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        西方蜜蜂工蜂不同蟲態(tài)發(fā)育的轉(zhuǎn)錄組學(xué)分析

        2022-06-15 06:05:40宋文菲胡宗文苗春輝余玉生楊爽李亞輝

        宋文菲 胡宗文 苗春輝 余玉生 楊爽 李亞輝

        摘要:【目的】基于轉(zhuǎn)錄組學(xué)對(duì)西方蜜蜂工蜂不同蟲態(tài)間的差異表達(dá)基因(DEGs)進(jìn)行篩選和功能注釋分析,揭示與工蜂生長(zhǎng)發(fā)育相關(guān)的信號(hào)通路,為深入解析工蜂生長(zhǎng)發(fā)育的分子調(diào)控機(jī)理提供基礎(chǔ)數(shù)據(jù)。【方法】以西方蜜蜂工蜂的3日齡幼蟲、1日齡蛹和1日齡羽化工蜂3個(gè)蟲態(tài)為研究對(duì)象,利用llumina NovaSeq 6000平臺(tái)進(jìn)行轉(zhuǎn)錄組測(cè)序,采用DESeq2篩選不同蟲態(tài)樣品間的表達(dá)差異基因,然后分別進(jìn)行GO功能注釋分析及KEGG信號(hào)通路富集分析,并通過(guò)實(shí)時(shí)熒光定量PCR進(jìn)行驗(yàn)證?!窘Y(jié)果】經(jīng)轉(zhuǎn)錄組測(cè)序,在西方蜜蜂工蜂3日齡幼蟲與1日齡蛹間篩選出4823個(gè)差異表達(dá)基因(51.86%上調(diào),48.14%下調(diào)),在1日齡蛹與1日齡羽化工蜂間篩選出3295個(gè)差異表達(dá)基因(57.51%上調(diào),42.49%下調(diào)),在3日齡幼蟲與1日齡羽化工蜂間篩選出5267個(gè)差異表達(dá)基因(52.95%上調(diào),47.05%下調(diào))。GO功能注釋分析結(jié)果顯示,3日齡幼蟲與1日齡蛹間的差異表達(dá)基因注釋到43個(gè)GO功能條目,1日齡蛹與1日齡羽化工蜂間的差異表達(dá)基因注釋到45個(gè)GO功能條目,3日齡幼蟲與1日齡羽化工蜂間的差異表達(dá)基因注釋到44個(gè)GO功能條目,主要涉及細(xì)胞過(guò)程、細(xì)胞部分及結(jié)合等。KEGG信號(hào)通路富集分析發(fā)現(xiàn),3日齡幼蟲與1日齡蛹間有2905個(gè)差異表達(dá)基因富集到332條KEGG信號(hào)通路上,其中17條KEGG信號(hào)通路呈顯著富集,涉及核糖體、氧化磷酸化和昆蟲激素生物合成等;1日齡蛹與1日齡羽化工蜂間有1644個(gè)差異表達(dá)基因富集到331條KEGG信號(hào)通路上,其中45條KEGG信號(hào)通路呈顯著富集,涉及氧化磷酸化、生熱作用和胰島素分泌等;3日齡幼蟲與1日齡羽化工蜂間有2958個(gè)差異表達(dá)基因富集到337條KEGG信號(hào)通路上,其中14條KEGG信號(hào)通路呈顯著富集,涉及核糖體、蛋白酶體和胰島素分泌等。6個(gè)隨機(jī)挑選差異表達(dá)基因的實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)果與轉(zhuǎn)錄組測(cè)序結(jié)果相符,即轉(zhuǎn)錄組測(cè)序結(jié)果可靠。【結(jié)論】昆蟲激素生物合成通路相關(guān)差異表達(dá)基因調(diào)控與西方蜜蜂工蜂各蟲態(tài)JH滴度變化規(guī)律一致,氧化磷酸化信號(hào)通路則與各蟲態(tài)的營(yíng)養(yǎng)攝入和活動(dòng)行為相關(guān),而胰島素分泌通路涉及各蟲態(tài)的營(yíng)養(yǎng)調(diào)控、脂肪體合成及細(xì)胞凋亡??梢?,昆蟲激素生物合成、胰島素分泌和氧化磷酸化3種信號(hào)通路在西方蜜蜂工蜂幼蟲、蛹和成蟲的發(fā)育調(diào)控中發(fā)揮著重要作用。

        關(guān)鍵詞:西方蜜蜂;工蜂;生長(zhǎng)發(fā)育;差異表達(dá)基因;信號(hào)通路;轉(zhuǎn)錄組測(cè)序

        中圖分類號(hào):S891? ? ? ? ? ? ? ? ? ? ? ? ? 文獻(xiàn)標(biāo)志碼: A 文章編號(hào):2095-1191(2022)03-0748-11

