| Temperature is one of the most important factors that restrict insect distributions.Seasonal variations and fluctuating in short period of temperature cause many challenges with the survival and the growth in arthropods.Insects have developed complete adapation mechanisms to to overcome the several of the environmental stress.In order to explore the cold temperature adaptation mechanism in rice water weevil(RWW),we have performed multi-omics,including mRNA transcriptome,miRNA transcriptome and 6-plex TMT proteomics.In addition,we have preceded2-DE experiment of rapid cold harden in RWW.The main results are shown as follow:To characterize changes in miRNAs in response to overwintering in L.oryzophilus,a comprehensive comparative analysis of microRNAs was performed involving an overwintering and a normal adult.High-throughput Illumina sequencing and bioinformatics analyses revealed 121 conserved and 14 potential novel microRNAs in two small libraries.The novel miRNAs exhibit low expression levels in both libraries.After the expression profiles of the miRNAs in the two libraries were normalized,36 miRNAs in L.oryzophilus were found to be differentially expressed in response to overwintering.In particular,14 conserved miRNAs and 6 novel miRNAs were up-regulated,while 15 conserved miRNAs and 1 novel miRNA were down-regulated.In addition,the expression patterns of 11 conserved and potentially novel miRNAs were confirmed by quantitative RT-PCRanalysis.Most importantly,this work provides a unique resource of characterized miRNAs for overwintering L.oryzophilus and contributes to studies of the functions of cold-related and other L.oryzophilus mi RNAs.Compared the methods of phenol extraction(PE)and TCA/acetone precipitation,we found that the PE protocol was optimal,resulting in good background and clear protein spots in 2-DE experiment.PE precipitation extraction was found to be more suitable than TCA for rice water weevil proteome analysis.6-plex tandem mass tags were performed on the overwintering and the summer adults.Altogether,822 proteins were finally quantified,136 of them showingsignificant variations in the overwintering with respect to summer samples.Specifically,50 proteins were up-regulated while 86 proteins were down-regulated between overwintering and summer samples.To further understand these DEPs,bioinformatics analysis such as KEGG and GO enrichment were performed.These differentially expressed proteins involved in chitin binding,ribosome,cictrate cycle,carbon metalism,microbial metabolism in diverse environments and purine metabolism were altered by selection on cold winter acclimatization.The alternations of these proteins suggest the importance of them in participate in the cold winter acclimatization.multi-omics differential expression analysis of miRNA,mRNA and protein suggest that Citrate synthase,Ferritin,Peptidase,Aldehyde dehydrogenase,Chitinase,Isocitrate dehydrogenase,ribosomal protein,ATP synthase,actin,ATPase,Translation initiation factor and Tubulin may response to the cold adaptation.To elucidate the rapid cold hardening(RCH)mechanism,a comparative proteomic analysis was applied with two-dimensional gel electrophoresis(2-DE)between RCH and control treatment samples.A total of 21 protein spots were found to be up-regulated in rice water weevil adults for the RCH condition.As an initial work,16 of these proteins were identified using MALDI TOF/TOF MS.In addition,the expression patterns of 9 proteins(including 2 HSPs that were not identified in the2-DE experiment)were confirmed by qPCR analysis.Using the geneMANIA app in cytoscape,we constructed a protein-protein interaction network analysis of these up-regulated proteins.Gene ontology(GO)function annotation shows that a“response to heat process,” including 6 HSPs,was related to the identified proteins.These up-regulated,RCH responsive proteins could possibly serve as potential biomarkers to study the molecular mechanisms of the L.oryzophilus response to cold stress. |
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