中華蜜蜂6日齡幼蟲腸道響應(yīng)球囊菌脅迫的差異表達(dá)基因分析

郭 睿，張 璐，徐細(xì)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(yīng)球囊菌脅迫的差異表達(dá)基因分析

郭 睿1*，張 璐1*，徐細(xì)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(xué)蜂學(xué)學(xué)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(dǎo)致白堊病，嚴(yán)重危害養(yǎng)蜂生產(chǎn)。本研究利用RNA-seq技術(shù)對(duì)健康及球囊菌脅迫的中蜂幼蟲腸道進(jìn)行深度測(cè)序，進(jìn)而對(duì)宿主的差異表達(dá)基因進(jìn)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(guò)濾得到186284296條有效讀段(clean reads)，差異表達(dá)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(gè)。Gene ontology(GO)富集分析結(jié)果顯示，上調(diào)基因富集在45個(gè)GO條目(term)，富集基因數(shù)最多的是細(xì)胞進(jìn)程、代謝進(jìn)程及催化活性，下調(diào)基因富集在32個(gè) GO term，富集基因數(shù)最多的是代謝進(jìn)程、單組織進(jìn)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(gè)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(gè)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(xì)胞免疫被顯著激活，體液免疫中的Toll-like與Jak-STAT信號(hào)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(yīng)答機(jī)制提供了重要的信息，也為解析中蜂幼蟲的球囊菌抗性機(jī)制奠定了基礎(chǔ)。

中華蜜蜂；幼蟲腸道；球囊菌；差異表達(dá)基因；免疫防御

蜜蜂是一種重要的社會(huì)學(xué)模式昆蟲，因其在發(fā)育學(xué)、神經(jīng)生物學(xué)、行為學(xué)和病原-宿主互作研究中的重要性而備受關(guān)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(bào)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(xì)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(guó)首次報(bào)道發(fā)現(xiàn)白惡病(Aronstein and Murray, 2010)，中國(guó)大陸1990年發(fā)生白堊病(Liang and Chen, 2008)。近年來(lái)，隨著養(yǎng)蜂活動(dòng)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(jiǎn)稱球囊菌)特異性侵染蜜蜂幼蟲而導(dǎo)致，可造成蜜蜂群勢(shì)的大幅下降，從而嚴(yán)重影響蜂蜜等產(chǎn)品的產(chǎn)量(Bailey, 1963)，據(jù)報(bào)道，白堊病可造成蜂蜜產(chǎn)量下降5-37%(Zaghlouletal., 2005)。近二十年來(lái)，國(guó)內(nèi)外學(xué)者在病原分類鑒定、形態(tài)學(xué)、病理學(xué)、流行病學(xué)、侵染過(guò)程、蜜蜂防御以及疾病防治等方面對(duì)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(cè)及侵染過(guò)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(yíng)養(yǎng)生態(tài)條件對(duì)球囊菌生長(zhǎng)及產(chǎn)孢的影響，結(jié)果表明營(yíng)養(yǎng)生態(tài)條件的變化對(duì)球囊菌的影響極大(Liangetal., 2001)；鄭志陽(yáng)等對(duì)健康和患病蜜蜂幼蟲血淋巴進(jìn)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(cè)到，但在健康幼蟲中檢測(cè)不到(Zhengetal., 2011)。

我國(guó)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(jiǎn)稱中蜂)基因組的公布(Parketal., 2015)，為中蜂的分子生物學(xué)研究提供了重要參考信息。Aronstein等利用cDNA-AFLP 對(duì)健康及球囊菌感染的西方蜜蜂Apismellifera幼蟲進(jìn)行了比較，結(jié)果表明差異表達(dá)基因(DEGs)參與了宿主的能量代謝和蛋白轉(zhuǎn)運(yùn)，其中的類幾丁質(zhì)編碼基因很可能參與了蜜蜂幼蟲對(duì)球囊菌的抵抗(Aronsteinetal., 2010)。Cornman等對(duì)來(lái)自培養(yǎng)基的球囊菌菌絲和來(lái)自蜜蜂幼蟲感染組織的球囊菌菌絲進(jìn)行了轉(zhuǎn)錄組測(cè)序，功能分析表明球囊菌的DEG參與了交配類型、細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)和應(yīng)激反應(yīng)。

