蜜蜂球囊菌基因结构优化及新基因鉴定
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摘要
为补充和完善蜜蜂球囊菌Ascosphaera apis基因组序列和功能注释信息,本研究利用RNA-seq技术对球囊菌菌丝和孢子进行深度测序,并基于高质量的转录组数据对已注释基因进行结构优化,对未注释基因进行预测、鉴定和分析。将测序得到的有效读段进行参考基因组比对和转录本重构;利用Cuffcompare软件将重构转录本与参考基因组进行比对。结果表明:共对101个已注释基因的5′端或3′端进行了延长;共鉴定出373个新基因,随机挑选10个新基因进行RT-PCR验证,其中8个能扩增出符合预期的目的片段,表明预测出的多数新基因真实存在;共计147个球囊菌新基因可注释到Nr和eggNOG数据库中,85个新基因注释到29个GO条目,66个新基因注释到33条代谢通路。本研究结果补充了球囊菌的基因结构和功能注释信息,也为新基因的功能研究打下了初步基础。
In order to supplement and improve the sequence and annotation information of Ascosphaera apis,the genome of A.apis mycelia and spore were sequenced via RNA-seq technology,followed by structural optimization of annotated genes and prediction and analysis of novel genes on the basis of high-quality transcriptome data in this study.Mapping of clean reads to the reference genome and reconstruction of transcripts were conducted using the generated clean reads.Cuffcompare software was used to compare the reconstructed transcripts with the reference genome.The results demonstrated that:The 5′or 3′end of 101 annotated genes were prolonged;A total of 373 novel genes were predicted,among which 10 novel genes were randomly selected for inverse transcription PCR identification and expected fragments were amplified from 8 genes suggesting the real existence of majority of predicted novel genes;A total of 147 novel genes have functional annotation in Nr and eggNOG databases,and 85 and 66 novel genes can be annotated to 29 GO terms and 33 KEGG pathways,respectively.The results not only provide supplement for improving structural and functional annotation of A.apis genes but also offer a primary foundation for functional study on these novel genes.
In order to supplement and improve the sequence and annotation information of Ascosphaera apis,the genome of A.apis mycelia and spore were sequenced via RNA-seq technology,followed by structural optimization of annotated genes and prediction and analysis of novel genes on the basis of high-quality transcriptome data in this study.Mapping of clean reads to the reference genome and reconstruction of transcripts were conducted using the generated clean reads.Cuffcompare software was used to compare the reconstructed transcripts with the reference genome.The results demonstrated that:The 5′or 3′end of 101 annotated genes were prolonged;A total of 373 novel genes were predicted,among which 10 novel genes were randomly selected for inverse transcription PCR identification and expected fragments were amplified from 8 genes suggesting the real existence of majority of predicted novel genes;A total of 147 novel genes have functional annotation in Nr and eggNOG databases,and 85 and 66 novel genes can be annotated to 29 GO terms and 33 KEGG pathways,respectively.The results not only provide supplement for improving structural and functional annotation of A.apis genes but also offer a primary foundation for functional study on these novel genes.
引文
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[11]郭睿,陈大福,黄枳腱,梁勤,熊翠玲,徐细建,郑燕珍,张曌楠,解彦玲,童新宇,侯志贤,江亮亮,刀晨.球囊菌胁迫中华蜜蜂幼虫肠道过程中病原的转录组学研究[J].微生物学报,2017,57(12):1865-1878Guo R,Chen D F,Huang Z J,Liang Q,Xiong C L,Xu X J,Zheng Y Z,Zhang Z N,Xie Y L,Tong X Y,Hou Z X,Jiang L L,Dao C.Transcriptome analysis of Ascosphaera apis stressing larval gut of Apis cerana cerana[J].Acta Microbiologica Sinica,2017,57(12):1865-1878(in Chinese)
[12] Venter J C,Adams M D,Myers E W,Li P W,Mural R J,Sutton G G.The sequence of the human genome[J].Science,2001,61(9):1207-8
