郭 睿，張 璐，徐細建，史秀麗，熊翠玲，鄭燕珍，付中民，黃枳腱，王鴻權(quán)，侯志賢，陳大福*

中華蜜蜂6日齡幼蟲腸道響應球囊菌脅迫的差異表達基因分析

郭 睿1*，張 璐1*，徐細建1，史秀麗2，熊翠玲1，鄭燕珍1，付中民1，黃枳腱1，王鴻權(quán)1，侯志賢1，陳大福1**

(1.福建農(nóng)林大學蜂學學院，福州350002; 2.新疆維吾爾自治區(qū)蜂業(yè)技術(shù)管理總站, 烏魯木齊 930001)

蜜蜂球囊菌特異性侵染蜜蜂幼蟲而導致白堊病，嚴重危害養(yǎng)蜂生產(chǎn)。本研究利用RNA-seq技術(shù)對健康及球囊菌脅迫的中蜂幼蟲腸道進行深度測序，進而對宿主的差異表達基因進行深入分析。本研究中，幼蟲腸道樣品的RNA-seq共得到191167730條原始讀段(raw reads)，經(jīng)過濾得到186284296條有效讀段(clean reads)，差異表達基因(DEG)分析結(jié)果顯示上調(diào)與下調(diào)基因的數(shù)量分別為4513和385個。Gene ontology(GO)富集分析結(jié)果顯示，上調(diào)基因富集在45個GO條目(term)，富集基因數(shù)最多的是細胞進程、代謝進程及催化活性，下調(diào)基因富集在32個 GO term，富集基因數(shù)最多的是代謝進程、單組織進程及催化活性， KEGG代謝通路(pathway)富集分析結(jié)果顯示上調(diào)基因富集在193個pathway，其中富集基因數(shù)最多的是核糖體、氨基酸的合成、碳代謝。下調(diào)基因富集在59個pathway，其中富集基因數(shù)最多的是甘氨酸、碳代謝以及二羧酸代謝。深入分析發(fā)現(xiàn)宿主的細胞免疫被顯著激活，體液免疫中的Toll-like與Jak-STAT信號通路也被球囊菌所激活。研究結(jié)果為揭示中蜂幼蟲在球囊菌入侵后期的脅迫應答機制提供了重要的信息，也為解析中蜂幼蟲的球囊菌抗性機制奠定了基礎。

中華蜜蜂；幼蟲腸道；球囊菌；差異表達基因；免疫防御

蜜蜂是一種重要的社會學模式昆蟲，因其在發(fā)育學、神經(jīng)生物學、行為學和病原-宿主互作研究中的重要性而備受關注(Galiziaetal., 2012; Begnaetal., 2012; Zayed, 2012; Foretetal., 2012; Kurzeetal., 2016)。蜜蜂作為最重要的授粉昆蟲，在農(nóng)業(yè)生產(chǎn)和生態(tài)維持中也發(fā)揮著不可替代作用(Committee on the Status of Pollinators in North Acerica, 2007)。據(jù)報道，蜜蜂為全球70%的作物和野生植物授粉(Kleinetal., 2007; Elke, 2010)。蜜蜂因其群遭受細菌、真菌及病毒等病原的侵襲。蜜蜂白堊病是一種最具代表性的致死性真菌病，1913年Massen在德國首次報道發(fā)現(xiàn)白惡病(Aronstein and Murray, 2010)，中國大陸1990年發(fā)生白堊病(Liang and Chen, 2008)。近年來，隨著養(yǎng)蜂活動及蜂產(chǎn)品全球貿(mào)易的快速發(fā)展，白堊病發(fā)病率逐年上升(Aizenetal., 2009)。白堊病是由蜜蜂球囊菌Ascosphaeraapis(簡稱球囊菌)特異性侵染蜜蜂幼蟲而導致，可造成蜜蜂群勢的大幅下降，從而嚴重影響蜂蜜等產(chǎn)品的產(chǎn)量(Bailey, 1963)，據(jù)報道，白堊病可造成蜂蜜產(chǎn)量下降5-37%(Zaghlouletal., 2005)。近二十年來，國內(nèi)外學者在病原分類鑒定、形態(tài)學、病理學、流行病學、侵染過程、蜜蜂防御以及疾病防治等方面對白堊病開展了一系列研究。本課題組也在球囊菌的生化、檢測及侵染過程等方面開展了較為系統(tǒng)的研究，如梁勤等從碳源、氮源、維生素、礦質(zhì)元素等方面研究了營養(yǎng)生態(tài)條件對球囊菌生長及產(chǎn)孢的影響，結(jié)果表明營養(yǎng)生態(tài)條件的變化對球囊菌的影響極大(Liangetal., 2001)；鄭志陽等對健康和患病蜜蜂幼蟲血淋巴進行 SDS-PAGE電泳和蛋白酶、酯酶的活性染色，發(fā)現(xiàn)健康蜜蜂幼蟲血淋巴中的蛋白含量豐富，主要由4種高分子質(zhì)量的蛋白組成，而患病幼蟲血淋巴中的蛋白含量很少，主要蛋白組分被降解，多種蛋白酶和酯酶的活性在患病幼蟲血淋巴中檢測到，但在健康幼蟲中檢測不到(Zhengetal., 2011)。

