杜宇，范小雪，蔣海賓，王杰，馮睿蓉，張文德，余岢駿，隆琦，蔡宗兵，熊翠玲，鄭燕珍,2，陳大福,2，付中民,2，徐國(guó)鈞,2，郭睿,2

微小RNA介導(dǎo)意大利蜜蜂工蜂對(duì)東方蜜蜂微孢子蟲(chóng)的跨界調(diào)控

杜宇1，范小雪1，蔣海賓1，王杰1，馮睿蓉1，張文德1，余岢駿1，隆琦1，蔡宗兵1，熊翠玲1，鄭燕珍1,2，陳大福1,2，付中民1,2，徐國(guó)鈞1,2，郭睿1,2

1福建農(nóng)林大學(xué)動(dòng)物科學(xué)學(xué)院（蜂學(xué)學(xué)院），福州 350002；2福建農(nóng)林大學(xué)蜂療研究所，福州 350002

【】東方蜜蜂微孢子蟲(chóng)（）感染意大利蜜蜂（，簡(jiǎn)稱(chēng)意蜂）導(dǎo)致蜜蜂微孢子蟲(chóng)病。本研究結(jié)合前期已獲得的miRNA和mRNA組學(xué)數(shù)據(jù)，通過(guò)生物信息學(xué)方法對(duì)意蜂工蜂中腸的差異表達(dá)miRNA（differentially expressed miRNA，DEmiRNA）靶向結(jié)合的東方蜜蜂微孢子蟲(chóng)的mRNA和差異表達(dá)mRNA（DEmRNA）進(jìn)行預(yù)測(cè)、數(shù)據(jù)庫(kù)注釋和調(diào)控網(wǎng)絡(luò)分析，以期在組學(xué)水平解析miRNA介導(dǎo)意蜂工蜂對(duì)東方蜜蜂微孢子蟲(chóng)的跨界調(diào)控機(jī)制。通過(guò)比較東方蜜蜂微孢子蟲(chóng)侵染7 d和10 d的意蜂工蜂中腸（AmT1、AmT2）和未受侵染的工蜂中腸（AmCK1、AmCK2）的miRNA組學(xué)數(shù)據(jù)篩選出宿主的顯著性DEmiRNA，通過(guò)比較侵染意蜂工蜂中腸的東方蜜蜂微孢子蟲(chóng)（NcT1、NcT2）和東方蜜蜂微孢子蟲(chóng)純凈孢子（NcCK）的mRNA數(shù)據(jù)篩選出病原的DEmRNA。利用TargetFinder軟件預(yù)測(cè)宿主顯著性DEmiRNA靶向結(jié)合的病原mRNA和DEmRNA。利用相關(guān)生物信息學(xué)工具對(duì)上述靶DEmRNA進(jìn)行GO和KEGG數(shù)據(jù)庫(kù)注釋。結(jié)合前期研究結(jié)果篩選出孢壁蛋白、極管蛋白、蓖麻毒素B凝集素、ABC轉(zhuǎn)運(yùn)蛋白、ATP/ADP移位酶和糖酵解/糖異生途徑等毒力因子和能量代謝通路相關(guān)的病原DEmRNA及與其存在靶向結(jié)合關(guān)系的宿主顯著性DEmiRNA，并構(gòu)建和分析二者的調(diào)控網(wǎng)絡(luò)。AmCK1 vs AmT1比較組中宿主的48條顯著上調(diào)miRNA和36條顯著下調(diào)miRNA分別靶向病原的1 345和1 046條mRNA；進(jìn)一步分析發(fā)現(xiàn)，宿主的47條顯著上調(diào)miRNA和34條顯著下調(diào)miRNA可分別靶向NcCK vs NcT1比較組中病原的584條顯著下調(diào)mRNA和265條顯著上調(diào)mRNA，它們可分別注釋到19和22個(gè)功能條目以及66和64條通路。AmCK2 vs AmT2比較組中宿主的56條顯著上調(diào)miRNA和51條顯著下調(diào)miRNA分別靶向病原的1 260和1 317條mRNA；進(jìn)一步分析發(fā)現(xiàn)，宿主的52條顯著上調(diào)miRNA和49條顯著下調(diào)miRNA可分別靶向NcCK vs NcT2比較組中病原的587條顯著下調(diào)mRNA和336條顯著上調(diào)mRNA，它們可分別注釋到20和23個(gè)功能條目以及64和65條通路。AmCK1 vs AmT1和AmCK2 vs AmT2比較組的8條共同顯著上調(diào)miRNA和1條共同顯著下調(diào)miRNA分別靶向NcCK vs NcT1和NcCK vs NcT2比較組中的144條共同顯著下調(diào)和10條共同顯著上調(diào)mRNA，可分別注釋到18和13個(gè)功能條目以及38和7條通路。此外，AmCK1 vs AmT1和AmCK2 vs AmT2比較組中宿主的顯著上調(diào)miRNA可靶向結(jié)合NcCK vs NcT1和NcCK vs NcT2比較組中與RNAi途徑，孢壁蛋白和蓖麻毒素B凝集素等毒力因子，糖酵解/糖異生途徑以及MAPK信號(hào)通路相關(guān)的病原下調(diào)表達(dá)mRNA。在東方蜜蜂微孢子蟲(chóng)的侵染過(guò)程中，意蜂工蜂中腸的DEmiRNA與病原的DEmRNA之間存在復(fù)雜的靶向結(jié)合關(guān)系以及潛在的跨界調(diào)控關(guān)系；宿主的DEmiRNA可能通過(guò)抑制或降解病原的RNAi途徑、毒力因子、糖酵解/糖異生通路、ATP/ADP移位酶、ABC轉(zhuǎn)運(yùn)蛋白及MAPK信號(hào)通路相關(guān)靶DEmRNA影響病原的侵染和增殖。

