Identification And Potential Application Of Related MicroRNAs To Cry Protein Defense In Chilo Suppressalis

Chilo suppressalis(Walker),belonging to the Lepidoptera Pyralidae family,is one of the most serious common pests on rice,occurring in the main rice-growing areas in our country.Crystal protein is an insecticidal protein produced by Bacillus thuringiensis(Bt)in the process of sporulation.It is widely used in insect control,and some cry genes has been successfully transferred to rice and can effectively control C.suppressalis.Studying the defense mechanism of insects against crystal protein is beneficial to the sustainable promotion and application of transgenic insect-resistant crops.Micro RNAs(miRNAs)are very important in biological processes.The target genes of insect miRNAs are key factors in the immune signaling pathway MAPK,and the results of our laboratory have shown that Csp38 plays an important role in the defense process of Cry1 Ca in C.suppressalis larvae.In this study,the middle intestine small RNA transcriptome was sequenced by Illumina Solexa sequencing technology,and was compared and analyzed between the group fed with Bt insecticidal protein Cry1 Ca and the control group without Cry1 Ca treatment.According to the target gene prediction,miRNA that may be involved in the Cry1 Ca defense response of C.suppressalis were screened,and whether the candidate miRNA participated in the defense process was functionally verified,then,the interaction between miRNA and target genes were confirmed by dual luciferase reporter assay.The research results provide a new theoretical basis and data support for analyzing the defense mechanism of insects against Cry proteins,at the same time,it is of great significance to enhance the toxicity of Bt to target insects and delay the development of insect resistance.The results of the study are as follows:1.The length of most micro RNA sequences was about between 22 nt,In the small RNA database,the proportion of the annotated sequences was very low,and they are only7.2% and 7.9%,respectively.After feeding with Cry1 Ca,the numbers of miRNA,sno RNA,sn RNA,r RNA and pi RNA were decreased.Compared with the control,358 miRNAs were up-regulated and 747 miRNAs were down-regulated in the treatment group;25 known miRNAs with the highest transcription level were selected for further analysis.The results showed that the transcription levels of these 25 miRNAs were significantly different between the Cry1 Ca treated group and the control,among which,3 miRNAs were significantly up-regulated and 22 miRNAs were significantly down-regulated.Among 66 novel miRNAs,23 were up-regulated and 43 were down regulated.The results of quantitative real-time PCR were consistent with the sequencing results.2.Select 10 candidate miRNAs from differentially expressed miRNAs,the mortality changes of C.suppressalis larvae toinsect-resistant transgenic rice T1C-19,T2A-1 and TT51 after Overexpression and knockdown of candidate miRNAs.Showed that knockdown csu-mi R-7322-5p could significantly reduce the mortality of C.suppressalis larvae against T1C-19 and TT51,overexpression csu-mi R-7322-5p could significantly increase the mortality of C.suppressalis larvae against T1C-19 and TT51;knockdown csu-mi R-3262 could significantly reduce the mortality of C.suppressalis larvae against T1C-19,overexpression csu-mi R-3262 could significantly increase the mortality of C.suppressalis larvae against TT51.Other miRNAs were not shown to be associated with larval mortality.3.Based on the existing genome sequence C.suppressalis in the laboratory.Using mi Randa,RNAhybrid and RNA22 used miRNA target prediction software packages,we predicted the csu-mi R-7322-5p can bind to the untranslated region of Csp38,and the minimum free energy(Energy)is-25.18 kcal/mol.The results of dual luciferase reporter assay and miRNA mimic/inhibitor feeding assays showed that Csp38 has a target relationship with csu-mi R-7322-5p,and csu-mi R-7322-5p has a post-transcriptional regulatory effect on Csp38.In this study,miRNA related to pest defense Cry insecticidal proteins were finally screened through Illumina Solexa,miRNA functional identification,and miRNA target gene identification.The miRNA can be used in production practice to improve the toxicity of Bt biopesticides,promote the sustainable application of Bt biopesticides,and provide the necessary resources for the biological control strategy of C.suppressalis.