Transcriptomal Study On The Galeruca Daurica Larvae In Response To Thermal Stress

Galeruca daurica?Joannis??Coleoptera:Chrysomelidae?is a new pest with serious outbreaks on the desert steppe in Inner Mongolia in recent years.Its larvae begin to occur as early as early April,when local temperature is not only low but also fluctuates sharply,and the lowest temperature sometimes falls to-10?.Our previous study show the stronger cold resistance in the larvae,but little is known about the mechanism of cold resistance.The larval transcriptomes were assembled using high-throughput sequencing technology.The raw reads were assembled,and annotated functionally in the NCBI databases,including Gene Ontology?GO?,Clusters of Orthologous Groups?COG?,and Kyoto Encyclopedia of Genes and Genome?KEGG?.The differentially expressed genes?DEGs?in the cold-and heat-stressed larvae were identified,and key DEGs and metabolic passways related to thermal stress were screened out.Meanwhile,two key DEGs related to cold resistance were cloned from G.daurica by rapid amplification of c DNA ends?RACE?,and bioinformatics analysis and expression profile were performed.The main results are as follow:1.The 2^{n d}-instar larvae of G.daurica were exposed to-10,-5,0,5,25?control?and 35?,respectively,for 1h,then allowed to recover at 25?for30 min.Six transcriptomes were obtained by high throughput sequencing.The32.67 Gb clean data were obtained through the sequencing quality control,and the Q30 base percentage of the samples was more than 85.75%.A total of30,915,046 raw reads were assembled into 22,732,767 contigs,which were further assembled with high integrity into 142,994 transcripts with a mean length of 1110 bp and 72,352 unigenes with a mean length of 793 bp.A total of 31,513 unigenes were further functionally annotated in the NCBI databases.Eleven DEGs were selected,and verified by real-time quantitative PCR.The m RNA expressions of nine DEGs were consistent with those obtained by digital gene expression profiles.2.Compared with the control?25??,718,865,1142,1474 and 1063DEGs were annotated functionally,respectively,from 5 thermal-stressed samples?-10,-5,0,5,and 35??.268 common DEGs were screened out from the cold-stressed samples?-10,-5,0,5??,and 38 common DEGs were obtained in the cold-and heat-stressed samples.The DEGs were mainly related to general function prediction only,transport&metabolism o f secondary metabolites,inorganic ions,carbohydrates and amino acids.The DEGs were enriched in the metabolic processes of biological processes and the catalytic activity of molecular functions by GO enrichment analysis.This showed that temperature influenced significantly the metabolism and catalytic activity in the G.daurica larvae.KEGG enrichment analysis also indicated that the DEGs were significantly enriched in the metabolic pathway,including pyruvate metabolism,glycine,serine and threonine metabolism,and purine metabolism.3.A serine protease gene was cloned from G.daurica by RACE technology,and named Gd SP?Gen Bank accession:MG797556?,which was 1110 bp in length with an open reading frame?ORF?of 969 bp,encoding a protein of 322 amino acids with the predicted molecular weight of 35.41 k D and p I of 5.61.The encoded protein shared typical structural features of serine proteases,with a transmembrane domain but no signal peptide.Homologous sequence alignment and phylogenetic analysis showed that Gd SP shared the highest amino acid sequence identity?30.53%?with Anoplophora glabripennis SP.RT-q PCR results showed that the expression levels of Gd SP had no significant difference among different temperature stresses whereas increased significantly after recovery from the stresses.4.A cuticular protein gene was cloned from G.daurica by RACE technology,and named Gd Abd?Gen Bank accession:MG874710?,which was708 bp in length with an ORF of 477 bp,encoding a protein of 158 amino acids with the predicted molecular weight of 16.98 k D and p I of 4.26.The encoded protein shared a typical consensus region of R&R consensus,with a transmembrane domain and a signal peptide.Homologous sequence alignment and phylogenetic analysis showed that Gd Abd shared the highest amino acid sequence identity?50.63%?with Leptinotarsa decemlineata Sg Abd-4.RT-q PCR results showed that rapid cold stress has no significant effects on the expression of Gd Abd while the recovery from cold shock elicits the up-regulated expression.