Comparison of gene expression in the Red Imported Fire Ant, Solenopsis invicta Buren, in different temperature conditions

Purpose: We aimed to provide a basis for the adaptive mechanism and a rich resource for the discovery and identification of novel genes involved in the cold and heat stress response in Solenopsis invicta. retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Solenopsis invicta was reared at lab condition, and incubated at 4 different temperature for 24h (10, 20, 30 and 40℃). RNA was extracted using Trizol reagent and Illumina sequencing was performed at Macrogen. Illumina short reads were quality-filtered and Illumina-based de novo transcriptome assembly was performed. Differential Gene Expression Analysis was studied for different tempearture conditions. Results: Totally 99,085 unigenes was obtained which at least 19,154 were annotated with gene descriptions, gene ontology terms, and metabolic pathways. 86 GO functional sub-groups and 23 EggNOG terms resulted. DEGs with log2FC≥10 were screened and were compared at different temperatures. We found 203, 48 and 66 specific DEGs co-regulated at 10, 20 and 40℃, respectively compare with 30℃. Comparing transcriptome profiles for differential gene expression resulted various DE proteins and genes, including cytochrome P450, NADH dehydrogenase subunit 1, cuticle protein and HSP which have previously been reported to be involved in cold and high temperature resistance. GO analysis revealed that antioxidant activity up-regulated under high temperature stress. Conclusions: we compared the transcriptomes of S. invicta under high- and low-temperature stresses using RNA-Seq technology based on the high-throughput sequencing. Comparative transcriptome analysis identified many genes and a large number of changes were discovered in metabolic pathway through the GO and KEGG enrichment analysis. Our data will facilitate further molecular investigations and genomic research. Many high- and low-temperature significantly up-regulated genes were first identified in this study. These newly found genes may be important and necessary to RIFA overwintering and its behavior for adaptation in new environment as well as quarantine area. Total RNA of Solenopsis invicta for four different treated temperature that 30℃ was used as control.

研究目的：本研究旨在为红火蚁（Solenopsis invicta）冷热胁迫响应相关适应性机制的解析提供理论依据，同时为发现和鉴定参与该响应的新型基因提供丰富资源；此外，本研究还旨在对比转录组谱分析（RNA-seq）、微阵列（microarray）与定量反转录聚合酶链反应（qRT–PCR）三种技术方法，并评估优化高通量数据分析的实验方案。
研究方法：将红火蚁于实验室条件下饲养，分别置于4种不同温度（10、20、30、40℃）下培养24小时，其中30℃作为对照。采用Trizol试剂提取总RNA，随后由Macrogen公司完成Illumina测序。对Illumina短读长序列进行质量过滤，并开展基于Illumina平台的从头转录组组装。针对不同温度处理组进行差异基因表达分析。
研究结果：本研究共获得99085条单基因（unigenes），其中至少19154条获得了基因注释、基因本体论（Gene Ontology, GO）术语及代谢通路注释。共得到86个GO功能亚组与23个EggNOG注释条目。筛选得到log2FC≥10的差异表达基因（DEGs），并在不同温度处理组间开展对比分析。相较于30℃对照组，分别在10、20、40℃处理组中鉴定得到203、48、66个共调控的特异性DEGs。通过对比转录组谱的差异基因表达情况，筛选得到多种差异表达蛋白与基因，包括此前已被报道参与冷热胁迫抗性的细胞色素P450（cytochrome P450）、NADH脱氢酶亚基1（NADH dehydrogenase subunit 1）、表皮蛋白（cuticle protein）及热休克蛋白（HSP）。GO分析显示，高温胁迫下抗氧化活性相关通路显著上调。
研究结论：本研究基于高通量测序技术的RNA-seq，对比分析了红火蚁在高低温胁迫下的转录组。通过GO与京都基因与基因组百科全书（KEGG）富集分析，共鉴定得到大量基因，且发现代谢通路存在大量表达变化。本研究的数据将为后续分子生物学研究与基因组学研究提供助力。本研究首次鉴定得到多个高低温显著上调表达的基因，这些新发现的基因对于红火蚁越冬、适应新环境以及检疫防控均可能具有重要的生物学意义。