目前，白堊病的相關(guān)研究主要集中在意蜂，有關(guān)球囊菌侵染中蜂的研究報(bào)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(xué)、分子生物學(xué)以及交叉感染實(shí)驗(yàn)證實(shí)確為A.apis(未發(fā)表數(shù)據(jù))。

本研究利用Illumina測(cè)序技術(shù)對(duì)對(duì)健康及球囊菌脅迫的中蜂6日齡幼蟲腸道進(jìn)行深度測(cè)序，得到宿主的差異表達(dá)基因(DEGs)，并通過(guò)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(yīng)球囊菌后期脅迫的應(yīng)答機(jī)制提供重要信息，也能為關(guān)鍵應(yīng)答基因的篩選及驗(yàn)證奠定基礎(chǔ)。

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(xué)蜂學(xué)學(xué)院教學(xué)蜂場(chǎng)，球囊菌菌株由福建農(nóng)林大學(xué)蜂學(xué)學(xué)院蜜蜂保護(hù)實(shí)驗(yàn)室保存并活化。

1.2 主要實(shí)驗(yàn)試劑及儀器

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

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

1.3 球囊菌活化、孢子純化及計(jì)數(shù)

按照本實(shí)驗(yàn)室已建立的方法對(duì)球囊菌進(jìn)行活化(Zhangetal., 2017)。按照J(rèn)ensen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(jì)數(shù)板對(duì)孢子進(jìn)行計(jì)數(shù)。

1.4 中蜂幼蟲的人工飼養(yǎng)及腸道樣品準(zhǔn)備

中蜂幼蟲的人工飼養(yǎng)參照王倩等(2009)的方法。從蜂學(xué)學(xué)院蜂場(chǎng)群勢(shì)較強(qiáng)的中蜂蜂群用移蟲針挑取2日齡幼蟲，放入無(wú)菌的24孔細(xì)胞培養(yǎng)板(每孔對(duì)應(yīng)1只幼蟲，孔內(nèi)加有35℃預(yù)溫的幼蟲飼料)，將24孔板放入恒溫恒濕培養(yǎng)箱，35℃，70%相對(duì)濕度(RH)條件下飼養(yǎng)。每隔24 h更換飼料。預(yù)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(duì)照組飼喂正常人工飼料。本次實(shí)驗(yàn)進(jìn)行3次生物學(xué)重復(fù)。

本研究的分析重點(diǎn)是中鋒幼蟲腸道在球囊菌脅迫后期的DEGs。鑒于后續(xù)將對(duì)宿主在球囊菌入侵全過(guò)程的脅迫應(yīng)答進(jìn)行研究，為降低測(cè)序成本，擬將健康中蜂4日齡幼蟲腸道作為唯一對(duì)照。因此，本研究中，分別剖取對(duì)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(gè)生物學(xué)重復(fù)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(kù)構(gòu)建及Illumina測(cè)序

利用RNAiso Reagent試劑盒抽提處理組和對(duì)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(guò)瓊脂糖凝膠電泳和NanoDrop ND-1000(NanoDrop, Wilmington, DE, USA)進(jìn)行檢測(cè)。cDNA文庫(kù)構(gòu)建參照張曌楠等的建庫(kù)方法(Zhangetal., 2017)。委托廣州基迪奧生物科技有限公司對(duì)上述幼蟲腸道樣品進(jìn)行雙端測(cè)序，測(cè)序平臺(tái)為Illumina HiSeq2500。

