| Cotton is attacked by a wide variety of pests throughout the growth period.The widespread planting of Bt transgenic cotton effectively controlled the outbreak of H.armigera.However,due to the vastly reduce the application of broad spectrum insecticides,Bt non-target pests mirid bugs has emerged as important pests and increasing harm.It is also hard to control because of its broad host range,great flight and dispersal capacity,as well as a high burst frequency.Insecticide treatments remain the main strategy for mirid bugs control.Nevertheless,frequent insecticide application is undesirable which caused the development of resistance and have a significant hazards to the environment and human health.Therefore,urgently needed an efficient strategy for mirid bugs control in the cotton fields.In recent years,plant-mediated RNAi is emerging as an efficient,eco-friendly and reliable alternative strategy for pest control.In this study,plant-mediated RNAi technology was used to create a new cotton germplasm to control mirid bugs,which provided a new strategy for the control of mirid bugs.The main results were as follows:1.The creation of a new germplasm of cotton resistant to Adelphocoris suturalisIn this study,plant mediated RNAi technology was used to target the As FAR gene,which affects the reproductive ability of A.suturalis,to create transgenic cotton with high expression of As FAR ds RNA(ds As FAR).After feeding on the transgenic cotton,RNAi effect was successfully induced,and the transcription levels of endogenous As FAR in A.suturalis were significantly suppressed.The field bioassay demonstrated that As FAR transgenic cotton suffered minor damage and there were on average 12-14 plant bugs per transgenic plant.Whereas the control plants were seriously damaged,and the average number of plant bugs in each cotton plant was more than 20.The results indicated that As FAR transgenic cotton has suppressed female fertility of A.suturalis,resulting in a significant decrease in offspring population and reducing the harm of A.suturalis.Bioassay of non-target insects showed that As FAR transgenic cotton only resistance to A.suturalis.There was no adverse effect on the growth and development of the non-target insect bollworm and aphid.The consecutive 2-year field trials demonstrated that there was no significant difference in agronomic traits between transgenic cotton and non-transgenic control in different generations.In addition,the expression of exogenous genes had no negative effect on cotton fiber quality.All the results showed that As FAR transgenic cotton had a high level of resistance to A.suturalis,had well and stable agronomic characteristics,no adverse effects on nontarget pests,which could be used as a ideal germplasm resource for the control of A.suturalis.Therefore,this study provides an alternative strategy for the management of A.suturalis.2.The creation of a new germplasm of cotton resistant to Apolygus lucorumIn this study,Al LIM gene of A.lucorum was selected as the target gene.Sequence analysis showed that Al LIM was highly specific.Subsequently,the gene was transferred into cotton,which successfully created transgenic cotton with high expression of ds Al LIM.Indoor bioassay results showed that A.lucorum after feeding on Al LIM transgenic cotton the relative expression level of Al LIM was significantly decreased,and serious defects occur during metamorphosis from larva to adult,eventually,due to the failure of molting,metamorphosis was completely blocked and died,the mortality rate was as high as 33% to 41%.In addition,a small number of A.lucorum molt to adult with abnormalities in the hind leg,tegmina and membranous wings.We performed deep RNA-Sequencing of the A.lucorum samples treated with ds Al LIM and ds GFP.A total of 4923 differentially expressed genes(DEGs)were detected and there were 2484 and 2439 genes with up-or down-regulation were observed respectively.KEGG pathway analysis showed that the signaling pathway associated with muscle cell growth and differentiation were significantly assigned.Based on the KEGG pathway analyses,we identified a diverse group of muscle-related genes that undergo differential transcriptional responses following ds Al LIM treatment.These results suggested that the Al LIM gene plays an important role in the development and formation of muscle in A.lucorum.Integrity of muscle structure and function is crucial for insect metamorphosis.Therefore,we speculate that depletion of Al LIM in A.lucorum leads to muscle development defect,making the muscle-dependent morphogenetic movements necessary for nymphal-adult molt were severely compromised,eventually leads to failure of molting,metamorphosis was completely blocked and increased mortality.Field bioassay showed that the transgenic plants only displayed minor damaged and the average number of A.lucorum per plant were 8.1-10.1,whereas the control plants were seriously damaged and the number of A.lucorum per plant was reached 21.5-24.1.Feeding on Al LIM transgenic cotton resulted in increased mortality and population decline of A.lucorum.Meanwhile,we also observed a small number of adult A.lucorum with abnormalities in the hind leg and membranous wings in the field.Overall,these results indicated that Al LIM transgenic cotton has a high level of resistance to A.lucorum.Bioassay of non-target insects showed that Al LIM transgenic cotton plants had no adverse effects on H.armigera and aphid,and also had no negative effects on helpful insect ladybird beetle.The consecutive 2-year field trials demonstrated that there was no significant difference in agronomic traits between transgenic cotton and nontransgenic control in different generations.In addition,the expression of exogenous genes had no negative effect on cotton fiber quality.All the results showed that Al LIM transgenic cotton has excellent effect on insect resistance,safe for non-target pests,beneficial insects and humans,perfect agronomic character and fiber quality,therefore,it can be used as an ideal germplasm resource for the control of A.lucorum. |
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