意大利蜜蜂；東方蜜蜂微孢子蟲(chóng)；微小RNA；跨界調(diào)控；調(diào)控網(wǎng)絡(luò)；免疫防御

0 引言

【研究意義】意大利蜜蜂（，簡(jiǎn)稱(chēng)意蜂）作為重要的經(jīng)濟(jì)昆蟲(chóng)和授粉昆蟲(chóng)在養(yǎng)蜂生產(chǎn)、科學(xué)研究和生態(tài)多樣性維持等方面具有不可替代的價(jià)值[1]。作為群居性昆蟲(chóng)，蜜蜂易遭受多種病原微生物的侵襲，其中東方蜜蜂微孢子蟲(chóng)（）是一種專(zhuān)性侵染成年蜜蜂中腸上皮細(xì)胞的單細(xì)胞真菌病原，可導(dǎo)致蜜蜂微孢子蟲(chóng)病，該病原還能與其他生物因子或非生物因子共同脅迫蜜蜂，嚴(yán)重危害蜜蜂健康和養(yǎng)蜂生產(chǎn)[2]。人們對(duì)于蜜蜂與東方蜜蜂微孢子蟲(chóng)的相互作用進(jìn)行了較多研究[3-5]，但對(duì)背后的分子機(jī)制還知之甚少。因此，探究微小RNA（microRNA，miRNA）介導(dǎo)意蜂工蜂對(duì)東方蜜蜂微孢子蟲(chóng)的跨界調(diào)控，不僅可為明確相關(guān)分子機(jī)制提供理論依據(jù)，也能加深對(duì)二者間互作的理解?！厩叭搜芯窟M(jìn)展】在蜂群中，東方蜜蜂微孢子蟲(chóng)的孢子通過(guò)糞-口或口-口途徑被蜜蜂宿主攝入體內(nèi)，病原增殖高度依賴宿主細(xì)胞的物質(zhì)和能量供應(yīng)[3-5]。長(zhǎng)期的協(xié)同進(jìn)化使二者間形成了獨(dú)特的互作關(guān)系，東方蜜蜂微孢子蟲(chóng)能抑制蜜蜂的免疫反應(yīng)，引起消化系統(tǒng)紊亂，縮短蜜蜂壽命，并影響其定位、學(xué)習(xí)記憶和歸巢能力等[3]。但對(duì)于蜜蜂能否跨界調(diào)控東方蜜蜂微孢子蟲(chóng)，相關(guān)研究還很滯后。miRNA是一類(lèi)長(zhǎng)度約為18—25 nt的高度保守的單鏈非編碼RNA（non-coding RNA，ncRNA），可通過(guò)靶向mRNA的3′ UTR抑制mRNA的翻譯或使其降解，從而發(fā)揮轉(zhuǎn)錄后水平的調(diào)控作用[6]。近期研究發(fā)現(xiàn)，miRNA不僅在原生細(xì)胞中發(fā)揮功能，還能在物種之間相互傳播，促進(jìn)不同物種之間的串?dāng)_、交流或信號(hào)干擾[7-10]。2012年，ZHANG等首次證實(shí)植物來(lái)源的miR-168a可通過(guò)胃腸道吸收進(jìn)入哺乳動(dòng)物的肝細(xì)胞，通過(guò)抑制小鼠低密度脂蛋白受體的表達(dá)適配器蛋白1（LDLRAP1），減弱血漿中低密度脂蛋白的清除[7]。該團(tuán)隊(duì)還發(fā)現(xiàn)植物蜂糧來(lái)源的miR-162a通過(guò)抑制蜜蜂幼蜂的卵巢和整體的生長(zhǎng)發(fā)育，阻止幼蟲(chóng)分化為蜂王并誘導(dǎo)趨向工蜂的發(fā)育過(guò)程[8]。目前被廣泛認(rèn)可的外源RNA介導(dǎo)的調(diào)控機(jī)制主要分為兩種，一種是在秀麗隱桿線蟲(chóng)（）[11]、赤擬谷盜（）[12]和褐飛虱（）[13]等物種體內(nèi)的系統(tǒng)性RNA干擾（RNAi）缺陷（SID）跨膜通道介導(dǎo)的遠(yuǎn)源dsRNA攝取，進(jìn)而導(dǎo)致體內(nèi)基因表達(dá)沉默，例如XU等[13]發(fā)現(xiàn)褐飛虱中外源性dsRNA通過(guò)siRNA途徑觸發(fā)基因沉默，SID-1是褐飛虱系統(tǒng)性RNAi必需的蛋白；另一種是miRNA可通過(guò)脫落囊泡（SV）、外泌體及凋亡小體等微囊泡（MV）腔室的包裹作用，以保護(hù)其在另一物種體內(nèi)不被外源RNase酶降解，并進(jìn)入到細(xì)胞體內(nèi)發(fā)揮跨界調(diào)控基因表達(dá)的作用[14-15]。MiRNA作為關(guān)鍵的效應(yīng)因子在宿主-病原互作中扮演關(guān)鍵角色[16-17]。CUI等[9]發(fā)現(xiàn)球孢白僵菌（）可將bba-milR-1裝載進(jìn)囊泡并轉(zhuǎn)運(yùn)到斯氏按蚊（）的細(xì)胞內(nèi)，跨界調(diào)控宿主基因和的表達(dá)，球孢白僵菌在侵染前期通過(guò)下調(diào)的表達(dá)抑制Toll信號(hào)通路，而在侵染后期通過(guò)下調(diào)的表達(dá)以逃避黑化反應(yīng)。MAYORAL等[10]通過(guò)印跡雜交證實(shí)沃爾巴克氏體（）的miRNA存在于埃及伊蚊（）純化的細(xì)胞層中，并作為效應(yīng)因子調(diào)節(jié)埃及伊蚊表達(dá)，促進(jìn)自身增殖。蜜蜂與微孢子蟲(chóng)的跨界調(diào)控研究迄今僅有一例報(bào)道，HUANG等[18]合成東方蜜蜂微孢子蟲(chóng)的siRNA并飼喂給被該病原侵染的西方蜜蜂（），通過(guò)深度測(cè)序和比較分析發(fā)現(xiàn)在侵染后1—6 d分別有7條宿主miRNA和5條病原miRNA發(fā)生差異表達(dá)，進(jìn)一步推測(cè)東方蜜蜂微孢子蟲(chóng)miRNA可能被轉(zhuǎn)運(yùn)到宿主細(xì)胞質(zhì)調(diào)控宿主的新陳代謝和免疫應(yīng)答。筆者團(tuán)隊(duì)前期已對(duì)東方蜜蜂微孢子蟲(chóng)侵染意蜂工蜂過(guò)程中宿主的侵染應(yīng)答機(jī)制和病原的侵染機(jī)制進(jìn)行了進(jìn)行一系列探索，系統(tǒng)解析了意蜂工蜂中腸的mRNA差異表達(dá)譜和免疫應(yīng)答[19]，miRNA差異表達(dá)譜及調(diào)控網(wǎng)絡(luò)[20]，差異表達(dá)lncRNA的多種調(diào)控方式及潛在功能[21]，以及東方蜜蜂微孢子蟲(chóng)的高表達(dá)基因[22]、可變剪接基因[23]、差異基因[24]、差異miRNA的表達(dá)譜[25]?！颈狙芯壳腥朦c(diǎn)】目前，有關(guān)意蜂與東方蜜蜂微孢子蟲(chóng)之間的跨界調(diào)控研究極為有限。筆者團(tuán)隊(duì)前期已對(duì)東方蜜蜂微孢子蟲(chóng)侵染意蜂工蜂過(guò)程中宿主的miRNA差異表達(dá)譜和病原的mRNA差異表達(dá)譜分別進(jìn)行解析，可為進(jìn)一步探究宿主差異表達(dá)miRNA（differentially expressed miRNA，DEmiRNA）跨界調(diào)控病原差異表達(dá)mRNA（DEmRNA）提供必要的數(shù)據(jù)基礎(chǔ)?！緮M解決的關(guān)鍵問(wèn)題】通過(guò)生物信息學(xué)方法預(yù)測(cè)意蜂工蜂中腸DEmiRNA靶向結(jié)合的東方蜜蜂微孢子蟲(chóng)DEmRNA，對(duì)靶DEmRNA進(jìn)行數(shù)據(jù)庫(kù)注釋和相關(guān)分析，進(jìn)一步構(gòu)建宿主DEmiRNA與病原DEmRNA的調(diào)控網(wǎng)絡(luò)，并對(duì)調(diào)控網(wǎng)絡(luò)中的病原DEmRNA進(jìn)行分析和探討，以期在組學(xué)水平解析DEmiRNA介導(dǎo)意蜂工蜂對(duì)東方蜜蜂微孢子蟲(chóng)的跨界調(diào)控，為闡明背后的分子機(jī)制打下基礎(chǔ)。

