| Rice is one of the China's main food crops.Enhancing rice's own resistance through transgenic breeding methods provides an effective solution to pest and disease management.At present,China has developed a series of transgenic Bt rice that is in the critical period of approval for commercial planting.The evaluation of environmental safety and food safety is very urgent.In recent years,considerable progresses have been made on the genetic drift of some transgenic rice lines,the risk of resistance to target pests,the effects of non-target organisms and arthropod communities in rice fields,and the residues of toxic proteins in soil.These works have accumulated a certain amount of experience and technics.The core of environmental risk assessment of transgenic Bt rice is to track the environmental behavior of foreign proteins and the ecological effects on non-target orgnisms.In this paper,four parts of the research work were carried out using two Bt rice lines developed by Huazhong Agricultural University containing cry1Ab/1Ac and cry2 A genes respectively:(1)bioassay for the environmental retention period of Bt proteins using a sensitive target Chilo suppressalis Walker,and comparsion with the results of ELISA;(2)transfer of foreign proteins in the food chain with sensitive and non-sensitive targets as nodes,and influence on the third trophic level(natural enemies);(3)the transmission of Bt proteins in the aquatic food chain and its biological effects;(4)the decay process of Bt rice and non-Bt rice straw and the associated bacterial and fungal communities.The main results are as follows: 1.Bioassay of environmental retention time of Bt proteins in Bt straw residuesThe lethal rates of cry1Ab/1Ac and cry2 A transgenic rice straw at the yellow ripe stage were 85% and 73% on the newly hatched larvae of C.suppressalis Walker.At this stage,the ELISA results showed that the contents of Bt protein in the two kinds of straw were about 4700 ng/g(fresh weight)and 2800 ng/g(fresh weight),respectively.The lethal rates after adding the two kinds of straw in the yellow ripe stage into the artificial diet were 46% and 35%(1000g artificial diet added 30 g dry weight).Based on these results,a series of Bt straw additions(from 30 g to 0.375 g)were set up.Using larva mortality as indicator,the results showed that the reaction limit of Bt straw addition was 7.5 g/1000 g artificial diet.Using larva growth as indicator,the reaction limit of Bt straw addition was 0.75 to 0.375 g/1000 g of artificial diet.The growth inhibition rate is 20-40 times more sensitive than mortality to Bt protein in artificial diet.The lower limit of detection of Bt protein in straw residue by the inhibitory rate of growth was about 10 ng/g.The half-life of Bt protein in Bt rice straw measured by ELISA method was about 9 d to 17 d,and the 90%-life of Bt protein was close to 80 d.The bioassay results showed that the half-life of Bt proteins in Bt rice straw was about 5 d to 10 d,and the 90%-life was 13 d to 20 d.The bioassay of the Bt protein environment retention period is much shorter than that of the ELISA assay.Therefore,the environmental risk of Bt proteins that remain in the environment with rice straw residues is much lower than originally estimated.2.Transfer and biological effects of Bt foreign proteins in the food chain with sensitive and non-sensitive targetsWe studied the effects of Bt rice on the growth and development of three herbivorous insects,C.suppressalis Walker,Mythimna separate Walker and Nilaparvata lugens St?l.The survival rate and body weight of C.suppressalis feeding on Bt rice were significantly lower than that of the control(p<0.05),but there was no significant difference of the M.separate and N.lugens between Bt treatments and non-Bt treatment.Lycosa pseudoamulata were fed with three herbivorous insects that feeding on Bt rice seedlings,and the effects on the growth and development of L.pseudoamulata were tested.We found that the survival and body weight of L.pseudoamulata preyed C.suppressalis feeding on Bt rice was significantly lower than that of the control,whereas the survival and body weight of L.pseudoamulata preyed M.separate and N.lugens feeding on Bt rice were not significant different from the control.We measured the enrichment of Bt protein along the food chain in the second and third trophic levels.The results showed that Bt protein was detected in three herbivorous insects at concentrations of 40 ng/g,45 ng/g,25 ng/g,also detected trace amounts of Bt protein in the natural enemy L.pseudoamulata in concentrations of 9 ng/g,6 ng/g,8 ng/g.Based on the above results,whether the Bt rice have a negative impact on the third trophic level(ie natural enemies)lies in whether the predators prey Bt rice sensitive targets or non-sensitive targets.Under normal circumstances,in addition to preying on sensitive targets of Bt rice,natural enemies also prey on Bt rice sensitive targets.In this cense,Bt rice is safe to natural enemies.3.Transfer and biological effects of Bt foreign proteins in the aquatic food chainWe studied the transmission law and biological effects of Bt rice exogenous proteins along the food chain “rice-Daphnia magna-Brachydanio rerio” and “rice-Procambarus clarkii”.The effects of Bt rice on the growth & development and three protective enzymes in D.magna,B.rerio and P.clarkii were analyzed.The ELISA method was used to detect the enrichment of Bt protein in each trophic level.The results showed that there was no significant difference in the survival rate,fecundity,and three protective enzyme activities of D.magna fed with transgenic rice compared with the control rice.The results showed that there was no significant difference in the survival rate,fecundity,and three protective enzyme activities of D.magna fed with transgenic rice compared to control rice(p<0.05).After 10 days of starvation of B.rerio treated with D.magna cultured in transgenic rice culture medium,there was no significant difference in survival rate,body length and protective enzyme activity in B.rerio from the transgenic group compared to the control treatment(p>0.05).There was no significant difference in the survival rate,body weight gain,body length growth,and protective enzyme activities in the P.clarkii treated with transgenic rice for 8 weeks(p>0.05).There was no significant difference(p>0.05)in viability,body weight gain,body length growth,and protective enzyme activities in the P.clarkii fed the transgenic rice for 8 weeks compared to the control and the the blank control.The concentration of Bt detected in D.magna is approximately 50 ng/g,the concentration of Bt detected in B.rerio is approximately 11 ng/g,and the concentration of Bt detected in P.clarkii is approximately 5 ng/g.From the above results,it can be seen that Bt rice is safe for these aquatic organisms.4.Bt rice and non-Bt rice straw decaying characteristics and microbial diversity in the processIt is one of the important contents of environmental risk assessment whether there are differences in the decaying characteristics of transgenic insect-resistant rice straw after returning to the field compared with non-Bt straw.This experiment used Minghui63 rice straw with cry1Ab/1Ac and cry2 A genes as material.The decay rate of straw in the field is compared to non-transgenic Minghui 63.We compared the diversity of microorganisms(including bacteria and fungi)in transgenic and non-transgenic straw by the second generation sequencing method.The results showed that there was no significant difference in residue,carbon content,nitrogen content,and carbon-nitrogen ratio of straw between the two transgenic lines and non-Bt control.There were detectable differences in the types and diversity of bacteria and fungi between transgenic and non-transgenic lines,and between Minghui 63 rice with cry1Ab/1Ac and cry2 A transgenes.The difference between these microscopic species and diversity may be due to expressing of Bt protein,or it may be due to the transgenic event itself,but the results of this study can determine that microscopic differences in microbes did not affect the decaying of Bt rice straw. |
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