Study On Environmental Behavior And Bioeffects Of Bt Transgenic Rice And Expressed Products Of Its Exogenous Gene

Great progress has been made on insect-resistant transgenic plants breeding home and abroad. China might become one of the countries to approve the commercialized planting of genetically modified rice, especially Bt transgenic rice. Therefore, it was imperative to research on environmental safety assessments of Bt transgenic rice. Expressed products of exogenous genes from Bt transgenic plants might be a kind of environmental pollutant. Studies on environmental behavior of such biology macromolecular in agroecosystems have been the focus in the world and are of great scientific and practical significance. However, it is well-known that for low level of expression of Bt plants, it is so difficult to get enough Cry1Ab purified protein that present studies mostly adopted plant tissues or Bt protein derived from Bacillus thuringiensis, which could not reflect actual environmental behavior of Bt protein from Bt plants. In addition, insertion of exogenous genes throws into confusion original gene-net, which might change physicochemical process and metabolism of Bt transgenic plants and bring pleiotropic effects. Few reports were found on whether response ability of transgenic plants towards such adversity as heavy-metal pollution. Ke-Ming-Dao rice (KMD) is a kind of Bt transgenic plant harbored with cry1Ab gene and selective marker genes (hpt and nptII) and highly-resistant for seven kinds of lepidoptera insects. At present, there are few reports related to environmental behavior and bioeffects of Bt transgenic rice and expressed products (Cry1Ab, HPT and NPTII protein) of its exogenous gene.Based on developing methods of purification and extraction of Cry1Ab protein from KMD straws and soil as well as improving ELISA method of HPT protein, the following subjects were carried out: (1) Expression, exudation and residue in rhizosphere of Cry1Ab protein from Bt transgenic rice; (2) Adsorptive and desorptive behavior of the Cry1Ab purified protein in bentonite, kaolinite, humic acid and soils; (3) Degradation of Cry1Ab protein and KMD straws in aquatic solution and soils; (4) Characteristics of expression, root exudation and residue in rhizosphere soil of HPT and NPTII protein; (5) Modified accumulation of selected elements in Bt transgenic rice. The results were appended following:1. Purification and extraction of Cry1Ab protein from KMD straws and soilIt was important to develop an effectively extracted method for studies on environmental behavior and fate of Cry1Ab protein in soil. A method of extracting and purifying Cry1Ab protein from Bt transgenic rice was improved. A solution of 0.1mol·L^-1 Na₂CO₃-NaHCO₃+5mmol·L^-1 DTT effectively extracted most of Cry1Ab protein presented in the tissue of cry1Ab transgenic rice. Cry1Ab crude protein was obtained after pretreated with ammonium sulfate precipitation, ultra-filtration, desalinization and freeze-drying concentration. The dialysed crude protein was further separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The purity and the bioactivity of the Cry1Ab protein were determined by SDS-PAGE and larvicidal assay, respectively. The purity of the protein with insecticidal activity remained was higher than 80% by this method.Efficiency of present methods on Cry1Ab protein extraction from soil was 4.6-35%. Thus, it is important to improve the extraction efficiency. By the new method, extraction recovery of Cry1Ab protein in soils amended with the purified protein and KMD straws were 46.18-81.73% and 47.72-82.25%, respectively.2. Development of HPT-ELISA method for detection of HPT proteinHpt gene fragment was inserted into the prokaryotic vector pGEX-KG, and transferred into E. coli BL21 for the expression of HPT fusion protein. After overnight cleavage with Thrombin and purification by liquid chromatography, the final HPT proteins were obtained with purity of 90%. The analysis of MALDI-TOF-MS demonstrated that molecular weight is 39.4kD. The HPT protein was used to immunize rabbit for the preparation of the polyclonal antibodies with high titer. The specificity of HPT-antibody (HPT-Ab) was detected by Western blot, which showed specific binding reaction between the antibodies to the purified HPT protein. Then the double-Ab sandwich ELISA method for HPT protein was established with the sensitivity of 0.31ng·mL^-1. The method was applied to determine HPT content in Bt transgenic rice.3. Expression, root exudation of Cry1Ab protein from KMD and its residue in rhizosphere soilCry1Ab protein in the shoot and root of KMD were 3.23～8.22μg·g^-1FW and 0.68-0.89μg·g^-1FW from early tillering to maturing stage, respectively. The contents of the Cry1Ab protein in root exudates were 1.66-48.02ng/individual·day during the whole growth, and its residue in rhizosphere soil was less than detectable limit (0.5ng·g^-1 air-dried soil). It was showed that Cry1Ab protein was not transferred into rhizosphere soil. Considering higher expression of Cry1Ab protein in KMD shoots, KMD straws should not be returned to soil.4. Adsorption and desorption of Cry1Ab protein on kaolinite, bentonite, humic acid, and soilsAdsorption of Cry1Ab protein on kaolinite, bentonite and humic acid was 131.27-1731.44ng·g^-1, 107.09-1846.42ng·g^-1 and 78.02-1066. 