1 材料與方法

試驗(yàn)于2017年9月至2019年10月在福建農(nóng)林大學(xué)動(dòng)物科學(xué)學(xué)院（蜂學(xué)學(xué)院）蜜蜂保護(hù)實(shí)驗(yàn)室完成。

1.1 供試生物材料

意蜂工蜂取自福建農(nóng)林大學(xué)動(dòng)物科學(xué)學(xué)院（蜂學(xué)學(xué)院）教學(xué)蜂場(chǎng)。東方蜜蜂微孢子蟲(chóng)感染的意蜂外勤蜂取自福州市閩侯縣荊溪源安養(yǎng)蜂場(chǎng)。

1.2 意蜂工蜂中腸的miRNA組學(xué)數(shù)據(jù)來(lái)源

筆者團(tuán)隊(duì)前期通過(guò)Percoll不連續(xù)密度梯度離心法對(duì)東方蜜蜂微孢子蟲(chóng)孢子進(jìn)行純化，并對(duì)意蜂工蜂進(jìn)行飼喂接種及中腸樣品的剖取[21]。前期已分別抽提東方蜜蜂微孢子蟲(chóng)侵染7 d和10 d的工蜂中腸樣品（AmT1、AmT2）和未受侵染的工蜂中腸樣品（AmCK1、AmCK2）的總RNA，并委托廣州基迪奧生物科技有限公司通過(guò)Illumina MiSeq平臺(tái)對(duì)建好的cDNA文庫(kù)進(jìn)行單端測(cè)序。

筆者團(tuán)隊(duì)已對(duì)測(cè)序數(shù)據(jù)進(jìn)行過(guò)濾和質(zhì)控[26]：（1）剔除原始讀段（raw reads）中含5′接頭序列、含polyA、低質(zhì)量的reads和剪切掉3′接頭序列后的＜18或＞30個(gè)核苷酸的序列，得到高質(zhì)量的有效序列標(biāo)簽（clean tags）；（2）利用Bowite軟件[27]將獲得的clean tags比對(duì)GeneBank及Rfam（11.0）數(shù)據(jù)庫(kù)，過(guò)濾比對(duì)上rRNA、scRNA、snoRNA和tRNA的clean tags，得到未注釋的tags（unannotated tags）；（3）比對(duì)東方蜜蜂微孢子蟲(chóng)參考基因組（assembly ASM98816v1）（https://www.ncbi.nlm.nih.gov/genome/931?genome_assembly_id=230435），去除比對(duì)上的數(shù)據(jù)（即為東方蜜蜂微孢子蟲(chóng)的數(shù)據(jù)）；（4）將剩余數(shù)據(jù)繼續(xù)比對(duì)西方蜜蜂參考基因組（assembly Amel_4.5）（http://www. ncbi.nlm.nih.gov/genome/48?genome_assembly_id=22683），剔除比對(duì)上基因組外顯子、內(nèi)含子和重復(fù)序列的clean tags，剩余比對(duì)上的數(shù)據(jù)（mapped tags）可用于后續(xù)分析。

筆者團(tuán)隊(duì)前期已利用miRDeep2軟件[28]將上述剩余的mapped tags與miRBase數(shù)據(jù)庫(kù)中收錄的miRNA前體序列進(jìn)行比對(duì)，獲得已知miRNA序列。同時(shí)，將未比對(duì)上的tags比對(duì)基因組，得到可能的前體序列，根據(jù)tags在前體序列上的分布信息和前體結(jié)構(gòu)能量信息，采用貝葉斯模型經(jīng)打分實(shí)現(xiàn)novel miRNA的鑒定。利用每百萬(wàn)標(biāo)簽序列（tags per million，TPM）公式（TPM=T×106/N，T表示miRNA的tags，N表示總miRNA的tags）對(duì)miRNA進(jìn)行表達(dá)量的歸一化處理。按照|log2fold change (FC)|≥1且≤0.05的標(biāo)準(zhǔn)篩選AmCK1 vs AmT1和AmCK2 vs AmT2比較組的顯著性DEmiRNA，用于本研究中靶向東方蜜蜂微孢子蟲(chóng)的mRNA和DEmRNA的預(yù)測(cè)和分析。

1.3 東方蜜蜂微孢子蟲(chóng)的mRNA組學(xué)數(shù)據(jù)來(lái)源

筆者團(tuán)隊(duì)前期按照1.2中的方法對(duì)意蜂工蜂進(jìn)行飼喂接種及中腸樣品制備，并利用基于鏈特異性cDNA建庫(kù)的RNA-seq技術(shù)對(duì)接種的中腸樣品進(jìn)行測(cè)序，得到同時(shí)包含宿主數(shù)據(jù)和病原數(shù)據(jù)的混合mRNA組學(xué)數(shù)據(jù)[29]。將上述混合數(shù)據(jù)連續(xù)比對(duì)核糖體數(shù)據(jù)庫(kù)、西方蜜蜂基因組（assembly Amel_4.5）和東方蜜蜂微孢子蟲(chóng)基因組（assembly ASM98816v1），篩濾得到處于侵染過(guò)程的病原mRNA組學(xué)數(shù)據(jù)[20]。其中，將侵染8日齡（即侵染后7 d）工蜂中腸內(nèi)的東方蜜蜂微孢子蟲(chóng)設(shè)為NcT1（NcT1-1、NcT1-2和NcT1-3為3個(gè)生物學(xué)重復(fù)），侵染11日齡（即侵染后10 d）工蜂中腸內(nèi)的東方蜜蜂微孢子蟲(chóng)設(shè)為NcT2（NcT2-1、NcT2-2和NcT2-3為3個(gè)生物學(xué)重復(fù)）。筆者團(tuán)隊(duì)前期也已利用基于鏈特異性cDNA建庫(kù)的RNA-seq技術(shù)對(duì)東方蜜蜂微孢子蟲(chóng)的純凈孢子（NcCK：NcCK-1、NcCK-2和NcCK-3）進(jìn)行深度測(cè)序，獲得了高質(zhì)量的mRNA組學(xué)數(shù)據(jù)[30]。測(cè)序原始數(shù)據(jù)已上傳NCBI SRA數(shù)據(jù)庫(kù)，Bioproject號(hào)分別為PRJNA395264（NcCK）和PRJNA406998（NcT1和NcT2）。

筆者團(tuán)隊(duì)前期已采用FPKM（Fragments Per Kilobase of transcript per Million fragments mapped）算法計(jì)算和歸一化基因表達(dá)量；利用edgeR軟件[31]篩選NcCK vs NcT1和NcCK vs NcT2比較組的顯著性DEmRNA，篩選標(biāo)準(zhǔn)為|log2FC|≥1且≤0.05。上述病原DEmRNA可用于本研究中宿主顯著性DEmiRNA的靶向預(yù)測(cè)及分析。

1.4 意蜂工蜂中腸顯著性DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)mRNA和DEmRNA的預(yù)測(cè)及分析

利用TargetFinder軟件[32]預(yù)測(cè)AmCK1 vs AmT1和AmCK2 vs AmT2比較組中顯著性DEmiRNA靶向結(jié)合的東方蜜蜂微孢子蟲(chóng)mRNA，以及NcCK vs NcT1和NcCK vs NcT2比較組的顯著性DEmRNA，采用默認(rèn)參數(shù)。利用OmicShare在線工具集合（www. omicshare.com）的相關(guān)工具對(duì)上述靶標(biāo)mRNA和DEmRNA進(jìn)行GO（Gene Ontology）和KEGG（Kyoto Encyclopedia of Genes and Genomes）數(shù)據(jù)庫(kù)注釋?zhuān)捎媚J(rèn)參數(shù)。

