The Analysis Of MRNA And MicroRNA Of Different Development Pieris Rapae By High Through Sequencing

Pieris rapae(lepidoptera: Pieridae) is one of the most destructive pest in agriculture, mainly feeding on Cruciferous vegetables. P. rapae and its natural enemy Pteromalus puparum provide an important physiological interaction model, serving as a perfect model to study innate immunity.Since the nuclear genome remains unavailable, functional transcriptome information may lead to a global vision of gene expression and a deep understanding of the biology of this insect, as well as the development of integrated control strategies. Hence, in current thesis, we explored the genes related to the development by illumina sequencing the mRNA and small RNA of P. rapae from six stages. In total, 282,237,200 and 165,434,625 raw reads were acquired from mRNA and small RNA, respectively.In mRNA transcription analysis, transcriptome assembly, annotation, and development related genes screening were performed. de novo assembly method was performed to generate96,096 unigenes and 196,202 transcripts. N50 and average length of the transcripts were 2269 bp and 1206.03 bp, respectively. 31,774(33%) unigenes were annotated by blastx against NCBI non-redundant(NR) protein database with and E-value cut-off 1e-5. We found most of the sequences aligned to the top five Lepidoptera species, indicating the reliable assembly transcriptome. Next, gene predicted by NR database were all annotated by GO, COG/KOG and KEGG databases. We found 849 genes involved in seven main development related pathways,such as Hippo, Notch and JAK2, etc. Compared the gene expression between adjacent life stages,20,477 unigenes were detected as significantly differential expressed genes, mostly found from the egg stage compared with the L1 stage. Interestingly, cuticle genes were detected significantly deferentially express across all the life stages. Furthermore, plenty of stage-specific expressed genes were found, which could be used as stage markers. Lastly, heat shock protein(Hsp)homologous evolution indicated that the closed homologous Hsps have similar expression patterns.In small RNA analysis, database blast and annotation, conserved and novel miRNA identification, pre-miRNA prediction, and expression level were performed. there were57,219,920 reads with the length distribution from 18 nt to 24 nt. First, We obtained 53,508,931 high quality reads above the thread of Q30. Among them, 121,695 reads were found to beunique sequences, 78,316 of which were annotated to the non-coding RNA database(NCBI and Rfam) of rRNA, tRNA, snRNA, snoRNA, siRNA, and miRNA. rRNA occupied the largest proportion, followed by tRNA and miRNA. Then 2458 candidate mi RNAs and 43,379un-annotated sequences were aligned to 111 known miRNA in the database of miRBase, hitting2311 sequences. After redundant sequences were removed, 143 conserved mature miRNAs and 54 conserved pre-mi RNA were identified. The remaining 43,526 sequences were used to predict novel miNRA. 18 novel pre-miRNA and 21 novel mature miRNAs were identified. 3 of the pre-miRNAs contain miRNA*. Compared with two successive developmental stages, 78 miRNAs were found to significantly differential express.In terms of the analysis of miRNA regulation, target genes prediction, development related target genes screening, and expression correlation were performed. the identified miRNAs were used to predicted their targets by using the P. rapae m RNA transcriptome s as references. 6198 and 6959 sequences were predicted as potential targets with the software of RNAHybrid and miranda, respectively, resulting in 5,589 unigene targets. All the target unigenes have been annotated to Nr database. Finally, 20 miRNAs and 37 unigenes were found to related with the development of P. rapae according to the insect development pathways in KEGG and the highly negative correlation of their expression.In summary, it is the first time that massive P. rapae genes annotations and expressions were provided through illumina sequencing, including 16,500 genes with full open reading frames, 143 conserved miRNA and 21 novel miRNA. Cuticular protein genes were found to be changed frequently during the P. rapae development and hsp genes with close evolutionary relationships had similar expression pattern. This study provides comprehensive sequence resources of development-related genes of P. rapae, and our findings will lay the foundation for future functionally genomics, developmental biology and gene regulation of P. rapae.