我國養(yǎng)蜂生產(chǎn)的主要蜂種是意大利蜜蜂和中華蜜蜂。中華蜜蜂Apisceranacerana(簡稱中蜂)基因組的公布(Parketal., 2015)，為中蜂的分子生物學研究提供了重要參考信息。Aronstein等利用cDNA-AFLP 對健康及球囊菌感染的西方蜜蜂Apismellifera幼蟲進行了比較，結(jié)果表明差異表達基因(DEGs)參與了宿主的能量代謝和蛋白轉(zhuǎn)運，其中的類幾丁質(zhì)編碼基因很可能參與了蜜蜂幼蟲對球囊菌的抵抗(Aronsteinetal., 2010)。Cornman等對來自培養(yǎng)基的球囊菌菌絲和來自蜜蜂幼蟲感染組織的球囊菌菌絲進行了轉(zhuǎn)錄組測序，功能分析表明球囊菌的DEG參與了交配類型、細胞內(nèi)信號轉(zhuǎn)導和應激反應。

目前，白堊病的相關研究主要集中在意蜂，有關球囊菌侵染中蜂的研究報道極少。前期我們發(fā)現(xiàn)中蜂蜂群偶爾可見白堊病患病幼蟲，從患病中蜂幼蟲上分離培養(yǎng)真菌病原，經(jīng)形態(tài)學、分子生物學以及交叉感染實驗證實確為A.apis(未發(fā)表數(shù)據(jù))。

本研究利用Illumina測序技術(shù)對對健康及球囊菌脅迫的中蜂6日齡幼蟲腸道進行深度測序，得到宿主的差異表達基因(DEGs)，并通過Gene ontology(GO)和KEGG代謝通路(pathway)富集分析深入研究DEGs。研究結(jié)果可為揭示中蜂幼蟲腸道響應球囊菌后期脅迫的應答機制提供重要信息，也能為關鍵應答基因的篩選及驗證奠定基礎。

1 材料與方法

1.1 生物材料

本研究中使用的中蜂幼蟲取自福建農(nóng)林大學蜂學學院教學蜂場，球囊菌菌株由福建農(nóng)林大學蜂學學院蜜蜂保護實驗室保存并活化。

1.2 主要實驗試劑及儀器

DNaseI和Oligotex mRNA Kits Midi試劑盒購自德國Qiagen公司，Dynal M280磁珠購自Invitrogen公司，DNA ligase購自美國Thermo公司，RNA Reagent抽提試劑盒、Ex Taq polymerase及Superscript II reverse transcriptase均購自日本TaKaRa公司，純化cDNA的Acpure beads為美國Agencourt產(chǎn)品，cDNA文庫構(gòu)建試劑盒TruSeqTMDNA SAcple Prep Kit-Set A為美國Illumina公司產(chǎn)品。其它試劑均為國產(chǎn)分析純。

倒置顯微鏡為中國上海光學儀器五廠產(chǎn)品，超凈工作臺為中國蘇州安泰空氣技術(shù)有限公司產(chǎn)品，恒溫恒濕氣候箱購自中國寧波江南儀器廠，凝膠成像系統(tǒng)為中國上海培清科技有限公司產(chǎn)品，PCR儀為美國Bio Rad公司產(chǎn)品，超低溫冰箱為中國中科美菱公司產(chǎn)品。