1.6 數(shù)據(jù)分析

對(duì)于下機(jī)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(jìn)行測(cè)序飽和度分析。使用短 reads 比對(duì)工具 bowtie(Langmeadetal., 2009)將clean reads映射(mapping)到核糖體數(shù)據(jù)庫(kù)(最多允許5個(gè)錯(cuò)配)，去除比對(duì)上核糖體的 reads，將保留下來(lái)的數(shù)據(jù)用于轉(zhuǎn)錄組的組裝及分析，進(jìn)而利用SOAP aligner/soap2軟件(Hurgobin, 2016)將未比對(duì)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(kù)，SRA號(hào): SRA456721)。

利用FPKM(Fragments Per Kilobase of transcript per Million mapped reads)法計(jì)算基因表達(dá)量。利用R軟件(version 2.16.2)計(jì)算各樣品之間的相關(guān)性系數(shù)。利用edgeR軟件(Robinsonetal., 2010)進(jìn)行DEGs分析。DEGs的篩選標(biāo)準(zhǔn)為FDR ≤ 0.05且|log2Fold change | ≥ 1。將DEGs向GO數(shù)據(jù)庫(kù)(http://www.geneontology.org/)的各條目(term)mapping，并計(jì)算每個(gè)term的基因數(shù)，從而得到具有某個(gè)GO功能的基因列表及基因數(shù)目統(tǒng)計(jì)，然后應(yīng)用超幾何檢驗(yàn)，找出與整個(gè)基因組背景相比，在差異表達(dá)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(yīng)用超幾何檢驗(yàn)，找出與整個(gè)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(píng)估

中蜂幼蟲腸道樣品的轉(zhuǎn)錄組測(cè)序共測(cè)得191167730條raw reads，經(jīng)過(guò)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(cè)序量的增多，檢測(cè)到的基因數(shù)也隨之上升、增長(zhǎng)速度趨于平緩，說(shuō)明本研究的測(cè)序深度檢測(cè)到的基因數(shù)趨于飽和(附圖1)。AcCK與AcT的組內(nèi)各生物學(xué)重復(fù)之間的相關(guān)性均在0.96以上，說(shuō)明樣本的重復(fù)性高(圖1)。上述結(jié)果說(shuō)明本研究的轉(zhuǎn)錄組數(shù)據(jù)質(zhì)量良好，可用于進(jìn)一步分析。

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

圖1 各幼蟲腸道樣品不同生物學(xué)重復(fù)間的相關(guān)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(guān)性; B: AcCK-2與AcCK-3間的相關(guān)性; C: AcCK-1與AcCK-3間的相關(guān)性; D: AcT-1與AcT-2間的相關(guān)性; E: AcT-2與AcT-3間的相關(guān)性; F: AcT-1與AcT-3間的相關(guān)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(gè)基因差異表達(dá)，其中，上調(diào)基因和下調(diào)基因的數(shù)量分別為4513和385個(gè)(表2)，上調(diào)基因的數(shù)量遠(yuǎn)遠(yuǎn)多余下調(diào)基因，說(shuō)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(dá)。