1.5 意蜂工蜂中腸顯著性DEmiRNA與東方蜜蜂微孢子蟲(chóng)mRNA和DEmRNA的調(diào)控網(wǎng)絡(luò)構(gòu)建及分析

根據(jù)1.4中預(yù)測(cè)出的意蜂工蜂中腸顯著性DEmiRNA與東方蜜蜂微孢子蟲(chóng)mRNA和顯著性DEmRNA的靶向結(jié)合關(guān)系，構(gòu)建二者之間的調(diào)控網(wǎng)絡(luò)，并利用Cytoscape軟件[33]可視化調(diào)控網(wǎng)絡(luò)。根據(jù)前人在微孢子蟲(chóng)和筆者所在課題組在東方蜜蜂微孢子蟲(chóng)方面的研究結(jié)果[34-39]，孢壁蛋白、極管蛋白、蓖麻毒素B凝集素、糖酵解/糖異生途徑以及ABC轉(zhuǎn)運(yùn)蛋白和ATP/ADP轉(zhuǎn)位酶與微孢子蟲(chóng)的侵染和增殖活動(dòng)關(guān)系密切，篩選與上述蛋白和途徑相關(guān)的病原DEmRNA及存在靶向結(jié)合關(guān)系的宿主顯著性DEmiRNA，并構(gòu)建、分析及可視化調(diào)控網(wǎng)絡(luò)。

2 結(jié)果

2.1 意蜂工蜂中腸DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)mRNA和DEmRNA的預(yù)測(cè)及分析

AmCK1 vs AmT1比較組中84條顯著性DEmiRNA靶向結(jié)合東方蜜蜂微孢子蟲(chóng)的1 620條mRNA，其中宿主的48條顯著上調(diào)miRNA和36條顯著下調(diào)miRNA分別靶向病原的1 345和1 046條mRNA。進(jìn)一步分析發(fā)現(xiàn)，宿主的47條顯著上調(diào)miRNA可靶向NcCK vs NcT1比較組中病原的584條顯著下調(diào)mRNA，34條顯著下調(diào)miRNA可靶向病原的265條顯著上調(diào)mRNA（圖1-A、1-B）。AmCK2 vs AmT2比較組中107條顯著性DEmiRNA共靶向結(jié)合東方蜜蜂微孢子蟲(chóng)的1 717條mRNA，其中宿主的56條顯著上調(diào)miRNA和51條顯著下調(diào)miRNA分別靶向病原的1 260和1 317條mRNA。進(jìn)一步分析發(fā)現(xiàn)，宿主的52條顯著上調(diào)miRNA可靶向NcCK vs NcT2比較組中病原的587條顯著下調(diào)mRNA，49條顯著下調(diào)miRNA可靶向病原的336條顯著上調(diào)mRNA（圖1-C、1-D）。

進(jìn)一步分析發(fā)現(xiàn)，AmCK1 vs AmT1和AmCK2 vs AmT2比較組包含8條共同顯著上調(diào)miRNA，可靶向NcCK vs NcT1和NcCK vs NcT2比較組中92條共同顯著上調(diào)mRNA和144條共同顯著下調(diào)mRNA；此外，1條共同顯著下調(diào)miRNA可靶向病原的10條共同顯著上調(diào)mRNA和16條共同顯著下調(diào)mRNA（圖2）。

2.2 意蜂工蜂中腸DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)mRNA和DEmRNA的功能注釋

GO數(shù)據(jù)庫(kù)注釋結(jié)果顯示，AmCK1 vs AmT1比較組中顯著性DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)的mRNA可注釋到25個(gè)功能條目，包括代謝進(jìn)程（312）、催化活性（279）和結(jié)合（274）等；AmCK2 vs AmT2比較組中顯著性DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)的mRNA可注釋到25個(gè)功能條目，包括代謝進(jìn)程（322）、催化活性（284）和細(xì)胞進(jìn)程（277）等。

AmCK1 vs AmT1中顯著上調(diào)miRNA靶向NcCK vs NcT1中的584條顯著下調(diào)mRNA，涉及代謝進(jìn)程（84）和催化活性（82）等19個(gè)功能條目；宿主的顯著下調(diào)miRNA靶向病原的265條顯著上調(diào)mRNA，涉及代謝進(jìn)程（70）和結(jié)合（63）等22個(gè)功能條目。AmCK2 vs AmT2中顯著上調(diào)miRNA靶向NcCK vs NcT2中病原的587條顯著下調(diào)mRNA，涉及代謝進(jìn)程（76）和催化活性（75）等20個(gè)功能條目；宿主的顯著下調(diào)miRNA靶向病原的336條顯著上調(diào)mRNA，涉及代謝進(jìn)程（76）和結(jié)合（69）等23個(gè)功能條目。進(jìn)一步分析結(jié)果顯示，AmCK1 vs AmT1和AmCK2 vs AmT2比較組8條共同顯著上調(diào)miRNA可靶向NcCK vs NcT1和NcCK vs NcT2比較組中144條共同顯著下調(diào)mRNA，涉及代謝進(jìn)程（20）等18個(gè)功能條目（圖3）；而1條宿主的共同顯著下調(diào)miRNA可靶向10條病原的共同顯著上調(diào)mRNA，涉及代謝進(jìn)程（4）等13個(gè)功能條目（圖3）。括號(hào)內(nèi)的數(shù)字表示注釋在該條目的mRNA數(shù)量。

2.3 意蜂工蜂中腸顯著性DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)mRNA和顯著性DEmRNA的通路注釋

KEGG數(shù)據(jù)庫(kù)注釋結(jié)果顯示，AmCK1 vs AmT1比較組中顯著性DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)的mRNA可注釋到84條通路，包括代謝途徑（107）、次生代謝物的生物合成（40）和核糖體（39）等；AmCK2 vs AmT2比較組中顯著性DEmiRNA靶向東方蜜蜂微孢子蟲(chóng)的mRNA可注釋到84條通路，包括代謝途徑（118）、核糖體（50）和次生代謝物的生物合成（48）等。

AmCK1 vs AmT1中顯著上調(diào)miRNA靶向NcCK vs NcT1中的顯著下調(diào)mRNA可注釋到代謝途徑（35）和核糖體在真核生物中的生物合成（17）等66條通路；宿主的顯著下調(diào)miRNA靶向病原的顯著上調(diào)mRNA可注釋到代謝途徑（29）和次生代謝物的生物合成（16）等64條通路。AmCK2 vs AmT2中顯著上調(diào)miRNA靶向NcCK vs NcT2中的顯著下調(diào)mRNA可注釋到代謝途徑（34）和細(xì)胞周期（15）等64條通路；宿主的顯著下調(diào)miRNA靶向病原的顯著上調(diào)mRNA可注釋到代謝途徑（35）和次生代謝物的生物合成（19）等65條通路。此外，AmCK1 vs AmT1和AmCK2 vs AmT2比較組的8條共同顯著上調(diào)miRNA可靶向NcCK vs NcT1和NcCK vs NcT2比較組中144條共同顯著下調(diào)mRNA，可注釋到代謝途徑（10）和RNA轉(zhuǎn)運(yùn)（5）等38條通路；1條宿主的共同顯著下調(diào)miRNA可靶向10條病原的共同顯著上調(diào)的mRNA，可注釋到代謝途徑（2）和抗生素的生物合成（1）等7條通路。括號(hào)內(nèi)的數(shù)字表示注釋在該通路的mRNA數(shù)量。