1.3 球囊菌活化、孢子純化及計數(shù)

按照本實驗室已建立的方法對球囊菌進行活化(Zhangetal., 2017)。按照Jensen等(2013)的方法純化球囊菌孢子，將高濃度孢子溶液梯度稀釋后用血球計數(shù)板對孢子進行計數(shù)。

1.4 中蜂幼蟲的人工飼養(yǎng)及腸道樣品準備

中蜂幼蟲的人工飼養(yǎng)參照王倩等(2009)的方法。從蜂學學院蜂場群勢較強的中蜂蜂群用移蟲針挑取2日齡幼蟲，放入無菌的24孔細胞培養(yǎng)板(每孔對應1只幼蟲，孔內(nèi)加有35℃預溫的幼蟲飼料)，將24孔板放入恒溫恒濕培養(yǎng)箱，35℃，70%相對濕度(RH)條件下飼養(yǎng)。每隔24 h更換飼料。預先配制添球囊菌孢子的人工飼料，混勻后調(diào)整孢子終濃度至為1×107孢子/mL，飼喂處理組3日齡幼蟲，對照組飼喂正常人工飼料。本次實驗進行3次生物學重復。

本研究的分析重點是中鋒幼蟲腸道在球囊菌脅迫后期的DEGs。鑒于后續(xù)將對宿主在球囊菌入侵全過程的脅迫應答進行研究，為降低測序成本，擬將健康中蜂4日齡幼蟲腸道作為唯一對照。因此，本研究中，分別剖取對照組4日齡幼蟲腸道(AcCK)和處理組6日齡幼蟲腸道(AcT)，AcCK與AcT的三個生物學重復分別為AcCK-1、AcCK-2、AcCK-3和AcT-1、AcT-2、AcT-3。每剖取一只幼蟲腸道，迅速將腸道移至RNA Free的EP管，液氮速凍，待一組腸道樣品(7只幼蟲腸道)集齊后，轉(zhuǎn)移保存于-80℃。

1.5 cDNA文庫構(gòu)建及Illumina測序

利用RNAiso Reagent試劑盒抽提處理組和對照組上述6頭幼蟲腸道的總RNA，然后用RNase-free DNaseI去除基因組DNA殘留。RNA的質(zhì)量通過瓊脂糖凝膠電泳和NanoDrop ND-1000(NanoDrop, Wilmington, DE, USA)進行檢測。cDNA文庫構(gòu)建參照張曌楠等的建庫方法(Zhangetal., 2017)。委托廣州基迪奧生物科技有限公司對上述幼蟲腸道樣品進行雙端測序，測序平臺為Illumina HiSeq2500。

1.6 數(shù)據(jù)分析

對于下機數(shù)據(jù)，利用Perl腳本去除含有adaptor、未知核苷酸比例大于5%和低質(zhì)量reads，獲得有效讀段(clean reads)。利用R軟件(Version 2.16.2)進行測序飽和度分析。使用短 reads 比對工具 bowtie(Langmeadetal., 2009)將clean reads映射(mapping)到核糖體數(shù)據(jù)庫(最多允許5個錯配)，去除比對上核糖體的 reads，將保留下來的數(shù)據(jù)用于轉(zhuǎn)錄組的組裝及分析，進而利用SOAP aligner/soap2軟件(Hurgobin, 2016)將未比對上核糖體的 reads mapping到中鋒幼蟲腸道參考轉(zhuǎn)錄組(前期已組裝并注釋，原始數(shù)據(jù)已上傳NCBI SRA數(shù)據(jù)庫，SRA號: SRA456721)。

利用FPKM(Fragments Per Kilobase of transcript per Million mapped reads)法計算基因表達量。利用R軟件(version 2.16.2)計算各樣品之間的相關性系數(shù)。利用edgeR軟件(Robinsonetal., 2010)進行DEGs分析。DEGs的篩選標準為FDR ≤ 0.05且|log2Fold change | ≥ 1。將DEGs向GO數(shù)據(jù)庫(http://www.geneontology.org/)的各條目(term)mapping，并計算每個term的基因數(shù)，從而得到具有某個GO功能的基因列表及基因數(shù)目統(tǒng)計，然后應用超幾何檢驗，找出與整個基因組背景相比，在差異表達基因中顯著富集的GO條目。KEGG(pathway)顯著性富集分析以KEGG pathway為單位，應用超幾何檢驗，找出與整個基因組背景相比，在DEGs中顯著性富集的pathway。

