Study On Environmental Persistence Of Exogenous Gene And Its Expressed Products Derived From Bt Rice

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.In this paper, mainly through ELISA and isotopic labeling, transgenic rice Huachi B6 and TT51 with, as well as their corresponding parents ((Jiazao 935 & Minghui 63)) and relative varieties ((Zhongjiu B & R9311)), and methods of Enzyme-linked immunosorbent assay (ELISA) and isotopic labeling technique were used in laboratory and field tests to evaluate:(1) expression of Bt-transgenic rice exogenous proteins gene in Bt-transgenic rice and its residue in rhizosphere soil; (2) the residue of Bt protein in rhizosphere soil after transgenic rice straw returned into soil; (3) the effect of transgenic rice TT51 straw degradation to on soil culturable microorganisms; (4) degradation of Bt gene in aerobic soil and the possibility of horizontal transfer to soil microorganismbacteria. The main results are as follows:(1) Expression of Bt-transgenic rice exogenous proteins and its residue in rhizosphere soilThe field experiment showed that the Bt transgenegene did not cause a significant differences in biomass (fresh weight, FW) between Bt rice (Huachi B6,TT51) and non-Bt rice ((Jiazao 935,, Minghui 63,, Zhongjiu B,, R9311)) ((p > 0.05)), but the growing of TT51 was grew much better than Huachi B6. In During the different stages, the amount contents of Bt protein in Bt transgenic rice (Huachi B6,& TT51) were significantly different, the content level of overall performance following the order ofwas seeding filling> heading> mature> flourish tilling. In different organsparts of rice plants, the contents of the overall performance of Bt protein in Bt transgenic rice (Huachi B6, TT51) was showed spike> shoot (except for spike)> root. From the flourish tilling to mature stage, the amount ofexpressed Bt protein in Huachi B6 shoot, root and spike were 7.80～18.32μg/g FW,1.16-5.82μg/g FW and 10.75～23.67μg/g FW, respectively, while in TT51, they were 2.27～3.26μg/g FW,0.52～1.15μg/g FW and 3.21～5.27μg/g FW, respectively. Furthermore, Bt protein inpaddy polished rice of transgenic rice Huachi B6 and TT51 were detected containing Bt protein, were the content were 9.09μg/g FW and 3.81μg/g FW, respectively. It, which means that insecticidal protein in different organs parts of TT51 plants was significantly lower than Huachi B6. In addition, rice of Bt transgenic rice--Huachi B6 and TT51 were detected containing Bt protein, the protein content in rice were 9.09μg/g FW and 3.81μg/g FW respectively. As long as the rice plants dosen't do not residue return into the soil, Bt protein won't did not cause serious residue in soil during the growth period of Bt transgenic rice (Huachi B6,& TT51).(2) The residue of Bt protein in rhizosphere soil after transgenic rice straw returned into soilDuring rice straw process,98-128 d after the Huachi B6 straw of Huachi B6 returned into soil, the degradation in soil of CrylAb protein derived from rice strawin soil degradation was relatively fastrapid, after then the degradation slow downdeclined. While 98-195 d after the TT51 straw returned into soil, the Cry1Ab/Cry1Ac protein in soil remained at 0.35-0.60 ng/g fresh soil, the degradation was relatively flat. About 195 d after straw returned into field, the Bt protein in soil was only 0.35 ng/g fresh soil, close to around the detection limit (0.25 ng/g fresh soil), which indicated that after a certain time for the straw to full degradation, the protein wasn't will not cause a serious residue in the soil. After measuring soil microorganisms, we found that straw returned would promote soil bacterium, fungi and actinomycetes growth, but won't will not significantly effect affect soil denitrifying bacteria, hydrolytic-fermentative bacteria and methanogenic bacteria. In addition, the promote of Bt transgenic rice TT51 on soil microorganisms was higher than Huachi B6, especially in during the later time.After studying the dDegradation of exogenous protein of TT51 straw in aerobic/flooded soil, we found thatshowed that, under 150 d aerobic conditions, degradation of Bt protein in soils were in accord with first order kinetics equation C= C0exp (-kt) (r= 0.933～0.982, p<0.01). The half-lives were 34.5 d (coastal saline soil), 41.8 d (fluvio-marine yellow loamy soil) and 103.5 d (paddy field on red sandstone soil), respectively. Under During 150 d of flooded conditionsincubation, degradation of Bt protein in soils were also in accord withfit for the first order kinetics equation. The degradation of Cry1Ab/Cry1Ac protein in straw was slow in weak acidic/neutral soil--paddy field on red sandstone soil and fluvio-marine yellow loamy soil, the half-lives were 165 d and 120 d, while the degradation was much faster in alkaline soil--coastal saline soil, with the half-lives was of 92 d.(3) Effects of transgenic rice TT51 straw degradation to on soil culturable microorganismsCompared with corresponding parents and relative varieties, after Bt transgenic rice straw was returned into soil, the variations of soil culturable microorganisms were evidently different.Under Aaerobic conditions, the added rice straw would be significantly enhanced the growth of bacterium bacteria in fluvio-marine yellow loamy soil and coastal saline soil, and there was no significant difference between Bt transgenic rice ((TT51)) and non-transgenic rice ((Minghui 63 and, R9311)). The growth of fungi and actinomycetes added rice straw would be were significantly enhanced the growth of fungi and actinomycetes in the three kinds of soils (p< 0.05). Compared with non-transgenic rice, Bt transgenic rice TT51's promotion on soil fungi and actinomycetes would be weremuch more obviously promotedsignificant, particular in the paddy field on red sandstone soil. As for Fflooded conditionsincubation, denitrifying bacteria were the add rice straw had no significantly affected impact on denitrifying bacteria in 3 kinds ofof the soils. The add rice straw could inhibit the growth of hydrolytic-fermentative bacteria were inhibited in paddy field on red sandstone soil and coastal saline soil (TT51< CK1, CK2), but it also could was promoted its growth in fluvio-marine yellow loamy soil (TT51> CK1, CK2). At the same time, the add rice straw had no significant impact on methanogenic bacteria were not impacted in paddy field on red sandstone soil, while which was but would be significantly enhanced the growth of methanogenic bacteria in fluvio-marine yellow loamy soil and saline soil (TT51> CK1, CK2).(4) Bt-gene degradation in soil and the possibility of horizontal transfer to soil bacteriaDuring the 30 d incubation of aerobic experiment, it was indicated that the 35S-Bt gene was not horizontally transferred into soil microorganisms. The aerobic soil degradation dynamics significantly followed the equation C= C0exp (-kt) (r= 0.952～0.990, p<0.01), and the half-lives in two soils were 3.53 d for the fluvio-marine yellow loamy soil and 5.11 d for the fluvo-aquic soil, respectively, which means that Bt gene was more easily degradable in the weak acidic soil.