| Plodia interpunctella, distributed all over the world, is one of the important pests in China. To date, most researches are about the physiological and biochemical, while the genetic studies are rare. In this paper, transcriptome sequencing was used to analysis the RNA expression level of Plodia interpunctella, assembled by De novo. A deeper study was made on growth and development of the pest by the method of molecular biology. Bt protein had an obvious effect on lepidoptera insects, but its mechanism was not clear. Three Bt toxic protein receptor genes were screened and identified based on the data of transcriptome sequencing to analysis. The stability of reference genes and microsatellites were studied also.1. The result of transcriptome sequencing obtained 84938 unigenes. The number of unigenes annotated to NR, Swiss-Prot, KEGG, COG and GO was 42222,34681,18838 and 21193 respectively. Totally 42399 Unigenes were annotated. There were 42364 CDS mapped to protein database, while 2505 predicted. The unigenes matched with Danaus plexippus was 54.10%. Classifying the function of the genes, a total of 21193 unigenes got annotations through GO database. GO has three ontologies:molecular function, cellular component and biological process. The main process of biological process was cellular, metabolic and single-organism Cell, cell part and organelle were the primary kinds in the cellular component. The molecular function also had three main categories:binding, catalytic activity and transporter activity.18838 unigenes annotated to COG database, were classified to 26 categories. KEGG database annotated 31638 unigenes.29323 unigenes of them had relationship with 252 pathways, mainly involved in metabolic pathways, focal adhesion and regulation of actin cytoskeleton.2. The digital gene expression of different development stages showed:762 genes were up-regulated and 903 were down-regulated in the egg vs. third-instar larvae. Between the third-instar larvae and pupae 1338 genes found significantly different The expression of 2174 genes was significantly different in pupae vs. adult. Of these genes 1299 were up-regulated, while 875 were down-regulated.In stage of egg, it is found that the highly expressed genes were involved in embryology and cell growth. And these genes were expressed significantly decreased in larval. The genes highly expressed in larvae included yolk protein, storage protein provided nutrients. These genes had a low expression in other stages. Most of the digestive enzyme highly expressed in pupae compared with larvae. And mostly related to cuticular protein had a significantly increased expression. In adult, the highest expressed genes were follicular epithelium yolk protein and vitellogenin related to oviposition. In addition, genes were up-regulated significantly associated with tentacles and smell. Based on the transcriptome data,21 genes encoding CYP450s,20 genes encoding GSTs and 6 genes encoding AchE were detected.3. Based on the transcriptome sequencing data,10 genes annotated to APN were screened,7 of these genes with complete open reading frame. Average length was 2087nt. Unigene19019_Plodia was a complete sequence with a length of 3426nt. Its open reading frame length was 2858nt encoding 953 amino acids. Six genes of cadherin had an average length of 8826nt including 5 genes with full open reading frame encoding amino acid up to 3473 and 1491 amino acids at least. Most of the genes had a higher homology with Bombyx mori.3 genes annotated to ALP with complete open reading frame had an average length of 2369nt. The average length of open reading frame was 1606nt encoding about 510 amino acids.4. Eight reference genes were chosen to analysis the stability with two kinds of software via qRT-PCR. Then, the expression levels of peroxidase (POD) were analyzed by using different reference genes. Based on the results of geNorm and NormFinder analysis, the most stable gene was Eflα in different developmental stages, while, in different strains the most stable genes were β-Actin and Eflα. Considering the results of the software Eflα considered the most appropriate reference gene in different developmental stages, while, Eflα and β-Actin were the most suitable reference genes in different strains of Plodia interpunctella.5. The transcriptome database of Plodia interpunctella was used to identify the microsatellite or simple sequence repeats (SSRs) and to analyze the SSR loci information. A total of 3173 SSRs were identified in 37246 unigenes, with one SSR per 13380bp.Howere different types of repeat SSRs had different distribution considerably. Except nucleotide, among all SSR motifs, AT/AT was the most frequent repeat motif, and binucleotide repeat had a highest quantity. |
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