2 結(jié)果與分析

2.1 RNA-seq數(shù)據(jù)質(zhì)控與評估

中蜂幼蟲腸道樣品的轉(zhuǎn)錄組測序共測得191167730條raw reads，經(jīng)過濾得到186284296條clean reads，各樣品clean reads數(shù)均在26509638 (97.96%)以上(表1)。兩端平均Q20為98.38%，兩端平均Q30為為95.94%。隨著測序量的增多，檢測到的基因數(shù)也隨之上升、增長速度趨于平緩，說明本研究的測序深度檢測到的基因數(shù)趨于飽和(附圖1)。AcCK與AcT的組內(nèi)各生物學重復之間的相關性均在0.96以上，說明樣本的重復性高(圖1)。上述結(jié)果說明本研究的轉(zhuǎn)錄組數(shù)據(jù)質(zhì)量良好，可用于進一步分析。

表1 RNA-seq數(shù)據(jù)統(tǒng)計Table 1 Overview of RNA-seq data

圖1 各幼蟲腸道樣品不同生物學重復間的相關性.Fig.1 Pearson correlation between every two biological repeats within each Apis cerana cerana larval gut sample 注: A: AcCK-1與AcCK-2間的相關性; B: AcCK-2與AcCK-3間的相關性; C: AcCK-1與AcCK-3間的相關性; D: AcT-1與AcT-2間的相關性; E: AcT-2與AcT-3間的相關性; F: AcT-1與AcT-3間的相關性.Note: A: parson correlations between AcCK-1 and AcCK-2; B: pearson correlations between AcCK-2 and AcCK-3; C: pearson correlations between AcCK-1 and AcCK-3; D: pearson correlations between AcT-1 and AcT-2; E: pearson correlations between AcT-2 and AcT-3; F: pearson correlations between AcT-1 and AcT-3.

2.2 DEGs分析

DEGs分析結(jié)果顯示，在AcCK vs AcT中共有4898個基因差異表達，其中，上調(diào)基因和下調(diào)基因的數(shù)量分別為4513和385個(表2)，上調(diào)基因的數(shù)量遠遠多余下調(diào)基因，說明在球囊菌脅迫后期，中蜂幼蟲腸道的絕大多數(shù)基因被病原激活表達。

表2 差異表達基因統(tǒng)計Table2 Summary of DEGs

2.3 DEGs的GO分類

DEGs的GO分類結(jié)果顯示，這些DEGs分為三類：生物學進程(biological process)、細胞組分(cellular component)和分子功能(molecular function)，上調(diào)基因分布于45個 GO term上，富集基因數(shù)最多的是細胞進程(cellular process)、代謝進程(metabolic process)、催化活性(catalytic activity)(圖2)。下調(diào)基因分布于32個 GO term上，富集基因數(shù)最多的是代謝進程(metabolic process)、單組織進程(single-organism process)、催化活性(catalytic activity)(圖2)。

2.4 DEGs的KEGG pathway富集分析

DEGs的KEGG pathway富集分析結(jié)果顯示，上調(diào)基因富集在193個pathway，其中富集基因數(shù)最多的是核糖體(ribosome)、氨基酸生物合成(biosynthesis of Acino acids)以及碳代謝(carbon metabolism)。下調(diào)基因富集在59個pathway，其中富集基因數(shù)最多的是甘氨酸(biosynthesis of acino acids)，碳代謝(carbon metabolism)，二羧酸代謝(glyoxylate and dicarboxylate metabolism)(圖3)。上述結(jié)果表明球囊菌侵染對中蜂幼蟲腸道的物質(zhì)代謝產(chǎn)生較大影響。進一步分析結(jié)果顯示，有2、17、20、29和52個上調(diào)基因富集在凋亡(apoptosis)、溶酶體(lysosome)、泛素介導的蛋白水解(ubiquitin mediated proteolysis)、吞噬體(phagosome)和內(nèi)吞作用(endocytosis)，說明宿主的細胞免疫在脅迫后期被顯著激活；分別有1和2個上調(diào)基因富集在中的Toll-like受體信號通路與Jak-STAT信號通路，說明宿主的此二條體液免疫通路在球囊菌脅迫后期被激活(圖4)。

