| Eucryptorrhynchus scrobiculatus is an strong host specialty of Ailanthus altissima (Mill.) Swingle and its variation Ailanthus altissima (Mill.) var. Qiantouchun, which is broadly distributed in China. E. chinensis larvae cause the most extensive damage by feeding on A. altissima roots and the damaged A. altissima became decreasing and slowing, even dead. Studies of E. scrobiculatus mainly focus on its biology, ecology and control measures, and no studies have examined its whole genome and transcriptome.To enrich the genetic information and to explore the mechanism of growth and development and seek prevention strategy at the molecular level, this study took examples of comparative transcriptome studies of other non-model species, transcriptome of E. scrobiculatus (include larvae, pupae, and adults) had been sequenced by Illumina HiSeq 2500 high-throughput sequencing platform and we did global analysis of E. scrobiculatus All-Unigenes by using bioinformatics method. Besides, We found a large number of differentially expressed genes in pairwise comparisons of the three developmental stages and carried analysis about identification of related functions and metabolic pathway of the Unigenes involved in. The main study results are as follows:1. The statistics of sequence data and assembly resultAccording to the transcriptome sequencing of E. scrobiculatus (include larvae, pupae, and adults), we obtained 50.53 Gb of clean data, including more than 4.66 Gb for each sample, the clean data quality value was≥30 for more than 90.28% of the cycle and the GC content for each sample was approximately 43%. After de novo assembly, we gained 65,186 unigenes with a mean length of 1,182.92 nt and an N50 length of 3,254 nt, including 17,590 unigenes longer than 1,000 nt.2. Annotation of All-Unigene(1) We have annotated the 65,186 All-Unigene, through blasting against various database, totally 22,116 Unigenes can be matched with above databases, accounting for 33.93% of the total Unigenes. 7,272 Unigenes is annotated to COG,11,136 to the GO,6,230 to the KEGG pathway and 13,491 to the KOG,15,041 to the Pfam,13,135 to the Swiss-Prot and 21,742 to the NR. Based on Nr database queries, we observed that a high proportion of E. scrobiculatus sequences had high similarity with sequences of Dendroctonus ponderosae (49.41%). It is speculated that the unannotated Unigenes could be specific transcript of E. scrobiculatus or undiscovered new transcript.(2) 11,136 Unigenes were annotated to GO database and they were classed into three categories: cellular component, molecular function, biological process. In biological process, maximum genes were involved in metabolism process, which indicated the life activities were vigorous. In molecular function, maximum genes were involved in binding and catalytic activity and this showed the high biologic activity. We identified 6,230 Unigene sequences in 180 KEGG pathways. Most genes were annotated to "Ribosome", followed by "Protein processing in endoplasmic reticulum". KEGG analysis offered help for exploring the molecular regulatory mechanism of growth and development, physiological metabolism, signal transduction, etc.3. The structural analysis of All-Unigene(1) According to the analysis of All-Unigene, we obtained 53,617 CDS. The most CDS were 100-300 nt (28,881,53.87%). The least CDS were over 2000 nt (3,825,7.13%).(2) This study analyzed the microsatellites in the unigenes (the length≥1 kb), a total of 1,823 SSR were identified and the frequency was 10.36%. Tri-nucleotide repeats were the most abundant (37.27%). The most SSR were less than 20 bp and the microsatellites over 20 bp were only 12.70%. There were significant negative correlation between the frequency and the length of microsatellites in E. scrobiculatus transcriptome.4. Differentially expressed genes analysis(1) We found a large number of differentially expressed genes in pairwise comparisons of the three developmental stages (FDR<0.01 and FC≥2). The differentially expressed genes between adults and pupae were the least. These differentially expressed genes provided useful information for identifying the genes involved in growth and development and studying these interactions.(2) The results of GO function classification of differentially expressed genes in pairwise comparisons of the three developmental stages showed that the most differentially expressed genes between larvae and adults were involved in activity and binding, which meant that physiological functions of adults were perfected, larvae were in growth and development stage and need all kinds of proteins to meet growth, so molecular activity were strong. The most differentially expressed genes between pupae and larvae were involved in metabolic process. The most differentially expressed genes between pupae and adults were involved in activity of enzyme, which showed the performed life activity of E. scrobiculatus were changing.(3) The results of annotation of differentially expressed genes in pairwise comparisons of the three developmental stages in KEGG pathway showed that there were 218 differentially expressed genes involved in 102 pathway between larvae and adults. The most up-regulated genes in the adult stage were involved in signal transduction, immune response and hormone synthesis and this showed that the circulation efficiency of information and materials were improving, immune systems were perfected, reproductive system were growing. There were more differentially expressed genes involved in "Lysosome", "Phagosome", "Amino sugar and nucleotide sugar metabolism", "Endocytosis", "Protein processing in endoplasmic reticulum", "Metabolism of xenobiotics by cytochrome P450" between pupae and larvae. Between pupae and adults, the genes related CYP3A4 were up-regulated in the adult stage in "Metabolism of xenobiotics by cytochrome P450", suggesting that it could be involved in the biosynthesis of the insect sex hormones. |
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