A：AmCK1 vs AmT1中顯著下調(diào)miRNA及其靶向NcCK vs NcT1中顯著上調(diào)mRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of significantly down-regulated miRNAs in AmCK1 vs AmT1 and their target significantly up-regulated mRNAs in NcCK vs NcT1；B：AmCK1 vs AmT1中顯著上調(diào)miRNA靶向NcCK vs NcT1中顯著下調(diào)mRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of significantly up-regulated miRNAs in AmCK1 vs AmT1 and their target significantly down-regulated mRNAs in NcCK vs NcT1；C：AmCK2 vs AmT2中顯著下調(diào)miRNA靶向NcCK vs NcT2中顯著上調(diào)mRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of significantly down-regulated miRNAs in AmCK2 vs AmT2 and their target significantly up-regulated mRNAs in NcCK vs NcT2；D：AmCK2 vs AmT2中顯著上調(diào)miRNA靶向NcCK vs NcT2中顯著下調(diào)mRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of significantly up-regulated miRNAs in AmCK2 vs AmT2 and their target significantly down-regulated mRNAs in NcCK vs NcT2

圖2 AmCK1 vs AmT1和AmCK2 vs AmT2比較組的共同顯著上調(diào)（下調(diào)）miRNA靶向NcCK vs NcT1和NcCK vs NcT2比較組的共同顯著下調(diào)（上調(diào)）mRNA的調(diào)控網(wǎng)絡(luò)

2.4 意蜂工蜂中腸顯著性DEmiRNA及其靶向的東方蜜蜂微孢子蟲(chóng)毒力因子/侵染因子相關(guān)DEmRNA的調(diào)控網(wǎng)絡(luò)

在AmCK1 vs AmT1比較組中，分別有13條顯著上調(diào)miRNA（miR-8212-y、miR-374-y和miR-590-y等）和9條顯著下調(diào)miRNA（miR-291-y、miR-409-y和miR-326-y等）靶向4條ABC轉(zhuǎn)運(yùn)蛋白編碼基因相關(guān)的顯著DEmRNA，分別有7條顯著上調(diào)miRNA（ame-miR-6052、miR-501-y和miR-767-x等）和1條顯著下調(diào)miRNA（miR-381-y）靶向3條ATP/ADP轉(zhuǎn)位酶編碼基因相關(guān)的顯著DEmRNA（圖4-A、表1、表2）。分別有9條顯著上調(diào)miRNA（miR-193-y、ame-miR-193和miR-590-y等）和1條顯著下調(diào)miRNA（miR-451-x）靶向NcCK vs NcT1比較組中的3條極管蛋白編碼基因相關(guān)的顯著DEmRNA，分別有5條顯著上調(diào)miRNA（miR-941-y、miR-16-y和novel-m0007-5p等）和1條顯著下調(diào)miRNA（miR-291-y）靶向1條孢壁和錨定吸盤(pán)復(fù)合蛋白編碼基因和1條孢壁蛋白編碼基因相關(guān)的顯著DEmRNA，分別有8條顯著上調(diào)miRNA（ame-miR-6052、miR-8212-y和miR-144-x等）和6條顯著下調(diào)miRNA（miR-409-y、miR-294-y和miR-291-x等）靶向7條蓖麻毒素B凝集素編碼基因相關(guān)的顯著DEmRNA（圖4-B、表1、表2）。分別有21條顯著上調(diào)miRNA（miR-8232-x、miR-144-x和miR-767-x等）和10條顯著下調(diào)miRNA（miR-291-y、miR-381-y和miR-462-y等）靶向10條糖酵解/糖異生途徑相關(guān)的顯著DEmRNA（圖4-C、表1、表2）。此外，分別有7條顯著上調(diào)miRNA（miR-224-x、novel-m0007-5p和miR-16-y等）和2條顯著下調(diào)miRNA（miR-155-x和miR-291-x）靶向MAPK信號(hào)通路相關(guān)的5條顯著DEmRNA（表1、表2）。

1：代謝進(jìn)程Metabolic process；2：細(xì)胞進(jìn)程Cellular process；3：?jiǎn)我唤M織進(jìn)程Single-organism process；4：應(yīng)激反應(yīng)Response to stimulus；5：信號(hào)Signaling；6：生物調(diào)節(jié)進(jìn)程Biological regulation process；7：定位Localization；8：生物調(diào)節(jié)Biological regulation；9：細(xì)胞成分組織或生物合成Cellular component organization or biogenesis；10：細(xì)胞Cell；11：細(xì)胞組件Cell part；12：細(xì)胞器Organelle；13：細(xì)胞膜組件Cell membrane part；14：細(xì)胞膜Cell membrane；15：高分子復(fù)合物Macromolecular complex；16：細(xì)胞器組件Organelle part；17：催化活性Catalytic activity；18：結(jié)合Binding

A：宿主的顯著性DEmiRNA與病原的ABC轉(zhuǎn)運(yùn)蛋白、ATP/ADP轉(zhuǎn)位酶相關(guān)DEmRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of hostsignificant DEmiRNAs and pathogen DEmRNAs associated with ABC transporter and ATP/ADP translocase；B：宿主的顯著性DEmiRNA與病原的蓖麻毒素B凝集素、孢壁蛋白和極管蛋白相關(guān)DEmRNA的調(diào)控網(wǎng)絡(luò)Regulatory network ofhost significant DEmiRNAs and pathogen DEmRNAs associated with ricin B lectin, spore wall protein and polar tube protein；C：宿主的顯著性DEmiRNA與病原的糖酵解/糖異生途徑相關(guān)DEmRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of hostsignificant DEmiRNAs and pathogen DEmRNAs associated with glycolysis/gluconeogenesis pathway

表2 靶向NcCK vs NcT1中病原毒力因子/侵染因子相關(guān)下調(diào)DEmRNA的AmCK1 vs AmT1中宿主顯著上調(diào)miRNA的信息概要