[13] Gerrard D T,Fricke C,Edward D A,Edwards D R,Chapman T.Genome-wide responses of female fruit flies subjected to divergent mating regimes[J].PLoS One,2013,8(6):e68136
[14] Elsik C G,Worley K C,Bennett A K,Beye M,Camara F,Childers C P.Finding the missing honey bee genes:Lessons learned from a genome upgrade[J].BMC Genomics,2014,15(1):86
[15]陈大福,郭睿,熊翠玲,梁勤,郑燕珍,徐细建,张曌楠,黄枳腱,张璐,王鸿权,解彦玲,童新宇.中华蜜蜂幼虫肠道响应球囊菌早期胁迫的转录组学.中国农业科学,2017,50(13):2614-2623Chen D F,Guo R,Xiong C L,Liang Q,Zheng Y Z,Xu X J,Zhang Z N,Huang Z J,Zhang L,Wang H Q,Xie Y L,Tong X Y.Transcriptome of Apis cerana cerana larval gut under the stress of Ascosphaera apis[J]. Scientia Agricultura Sinica,2017,50(13):2614-2623(in Chinese)
[16]穆彩琴,张瑞娟,屈聪玲,韩渊怀,王兴春,杨致荣.基于RNA-Seq技术的谷子新基因发掘及基因结构优化[J].植物生理学报,2016,52(7):1066-1072Mu C Q,Zhang R J,Qu C L,Han Y H,Wang X C,Yang Z R.Identification of novel genes and optimization of annotated genes in foxtail millet by RNA-Seq technology[J].Plant Physiology Journal,2016,52(7):1066-1072(in Chinese)
[17]韩昆鹏,段炼,李婷婷,王金玉,张涛,李国辉,张跟喜,薛倩.京海黄鸡卵巢转录组研究:基因结构分析与新基因发掘注释[J].中国畜牧兽医,2016,43(4):854-861Han K P,Duan L,Li T T,Wang J Y,Zhang T,Li G H,Zhang G X,Xue Q.Study on RNA-Seq of Jinghai Yellow chicken ovary:Analysis of gene structure and mining new genes[J]. China Animal Husbandry&Veterinary Medicine,2016,43(4):854-861(in Chinese)
[18] Chen S Y,Deng F L,Jia X B,Li C,Lai S J. A transcriptome atlas of rabbit revealed by PacBio singlemolecule long-read sequencing[J].Scientific Reports,2017,7:7648
[19] Jiao Y,Peluso P,Shi J,Liang T,Stitzer M C,Bo W.Improved maize reference genome with single-molecule technologies[J].Nature,2017,546(7659):524-527
[20] Mukhopadhyay R,Joaquin J,Hogue R,Kilaru A,Jospin G,Mars K.Complete genome sequence of a Paenalcaligenes hominis strain isolated from a paraplegic patient with neurogenic bladder using single-molecule real-time sequencing technology[J].Genome Announcements,2017,5(17):e00252-17
[21] Song C X,Clark T A,Lu X Y,Kislyuk A,Dai Q,Turner S W,He C,Korlach J.Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine[J].Nature Methods,2012,9(1):75-77
[2] Aizen M A,Garibaldi L A,Cunningham S A,Klein A M.How much does agriculture depend on pollinators?Lessons from long-term trends in crop production[J].Annals of Botany,2009,103(9):1579-1588
[3]郑志阳,李江红,梁勤,陈大福.蜜蜂球囊菌分泌多种胞外酶侵染蜜蜂幼虫[J].福建农林大学学报:自然科学版,2011,40(3):280-284Zheng Z Y,Li J H,Liang Q,Chen D F.Ascosphaera apis secretes multiple extracellular enzymes to infect honeybee larvae[J].Journal of Fujian Agriculture and Forestry University:Natural Science Edition,2011,40(3):280-284(in Chinese)
[4] Qin X,Evans J D,Aronstein K A,Murray K D,Weinstock G M. Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis[J].Insect Molecular Biology,2006,15(5):715-718
[5] Aronstein K A,Murray K D,de León J H,Qin X,Weinstock G M.High mobility group(HMG-box)genes in the honeybee fungal pathogen Ascosphaera apis[J].Mycologia,2007,99(4):553-561
[6] Cornman R S,Bennett A K,Murray K D,Evans J D,Elsik C G,Aronstein K.Transcriptome analysis of the honey bee fungal pathogen,Ascosphaera apis:Implications for host pathogenesis[J].BMC Genomics,2012,13(1):285
[7]李汶东,熊翠玲,王鸿权,侯志贤,童新宇,张璐,付中民,郑燕珍,陈大福,郭睿.基于RNA-seq数据大规模挖掘蜜蜂球囊菌的SSR分子标记[J].福建农林大学学报:自然科学版,2017,46(4):434-438Li W D,Xiong C L,Wang H Q,Hou Z X,Tong X Y,Zhang L,Fu Z M,Zheng Y Z,Chen D F,Guo R.Large scale development of SSR molecular markers of Ascosphaeraapis based on RNA-seq data[J].Journal of Fujian Agriculture and Forestry University:Natural Science Edition,2017,746(4):434-438(in Chinese)
