| Dendroctonus valens Le Conte is an important invasive forest pest in China.Adults and larvae mainly winter at the trunks and roots of Pinus tabuliformis and Pinus sylvestris;larvae,in particular,result in pine weakness or even death.Since the species was introduced from the United States to Shanxi in 1998,its distribution has spread northward.In 2017,it invaded a large area at the junction of Liaoning,Inner Mongolia and Hebei provinces,showing strong cold tolerance.To identify genes relevant to cold tolerance and the process of overwintering,we sequenced the transcriptomes of wintering and nonwintering adult and larval D.valens using the Illumina Hi Seq platform.The differential transcriptomics analysis methods of other non-model organisms were used to compare the differentially expressed genes of adult and larvae in two periods,and the identification of related functions and the preliminary analysis of their involvement in metabolic pathways were carried out,the genes related to cold tolerance were preliminarily screened,and transcriptome data were verified.The main results are as follows:1.All the genetic information at transcription level of D.valens in different periods was obtained.After sequencing 12 samples of D.valens adults and larvae in different periods in January and May,a total of 86.32 Gb of clean data was obtained,and the clean data of each sample reached more than 6.14 Gb,Q30?93.21%,Q20?97.82%,and the average GC content accounted for 42.98%.Trinity was used to assemble all clean reads from scratch,and 110932 transcripts were obtained.After clustering and deredundancy,58956 unigenes were obtained.The average transcript length after assembly was 1163.73 bp,N50 was 2101 bp,and the BUSCO score was 96.3%.The assembled sequences were compared with sequence data in the Nr,Pfam,COG,Swiss-Prot,KEGG,and GO databases.Functional annotations were obtained for 28218 sequences,accounting for approximately 47.86% of all unigene sequences in the transcriptome.2.The transcriptional differences between D.valens adults and larvae before and after overwintering were identified.Compared with samples collected in Non-wintering period,8260 DEGs were obtained from adult screening and 4996 DEGs were obtained from larvae screening.There were 1189 differential genes co-expressed by adults and larvae,and 92 DEGs were up-regulated in both adults and larvae.DEGs enrichment analysis showed that 720 genes in the common differentially expressed gene set received GO annotations,which were mainly enriched in carbohydrate metabolic process,peptide metabolic process,intracellular organelle,hydrolase activity,acting on glycosyl bonds andhydrolase activity,hydrolyzing O-glycosyl compounds and other items,it showed that temperature has a greater effect on the metabolic activity of D.valens.A KEGG enrichment analysis revealed that common DEGs could be enriched into three significantly pathways,including pentose and glucuronate interconversions,galactose metabolism were related to carbohydrate metabolism and lysosome related to transport catabolism.It is indicated that metabolic regulation is very important in dealing with low temperature.3.Genes that may be related to cold tolerance were screened preliminarily.we selected 112 candidate genes related to cold tolerance.In total,44 genes encoded enzymes and proteins related to the degradation of cryoprotectants,including 21 genes involved in the synthesis and metabolism of lowmolecular-weight polyols,10 genes involved in trehalose synthesis,and 13 genes involved in lowmolecular-weight amino acids.In addition,one gene encoding the cytoprotective protein HSP70,11 genes encoding cytoskeletal proteins,1 gene with cuticular functions,1 gene involved in antioxidant defense,15 genes related to signal transduction,6 genes related to apoptosis,and 31 genes associated with other processes related to cold tolerance were annotated.4.The reliability of transcriptome data is verified.15 candidate genes that may be related to cold tolerance were randomly selected for q PCR verification.The results showed that the expression trend of the 15 differentially expressed genes were completely consistent with the RNA-seq sequencing results,confirming the transcriptome data is reliable for subsequent analysis.In summary,this study obtained transcriptome data of adult and larvae of the red fat larvae at different stages,and screened out genes that may be related to cold tolerance.The results of the study enriched the genetic information of D.valens,it also provided a foundation for further molecular and functional studies of this trait and provided a theoretical reference for the development of a prevention and control strategy. |
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