在AmCK2 vs AmT2比較組中，分別有8條顯著上調(diào)miRNA（miR-28-y、miR-8924-y和miR-8212-y等）和10條顯著下調(diào)miRNA（miR-142-y、miR-8159-x和miR-2184-x等）可靶向3條ABC轉(zhuǎn)運(yùn)蛋白編碼基因相關(guān)的顯著DEmRNA；分別有6條顯著上調(diào)miRNA（novel-m0009-3p、miR-706-x和miR-1332-y等）和7條顯著下調(diào)miRNA（miR-462-x、miR-144-x和miR-8159-x等）可靶向3條ATP/ADP轉(zhuǎn)位酶編碼基因相關(guān)的顯著DEmRNA（圖5-A）。分別有8條顯著上調(diào)miRNA（miR-424-x、miR-318-y和miR-4217-y等）和7條顯著下調(diào)miRNA（miR-144-x、miR-8159-x和miR-142-x等）可靶向NcCK vs NcT2比較組中的3條極管蛋白編碼基因相關(guān)的顯著DEmRNA；分別有7條顯著上調(diào)miRNA（miR-182-x、miR-5119-y和miR-138-x等）和9條顯著下調(diào)miRNA（miR-462-x、miR-142-y和miR-5112-x等）可靶向1條孢壁和錨定吸盤(pán)復(fù)合蛋白編碼基因、2條孢壁蛋白前體編碼基因及1條孢壁蛋白編碼基因相關(guān)的顯著DEmRNA；分別有5條顯著上調(diào)miRNA（miR-547-x、miR-4577-y和miR-8212-y等）和9條顯著下調(diào)miRNA（miR-144-x、miR-142-y和miR-223-y等）可靶向6條蓖麻毒素B凝集素編碼基因相關(guān)的顯著DEmRNA（圖5-B）。分別有14條顯著上調(diào)miRNA（miR-222-y、miR-221-z和miR-1332-y等）和19條顯著下調(diào)miRNA（miR- 8159-x、miR-2184-x和miR-8271-y等）可靶向病原的12條糖酵解/糖異生途徑相關(guān)的顯著DEmRNA（圖5-C）。此外，分別有5條顯著上調(diào)miRNA（miR-4796-y、miR-1332-y和miR-4217-y等）和5條顯著下調(diào)miRNA（miR-142-y、miR-8159-x和miR-731-x等）可靶向MAPK信號(hào)通路相關(guān)的5條顯著DEmRNA。

A：宿主的顯著性DEmiRNA與病原的ABC轉(zhuǎn)運(yùn)蛋白、ATP/ADP轉(zhuǎn)位酶相關(guān)DEmRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of hostsignificantDEmiRNAs and pathogen DEmRNAs associated with ABC transporter and ATP/ADP translocase；B：宿主的顯著性DEmiRNA與病原的蓖麻毒素B凝集素、孢壁蛋白和極管蛋白相關(guān)DEmRNA的調(diào)控網(wǎng)絡(luò)Regulatory network ofhost significant DEmiRNAs and pathogen DEmRNAs associated with ricin B lectin, spore wall protein and polar tube protein；C：宿主的顯著性DEmiRNA與病原的糖酵解/糖異生途徑相關(guān)DEmRNA的調(diào)控網(wǎng)絡(luò)Regulatory network of hostsignificant DEmiRNAs and pathogen DEmRNAs associated with glycolysis/gluconeogenesis pathway

3 討論

隨著相關(guān)研究的增多和深入，有關(guān)miRNA在宿主和病原互作中的媒介作用被廣泛報(bào)道[16-17,40-42]。前期研究中，筆者團(tuán)隊(duì)一方面解析了意蜂工蜂中腸響應(yīng)東方蜜蜂微孢子蟲(chóng)侵染的miRNA差異表達(dá)譜及DEmiRNA介導(dǎo)的宿主免疫應(yīng)答[20]；另一方面解析了東方蜜蜂微孢子蟲(chóng)侵染意蜂工蜂過(guò)程中的mRNA差異表達(dá)譜以及毒力因子、侵染因子及相關(guān)DEmRNA在病原侵染中的作用[24]。利用已獲得的高質(zhì)量miRNA和mRNA組學(xué)數(shù)據(jù)，本研究進(jìn)一步探究miRNA介導(dǎo)意蜂工蜂與東方蜜蜂微孢子蟲(chóng)間的相互作用。

東方蜜蜂微孢子蟲(chóng)能夠控制蜜蜂的物質(zhì)代謝、能量代謝和免疫防御等[43]。但關(guān)于蜜蜂是否能夠通過(guò)差異表達(dá)miRNA調(diào)控東方蜜蜂微孢子蟲(chóng)基因表達(dá)的研究未見(jiàn)報(bào)道。本研究中，AmCK1 vs AmT1比較組中的顯著上調(diào)miRNA靶向NcCK vs NcT1比較組中的顯著下調(diào)mRNA可注釋到11條碳水化合物代謝通路、8條脂質(zhì)代謝通路、3條氨基酸代謝通路及2條能量代謝通路。AmCK2 vs AmT2比較組中顯著上調(diào)miRNA靶向NcCK vs NcT2比較組中的顯著下調(diào)mRNA可注釋到10條碳水化合物代謝通路、8條脂質(zhì)代謝通路、2條氨基酸代謝通路及2條能量代謝通路。此外，AmCK1 vs AmT1和AmCK2 vs AmT2比較組的共同顯著上調(diào)miRNA靶向NcCK vs NcT1和NcCK vs NcT2比較組的共同顯著下調(diào)mRNA可注釋到8條碳水化合物代謝通路、2條脂質(zhì)代謝通路以及2條能量代謝通路。以上結(jié)果表明被東方蜜蜂微孢子蟲(chóng)感染的意蜂工蜂中腸可能通過(guò)合成與分泌miRNA跨界抑制或降解病原相關(guān)mRNA，進(jìn)而影響中腸細(xì)胞內(nèi)寄生的病原的糖類(lèi)、脂質(zhì)、蛋白和遺傳物質(zhì)等物質(zhì)代謝途徑，以及氧化磷酸化和甲烷代謝等能量代謝途徑，體現(xiàn)出二者之間存在密切的相互作用。

RNAi是一種RNA介導(dǎo)的特異性基因沉默機(jī)制，已被大量研究證實(shí)在動(dòng)物、植物、昆蟲(chóng)和微孢子蟲(chóng)等生物中發(fā)揮基因表達(dá)調(diào)節(jié)作用，具有防控病蟲(chóng)害的應(yīng)用潛力[12-13,44-45]。多數(shù)微孢子蟲(chóng)在進(jìn)化過(guò)程已失去RNAi途徑，但東方蜜蜂微孢子蟲(chóng)保留了該途徑的3個(gè)關(guān)鍵基因即、和（）的同源序列[46]。HUANG等[18]針對(duì)設(shè)計(jì)特異性siRNA，并混入飼料對(duì)東方蜜蜂微孢子蟲(chóng)感染的西方蜜蜂工蜂進(jìn)行飼喂，發(fā)現(xiàn)病原的孢子載量顯著降低，并且超過(guò)10%的病原蛋白編碼基因發(fā)生顯著性差異表達(dá)。本研究發(fā)現(xiàn)，在AmCK1 vs AmT1比較組中，宿主的miR-30-x、miR-30-y和miR-196-x顯著上調(diào)表達(dá)且靶向NcCK vs NcT1比較組中病原2個(gè)顯著下調(diào)表達(dá)的-1，包括XM_002995496.1（=3.68E-34, log2FC=-2.6）和XM_002996709.1（=3.39E-34, log2FC=-3.0），說(shuō)明意蜂工蜂中腸可能通過(guò)差異表達(dá)相應(yīng)的miRNA對(duì)病原的RNAi途徑相關(guān)的部分mRNA進(jìn)行表達(dá)調(diào)控，進(jìn)而影響東方蜜蜂微孢子蟲(chóng)的RNAi途徑。