3 結(jié)論與討論

白堊病是養(yǎng)蜂生產(chǎn)的一大頑疾，每年給養(yǎng)蜂業(yè)造成巨大損失。前人研究主要集中在意蜂幼蟲白堊病的諸多方面，而球囊菌侵染中蜂幼蟲的研究進展幾無報道。中蜂白堊病蟲尸在自然蜂群中僅偶爾可見，本課題組前期已從自然蜂群中蜂白堊狀蟲尸上分離得到球囊菌，通過形態(tài)學和分子生物學手段證明該病原即為A.apis(未發(fā)表數(shù)據(jù))。前期研究中，我們組裝并注釋了中蜂幼蟲腸道的參考轉(zhuǎn)錄組并開發(fā)出15個SSR分子標記(Xiongetal., 2017)，在此基礎上，本研究利用RNA-seq技術(shù)對健康及球囊菌脅迫的中蜂幼蟲腸道進行轉(zhuǎn)錄組測序，進而對宿主響應球囊菌脅迫的DEGs進行深入分析。腸道是昆蟲的重要免疫器官，在抵御病原微生物入侵過程中發(fā)揮重要作用。本研究選擇中蜂幼蟲腸道作為測序?qū)ο?，其轉(zhuǎn)錄組變化能更為精確地反映宿主響應球囊菌脅迫的應答表現(xiàn)。

我國養(yǎng)蜂生產(chǎn)中的常用蜂種是意蜂和中蜂，中蜂作為我國養(yǎng)蜂生產(chǎn)的主要蜂種之一，較意蜂具有更強的球囊菌抗性。本研究發(fā)現(xiàn)球囊菌脅迫的中蜂6日齡幼蟲腸道上調(diào)基因的數(shù)量(4513 unigenes)遠遠多于下調(diào)基因(385 unigenes)，宿主的絕大多數(shù)基因被球囊菌激活表達，說明宿主響應脅迫的應答活躍，這或許與中蜂幼蟲的球囊菌抗性密切相關。球囊菌被 意幼蟲攝入中腸后，在整個幼蟲期因中腸缺氧而不萌發(fā)，至幼蟲期結(jié)束進入蛹期后，此時蜜蜂的中后腸接通，球囊菌孢子伴隨蛹便進入后腸，在氧氣的刺激下在此迅速萌發(fā)生長，1-2 d內(nèi)菌絲即突破體表而導致宿主死亡(Lietal., 2012)。本研究中，下調(diào)基因共富集在59個pathway，其中的49個pathway與新陳代謝相關，包括物質(zhì)代謝(如氨基酸生物合成和半乳糖代謝)和能量代謝(如氮代謝)，說明球囊菌在脅迫后期通過病原-宿主互作對中蜂幼蟲腸道的新陳代謝系統(tǒng)產(chǎn)生較強抑制。