[8]杜宇,熊翠玲,史秀丽,郑燕珍,付中民,徐细建,陈大福,郭睿.意大利蜜蜂6日龄幼虫肠道内球囊菌的差异表达基因分析[J].浙江农业学报,2017,29(7):1119-1128Du Y,Xiong C L,Shi X L,Zheng Y Z,Fu Z M,Xu X J,Chen D F,Guo R.Transcriptome analysis of differentially expressed genes in Ascosphaera apis stressing the 6-day-old larval gut of Apis mellifera ligustica[J].Acta Agriculturae Zhejiangensis,2017,29(7):1119-1128(in Chinese)
[9]陈大福,王鸿权,李汶东,熊翠玲,郑燕珍,付中民,徐细建,黄枳腱,郭睿.胁迫中华蜜蜂幼虫肠道的球囊菌及其体外培养的高表达基因分析[J].福建农林大学学报:自然科学版,2017,46(5):562-568Chen D F,Wang H Q,Li W D,Xiong C L,Zheng Y Z,Fu Z M,Xu X J,Huang Z J,Guo R.Analysis of highly expressed genes of Ascosphaera apis infecting the gut of Apis cerana ceranalarvae and its in vitro culture[J].Journal of Fujian Agriculture and Forestry University:Natural Science Edition,2017,46(5):562-568(in Chinese)
[10]陈大福,郭睿,熊翠玲,梁勤,郑燕珍,徐细建,黄枳腱,张曌楠,张璐,李汶东,童新宇,席伟军.胁迫意大利蜜蜂幼虫肠道的球囊菌的转录组分析[J].昆虫学报,2017,60(4):401-411Chen D F,Guo R,Xiong C L,Liang Q,Zheng Y Z,Xu X J,Huang Z J,Zhang Z N,Zhang L,Li W D,Tong X Y,Xi W J.Transcriptomic analysis of Ascosphaera apis stressing larval gut of Apis mellifera ligustica(Hyemenoptera:Apidae)[J].Acta Entomologica Sinica,2017,60(4):401-411(in Chinese)
[11]郭睿,陈大福,黄枳腱,梁勤,熊翠玲,徐细建,郑燕珍,张曌楠,解彦玲,童新宇,侯志贤,江亮亮,刀晨.球囊菌胁迫中华蜜蜂幼虫肠道过程中病原的转录组学研究[J].微生物学报,2017,57(12):1865-1878Guo R,Chen D F,Huang Z J,Liang Q,Xiong C L,Xu X J,Zheng Y Z,Zhang Z N,Xie Y L,Tong X Y,Hou Z X,Jiang L L,Dao C.Transcriptome analysis of Ascosphaera apis stressing larval gut of Apis cerana cerana[J].Acta Microbiologica Sinica,2017,57(12):1865-1878(in Chinese)
[12] Venter J C,Adams M D,Myers E W,Li P W,Mural R J,Sutton G G.The sequence of the human genome[J].Science,2001,61(9):1207-8
[13] Gerrard D T,Fricke C,Edward D A,Edwards D R,Chapman T.Genome-wide responses of female fruit flies subjected to divergent mating regimes[J].PLoS One,2013,8(6):e68136
[14] Elsik C G,Worley K C,Bennett A K,Beye M,Camara F,Childers C P.Finding the missing honey bee genes:Lessons learned from a genome upgrade[J].BMC Genomics,2014,15(1):86
[15]陈大福,郭睿,熊翠玲,梁勤,郑燕珍,徐细建,张曌楠,黄枳腱,张璐,王鸿权,解彦玲,童新宇.中华蜜蜂幼虫肠道响应球囊菌早期胁迫的转录组学.中国农业科学,2017,50(13):2614-2623Chen D F,Guo R,Xiong C L,Liang Q,Zheng Y Z,Xu X J,Zhang Z N,Huang Z J,Zhang L,Wang H Q,Xie Y L,Tong X Y.Transcriptome of Apis cerana cerana larval gut under the stress of Ascosphaera apis[J]. Scientia Agricultura Sinica,2017,50(13):2614-2623(in Chinese)
[16]穆彩琴,张瑞娟,屈聪玲,韩渊怀,王兴春,杨致荣.基于RNA-Seq技术的谷子新基因发掘及基因结构优化[J].植物生理学报,2016,52(7):1066-1072Mu C Q,Zhang R J,Qu C L,Han Y H,Wang X C,Yang Z R.Identification of novel genes and optimization of annotated genes in foxtail millet by RNA-Seq technology[J].Plant Physiology Journal,2016,52(7):1066-1072(in Chinese)
[17]韩昆鹏,段炼,李婷婷,王金玉,张涛,李国辉,张跟喜,薛倩.京海黄鸡卵巢转录组研究:基因结构分析与新基因发掘注释[J].中国畜牧兽医,2016,43(4):854-861Han K P,Duan L,Li T T,Wang J Y,Zhang T,Li G H,Zhang G X,Xue Q.Study on RNA-Seq of Jinghai Yellow chicken ovary:Analysis of gene structure and mining new genes[J]. China Animal Husbandry&Veterinary Medicine,2016,43(4):854-861(in Chinese)
[18] Chen S Y,Deng F L,Jia X B,Li C,Lai S J. A transcriptome atlas of rabbit revealed by PacBio singlemolecule long-read sequencing[J].Scientific Reports,2017,7:7648
[19] Jiao Y,Peluso P,Shi J,Liang T,Stitzer M C,Bo W.Improved maize reference genome with single-molecule technologies[J].Nature,2017,546(7659):524-527
[20] Mukhopadhyay R,Joaquin J,Hogue R,Kilaru A,Jospin G,Mars K.Complete genome sequence of a Paenalcaligenes hominis strain isolated from a paraplegic patient with neurogenic bladder using single-molecule real-time sequencing technology[J].Genome Announcements,2017,5(17):e00252-17
[21] Song C X,Clark T A,Lu X Y,Kislyuk A,Dai Q,Turner S W,He C,Korlach J.Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine[J].Nature Methods,2012,9(1):75-77