Screening Of Endogenous Mirna In Striped Stemborer Chilo Suppressalis And Development Of Insect-Resistant Rice

Rice（Oryza sativa L.）yield has an important impact on national food security and social stability of China.Striped stem borer（Chilo suppressalis Walker,SSB）has been recognized as one of major pests for rice production in the world.It is urgent to develop appropriate strategies for the effective control of SSB so as to promote rice yield.The application of pesticides is a commonly used strategy for pest control,but due to the negative impacts on the environment and the emergence of resistant pests,it is necessary to develop alternative and ecologically friendly pest control strategies.Breeding of transgenic rice with the expression of Bacillus thuringiensis（Bt）toxins is regarded as a powerful strategy to control SSB.However,resistance of pests to transgenic Bt plants may be gradually evolved,and Bt toxins have a very limited number of target insects.Hence,novel strategies controlling pests must be continuously developed.The rapid development of RNA interference（RNAi）technology provides great new opportunities for pest control.Currently,the target genes using for RNAi-based transgenic crops are mostly protein-coding genes.The use of insect-specific microRNA（miRNA）instead of protein-coding genes for pest control can eliminate or reduce the risk to non-target organisms.In this study,we selected the sequences of SSB miRNA that are specifically expressed in the midgut as target genes to construct the artificial microRNA（amiRNA）expression vectors,which were subsequently transformed into rice through Agrobacterium-mediated genetic transformation,resulting in the generation of transgenic rice plants carrying high amiRNA expression.The insect resistance of the amiRNA rice was evaluated through SSB feeding test using rice stem.The following main results were obtained.（1）Selection of SSB miRNA candidates expressed in the midgut.Eleven novel miRNAs（csu-novel-miR36、csu-novel-miR38、csu-novel-miR47、csu-novel-miR51、csu-novel-miR53、csu-novel-miR57、csu-novel-miR67、csu-novel-miR78、csu-novel-miR82、csu-novel-miR92 and csu-novel-miR96）specifically expressed in the midgut were selected from 106 novel SSB miRNAs identified in our previous research.（2）Evaluation of insect resistance of transgenic amiRNA rice.The eleven amiRNA expression vectors,including csu-novel-miR36、csu-novel-miR38、csu-novel-miR47、csu-novel-miR51、csu-novel-miR53、csu-novel-miR57、csu-novel-miR67、csu-novel-miR78、csu-novel-miR82、csu-novel-miR92and csu-novel-miR96,were transferred to ZH11（japonica rice）by Agrobacterium-mediated transformation.The stems were collected from T₀amiRNA rice plants with high amiRNA expression levels at the booting stage.The results revealed that the SSB larvea fed with three trangenic lines showed obvio us decreases in average body weight after 7 d and 10 d.The average body weight of the larvae fed on stem cuttings of csu-53 rice was 21.99%lower at 7 d and 31.94%lower at10 d compared with that of larvae fed on the control stem cuttings,indicating that csu-53rice had higher resistance to SSB than the other two amiRNA（csu-78 and csu-82）rice.To confirm the efficacy of csu-novel-miR53,the miRNA mimics of csu-novel-miR53（agomir-53）,agomir negative control（agomir-NC）,the antisense sequence of csu-novel-miR53（antagomir miR-53,antagomir-53）,and antagomir negative control（antagomir-NC）were generated to feed SSB larva in vitro via artificial diet-based delivery.The results revealed that agomir-53 led to significant growth inhibition and lethality of SSB larvae compared with agomir-NC,antagomir-53 and antagomir-NC.The larvae fed with agomir-53 diet showed 47.91%lower average body weight at 5 d and21.80%lower average body weight at 10 d than those fed with the agomir-NC control.However,the larvae fed with agomir-NC,antagomir-53 and antagomir-NC diets were not significantly different in body weight.The mortality of SSB larvae fed on agomir-53 diet was 46.46%higher at 5 d and 39.35%higher at 10 d than that of larvae fed on the agomir-NC control.Consistently,larvae fed with agomir-NC,antagomir-53 and antagomir-NC diets were not significantly different in mortality.（3）Screening and insect resistance evaluation of single copy-inserted transgenic rice families.Three transgenic rice lines with single-copy insertion and effective expression of amiR53,namely CS-2,CS-9 and CS-27,were selected from the previously transformed csu-53 rice plants via Southern blotting,expression detection and germination test.The homozygous lines of CS-2,CS-9 and CS-27 were acquired through selfing.In the consecutive feeding experiment,newly hatched SSB larvae were fed with the stem cuttings of CS-2,CS-9 and CS-27 until pupation.As a result,the SSB larvae fed on transgenic csu-53 rice exhibited a slower increase in average body weight compared with those fed on the WT stem cuttings at 5 d after infestation,and the gap in average body weight showed constant increases until pupation.The average pupation time of larvae fed on the stem cuttings of CS-2,CS-9 and CS-27 was all delayed by approximately seven days relative to that of larvae fed with the WT.Relative to that of larvae fed with the WT,the average pupal weight was reduced by approximately 16.5%,and the eclosion rate was decreased by about 43.2%in average.Taken together,our results indicated that the csu-53 transgenic plants have delayed development and normal growth of SSB.（4）Non-target pests resistance evaluationThe evaluation of insect resistance on rice leaf folder（Cnaphalocrocis medinalis）and brown planthoppe（Nilaparvata lugens）showed that the csu-53 plants had no resistance to the two species of non-target pests.（5）Confirmation of the target gene of csu-novel-miR53.Transcriptome analysis identified 28 differentially expressed genes（DEGs）,which were then validated by expression detection.As a result,among the DEGs confirmed by expression detection,only DN90065＿c0＿g12 contained a predicted target site of csu-novel-miR53,indicating that this gene might be the direct target gene.Ds RNA targeting DN90065＿c0＿g12 was then designed and synthesized for in vitro feeding of SSB larvae via artificial diet-based delivery.The analysis results demonstrated that the expression level of DN90065＿c0＿g12 in SSB lavae fed on the DN90065＿c0＿g12 dsRNA diet was supressed with a silencing efficiency of 78.6%on the 7^thd and 39.4%on the 10^thd.The suppressed expression level of the target gene was consistent with the larval phenotype that the average body weight of larvae fed on the DN90065＿c0＿g12 dsRNA diet was approximately 31.7%lower on the 7^thd and 41.3%lower on the 10^thd than that of the larvae fed on the WT control（GFP dsRNA）,respectively.These results indicated that DN90065＿c0＿g12 is the most likely target gene of csu-novel-miR53.In conclusion,we utilized an insect endogenous miRNA（csu-novel-miR53）specifically expressed in the midgut to confer amiRNA-mediated SSB resistance based on the expression in transgenic rice,and obtained three SSB-resistant transgenic rice lines with single-copy insertion and effective expression of amiR53.Our findings present an alternative approach for developing genetically engineered insect-resistant crops for non-model insects.