Effects Of Transgenic Rice And Soybean On Rhizosphere Microbes

The consistent and substantial, economic, environmental and welfare benefits were generated from biotech crops over the last fourteen years. However, at the same time, potential risk to environment originated from transgenic crops is existed. Rice and soybean are two important food and economics crops. Study of insect-resistant rice and disease-resistant soybean was developed earlier in domestic. Comprehensive and exactly environmental safety assessment of transgenic soybean and rice was important guarantee of commercial plantantion. Classical counting, DGGE and fluorescence labeling were used to investigate the effect of both transgenic crops and genetically engineered Bacillus thuringiensis strain on native microbes in the soil. Furthermore, a detection system of horizontal transfer of foreign gene was established by Acinetobacter baylyi ADP1 as indicator.1. Effects of the community structure of microbes in rhizosphere soils of transgenic soybean and rice:G0431 and G0433 were genetically modified soybean by transformation of two anti-fungus genes,"chi"(chitinase gene from bean) and"rip"(barley ribosome-inactivating protein gene) into Heinong 35 and Jilin30 natural strain, respectively, and MF86 was genetically modified rice by transformation of two insecticidal genes, cry1Ac (from Bacillus thuringiensis) and sck (modified Cowpea Trypsin Inhibitor gene) into Minghui 86 natural strain. Classical plate counting and denaturing gradient gel electrophoresis were executed to analyze the numbers of culturable microbes in rhizosphere soil and the community structure of bacteria and fungi. The results showed that there was no significant difference between transgenic crops and their host plant in the number of the culturable soil microorganism. Moreover, the results of cluster analysis of DGGE profile of bacteria and fungus indicated that the time was the main factor which affected the community structure in rhizosphere soil microbes. In the year of 2008, the Shannon's index of the bacterial community of the soybean rhizosphere was 5.32-5.37 and the evenness index was 0.91-0.95; the Shannon's index of the fungi community of the soybean rhizosphere was 4.78-4.91and the evenness index was 0.81-0.89; the Shannon's index of the bacterial community of the rice rhizosphere soil was 5.11-5.16 and the evenness index was 0.93-0.99; the Shannon's index of the fungi community of the rice rhizosphere was 4.78-5.14, and the evenness index was 0.66-0.95. In the year of 2009, the Shannon's index of the bacterial community of the soybean rhizosphere was 5.40-5.45 and the evenness index was 0.93-0.97; the Shannon's index of the fungi community of the soybean rhizosphere was 5.21-5.26 and the evenness index was 0.96-1.00; the Shannon's index and evenness index of the bacteria community of the rice rhizosphere soil were 5.28-5.35 and 0.88-0.95, respectively; while the Shannon's index and evenness index of the fungi community of the rice rhizosphere soil were 4.99-5.39 and 0.67-0.98. In conclusion, there was no remarkable difference in the rhizosphere soil between the transgenic and non-transgenic crops in the numbers of culturable bacteria (including Bacillus and fluorescent Pseudomonas), fungi and actinomycetes. All the results demonstrated that both transgenic soybean and rice tested were no significant effect to native microorganisim in the field. 2. Detection system of horizontal transfer for foreign gene using Acinetobacter baylyi ADP1 as indicator.An analytical method was established using Acinetobacter baylyi ADP1 as indicator to investigate whether the foreign gene from rice could be transferred into native microbes in soil. Acinetobacter baylyi ADP1 integrates foreign DNA into the chromosome with a high effficiency. PCR identification of 5.0×10~6 transformants obtained by transforming Acinetobacter baylyi ADP1 with sonicated genomic DNA of transgenic rice MF86, no positive transformant containing Bt cry gene fragment was found. This result indicated that Bt cry gene, one of foreign gene from transgenic rice could not be transferred into Acinetobacter baylyi ADP1 or the transformation frequency was only lower than 10-6 possibly.3. Dectection of the horizontal transfer of recombinant plasmid in fermentation supernatant of engineered Bacillus thuringiensis strain:In the anaphase on the fermentation, after cell lysis, the recombinant plasmid DNA in engineered Bacillus thuringiensis strain entered supernatant, and was confirmed by both PCR and transformation. It was an important aspect to evaluate the environmental safety of engineered Bt, whether the plasmid haboring foreign gene could transfer into familiar microbes to lead to adverse influence. There was no positive transformant after the different strains were transformed with DNA prepared from fermentation supernatant and sterile fermentation supernatant that treated with different placement time. The results indicated that the recombined plasmid harboring foreign gene from B. thuringiensis engineered strain couldn't transfer into other environmental microbes or the transformation frequency was only lower than 10-8 possibly, and this conclusion demonstrated again that B. thuringiensis engineered strain is safe.4. Monitoring of spread of B. thuringiensis engineered strain into rice plant:In order to detect if engineered Bacillus thuringiensis can enter the interior rice through root, a gfp-labeled B. thuringiensis strain HD73 (pSTK-gfp) was constructed. Green fluorescence was observed all through the life cycle of HD73 (pSTK-gfp) under the laser scanning confocal microscope (LSCM). Classical plate counting and LSCM were used to analyze the HD73 (pSTK-gfp) after inoculating into the root of the rice (Nipponbare). There were no positive strain (HD73-pSTK-gfp) detected from inner of the rice. The results showed that the spread ability of engineered Bt was very limited. So, we could conclude that B. thuringiensis engineered strain was safe in this level.