表2 差異表達(dá)基因統(tǒng)計(jì)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(xué)進(jìn)程(biological process)、細(xì)胞組分(cellular component)和分子功能(molecular function)，上調(diào)基因分布于45個(gè) GO term上，富集基因數(shù)最多的是細(xì)胞進(jìn)程(cellular process)、代謝進(jìn)程(metabolic process)、催化活性(catalytic activity)(圖2)。下調(diào)基因分布于32個(gè) GO term上，富集基因數(shù)最多的是代謝進(jìn)程(metabolic process)、單組織進(jìn)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(gè)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(gè)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(duì)中蜂幼蟲腸道的物質(zhì)代謝產(chǎn)生較大影響。進(jìn)一步分析結(jié)果顯示，有2、17、20、29和52個(gè)上調(diào)基因富集在凋亡(apoptosis)、溶酶體(lysosome)、泛素介導(dǎo)的蛋白水解(ubiquitin mediated proteolysis)、吞噬體(phagosome)和內(nèi)吞作用(endocytosis)，說(shuō)明宿主的細(xì)胞免疫在脅迫后期被顯著激活；分別有1和2個(gè)上調(diào)基因富集在中的Toll-like受體信號(hào)通路與Jak-STAT信號(hào)通路，說(shuō)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(jìn)展幾無(wú)報(bào)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(guò)形態(tài)學(xué)和分子生物學(xué)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(gè)SSR分子標(biāo)記(Xiongetal., 2017)，在此基礎(chǔ)上，本研究利用RNA-seq技術(shù)對(duì)健康及球囊菌脅迫的中蜂幼蟲腸道進(jìn)行轉(zhuǎn)錄組測(cè)序，進(jìn)而對(duì)宿主響應(yīng)球囊菌脅迫的DEGs進(jìn)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(guò)程中發(fā)揮重要作用。本研究選擇中蜂幼蟲腸道作為測(cè)序?qū)ο?，其轉(zhuǎn)錄組變化能更為精確地反映宿主響應(yīng)球囊菌脅迫的應(yīng)答表現(xiàn)。

我國(guó)養(yǎng)蜂生產(chǎn)中的常用蜂種是意蜂和中蜂，中蜂作為我國(guó)養(yǎng)蜂生產(chǎn)的主要蜂種之一，較意蜂具有更強(qiáng)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(yuǎn)遠(yuǎn)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(dá)，說(shuō)明宿主響應(yīng)脅迫的應(yīng)答活躍，這或許與中蜂幼蟲的球囊菌抗性密切相關(guān)。球囊菌被 意幼蟲攝入中腸后，在整個(gè)幼蟲期因中腸缺氧而不萌發(fā)，至幼蟲期結(jié)束進(jìn)入蛹期后，此時(shí)蜜蜂的中后腸接通，球囊菌孢子伴隨蛹便進(jìn)入后腸，在氧氣的刺激下在此迅速萌發(fā)生長(zhǎng)，1-2 d內(nèi)菌絲即突破體表而導(dǎo)致宿主死亡(Lietal., 2012)。本研究中，下調(diào)基因共富集在59個(gè)pathway，其中的49個(gè)pathway與新陳代謝相關(guān)，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(shuō)明球囊菌在脅迫后期通過(guò)病原-宿主互作對(duì)中蜂幼蟲腸道的新陳代謝系統(tǒng)產(chǎn)生較強(qiáng)抑制。

