The Long-term Effects Of Bt Rice On Nature Enemies And Safety Assessment Exploration Of RNAi-mediated Transgenic Rice

It is fully accepted that rigorous and systematical environment and food safety assessments for transgenic crops is necessary before their commercial planting.The safety assessments of transgenic crops on non-target organisms(NTOs)is an important part of their environmental safety evaluation.Hence,this thesis focused on the safety assessments of Bt(Bacillus thuringiensis)rice and RNAi-based transgenic rice on NTOs,especially on natural enemies,combined with the methods in ecology,molecular biology and bioinformatics.These studies will support the industrialization process of Bt rice with strong security data,and also provide theoretical basis and technical reserves for the establishment of safety assessments system for RNAi-based transgenic crops.The main results are described as follows:1.Assessment of the long-term ecological effects of Cry1Ab rice on important natural enemy communitiesBy analyzing the data from arthropod surveys among 10 years,the PRC results showed that the natural enemy communities in Cry1Ab rice fields was similar with them in non-transgenic controls.Further analysis suggested that Cry1Ab rice had no long-term impacts on the structure of the spider sub-community,an important predator in paddy fields,whilst chemical insecticides exhibit negative impacts.And the biological control of rice planthoppers by spiders was enhanced in insecticide-free Bt rice fields.It could conclude that Cry1Ab rice had no significant effects on natural enemy communities and it could represent a powerful tool for integrated pest management in paddy fields.2.Assessment of the effects of transgenic miR-14 rice on the non-target enemy,Cotesia chilonisTaken the Tier-1 safety assessment method of Bt crop as a reference,the effects of a high concentration of miRNA directly exposed to the NTO,Cotesia chilonis,were evluated.The results showed that C.chilonis could obtain exogenous miRNA through direct feeding and the hemolymph of Chilo suppressalis.The development time of C.chilonis was significantly affected when miR-14 injected into parasitized C.suppressalis larva,while the high concentration miR-14 direct feeding had no significant effects on the biological parameters of C.chilonis.Combined with the real conditions in rice paddy,it could be inferred that transgenic miR-14 rice had no significant negative effects on the important non-target parasitoid,C.chilonis.3.Assessment of the effects of ds RNAs which target on Nilaparvata lugens on three NTOsAt first,the method of ds RNA feeding with the artificial diet to N.lugens was established.Based this method,a total of 36 different ds RNA fragments of N.lugens were tested,among which 25 ds RNA fragments significantly reduced the survival rates of N.lugens.Four ds RNA fragments(ds Nl Na,ds Nlsft,ds Nlvatp and ds Nlaup)with high lethal efficiency to N.lugens were selected to evaluate their effects on three NTOs(Tribolium castaneum,Cyrtorhinus lividipennis and Habrobracon hebetor).Combined the results of sequence similarity,biological parameters,and gene transcription levels,it showed that ds Nl Na might have some non-target effects on T.castaneum and H.hebetor,even though it shared relatively low sequence similarity with the homologous genes from three NTOs.While ds Nlvatp has a continuous 19-23nucleotides matches to all three NTOs,it only have non-target effect on T.castaneum when fed with high concentration.In addition,the survival rate of T.castaneum was significant affected by the ds Nlaup continuously feeding.However,the biological parameters and relevant m RNA expressions of three NTOs were all neither affected by the high concentration feeding,nor LC₅₀continuously feeding of ds Nlsft.In conclusion,the effect of ds RNAs targeted on N.lugens on NTOs is not entirely depended on the continuous 21 nucleotides matches to the homologous genes from NTOs,but also on the taxonomic status of the NTO and the gene family of the ds RNA.Thus,the safety assessments of ds RNA on NTOs should take both ds RNA fragment and NTO species into considication case by case.Based on these results,ds Nlsft has the potential to be applied in the future for its relatively higher controlling efficiency to N.lugens and safety to NTOs.