基于PacBio Iso-Seq构建低温胁迫后中华蜜蜂工蜂全长转录组

Construction of the full-length transcriptome of the Chinese honey bee(Apis cerana cerana)workers under low temperature stress based on PacBio Iso-Seq

【目的】中华蜜蜂Apis cerana cerana是东方蜜蜂Apis cerana在中国地区分布最广泛的亚种,在地理适应和生态多样性方面有显著优势。中华蜜蜂的低温适应性优于西方蜜蜂A.mellifera,可用于高海拔高山地区和冬季蜜源的采集,该性能在生产方面有重要作用,探究中华蜜蜂低温耐受性可有助于优良品系的筛选,为农业经济创造更大价值。【方法】收集分别在常温(25.0±0.2)℃(对照)和低温(4.0±0.2)℃下处理6 h时的中华蜜蜂3,10和21日龄工蜂6组样本,进行转录组二代测序(Illumina RNA-Seq)和全长转录组三代测序(PacBio Iso-Seq),利用二代测序数据对三代测序数据进行校正,基于高质量的测序数据对比NR,NT,Pfam,KOG/COG,Swiss-Prot,KEGG和GO数据库,对新转录本和新基因进行功能注释。【结果】中华蜜蜂工蜂6组样本全长转录组测序分别获得71.17,42.76,58.82,53.45,40.33和42.79 Gb读段,平均N50达到205895 bp,质控后得到164194条一致性序列;通过比对到东方蜜蜂参考基因组可获得21519条新转录本,与中华蜜蜂物质代谢、神经系统和信号转导等密切相关,且不同日龄中华蜜蜂受低温胁迫后,物质代谢、细胞发育、寿命及神经发育等受到影响,同时还鉴定到28647条长链非编码RNA(long non-coding RNA,LncRNA),其中lincRNA和sense_intronic两种类型LncRNA的占比最多,分别为35%和45%。【结论】本研究利用矫正后的中华蜜蜂工蜂PacBio Iso-Seq转录组数据鉴定到大量未注释的新基因和新转录本,并对其进行了功能注释,分析了LncRNA的数量和类型,研究结果完善了东方蜜蜂参考基因组,为中华蜜蜂抗寒机制的探究提供了良好的数据背景。

【Aim】The Chinese honey bee(Apis cerana cerana)is the most widely distributed subspecies of the eastern honey bee(Apis cerana)in China,with significant advantages in geographic adaptation and ecological diversity.The cold resistance of A.c.cerana is superior to that of the western bee(A.mellifera)and can be used for beekeeping at high altitudes and collecting winter honey sources,this performance plays an important role in production.Exploring the low temperature tolerance of A.c.cerana can help to select excellent strains and create greater value for agricultural economy.【Methods】Samples of six groups of A.c.cerana workers of the 3-,10-and 21-day-old subjected to normal temperature[(25.0±0.2)℃]treatment(the control group)and low temperature[(4.0±0.2)℃]treatment,respectively,for 6 h were collected and used for transcriptome sequencing by Illumina RNA-Seq and for full-length transcriptome sequencing by PacBio Iso-Seq.The Illumina RNA-Seq data were used to correct the PacBio Iso-Seq data.Based on the high-quality sequencing data,functional annotation of novel transcripts and genes were conducted using NR,NT,Pfam,KOG/COG,Swiss-Prot,KEGG and GO databases.【Results】A total of 71.17,42.76,58.82,53.45,40.33 and 42.79 Gb reads of the full-length transcriptome sequencing of A.c.cerana workers in six groups were obtained,respectively,with an average N50 of 205895 bp,yielding 164194 consistent sequences after quality control.A total of 21519 novel transcripts were obtained by alignment to the A.cerana reference genome,and were closely related to the material metabolism,nervous system,and signal transduction of A.c.cerana.The material metabolism,cell development,lifespan,and neural development of A.c.cerana at different day-old were affected by low temperature stress.Furthermore,28647 long non-coding RNAs(LncRNAs)were identified,among them lincRNA and sense_intronic were the most abundant lncRNAs,accounting for 35%and 45%,respectively.【Conclusion】This study identified a large number of unannotated novel genes and transcripts using the corrected the PacBio Iso-Seq data and conducted functional annotation of A.c.cerana workers.The quantity and types of LncRNA were also analyzed.These results complement the reference genome of A.cerana and provide a good data background for exploring the cold resistance mechanism of A.c.cerana.