could be selected effectively with kanamycin at 150 mg/L.</p>

Automatic separation system for marine meiobenthos based on laser-induced fluorescence technology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

An automatic system for marine meiobenthos separation was developed by using laser-induced fluorescence technology. Rose Bengal was used as organism dye and the spectrums of Rose Bengal were measured. Laser-induced fluorescence system was established to detect marine meiobenthos in sediments. Data obtained from experiments were analyzed by using a mathematical model. The results showed that laser-induced fluorescence technology worked well in the system. The system could select the meiobenthos efficiently and precisely.