圖2 差異表達基因的GO分析Fig.2 GO analysis of DEGs between AcCK and AcT注: A: 上調(diào)基因; 1: 行為; 2: 生物附著; 3: 生物調(diào)控; 4: 細胞成分組織或生物合成; 5: 細胞進程; 6: 發(fā)展進程; 7: 生長; 8: 免疫系統(tǒng)進程; 9: 定位; 10: 運行; 11: 代謝進程; 12: 多組織進程; 13: 多細胞組織進程; 14: 生殖; 15: 生殖進程; 16: 應激; 17: 信號; 18: 單一有機體進程; 19: 細胞; 20: 細胞接合; 21: 細胞零件; 22: 細胞外基質(zhì); 23: 細胞外區(qū)域; 24: 細胞外區(qū)域組分; 25: 大分子復合物; 26: 細胞膜; 27: 細胞膜組分; 28: 細胞膜膜蛋白; 29: 細胞器; 30: 細胞器組分; 31: 突觸; 32: 突觸組分; 33: 病毒; 34: 病毒組分; 35: 抗氧化劑活性; 36: 結(jié)合; 37: 催化活性; 38: 電子載體活性; 39: 酶的調(diào)節(jié); 40: 脒基核苷酸交換因子活性; 41: 分子功能調(diào)節(jié); 42: 分子轉(zhuǎn)導活性; 43: 核酸結(jié)合轉(zhuǎn)化因素; 44: 分子結(jié)構(gòu)活性; 45: 運輸活性.B: 下調(diào)基因; 1: 生物附著; 2: 生物調(diào)控; 3 細胞成分組織或生物合成; 4: 細胞進程; 5: 發(fā)展進程; 6: 定位; 7: 運行; 8: 代謝進程; 9: 多細胞組織進程; 10: 應激; 11: 信號; 12: 單一有機體進程; 13: 細胞; 14: 細胞零件; 15: 細胞外基質(zhì); 16: 細胞外基質(zhì)組成; 17: 細胞外區(qū)域; 18: 細胞外區(qū)域組分; 19: 大分子復合物; 20: 細胞膜; 21: 細胞膜組分; 22: 細胞器; 23: 細胞器組分; 24: 病毒; 25: 病毒組分; 26: 結(jié)合; 27: 催化活性; 28: 酶的調(diào)節(jié); 29: 分子功能調(diào)節(jié); 30: 分子轉(zhuǎn)導活性; 31: 分子結(jié)構(gòu)活性; 32: 運輸活性。 Note: A: up-regulated genes; 1: behavior; 2: biological adhesion; 3: biological regulation; 4: cellular component organization or biogenesis; 5: cellular process; 6: developmental process; 7: growth; 8: immune system process; 9: localization; 10: locomotion; 11: metabolic process; 12: multi-organismal process; 13: multicellular organismal process; 14: reproduction; 15: reproductive process; 16: response to stimulus; 17: signaling; 18: single-organism process; 19: cell; 20: cell junction; 21: cell part; 22: extracellular matrix; 23: extracellular region; 24: extracellular region part; 25: macromolecular complex; 26: membrane; 27: membrane part; 28: membrane-enclosed lumen; 29: organelle; 30: organelle part; 31: synapse; 32: synapse part; 33: virion; 34: virion part; 35: antioxidant activity; 36: binding; 37: catalytic activity; 38: electron carrier activity; 39: enzyme regulator activity; 40: guanyl-nucleotide exchange factor activity; 41: molecular function regulator; 42: molecular transducer regulator; 43: nucleic acid binding transcription factor activity; 44: structural molecule activity; 45: transporter activity.B: down-regulated genes; 1: biological adhesion; 2: biological regulation; 3: cellular component organization or biogenesis; 4: cellular process; 5: developmental process; 6: localization; 7: locomotion; 8: metabolic process; 9: multicellular organismal process; 10: response to stimulus; 11: signaling; 12: single-organism process; 13: cell; 14: cell part; 15: extracellular matrix; 16: extracellular matrix part; 17: extracellular region; 18: extracellular region part; 19: macromolecular complex; 20: membrane; 21: membrane part; 22: organelle; 23: organelle part; 24: virion; 25: virion part; 26: binding; 27: catalytic activity; 28: enzyme regulator activity; 29: molecular function regulator; 30: molecular transducer regulator; 31: structural molecule activity; 32: transporter activity.

