| Carnation is an important ornamental garden plant. Florigene and Suntory produce a new carnation line with blue-purple flowers suitable for commercialization by introducing the dihydroflavonol-4-reductase (DFR) gene and the flavonoid -3',5'-hydroxylase (F3'5'H) gene into FE123 with white flowers via transformation method. Moonshade and Moonlite are two strains in the new carnation line.As the release of transgenic plants may create biosafety risks, the biosafety assessment of genetically modified plants is necessary. The genetic stability and the potential impact on soil microbial community are two important parts about the transgenic plants to assess the environmental safety. Transgenic carnation was the first biosafety assessment flower in China, so the genetic stability was tested, as well as the impact on soil microbial community structure.Degenerate primers were designed according to the full-length gene of F3'5'H in Petunia hybrida and a 1500 bp DNA fragment was cloned from the transgenic carnation, and the homology of two sequnences was 100% via BLAST. The prokaryotic expression vector was constructed, and an engineering strain of E. coli BL21 (DE3) (+F3'5'H) was obtained. SDS-PAGE analysis revealed that the strain was highly expressed F3'5'H recombinant protein by IPTG induction, representing about 30% of total bacterial proteins. F3'5'H recombinant protein with purified was as an antigen for antiserum. ELISA immunological analysis showed that the antiserum titer was 1:25600. Western blot results showed that the recombinant protein had a good binding activity of IgG, and antiserum also responsed to the protein expressed by F3'5'H extracted from Moonshade and Moonlite.The number of soil microbe from GM and non-transgenic carnation plants root was counted by colony counting method, and the results showed that there was no significant difference of soil microbe (bacteria and fungi) by the statistical analysis. It indicated that genetically modified carnation had yet to have a significant effect on the microbial quantity. In order to choose a better method for extracted the soil genomic DNA, we compared the lab method with kit method. The results showed that the amount of DNA extracted by lab method was higher than that obtained by kit method, but the purity is less, as well as the long time-consuming and the complicated procedures. Based on these results, the kit method was choosed for the follow-up test.In order to study whether there was an impact on the soil bacteria by the transgenic carnation, bacterial 16S rDNA gene clone libraries of GM and non-GM carnations were constructed. The results showed that bacterial diversity of these libraries was very rich, and 8 main groups were isolated. Alphaproteobacteria, Betaproteobacleria, Planctomycetes and Acidobacteria were shared with GM and non-GM carnation; A small amount of Verrucomicrobia strains were found in the GM carnation soil; The other two bacterias were distributed in GM and non-GM carnation soil. Overall, the results showed there was no significant difference between the transgenic and non-transgenic carnation carnation soil bacterial community structure, so it indicated that the cultivation of genetically modified carnation did not have a significant impact on the soil bacterial community structure. |
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