孢壁蛋白與微孢子蟲(chóng)的孢子發(fā)芽與侵染能力密切相關(guān)，并通過(guò)促使孢子黏附宿主細(xì)胞以調(diào)節(jié)家蠶微孢子蟲(chóng)（）的感染過(guò)程[47-48]。本研究發(fā)現(xiàn)，AmCK2 vs AmT2比較組中宿主的2條顯著上調(diào)miRNA靶向NcCK vs NcT2比較組中病原顯著下調(diào)的孢壁蛋白9編碼基因（圖5），暗示被東方蜜蜂微孢子蟲(chóng)侵染的意蜂工蜂中腸可能通過(guò)上調(diào)部分miRNA的表達(dá)量加強(qiáng)對(duì)東方蜜蜂微孢子蟲(chóng)孢壁蛋白編碼基因的抑制，從而一定程度上限制病原侵染和增殖。

蓖麻毒素B凝集素是一種通過(guò)識(shí)別并結(jié)合宿主細(xì)胞表面多糖蛋白配體和糖脂的半乳糖殘基的凝集素蛋白，并促進(jìn)病原體附著或感染宿主細(xì)胞[49-50]。LIU等[51]研究證實(shí)家蠶微孢子蟲(chóng)的蓖麻毒素在孢子黏附宿主細(xì)胞和提高孢子的感染性等方面扮演重要角色。本研究發(fā)現(xiàn)，AmCK1 vs AmT1和AmCK2 vs AmT2比較組中宿主的miR-196-x顯著上調(diào)且均靶向NcCK vs NcT1和NcCK vs NcT2比較組中下調(diào)表達(dá)的蓖麻毒素B凝集素編碼基因XM_002996297.1（表1、表2），暗示意蜂工蜂中腸在東方蜜蜂微孢子蟲(chóng)侵染的過(guò)程中通過(guò)上調(diào)表達(dá)miR-196-x對(duì)靶向的XM_002996297.1進(jìn)行持續(xù)抑制，在病原侵染的不同時(shí)間點(diǎn)通過(guò)選擇性差異表達(dá)不同的miRNA影響病原的蓖麻毒素B凝集素編碼基因的表達(dá)，從而阻遏東方蜜蜂微孢子蟲(chóng)對(duì)宿主細(xì)胞的黏附和侵染。

在長(zhǎng)期的協(xié)同進(jìn)化過(guò)程中，微孢子蟲(chóng)的線粒體已逐漸退化消失，取而代之的是一種稱(chēng)為紡錘剩體（mitosome）的細(xì)胞器[35]。微孢子蟲(chóng)在寄主細(xì)胞中生存和增殖需要源源不斷的能量和物質(zhì)供給，既能通過(guò)糖酵解/糖異生途徑將葡萄糖轉(zhuǎn)化為丙酮酸[52]，還可以通過(guò)ATP/ADP轉(zhuǎn)位酶和ABC轉(zhuǎn)運(yùn)蛋白竊取宿主合成的能量和物質(zhì)供自身能量需求[36]。本研究發(fā)現(xiàn)，AmCK1 vs AmT1比較組中宿主的miR-222-y等10條顯著上調(diào)miRNA靶向NcCK vs NcT1比較組中病原的糖酵解/糖異生途徑相關(guān)的2條顯著下調(diào)mRNA（乙酰輔酶A合成酶編碼基因XM_002995703.1和磷酸丙糖異構(gòu)酶編碼基因XM_002996794.1）（表1、表2、圖4）；AmCK2 vs AmT2比較組中宿主的miR-222-y和miR-221-z顯著上調(diào)表達(dá)且均靶向NcCK vs NcT2比較組中病原的糖酵解/糖異生途徑相關(guān)的顯著下調(diào)表達(dá)的XM_002995703.1（圖5）。上述結(jié)果表明在東方蜜蜂微孢子蟲(chóng)的侵染過(guò)程中，意蜂工蜂中腸可能通過(guò)上調(diào)miR-222-y和miR-221-z等miRNA的表達(dá)量，加強(qiáng)對(duì)病原糖酵解/糖異生途徑中乙酰輔酶A合成酶編碼基因（XM_002995703.1）的抑制，從而限制病原的能量代謝，影響病原的侵染與增殖。

ATP/ADP轉(zhuǎn)位酶和ABC轉(zhuǎn)運(yùn)蛋白參與微孢子蟲(chóng)對(duì)宿主物質(zhì)和能量的竊取[36]。ABC轉(zhuǎn)運(yùn)蛋白廣泛存在于真核細(xì)胞，能夠利用ATP的能量對(duì)胞內(nèi)的糖、核苷酸、氨基酸、多肽、蛋白質(zhì)等進(jìn)行跨膜轉(zhuǎn)運(yùn)[53]。PALDI等[36]研究發(fā)現(xiàn)通過(guò)RNAi敲減東方蜜蜂微孢子蟲(chóng)的ATP/ADP轉(zhuǎn)位酶基因可導(dǎo)致病原的增殖水平下降。本研究發(fā)現(xiàn)，AmCK1 vs AmT1比較組中宿主的miR-454-y和miR-144-x顯著上調(diào)且靶向NcCK vs NcT1比較組中顯著下調(diào)表達(dá)的ATP/ADP轉(zhuǎn)位酶編碼基因（XM_002996538.1）（表1、表2、圖4）；AmCK2 vs AmT2比較組中顯著上調(diào)表達(dá)的miR-1332-y等4條miRNA靶向結(jié)合NcCK vs NcT2比較組中顯著下調(diào)表達(dá)的ATP/ADP轉(zhuǎn)位酶編碼基因（XM_002996538.1）（圖5）。此外，AmCK1 vs AmT1比較組中宿主的miR-16-y等9條顯著上調(diào)miRNA靶向NcCK vs NcT1比較組中病原的3條顯著下調(diào)表達(dá)的ABC轉(zhuǎn)運(yùn)蛋白編碼基因（XM_002995069.1、XM_002996253.1和XM_002996675.1）（表1、表2、圖4）；AmCK2 vs AmT2比較組中宿主的6條顯著上調(diào)miRNA靶向NcCK vs NcT2比較組中病原的2條顯著下調(diào)表達(dá)的ABC轉(zhuǎn)運(yùn)蛋白編碼基因（圖5）。有趣的是，宿主的miR-28-y和miR-8212-y在2個(gè)比較組中均顯著上調(diào)表達(dá)且均靶向2個(gè)比較組中病原的下調(diào)表達(dá)的ABC轉(zhuǎn)運(yùn)蛋白編碼基因。以上結(jié)果說(shuō)明意蜂工蜂中腸在東方蜜蜂微孢子蟲(chóng)侵染過(guò)程的不同時(shí)間點(diǎn)差異表達(dá)不同的miRNA對(duì)ATP/ADP轉(zhuǎn)位酶編碼基因進(jìn)行跨界調(diào)控，從而抑制東方蜜蜂微孢子蟲(chóng)對(duì)宿主細(xì)胞的能量竊?。凰拗饕部赡芡ㄟ^(guò)差異表達(dá)不同的miRNA、持續(xù)差異表達(dá)相同的miRNA對(duì)病原的ABC轉(zhuǎn)運(yùn)蛋白編碼基因相關(guān)mRNA進(jìn)行抑制，進(jìn)而限制東方蜜蜂微孢子蟲(chóng)通過(guò)轉(zhuǎn)運(yùn)宿主的營(yíng)養(yǎng)物質(zhì)滿足增殖所需。