圖3 差異表達基因的KEGG pathway富集分析Fig.3 KEGG enrichment analysis of DEGs between AcCK and AcT注: A: 上調(diào)基因; 1: 酪氨酸代謝; 2: 糖酵解; 3: 苯丙氨酸代謝; 4: 調(diào)節(jié)細胞骨架; 5: 半胱氨酸和蛋氨酸代謝; 6: 核糖體; 7: 卵母細胞減數(shù)分裂; 8: 苯丙氨酸、酪氨酸、色氨酸代謝; 9: 組氨酸代謝; 10: 脂肪酸退化; 11: 色氨酸代謝; 12: 氨基糖和核苷酸代謝; 13: 酮體的減數(shù)分裂; 14: 丁酮代謝; 15: 頡氨酸、亮氨酸、異亮氨酸的減數(shù)分裂; 16: 丙氨酸、天冬氨酸、谷氨酸代謝; 17: 檸檬酸循環(huán); 18: 二氧代羧酸代謝; 19: 氨基酸生物合成; 20: 碳代謝.B: 下調(diào)基因; 1: 過氧物酶體; 2: 甘油酯代謝; 3: 淀粉與蔗糖的代謝; 4: 氰基氨基酸代謝 5: 磷酸戊糖途徑; 6: 鞘糖脂生物合成7: 丙氨酸、天冬氨酸和谷氨酸代謝; 8: 其他葡聚糖降解; 9: 半乳糖代謝、果糖與甘露糖代謝; 10: 葉酸碳庫; 11: 戊糖，葡萄糖醛酸轉(zhuǎn)換; 12: 溶酶體; 13: 纈氨酸、亮氨酸和異亮氨酸降解; 14: 粘多糖的降解; 15: 糖酵解和糖異生; 16: 氨基酸生物合成; 17: 甘氨酸; 18: 絲氨酸和蘇氨酸代謝; 19: 碳代謝作用; 20: 二羧酸代謝。 Note: A: up-regualted genes; 1: tyrosine metabolism; 2: glycolysis; 3: phenylalanine metabolism; 4: regulation of actin cytoskeleton; 5: cysteine and methionine metabolism; 6: ribosome; 7: oocyte meiosis; 8: phenylalanine, tyrosine and tryptophan biosynthesis; 9: histidine metabolism; 10: fatty acid degradation; 11: tryptophan metabolism; 12: amino sugar and nucleotide sugar metabolism; 13: synthesis and degradation of ketone bodies; 14: butanoate metabolism; 15: valine, leucine and isoleucine degradation; 16: alanine, aspartate and glutamate metabolism; 17: citrate cycle; 18: 2-oxocarboxylic acid metabolism; 19: biosynthesis of amino acids; 20: carbon metabolism.B: down-regulated genes; 1: peroxisome; 2: glycerolipid metabolism; 3: starch and sucrose metabolism; 4: cyanoamino acid metabolism; 5: pentose phosphate pathway; 6: glycosphingolipid biosynthesis-globo series; 7: alanine,aspartate and glutamate metabolism; 8: other glycan degradation; 9: galactose metabolism; 10: fructose and mammose metabolism; 11: one carbon pool by folate; 12: pentose and glucoronate interconversions; 13: lysosome; 14: valine,leucine and isoleucine degradation; 15: glycosaminoglycan degradation; 16: glycolysis; 17: biosynthesis of amino acids; 18: glycine,serine and threonine metabolism; 19: carbon metabolism; 20: glyoxylate and dicarboxylate metabolism.

圖4 富集在Toll-like和Jak-STAT信號通路的上調(diào)基因的表達量聚類Fig.4 Expression cluster of the up-regulated unigenes enriched in Toll-like and Jak-STAT signaling pathway

前期研究發(fā)現(xiàn)球囊菌接種感染后，意蜂幼蟲與中蜂幼蟲的預蛹(7 d)死亡率分別為70.83%和16.67%，說明后者具有較強的球囊菌抗性，可通過某種機制抵御球囊菌入侵。昆蟲中腸內(nèi)側(cè)有一層由幾丁質(zhì)和蛋白質(zhì)構(gòu)成的圍食膜，它作為第一道物理屏障能夠阻擋經(jīng)口攝入的病原微生物的入侵(Vuocoloetal., 2001; Pengetal., 1999; Wang and Granados, 2001)。角質(zhì)層蛋白是構(gòu)成圍食膜的主要成分之一。本研究中，有5個角質(zhì)層蛋白編碼基因表現(xiàn)為下調(diào)，說明球囊菌可通過抑制宿主角質(zhì)層蛋白編碼基因的表達來促進侵染。當物理屏障被病原突破后，昆蟲隨即啟動細胞免疫和體液免疫，例如細胞內(nèi)吞、黑化作用、吞噬作用、酶促降解及分泌抗菌肽等(Gliński and Jarosz, 2001; Glinski and Buczek, 2003)。本研究發(fā)現(xiàn)分別有2、17、20、29和52個上調(diào)基因富集在凋亡、溶酶體、泛素介導的蛋白水解、吞噬體和內(nèi)吞作用，僅有1和8個下調(diào)基因分別富集在吞噬體和溶酶體，說明中蜂幼蟲腸道的細胞免疫在球囊菌脅迫后期被顯著激活。同時，我們還發(fā)現(xiàn)有1和2個上調(diào)基因分別富集在Toll-like及Jak-STAT信號通路上，推測中蜂幼蟲的此二條免疫信號通路在抵御球囊菌入侵的過程中發(fā)揮關鍵作用。推測中蜂幼蟲的細胞和體液免疫在一定程度上賦予其較強的球囊菌抗性。

