| The Chinese white wax scale insect, Ericerus pela, can survive a very low temperature.And some overwintering individuals could supercool to below-30°C. The low temperature adaptability is closly related to the natural population distribution of E. pela. To assess the deep supercooling ability of E. pela, By employing the high-thoughout sequencing technology,quantitative real-time PCR, gene cloning, prokaryotic expression, and transgenic plant, I analyzed the transcriptome, proteome, differentially expressed genes compared with the before overwintering adults, the annual expression dynamics of the putative afp genes, the sequence characterics of the putative afp genes, and gene functions. Based on these results, I illuminated the molecular mechanism adaptability to extreme low temperature, and mined novel insect antifreeze protein gene from E. pela. The main results obtained are as follows:(1) A total of 53,517,657 clean reads were obtained. These reads were assembled into161,005 transcripts with mean length of 1,506 bp. Some 11,871 CDS were predicted to encode amino acids. There was no afp genes indentified. Gene Ontology classification and Kyoto Encyclopedia of Genes and Genomes showed that the greatest number of transcripts was involved in signal transduction and metabolic process. Of these groups, genes involved in MAPK signaling pathway, calcium signaling pathway, PI3K-Akt signaling pathway, and pathways related to biosynthesis of soluble sugars, sugar alcohols, and free amino acids were dominant.(2) Proteome analysis was performed to confirm the expression of the genes in transcriptome. The proteomics data was used to isolate and identify the proteins related to low temperature resistance of E. pela. FAD, PFK, dehydrated protein, heat shock protein is also found in the protein group. Go and KEGG analysis showed that most proteins were enriched in signal transduction and metabolism, which PPAR signaling pathway, MAPK signaling pathway,calcium signaling pathway were dominant. A total of 388 putative afp genes were identified by using the random forest approach in the proteome results.(3) The RNA sequencing techonology was employed to analyze the gene expression profile. The gene expression difference between ChangChun overwintering adults and KunMing before overwintering adults was analyzed. A total of 2,386 genes were expressed differently between ChangChun adults and KunMing adults. 1,124 differently expressed genes(DEGs) were up-regulated in ChangChun adults when compared with KunMing adults, and the other 1,262 DEGs were down-regulated. The DEGs were enriched in starch and sucrose metabolism, galactose metabolism, arginine and proline metabolism, glycine, serine and threonine metabolism.(4) A total of 39 putative afp genes were screened according to the with the analysis of proteomics analysis, DEGs, and the random forest approach predict results. I furether analyzed the gene expression profile dynamic across one year, and found 10 candidate genes with expression profile of the mountain shape, which was consistent with the expression pattern of insect afp genes.(5) The full length cDNA sequence of the 10 candidate afp genes were cloned, and their sequence characters, protein tertiary structure, and their phylogenetic relationships were analyzed. The bioinformatics analysis results showed that, the 10 putative afp genes had the sequence and structure characters of afp genes.(6) The prokaryotic expression vectors that contained the 10 putative afp genes separately were constructed by homologous recombination. The competent Escherichia coli BL21 cells were transformed with the 10 recombinant plasmids separately. The heterologous expressions of the 10 proteins were induced by IPTG. One antifreeze protein was successfully expressed,and it was expressed in the supernatant and in the cell largely and activity. The heterologous expression of one AFP had the thermal hysteresis activity of 0.97 °C.(7) The plant expression vector that contained the afp gene was constructed. Then the afp gene was integrated into the genome of tobacco with Agrobacterium tumefaciens-mediated transformation system. The supercooling point(SCP) test showed that, the SCP of the transgenic plant was 0.36℃ lower than that of the wild tobacco. However, the statistics result showed that the difference was not significant.In conclusion, the study was focus on the adaptability of E. pela to extreme low temperature in ChangChun City, and analyzed the molecular mechanism underlying the adaptability of E. pela to low temperature. In addition, afp genes of E. pela were mined. This study will better our understanding of the relationship between the low-temperature adaptation and the population distribution. And will help us design and produce more molecules with antifreeze activity. |
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