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Li P.,Biotech Research Institute of Shanghai Academy of Agricultural science | Li P.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Jia J.,Biotech Research Institute of Shanghai Academy of Agricultural science | Jia J.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 6 more authors.
Applied Biochemistry and Biotechnology | Year: 2013

Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives. © 2013 Springer Science+Business Media New York. Source


Li P.,Biotech Research Institute of Shanghai Academy of Agricultural science | Li P.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Yang S.F.,Biotech Research Institute of Shanghai Academy of Agricultural science | Yang S.F.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 10 more authors.
Genetics and Molecular Research | Year: 2015

The demand for molecular analysis of aquatic microbial communities in freshwater has highlighted the need for efficient methods of DNA extraction. The centrifugation method and filtration-membrane method are 2 widely used methods for extracting DNA. The objective of this study was to compare the extraction efficiency of 3 methods, including the centrifugation method, filtration-membrane method, and modified filtration-membrane method, by evaluating the quantity and purity of DNA extracts obtained from water. DNA extraction was analyzed by agarose gel electrophoresis, ultraviolet-spectroscopy, restriction enzyme digestion, and polymerase chain reaction. The results showed that the modified filtration-membrane method was the most efficient for extracting microbial DNA from freshwater with high integrity and purity and is suitable for molecular applications. © FUNPEC-RP. Source


Li P.,Biotech Research Institute of Shanghai Academy of Agricultural science | Li P.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Dong J.,Biotech Research Institute of Shanghai Academy of Agricultural science | Dong J.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 14 more authors.
European Journal of Soil Biology | Year: 2014

A field experiment was carried out to assess the impact of β-carotene transgenic rice with four synthetic genes on bacterial communities and enzyme activities in the rhizosphere. Soil samples from two neighboring fields cultivated with transgenic rice AH33 and its parental non-transgenic variety Zhonghua 11 were taken at seedling, tillering, heading and maturing stages. There were no significant differences in activities of catalase, sucrase and protease between AH33 and Zhonghua 11 at the same growth stage. Although soil urease activities of AH33 showed significant differences (. P < 0.05) compared with that of Zhonghua 11 at the heading stage, these differences were transient and disappeared at other stages, indicating that changes in soil enzyme activities were mainly related to plant age. Changes in the bacterial communities throughout different growth stages were monitored and analyzed using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library techniques. The resulting dendrograms of DGGE patterns showed four distinct clusters, which were identified to be correlating with the respective growth stages. No effect of the transgenic rice AH33 on dominant bacterial communities at the same growth stage was detectable. This indicated that the impact of crop growth stage outweighed possible alterations due to the genetic modifications. The analyses of DGGE and 16S rRNA gene clone libraries revealed that there were no significant differences (. P < 0.05) in the Shannon-Wiener diversity index (. H'), richness (. R), and evenness (. E) of rhizosphere soil between AH33 and Zhonghua 11 at any of the stages. Thus, there was no indication of altered overall bacterial diversity due to the genetic modification. Sequencing of PCR amplicons indicated that the DGGE profiles were mainly composed of Proteobacteria, Acidobacteria, Gemmatimonadetes, Chloroflexi and Verrucomicrobia, and in addition, the 16S rRNA gene clone libraries indicated the presence of Plantomycetes, Armatimonadetes, Actinobacteria, Chlorobi, Cyanobacteria and Fimicutes. In conclusion, our study based on the analysis of four different soil enzymes and the diversity of rhizosphere bacteria gives no indication that the genetic modification with the four synthetic genes and their expression in the transgenic rice AH33 has any specific effects, related to the genetic modification, on the soils in which they are cultivated. © 2014 Published by Elsevier Masson SAS. Source

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