10ng·g^-1, respectively. Their adsorption-rates were 41.34-79.95%, 26.60-69.90% and 30.34-45.83%, respectively. Adsorption of the protein on the three adsorbents increased as the amount of the protein increased, while the adsorptive rate decreased with increasing of the protein amounts. Their capacity of adsorption of the Cry1Ab protein was followed as the order kaolinte > bentonite > humic acid, and their capacity of desorption followed the order humic acid > kaolinte > bentonite.Adsorption rate of Cry1Ab protein in 5 soils was decreasing with increasing of its concentration. Adsorption rate in powdery-muddy paddy soil, fiuvio-marine yellow loamy and Coastal saline soil were 24.85% and 40.81%, 9.12% and 31.67%, 12.47% and 30.75%, respectively at the concentration of 782.55ng·mL^-1 and 125.64ng·mL^-1. Desorption rate on the soils dropped with content of soil-absorbed protein decreasing, which on powdery-muddy paddy soil was 12.95% and 5.88%, respectively. Results showed that adsorption amount of CrylAb protein in different soils was notably positively related with concentration of the protein (p<0.05) and organic matter of soil. 5. Degradation of KMD straws and Cry1Ab protein in aquatic solution and soilsDegradation of Cry1Ab protein from KMD was examined under both aerobic and flooded conditions in five paddy soils and in aqueous solutions. The hydrolysis of Cry1Ab protein in aqueous solutions was inversely correlated with the solution pH in the range of 4.0 to 8.0, and positively with the initial concentration of Cry1Ab protein. Under aerobic conditions, rapid degradation of Cry1Ab protein in paddy soils were in accord with first order kinetics equation C=C₀exp(-kt) with half-lives ranging from 19.6 to 41.3d. The degradation was mostly biotic and not related to any specific soil property. Degradation of the Cry1Ab protein was significantly prolonged under flooded conditions compared with aerobic conditions, with half lives extended to 45.9 to 141d. These results suggest that the toxin protein, when introduced into a paddy field upon harvest, will probably undergo rapid removal after the field is drained and exposed to aerobic conditions.Degradation of Cry1Ab protein of KMD straw in aerobic and flooded soil. Under aerobic conditions, the half-lives of the Cry1Ab protein in the soils amended with KMD straw (4%, w/w) ranged from 11.5 to 34.3d in accord with first order kinetics equation C=C₀exp(-kt). The results also showed that the Cry1Ab protein could be detected (detectable limit, 0.5ng g^-1·air-dried soil) after 146d and 138d incubation. However, the degradation of the Cry1Ab protein in Coastal saline soil was fastest with half-life 11.5d, and the Cry1Ab protein couldn't be found in the soil after 60d incubation. The residence time of the protein varied significantly in fiuvio-marine yellow loamy soil amended with KMD straw at the rate of 3, 4 and 7%, with half-lives of 9.9, 13.8 and 18.0d, respectively. Under flooded conditions, degradation of the Cry1Ab protein within incubation for 60d also followed the first order kinetics equation. The protein degraded more slowly in acidic soils with half life of 70d for Paddy field on quaternary red soil and 61.3d for paddy field on red sandstone soil, while faster in neutral and alkaline soils with half-life of 43.9d.6. HPT and/or NPTII protein in plants, root exudates and rhizosphere soil of KMDContents of HPT protein in the shoot and root of KMD were 24.59-60.12ng·g^-1FW and 15.67-33.13ng·g^-1FW from early tillering to maturing stage, respectively, and NPTII protein in corresponding parts was 1.61～2.99ng·g^-1FW and 3.86～9.97ng·g^-1FW. In polished rice of KMD, HPT content was 5.28ng·g^-1DW, while no NPTII protein was detected (lower than detectable limit of 0.31ng·mL^-1). Results also demonstrated that HPT and NPTII protein was not easy to transfer into soil environment via root exudates and no NPTII protein was observed in KMD rhizosphere soil. Therefore, field-growing KMD would not cause serious accumulation of HPT and/or NPTII protein and did not constitute hazards to agroecosystems.7. Modified accumulation of selected elements in KMDPot experiments were conducted under greenhouse conditions to evaluate the impact of heavy metal amendment on modified accumulation of Al, As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn in KMD and its wild-type. Results showed that under Cd, Cu, Pb and Zn pollution, comparability and significant differences were both found in accumulation of the 15 elements of KMD and its parent. At 5, 10 and 20mg Cd kg^-1 soil, cadmium contents in KMD grains reached 0.68, 0.68 and 1.45mg·kg^-1, respectively, which were significantly higher than the international Cd criteria of 0.2～0.4mg·kg^-1 for rice as specified by the Codex Alimentarius Commission of FAO/WHO. On account of the growing concern about the safety of foods and human health, the present results imply unsafety to cultivate Bt transgenic rice in heavy Cd polluted areas. Under Cu amendment, contents of Cr and Cd, toxic heavy metals in KMD straw were enhanced at different levels. Results also showed that significant accumulation was found in content of Cd in KMD straw under Cd stress, and adsorption of Pb was obviously promoted after Pb amendment. Thus, straws of KMD growing in paddy fields with high levels of heavy metal contamination, especially in Cd or Pb polluted areas, should not be returned back to the field.More work on heavy-metal accumulation of Bt transgenic rice and other wild-type varieties needs to be done under field condition for continuous experiment in different areas to vertify the above-mentioned results. Therefore, it was suggested that the scope of ecological risk assessments of Bt transgenic rice be widened and take into consideration study on other unexpected effects before commercialization.