目前尚無一種殺真菌劑被批準應用于養(yǎng)蜂生產(chǎn)(Galiziaetal., 2012)，一般通過選育抗病品系、改善養(yǎng)蜂管理和保持清潔衛(wèi)生來防治白堊病(Gilliac Metal., 1988)，但是效果并不理想。蜜蜂幼蟲在球囊菌脅迫過程中免疫應答機制及分子調(diào)控機制的缺失嚴重阻礙白堊病的有效治療。本研究利用二代測序技術(shù)對健康及球囊菌脅迫的中蜂幼蟲腸道進行測序，通過DEGs分析并對宿主的脅迫應答進行深入分析，研究結(jié)果不僅在轉(zhuǎn)錄組水平揭示了中蜂幼蟲在球囊菌入侵后期的脅迫應答，也為解析中蜂幼蟲的球囊菌抗性機制奠定了基礎。

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Analysis of the differentially expressed genes in the 6-day-old larval gut ofApisceranaceranaunder the stress ofAscosphaeraapis

GUO Rui1*, ZHANG Lu1*, XU Xi-Jian1, SHI Xiu-Li2, XIONG Cui-Ling1, ZHENG Yan-Zhen1, FU Zhong- Min1, HUANG Zhi-Jian1, WANG Hong-Quan1, HOU Zhi-Xian1, CHEN Da-Fu1**

(1.College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2.Apicultural technology management station in the Xinjiang Uygur Autonomous Region, Wulumuqi 930001, China)

Ascosphaeraapisspecially infect honeybee larvae and leads to chalkbrood, which causes a huge loss for apiculture.Here, untreated andA.apis-treatedApisceranaceranalarval guts were sequenced using RNA-seq technology, followed by analysis of differentially expressed genes (DEGs).In this study, Illumina sequencing of larval guts generated a total of 191,1677,30 raw reads, and after filtration, 186,284,296 clean reads were obtained.DEGs analysis showed that 4513 genes were up-regulated and 385 were down-regulated.GO enrichment analysis displayed that the up-regulated genes were enriched in 45 terms, among them the mostly enriched ones were cell process, metabolic process and catalyitic activity; the down-regulated genes were enriched in 32 terms, and the mostly enriched ones were metabolic process, single organism process as well as catalyitic activity.Furthermore, KEGG enrichment analysis suggested that the up-regualted genes were engaged in 193 pathways, the mostly enriched one was ribosome, followed by biosynthesis of acino acids and carbon metabolism; the down-regualted genes were involved in 59 pathways, and the mostly enriched ones were glycine, carbon metabolism as well as glyoxylate and dicarboxylate metabolism.Further analysis showed that the host’s cellular immune was activated and the humoral immunity such as Toll-like and Jak-STAT signaling pathways were significantly induced to activation.Findings in the present study can not only provide key information for uncovering the mechanism regulating theA.c.ceranalarvae’s responses toA.apisduring the late stage of stress, but also lay a foundation for clarifying theA.apis-resistance mechanism ofA.c.ceranalarvae.

Apisceranacerana; larval gut;Ascosphaeraapis; differentially expressed gene; immune defense

郭睿，張璐，徐細建,等.中華蜜蜂6日齡幼蟲腸道響應球囊菌脅迫的差異表達基因分析[J].環(huán)境昆蟲學報，2017,39(3):539-547.

現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設專項資金(CARS-45-KXJ7)；福建農(nóng)林大學科技發(fā)展資金(KF2015123)

*共同第一作者簡介：郭睿，男，1987年生，安徽六安人，講師，研究方向為蜜蜂分子生物學，E-mail: fafu_ruiguo@126.com；張璐，女，1996年生，河南濮陽人，本科生，研究方向為蜂學，E-mail: m17805949180@163.com

**通訊作者Authors for correspondence, E-mail: dfchen826@163.com

Received: 2017-03-04; 接受日期Accepted: 2017-05-02

S891

A

1674-0858(2017)03-0539-09