| RNA interference(RNAi)is a biological process in which small RNA(s RNA)molecules sequencespecifically silence gene expression at the transcriptional or post-transcriptional level.Because of its simplicity,efficiency and specificity,it has been widely used in pest management.A micro RNA(mi RNA)-mediated RNAi insect-resistant rice Csu260-16 provides an attractive control strategy for Chilo suppressalis.However,the focus of public attention is the environmental safety of genetically modified insect-resistant crops and its impact on non-target organisms,before the commercial application of genetically modified insect-resistant crops.As an important economic insect,the Italian honeybee(Apis mellifera)is an important pollinator in the rice field ecosystem and commonly used indicator species in the environmental safety assessment of transgenic insect-resistant crops.The rice leaf roller(Cnaphalocrocis medinalis)and the target pest C.suppressalis are both important lepidopteran pests in rice fields,causing serious losses to rice production,and their occurrence periods overlap.Therefore,whether RNAi insect-resistant rice Csu260-16 can control the damage of the rice leaf roller is also one of the focus of our attention.Based on this,the Italian honeybee adults and the rice leaf roller were used as the research objects in this study.The environment safety of the RNAi insect-resistant rice Csu260-16 to A.mellifer and rice leaf roller was systematically evaluated by bioinformatics analysis,bioassay and the determination of target gene expression level.The research results can provide a basis for the formulation of environmental safety assessment guidelines for RNAi insect-resistant crops and supply theoretical basis and science data for the environmental safety assessment of RNAi transgenic insect-resistant crops.The main results of this study are as follows:(1)Homology analysis of target gene sequence and prediction of the binding site of Csu-novel-260 and target gene: We cloned the full-length of C.suppressalis Csdib,A.mellifera Amdib,and C.medinalis Cmdib.The phylogenetic analysis revealed that the Cmdib gene and the Csdib gene in relationship were near with each other while they were far with the Amdib gene.In addition,the bioinformatics software mi Randa and RNAhybrid predicted that there was no potential binding site between the Amdib gene sequence and Csu-novel-260,while there was a potential binding site between the Cmdib gene sequence and Csu-novel-260.(2)RNAi toxicology bioassay: the results showed that the survival rate of A.mellifera adults in RNAi treatment group was not significantly different from that in blank control(CK)and negative control(NC),but the survival rate of A.mellifera adults in the three treatment groups was significantly higher than that in positive control group.This indicated that the pollen of RNAi insect-resistant rice Csu260-16 had no significant adverse effects on the Italian bee adults.Bioassay of in vitro rice tissue: The results showed that RNAi insect-resistant rice Csu260-16 significantly reduced the pupation rate of the C.medinalis,but had no significant effect on the survival rate and the body weight of the C.medinalis larvae.(3)The effect of Csu-novel-260 or Csu260-16 insect-resistant rice on the expression level of insect dib genes: The expression level of Amdib gene of the bees adults was not significantly affected after feeding the sugar water containing Csu-novel-260;The expression of Cmdib gene of C.medinalis was significantly suppressed after feeding the RNAi insect-resistant rice Csu260-16.Overall,these results suggest that RNAi insect-resistant rice Csu260-16 has significant resistance to C.medinalis and the impact of the Csu260-16 rice on A.mellifera is negligible.This study provides a broad map and scientific data for future studies involving the risk assessment of RNAi-based IRGE plants. |
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