意大利蜜蜂幼虫响应东方蜜蜂微孢子虫胁迫的高表达基因分析
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摘要
为探究意大利蜜蜂(Apis mellifera ligustica,简称意蜂)幼虫响应东方蜜蜂微孢子虫(Nosema ceranae)胁迫的高表达基因(HEG)的作用,利用RNA-seq技术对正常的意蜂4、5和6日龄幼虫(Am4CK、Am5CK和Am6CK)及N.ceranae胁迫的意蜂4、5和6日龄幼虫(Am4T、Am5T和Am6T)进行测序。共得到268 765 544条原始读段,经过滤得到的有效读段数为266 318 442条,各样品的Q30均在92.42%及以上。根据FPKM值≥15的标准从上述6个样品中分别筛选出2 460、2 692、2 908、2 367、2 549和2 270个HEG。Venn分析结果显示,去除与对照组共有的HEG,Am4T、Am5T和Am6T的共有HEG数为3个,特有HEG数分别为373、210和79个。GO分类结果显示,Am4T、Am5T和Am6T的特有HEG分别能够注释26、27和16个GO条目,注释基因数最多的分别是代谢进程、结合和结合。KEGG分析结果显示这些特有HEG可分别富集在145、138和68条代谢通路,富集基因数最多的分别是过氧化物酶体、碳代谢和内质网蛋白加工。进一步分析结果表明N.ceranae胁迫可导致宿主的部分物质代谢、能量代谢和免疫代谢通路水平提高。从6个样品的共有HEG中随机选取10个进行RT-PCR验证,均能成功扩增出目的条带,说明本研究中的HEG真实存在。研究结果揭示了意蜂幼虫响应N.ceranae胁迫的HEG表达谱及作用,为深入解析宿主的胁迫应答机制提供了重要信息。
To investigate the role of highly expressed genes(HEGs) in Apis mellifera ligustica larvae responding to Nosema ceranae stress,4,5 and 6 day-old normal larvae(Am4 CK,Am5 CK and Am6 CK) and N.ceranae-stressed larvae(Am4 T,Am5 T and Am6 T) were sequenced by RNA-seq technology.In total,268 765 544 raw reads were produced and 266 318 442 clean reads were obtained after filtering,with a Q30 of each sample ≥92.42%.Based on the criteria of FPKM ≥15,2 460,2 692,2 908,2 367,2 549 and 2 270 HEGs were screened out from above-mentioned six samples,respectively.Venn analysis showed three HEGs were shared by Am4 T,Am5 T and Am6 T after removing HEGs shared with control groups,and the numbers of specific HEGs were 373,210 and 79,respectively.GO classification suggested specific HEGs in Am4 T,Am5 T and Am6 T could be annotated to 26,27 and 16 terms,and the largest ones were metabolic process,binding and binding.KEGG analysis demonstrated these specific HEGs were enriched in 145,138 and 68 pathways,and the mostly enriched ones were peroxisome,carbon metabolism and endoplasmic reticulum protein processing.Further analysis indicated N.ceranae stress can lead to the enhancement of part of host's material,energy and immunity metabolism.Finally,10 HEGs were randomly selected from the shared HEGs for RT-PCR,and all could produce signal bands,suggesting the true existence of HEGs in our study.These results illuminate the expression pattern and potential role of HEGs in A.m.ligustica larvae responding to N.ceranae challenge,and offer important information for further investigation of mechanisms regulating host's responses to stress.
To investigate the role of highly expressed genes(HEGs) in Apis mellifera ligustica larvae responding to Nosema ceranae stress,4,5 and 6 day-old normal larvae(Am4 CK,Am5 CK and Am6 CK) and N.ceranae-stressed larvae(Am4 T,Am5 T and Am6 T) were sequenced by RNA-seq technology.In total,268 765 544 raw reads were produced and 266 318 442 clean reads were obtained after filtering,with a Q30 of each sample ≥92.42%.Based on the criteria of FPKM ≥15,2 460,2 692,2 908,2 367,2 549 and 2 270 HEGs were screened out from above-mentioned six samples,respectively.Venn analysis showed three HEGs were shared by Am4 T,Am5 T and Am6 T after removing HEGs shared with control groups,and the numbers of specific HEGs were 373,210 and 79,respectively.GO classification suggested specific HEGs in Am4 T,Am5 T and Am6 T could be annotated to 26,27 and 16 terms,and the largest ones were metabolic process,binding and binding.KEGG analysis demonstrated these specific HEGs were enriched in 145,138 and 68 pathways,and the mostly enriched ones were peroxisome,carbon metabolism and endoplasmic reticulum protein processing.Further analysis indicated N.ceranae stress can lead to the enhancement of part of host's material,energy and immunity metabolism.Finally,10 HEGs were randomly selected from the shared HEGs for RT-PCR,and all could produce signal bands,suggesting the true existence of HEGs in our study.These results illuminate the expression pattern and potential role of HEGs in A.m.ligustica larvae responding to N.ceranae challenge,and offer important information for further investigation of mechanisms regulating host's responses to stress.
引文
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[18] 熊翠玲,耿四海,周丁丁,等.感染意大利蜜蜂工蜂的东方蜜蜂微孢子虫及其纯化孢子的高表达基因分析[J].上海交通大学学报(农业科学版),2019(2):6-13.
[19] 郭睿,刀晨,熊翠玲,等.意大利蜜蜂工蜂中肠响应Nosema ceranae胁迫的高表达基因分析[J].环境昆虫学报,2018,40(5):1106-1112.
