Study On Transcriptome In Response To Cold Stress And HSP Expression Patterns In Eogystia Hippophaecolus

Seabuckthorn carpenter moth,Eogvstia hippophaecolus(Lepidoptera:Cossidae),is an important borer pest of sea buckthorn(Hippophae rhamnoides)in China,whose larvae bore the root of sea buckthorn,leading to death of the host plant.Larval E.hippophaecolus can endure ice formation in body,exhibiting highly cold tolerant ability.Using this characteristic of E.hippophaecolus as the starting point we conducte d this study,aiming to peek at the cold adaptation mechanism in E.hippophaecolus.We sequenced the E.hippophaecolus transcriptome using RNA-Seq technology and performed de novo assembly from the short paired-end reads.We investigated larval response to cold stress by comparing gene expression pro files between treatments.We studied the expression patterns of target genes.The main results are as follows:1.All the genetic information at transcription level of E.hippophaecolus was obtained.By Illumina HiSeq TM 2500 high throughput sequencing,39.00Gb raw data were obtained,of which each sample is more than 4.71Gb,with Q30 base percentage reaching or exceeding 88.30%.After de nove assembly,191,200 transcripts and 118,034 unigenes were acquired,N50 of which are 687 bp and 904 bp,respectively.BUSCO assessment result showed high quality of the transcriptome assembly.In total,22,161 unigenes were annotated by NR,Swiss-Prot,KEGG,GO and Pfam database.2.Microsatellites of E.hippophaecolus was exploited based on transcriptome.In order to exploit SSR information of E.hippophaecolus,we identify EST-SSR loci,analyze their features and design primers preliminarily according to the transcriptome of E.hippophaecolus.Results show 5,126 unigenes contain 7,499 SSR loci in total(51.41%).Mononucleotide repeats were predominant significantly,of which the occurrence frequency is 39.52%.There are 77 kinds of repeat motifs existing in E.hippophaecolus transcriptome.(A/T)n(73.74%)is the most frequent in all the repeat types,and next is(AT/AT)n(3.37%).Most of the SSR are less than ten times of repetition and are 10bp in length.3.Expression difference in E.hippophaecolus transcriptomes under low-temperature treatments was analysied.By comparing transcriptome profiles for differential gene expression,many differentially expressed proteins and genes were identified,of which heat shock proteins,cuticular proteins were found up-regrulated and have previously been reported to be involved in cold resistance of insects.Cytochrome oxidase subunit,NADH dehydrogenase subunit and ATP synthase subunits were found downregulated.Gene expression profile varied from different larval stages.Only seven common differentially expressed genes were shared by two groups.That indicated distinct mechanism of cold hardiness in insects among different development stages.4.The expression pattern of heat shock protein was clarified.The heat shock proteins were screened from E.hippophaecolus transcriptome as target genes.The phylogenetic analysis and expression pattern were carried out.The results showed that the small heat shock proteins were not conserved and the similarity among homologous sequences was low,but they all contained a typical alpha lens conserved domain structure.The phylogenetic analysis showed that the homology of the small heat shock protein in different orders was poor.In Lepidoptera,homology of heat shock proteins with similar molecular weights was higher than that with less similar molecular weights.Expression of the target genes in larvae under different cold processing temperatures,laval stages and cold processing hours was analysied by fluorescence quantitative PCR.The results showed that the expression of the target genes would upregulated as temperature growing down,but the trend is not identical.The expression of the target genes increased after certain cold treatment hours,but did not increase with treatment time increasing all the time.Larval stage had no significant impact on Eh3Shsp expression.Eh1 Shsp and Eh2Shsp were downregulated with the increase of larval age.In conclusion,small heat shock protein would be stimulated at low temperature and has a positive effect on cold tolerance of E.hippophaecolus,but the mechanism of regulation of genes are different.