The Environmental Risk Assay Of A Foreign Gene Horizontal Transfer From Bt Transgenic Populus Tomentosa Stub-D To Microorganisms

Samples of rhizosphere soil, roots, branches, leaves and long horn beetle (Apripona germari Hope)'s fecal material of 8 a old Bt transgenic Populus tomentosa Stub-D and litter layer and soil layers of different depths in Bt transgenic Populus tomentosa Stub-D plot were collected, together with samples of rhizosphere soil, roots, branches, leaves from nontransgenic Populus tomentosa Stub-D, and cultivable bacteria and fungi were isolated respectively from each sample. PCR was used with a pair of specific primers depending on 35S promoter and Bt Gene sequences to detect the exogenous Bt Gene fragment in the microorganisms isolated from all the samples, the results were as follow:(1)Using 2 pairs of specific primers to detect the exogenous genes in the 14 lines of transgenic Populus tomentosa Stub-D, it was found that the 14 strains all have got target exogenous genes of Bt and Nptâ…¡in their genomes.(2)Cultivable bacteria and fungi were isolated from all the samples mentioned above and 307 strains of fungi and 2526 strains of bacteria were obtained, of which concerning Bt transgenic Populus tomentosa Stub-D there were 78 strains of fungi and 95 strains of bacteria from rhizosphere soil, 24 strains of fungi and 36 strains of bacteria from branches, 16 strains of fungi and 25 strains of bacteria from racy leaves, 33 strains of fungi and 81 strains of bacteria from soil layers of different depths, 39 strains of fungi and 42 strains of bacteria from Apriona germari Hope s fecal material, and 2145 strains of bacteria from litter layer; and of which concerning nontransgenic Populus tomentosa Stub-D there were 60 strains of fungi and 39 strains of bacteria from rhizosphere soil, 16 strains of fungi and 19 strains of bacteria from branches, 22 strains of fungi and 22 strains of bacteria from racy leaves(3)PCR was used with a pair of specific primers to detect the exogenous gene on the origin of Bt transgenic Populus tomentosa Stub-D in the genomes of cultivable bacteria and fungi isolated from all the samples, and 4 strains of fungi and 7 strains of bacteria were proved positive with repeatable and stable results. Of the 4 strains of fungi there were 1 strain from rhizosphere soil of nontransgenic Populus tomentosa Stub-D, 1 from 20cm soil layer and 1 from Apriona germari Hope's fecal material; of the 7 strains of bacteria there were 2 strains from inside the roots of transgenic Populus tomentosa Stub-D, 2 from branche surface of transgenic Populus tomentosa Stub-D, 1 from inside the branches of transgenic Populus tomentosa Stub-D and 1 from 0cm soil layer.(4)PCR was used to amplify the conserved domains in genomes of the 6 strains of bacteria and 2 in 4 strains of fungi with positive PCR results, 16S rRNA sequences were for bacteria and 28S rDNA D1/D2 sequences and ITS sequences were for fungi. After DNA sequencing, the sequence information of PCR products were compaired with GenBank and the result showed all the bacteria are all in the same genus of Paenibacillus and 1 fungus is in Fusarium, the other is in Cladosporium, the results matched the morphological identification.(5)DNA equencing was used to detect the specific DNA sequence in the genome of the bacterium isolated from litter layer amplified by PCR with a pair of specific primers, and the result showed that between this sequence and Bt gene there was a low homogeneity of 58.34% in nucleotide, and the PCR result was negative, indicating that no real gene drift has been found in litter layer yet.