[20] Aronstein K A,Murray K D.Chalkbrood disease in honey bees[J].Journal of Invertebrate Pathology,2010,103(S1):20-29.
[21] 席琼,胡巢凤.NOD样受体在炎症反应中的调控作用[J].生命科学,2010,22(5):454-458.
[22] Nizet V,Johnson R S.Interdependence of hypoxic and innate immune responses[J].Nature Reviews Immunology,2009,9(9):609-617.
[23] 谢志坚,熊晓东,左筠.Fc-R介导巨噬细胞吞噬作用信号转导机制[J].国际免疫学杂志,2002,25(1):5-9.
[24] Chaimanee V,Chantawannakul P,Chen Y,et al.Differential expression of immune genes of adult honey bee(Apis mellifera) after inoculated by Nosema ceranae[J].Journal of Insect Physiology,2012,58(8):1090-1095.
[2] 段成鼎.中华蜜蜂与意大利蜜蜂比较生物学的研究[D].福州:福建农林大学,2003.
[3] Fries I,Feng F,Silva A D,et al.Nosema ceranae,n.sp.(microspora,nosematidae),morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana,(hymenoptera,apidae)[J].European Journal of Protistology,1996,32(3):356-365.
[4] Huang W F,Jiang J H,Chen Y W,et al.A Nosema ceranae isolate from the honey bee Apis mellifera[J].Apidologie,1995,38(1):30-37.
[5] Chen Y,Evans J D,Smith I B,et al.Nosema ceranae is a long-present and wide-spread microsporidian infection of the European honey bee(Apis mellifera) in the United States[J].Journal of Invertebrate Pathology,2008,97(2):186-188.
[6] Dussaubat C,Brunet J L,Higes M,et al.Gut pathology and responses to the microsporidium,Nosema ceranae,in the honey bee,Apis mellifera[J].PLoS One,2012,7(5):e37017.
[7] Garcíapalencia P,Martínhernández R,Gonzálezporto A V,et al.Natural infection by Nosema ceranae causes similar lesions as in experimentally infected caged-worker honey bees(Apis mellifera)[J].Journal of Apicultural Research,2010,49(3):278-283.
[8] Mayack C,Naug D.Energetic stress in the honeybee Apis mellifera from Nosema ceranae infection[J].Journal of Invertebrate Pathology,2009,100(3):185-188.
[9] Martín-Hernández R,Botías C,Barrios L,et al.Comparison of the energetic stress associated with experimental Nosema ceranae,and Nosema apis,infection of honeybees(Apis mellifera)[J].Parasitology Research,2011,109(3):605-612.
[10] Lecocq A,Jensen A B,Kryger P,et al.Parasite infection accelerates age polyethism in young honey bees[J].Scientific Reports,2016,6:22042.
[11] Mayack C,Naug D.Parasitic infection leads to decline in hemolymph sugar levels in honeybee foragers[J].Journal of Insect Physiology,2010,56(11):1572-1575.
[12] Vidau C,Diogon M,Aufauvre J,et al.Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae[J].PLoS One,2011,6(6):e21550.
[13] Shutler D,Head K,Burghermaclellan K L,et al.Honey bee Apis mellifera parasites in the absence of Nosema ceranae fungi and Varroa destructor mites[J].PLoS One,2014,9(6):e98599.
[14] Wu J Y,Smart M D,Anelli C M,et al.Honey bees(Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema,(Microsporidia) infection[J].Journal of Invertebrate Pathology,2012,109(3):326-329.
[15] Higes M,Martínhernández R,Garridobailón E,et al.Honeybee colony collapse due to Nosema ceranae in professional apiaries[J].Environmental Microbiology Reports,2010,1(2):110-113.
[16] Eiri D M,Guntima S,Matthew E,et al.Nosema ceranae can infect honey bee larvae and reduces subsequent adult longevity[J].PLoS One,2015,10(5):e0126330.
[17] Benvau L R,Nieh J C.Larval honey bees infected with Nosema ceranae have increased vitellogenin titers as young adults[J].Scientific Reports,2017,7(1):14144.
[18] 熊翠玲,耿四海,周丁丁,等.感染意大利蜜蜂工蜂的东方蜜蜂微孢子虫及其纯化孢子的高表达基因分析[J].上海交通大学学报(农业科学版),2019(2):6-13.
[19] 郭睿,刀晨,熊翠玲,等.意大利蜜蜂工蜂中肠响应Nosema ceranae胁迫的高表达基因分析[J].环境昆虫学报,2018,40(5):1106-1112.
[20] Aronstein K A,Murray K D.Chalkbrood disease in honey bees[J].Journal of Invertebrate Pathology,2010,103(S1):20-29.
[21] 席琼,胡巢凤.NOD样受体在炎症反应中的调控作用[J].生命科学,2010,22(5):454-458.
[22] Nizet V,Johnson R S.Interdependence of hypoxic and innate immune responses[J].Nature Reviews Immunology,2009,9(9):609-617.
[23] 谢志坚,熊晓东,左筠.Fc-R介导巨噬细胞吞噬作用信号转导机制[J].国际免疫学杂志,2002,25(1):5-9.
[24] Chaimanee V,Chantawannakul P,Chen Y,et al.Differential expression of immune genes of adult honey bee(Apis mellifera) after inoculated by Nosema ceranae[J].Journal of Insect Physiology,2012,58(8):1090-1095.