MAPK信號(hào)通路與真菌的交配、菌絲侵染、附著胞形成、細(xì)胞壁完整性、脅迫反應(yīng)和毒力等過(guò)程密切相關(guān)[54]。筆者團(tuán)隊(duì)前期研究發(fā)現(xiàn)蜜蜂球囊菌（）在侵染不同抗性蜜蜂幼蟲(chóng)的過(guò)程中，MAPK信號(hào)通路及富集基因的活躍程度存在差異，中華蜜蜂（）幼蟲(chóng)可能通過(guò)抑制該信號(hào)通路影響蜜蜂球囊菌的增殖，而蜜蜂球囊菌可能通過(guò)激活該通路促進(jìn)對(duì)意蜂幼蟲(chóng)的侵染，體現(xiàn)了二者互作的復(fù)雜性[55-56]。本研究發(fā)現(xiàn)，AmCK1 vs AmT1比較組中宿主的3條顯著上調(diào)miRNA（novel-m0007- 5p、miR-29-y和miR-16-y）靶向NcCK vs NcT1比較組中病原MAPK信號(hào)通路相關(guān)的2條顯著下調(diào)mRNA（HECTD蛋白編碼基因XM_002995842.1和磷脂酰肌醇-4-磷酸-5-激酶編碼基因XM_002996061.1）（表1、表2）；AmCK2 vs AmT2比較組中宿主顯著下調(diào)表達(dá)的miR-8159-x和miR-316-x共同靶向NcCK vs NcT2比較組中病原顯著下調(diào)表達(dá)的HECTD蛋白編碼基因XM_002995842.1。上述結(jié)果說(shuō)明意蜂工蜂中腸在東方蜜蜂微孢子蟲(chóng)侵染過(guò)程的不同時(shí)間點(diǎn)通過(guò)差異表達(dá)不同的miRNA抑制病原的MAPK信號(hào)通路，從而影響病原在宿主細(xì)胞內(nèi)的環(huán)境適應(yīng)，以及病原的細(xì)胞壁完整性和毒力等方面。

4 結(jié)論

在東方蜜蜂微孢子蟲(chóng)侵染意蜂工蜂中腸的過(guò)程中，宿主DEmiRNA與病原DEmRNA之間存在復(fù)雜的靶向結(jié)合關(guān)系和潛在的調(diào)控關(guān)系，宿主的DEmiRNA可能通過(guò)調(diào)控病原的RNAi途徑、毒力因子、糖酵解/糖異生途徑、ATP/ADP移位酶、ABC轉(zhuǎn)運(yùn)蛋白及MAPK信號(hào)通路相關(guān)DEmRNA的表達(dá)影響病原的侵染和增殖。

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MicroRNA-mediated Cross-kingdom Regulation ofworker to

DU Yu1, FAN XiaoXue1, JIANG HaiBin1, WANG Jie1, FENG RuiRong1, ZHANG WenDe1, YU KeJun1, LONG Qi1, CAI ZongBing1, XIONG CuiLing1, ZHENG YanZhen1,2, CHEN DaFu1,2, FU ZhongMin1,2, XU GuoJun1,2, GUO Rui1,2

1College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002;2Apitherapy Research Institute, Fujian Agriculture and Forestry University, Fuzhou 350002

【】infectsand causes microsporidiosis. In this study, to reveal the mechanism of miRNA-mediated cross-kingdom regulation ofworker to, prediction, GO and KEGG database annotation as well as regulatory network analysis ofmRNAs and differentially expressed mRNAs (DEmRNAs) targeted by differentially expressed miRNAs (DEmiRNAs) ofworkers’ midguts were conducted by bioinformatic approaches based on previously gained miRNA and mRNA omics data【】Significant host DEmiRNAs were screened out by comparison of miRNA omics data fromworkers’ midguts at 7 d and 10 d postinfection (AmT1, AmT2) and corresponding uninfected midguts (AmCK1, AmCK2). DEmRNAs of pathogen were screened out through comparison of mRNA omics data frominfectingworker’s midgut (NcT1 and NcT2) and pure fungal spores (NcCK). mRNAs and DEmRNAs oftargeted by significant host DEmiRNAs were predicted using TargetFinder software. GO and KEGG database annotations of aforementioned targets were conducted using related bioinformatics tools. On basis of our previous findings, pathogen DEmRNAs associated with spore wall protein, polar tube protein, ricin B lectin, ATP/ADP translocase, ABC transporters and glycolysis/gluconeogenesis, and their target significant DEmiRNAs of host were filtered out, followed by construction and investigation of regulatory network.【】In AmCK1 vs AmT1 comparison group, 48 significantly up-regulated miRNAs and 36 significantly down-regulated miRNAs could respectively target 1 345 and 1 046 mRNAs of; additionally, 47 significantly up-regulated miRNAs and 34 significantly down-regulated miRNAs of host could target 584 significantly down-regulated mRNAs and 265 significantly up-regulated mRNAs in NcCK vs NcT1; these targets were involved in 19 and 22 functional terms as well as 66 and 64 pathways. In AmCK2 vs AmT2 comparison group, 56 significantly up-regulated miRNAs and 51 significantly down-regulated miRNAs could respectively target 1 260 and 1 317 mRNAs of, additionally, 52 significantly up-regulated miRNAs and 49 significantly down-regulated miRNAs could target 587 significantly down-regulated mRNAs and 336 significantly up-regulated mRNAs in NcCK vs NcT2, which were engaged in 20 and 23 functional terms as well as 64 and 65 pathways. Further, eight common significantly up-regulated miRNAs and one common significantly down-regulated miRNA in AmCK1 vs AmT1 and AmCK2 vs AmT2 comparison groups could respectively target 144 common significantly down-regulated mRNAs and 10 common significantly up-regulated mRNAs in NcCK vs NcT1 and NcCK vs NcT2 comparison groups, which could be annotated to 18 and 13 functional terms as well as38 and seven pathways. Moreover,host significantly up-regulated miRNAs in AmCK1 vs AmT1 and AmCK2 vs AmT2 could target pathogen significantly down-regulated mRNAs in NcCK vs NcT1 and NcCK vs NcT2, associated with RNAi, virulence factors such as polar tube protein, spore wall protein and ricin B lectin, glycolysis/gluconeogenesis and MAPK signal pathway.【】Complex target binding relationship and potential cross-kingdom regulatory relationship exist between host DEmiRNAs and pathogen DEmRNAs during the infection ofworker with; host DEmiRNAs are likely to inhibit or degrade pathogen DEmRNAs associated with RNAi, virulence factor/infection factor, glycolysis/gluconeogenesis pathway, ATP/ADP translocase, ABC transporters, and MAPK signal pathway to affectinfection and proliferation.

;;microRNA; cross-kingdom regulation; regulatory network; immune defense

10.3864/j.issn.0578-1752.2021.08.019

2020-06-02；

2020-06-22

國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專(zhuān)項(xiàng)（CARS-44-KXJ7）、福建農(nóng)林大學(xué)杰出青年科研人才計(jì)劃（xjq201814）、福建農(nóng)林大學(xué)科技創(chuàng)新專(zhuān)項(xiàng)（CXZX2017342，CXZX2017343）、福建農(nóng)林大學(xué)優(yōu)秀碩士學(xué)位論文資助基金（杜宇）、福建省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃（202010389016，202010389158）

杜宇，E-mail：m18505700830@163.com。范小雪，E-mail：imfanxx@163.com。杜宇和范小雪為同等貢獻(xiàn)作者。通信作者郭睿，E-mail：ruiguo@fafu.edu.cn

（責(zé)任編輯 岳梅）