| Microdera punctipennis(Coleoptera: Tenebrionidae)is endemic to the Gurbantunggut Desert,Northwest part of China.The climate of the region are harsh,and the temperature difference between day and night,and the seasonal change varies greatly.Tenebrionidae insects are the dominant species that distributed in the region.They have a strong resistance to the environmental stresses,such as drought,high and low temperatures.Hence,there may have a variety of genes and molecular mechanisms that play important roles in stress response.Our previous work showed that several genes including antifreeze protein,heat shock protein and attacin play a role in the process of adaptation to the desert environmental in M.punctipennis.However,the stress response is a complicated process that usually the result of interaction of many different families of genes.The investigation of few genes from a few aspects could not explain the whole picture of the mechanism.In this dissertation,we have sequenced the transcriptome data sets of short term low temperature stressed and overwintering M.punctipennis adults by Next generation sequencing technology(NGS),and analyzed the sequencing data with the methods of bioinformatics.The main results are as follows:(1)Analysis of the transcriptome response to short term cold stress: the transcriptome sequenced reads from M.punctipennis exposed to 4℃ for 3 h and the control beetle reared at 25℃ were de novo assembled by using Trinity software,51,712 non-redundant transcripts with the average length of 984 bp were obtained,of which 514 unigenes were up-regulated and 91 down-regulated.Further analysis showed that stress-related GO terms,biological regulation and immune system were enriched significantly.Pathways such as purine metabolism,thiamine metabolism,and glycolysis/gluconeogenesis are significantly enriched.In the stress responsive GO term,the expression levels of eight abiotic strass related genes,including heat shock protein(Hsp90)gene,superoxide dismutase(SOD)gene,calnexin gene,etc.,were upregulated significantly.The transcriptome data of the desert beetle M.punctipennis revealed that the biological processes like metabolic process,stress response,biological regulation and immune system were significantly up-regulated during cold acclimation at 4℃.The metabolic pathways revealed by KEGG enrichment analysis showed that low temperature transcriptome of M.punctipennis was different from that of the other species.The results provide basic data for successive bioinformatics analysis,mining the key genes in cold tolerance of M.punctipennis as well as for elucidation of the molecular mechanisms of insect response to cold.(2)Transcriptome analysis of overwintering M.punctipennis: RNA-seq was performed on the winter adults and the control adults that were kept in laboratory at 30 ℃.A total of 175,247 unigenes were acquired with an average length of 645 bp.By using DESeq package,we identified 3,367 unigenes were up-regulated and 7,988 were down-regulated in the winter adults compared with the controls.To further our understanding of these differentially expressed genes(DEGs),we conducted Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses and found that the “ECM-receptor interaction”,“PI3K-Akt signaling pathway”,“Estrogen signaling pathway”,“Tight junction”,and “Regulation of actin cytoskeleton”,etc.might play important roles in M.punctipennis overwintering.Overall,the sequence data will provide basic information for subsequent bioinformatical analysis and mining of the genes responsible for cold tolerance in M.punctipennis,as well as for understanding the molecular mechanisms of desert beetle overwintering.(3)Analysis of transcription regulatory factors in the transcriptome: we report the identification of transcription factor(TF),transcription co-factor(Tco F)and chromatin remodeling factor(CRF)coding transcripts in M.punctipennis for the first time,as evidenced by deep RNA sequencing data.In total,2,607 TF-coding transcripts,957 Tco F-coding transcripts and 206 CRF-coding transcripts were identified in the transcriptome.M.punctipennis TF-coding transcripts distributed in 62 TF families,representing 1.06% of the assembled unigenes.Differential expression analysis showed that there were 61 and 81 TF-coding transcripts up-regulated at 4℃ and winter in M.punctipennis respectively.Among of them,most belong to the C2H2 type zinc finger(zf-C2H2)family.Meanwhile,44 Tco F-coding transcripts were significantly upregulated during winter in M.punctipennis.Our analysis identified multiple potential regulatory factors that may participate in cold stress response,and offer candidate TFs for further studies on their roles in low temperature acclimation and tolerance.The resources introduced in this work will also contribute to a better understanding of the transcriptional regulatory landscape of M.punctipennis at the genomic level.(4)Comparison of the transcriptome to the genomes of model insects: To further analyze the M.punctipennis transcriptome,we have obtained mitochondrion DNA sequences of M.punctipennis and eight other model insect to construct their phylogenetic relation,and compared M.punctipennis transcriptome assembly(unigenes)to those model insects’ genome and proteome.The phylogenetic tree and alignment result showed that T.castaneum is the closest relative to M.punctipennis among the existing model insects.Alignment of the unigenes to the T.castaneum genome and transcriptome showed that there are 16.41% of unigenes mapped to the T.castaneum genome,among of them approximately 12% of unigenes mapped to the untranslated region(5′-UTR and 3′-UTR)of T.castaneum transcripts,and 77 unigenes mapped to the non-coding RNA.By aligning the unigenes that have mapped to the T.castaneum protein sequences to their CDS,1218 orthologous sequences were identified.Among of them,we analyzed 363 orthologous sequences divergence rate between two species found that most of the M.punctipennis genes under the purification selection.Although,there are still few genes under the positive selection.By analyzing the divergence rate of chromosomes of T.castaneum,on which these orthologous sequences have distributed we found there were no significant differences among them.Overall,this study demonstrates the usefulness of next-generation sequencing for obtaining genomic resources for comparative genomic analysis of nonmodel insect species.(5)Bioinformatics’ analysis of the microsatellites in the transcriptome: the transcriptome data of the M.punctipennis was screened for simple sequences from 1 to 6 nucleotide repeating units,and analyzed their abundance and characteristics.The results showed that the distribution frequency of the microsatellites in the transcriptome was 12.40 kb,of which the single nucleotide repeats was accounted for 47.17%,the most abundant repeat type,and closely followed by trinucleotide repeats(39.81%).The di-,tetra-,penta-and hexanucleotide repeats account for 10.94%,0.90%,0.88% and 0.29% respectively.By aligning of the transcripts of M.punctipennis to the T.castaneum,we compared the microsatellite motifs of the homologous sequences and found that microsatellites in protein coding regions are highly species specific between the two insects.When comparing the microsatellite motifs and the repetition times of the different lengths of the microsatellites between the cold responsive transcripts and the total transcripts of M.punctipennis,we found no significant differences between them.The results of this study could provide a basic data for further study of genetic structure and diversity of population of M.punctipennis. |
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