圖2 差異表達(dá)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(xì)胞成分組織或生物合成; 5: 細(xì)胞進(jìn)程; 6: 發(fā)展進(jìn)程; 7: 生長(zhǎng); 8: 免疫系統(tǒng)進(jìn)程; 9: 定位; 10: 運(yùn)行; 11: 代謝進(jìn)程; 12: 多組織進(jìn)程; 13: 多細(xì)胞組織進(jìn)程; 14: 生殖; 15: 生殖進(jìn)程; 16: 應(yīng)激; 17: 信號(hào); 18: 單一有機(jī)體進(jìn)程; 19: 細(xì)胞; 20: 細(xì)胞接合; 21: 細(xì)胞零件; 22: 細(xì)胞外基質(zhì); 23: 細(xì)胞外區(qū)域; 24: 細(xì)胞外區(qū)域組分; 25: 大分子復(fù)合物; 26: 細(xì)胞膜; 27: 細(xì)胞膜組分; 28: 細(xì)胞膜膜蛋白; 29: 細(xì)胞器; 30: 細(xì)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(dǎo)活性; 43: 核酸結(jié)合轉(zhuǎn)化因素; 44: 分子結(jié)構(gòu)活性; 45: 運(yùn)輸活性.B: 下調(diào)基因; 1: 生物附著; 2: 生物調(diào)控; 3 細(xì)胞成分組織或生物合成; 4: 細(xì)胞進(jìn)程; 5: 發(fā)展進(jìn)程; 6: 定位; 7: 運(yùn)行; 8: 代謝進(jìn)程; 9: 多細(xì)胞組織進(jìn)程; 10: 應(yīng)激; 11: 信號(hào); 12: 單一有機(jī)體進(jìn)程; 13: 細(xì)胞; 14: 細(xì)胞零件; 15: 細(xì)胞外基質(zhì); 16: 細(xì)胞外基質(zhì)組成; 17: 細(xì)胞外區(qū)域; 18: 細(xì)胞外區(qū)域組分; 19: 大分子復(fù)合物; 20: 細(xì)胞膜; 21: 細(xì)胞膜組分; 22: 細(xì)胞器; 23: 細(xì)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(dǎo)活性; 31: 分子結(jié)構(gòu)活性; 32: 運(yùn)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(dá)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(xì)胞骨架; 5: 半胱氨酸和蛋氨酸代謝; 6: 核糖體; 7: 卵母細(xì)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(guò)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(kù); 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(hào)通路的上調(diào)基因的表達(dá)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(yù)蛹(7 d)死亡率分別為70.83%和16.67%，說(shuō)明后者具有較強(qiáng)的球囊菌抗性，可通過(guò)某種機(jī)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(gè)角質(zhì)層蛋白編碼基因表現(xiàn)為下調(diào)，說(shuō)明球囊菌可通過(guò)抑制宿主角質(zhì)層蛋白編碼基因的表達(dá)來(lái)促進(jìn)侵染。當(dāng)物理屏障被病原突破后，昆蟲隨即啟動(dòng)細(xì)胞免疫和體液免疫，例如細(xì)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(gè)上調(diào)基因富集在凋亡、溶酶體、泛素介導(dǎo)的蛋白水解、吞噬體和內(nèi)吞作用，僅有1和8個(gè)下調(diào)基因分別富集在吞噬體和溶酶體，說(shuō)明中蜂幼蟲腸道的細(xì)胞免疫在球囊菌脅迫后期被顯著激活。同時(shí)，我們還發(fā)現(xiàn)有1和2個(gè)上調(diào)基因分別富集在Toll-like及Jak-STAT信號(hào)通路上，推測(cè)中蜂幼蟲的此二條免疫信號(hào)通路在抵御球囊菌入侵的過(guò)程中發(fā)揮關(guān)鍵作用。推測(cè)中蜂幼蟲的細(xì)胞和體液免疫在一定程度上賦予其較強(qiáng)的球囊菌抗性。

目前尚無(wú)一種殺真菌劑被批準(zhǔn)應(yīng)用于養(yǎng)蜂生產(chǎn)(Galiziaetal., 2012)，一般通過(guò)選育抗病品系、改善養(yǎng)蜂管理和保持清潔衛(wèi)生來(lái)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(guò)程中免疫應(yīng)答機(jī)制及分子調(diào)控機(jī)制的缺失嚴(yán)重阻礙白堊病的有效治療。本研究利用二代測(cè)序技術(shù)對(duì)健康及球囊菌脅迫的中蜂幼蟲腸道進(jìn)行測(cè)序，通過(guò)DEGs分析并對(duì)宿主的脅迫應(yīng)答進(jìn)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(yīng)答，也為解析中蜂幼蟲的球囊菌抗性機(jī)制奠定了基礎(chǔ)。

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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(xì)建,等.中華蜜蜂6日齡幼蟲腸道響應(yīng)球囊菌脅迫的差異表達(dá)基因分析[J].環(huán)境昆蟲學(xué)報(bào)，2017,39(3):539-547.

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

*共同第一作者簡(jiǎn)介：郭睿，男，1987年生，安徽六安人，講師，研究方向?yàn)槊鄯浞肿由飳W(xué)，E-mail: fafu_ruiguo@126.com；張璐，女，1996年生，河南濮陽(yáng)人，本科生，研究方向?yàn)榉鋵W(xué)，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