        Transcriptome analysis of development of different stages in Apis mellifera worker bees

        SONG Wen-fei HU Zong-wen MIAO Chun-hui YU Yu-sheng YANG Shuang LI Ya-hui

        (1College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan? 650201, China;

        2Sericulture and Apiculture Research Institute, Yunnan Academy of Agricultural Sciences,

        Mengzi, Yunnan? 661101, China)

        Abstract:【Objective】To screen and functional annotation analysis of differentially expressed genes (DEGs) among different stages of Apis mellifera worker bees based on transcriptomics, reveal signaling pathways related to development of worker bees,so as to provide basic data for in-depth analysis of the molecular regulation mechanism of growth and deve-lopment of worker bees. 【Method】The 3-day-old larvae,1-day-old pupae and 1-day-old eclosion worker bees were takenas the research objects. Transcriptome sequencing was performed by Illumina NovaSeq 6000 platform,and screening of DEGs among different worker bees samples by DESeq2. GO functional annotation analysis and KEGG signaling pathway enrichment analysis were performed, and then real-time quantative PCR (qRT-PCR) verification was conducted. 【Result】After transcriptome sequencing, 4823 DEGs (51.86% up-regulated, 48.14% down-regulated) were screened between 3-day-old larvae and 1-day-old pupae of A. mellifera worker bees. 3295 DEGs were screened between 1-day-old pupae and 1-day-old eclosion worker bees (57.51% up-regulated, 42.49% down-regulated), 5267 DEGs (52.95% up-regulated, 47.05% down-regulated) were screened between 3-day-old larvae and 1-day-old eclosion worker bees. The annotated GO function entries in the GO database of the three instar differential genes were 43 (between 3-day-old larvae and 1-day-old pupae), 45 (between 1-day-old pupae and 1-day-old eclosion worker bees), and 44 (between 3-day-old larvae and 1-day-old eclosion worker bees) respectively, mainly involving cellular process, cell part, binding, etc. KEGG signaling pathway enrichment analysis results showed that 2905 DEGs were enriched in 332 KEGG signaling pathways between 3-day-old larvae and 1-day-old pupae, of which 17 KEGG signaling pathways were significantly enriched, involving ribosomes, oxidation phosphorylation and insect hormone biosynthesis.1644 DEGs were enriched in 331 KEGG signaling pathways between 1-day-old pupae and 1-day-old eclosion worker bees, of which 45 KEGG signaling pathways were significantly enriched, involving oxidative phosphorylation, thermogenesis and insulin secretion. 2958 DEGs were enriched in 337 KEGG signaling pathways between 3-day-old larvae and 1-day-old eclosion worker bees, of which 14 KEGG signaling pathways were significantly enriched, involving ribosomes, proteasomes and insulin secretion. The qRT-PCR results of 6 randomly selected DEGs were consistent with the transcriptome sequencing results, indicating that the transcriptome sequencing results were reliable. 【Conclusion】The regulation of DEGs related to insect hormone biosynthesis pathway is consistent with the change rule of juvenile hormone(JH) titer in different stages of A. mellifera worker bees. The oxidative phosphorylation signaling pathway is related to the nutrient intake and activity behavior of different stages, and the insulin secretion pathway involves in the regulation of nutritional regulation, fat body synthesis and apoptosis of diffe-rent stages. The results showsthat three signaling pathways of insect hormone biosynthesis, insulin secretion and oxidative phosphorylation play important roles in the developmental regulation of larvae, pupae and adult of A. mellifera worker bees.4293EE6F-6C74-4C82-858F-238197A4E9A1

        Key words:Apis mellifera;worker bees; growth and development; differentially expressed genes;signaling pathway; transcriptome sequencing

        Foundation items:National Modern Agriculture Industry Technology System(Honey Bee) Construction Project(CARS-44-SYZ16); Yunnan Province Science and Technology Plan Project (202105AF150052); Yunan Science and Technology Mission Funding Project (202204BI090013)

        0 引言

        【研究意義】西方蜜蜂(Apis mellifera L.)是一類以雌性為主的社會(huì)性昆蟲,受級(jí)型分化影響雌性單元分化為蜂王和工蜂,蜂王負(fù)責(zé)繁殖后代和維持秩序,而數(shù)量最多的工蜂承擔(dān)著覓食、哺育及筑巢等職能分工(Amdam and Seehuus,2006;Barchuk et al.,2007;宋文菲等,2021)。工蜂在生長(zhǎng)發(fā)育過(guò)程中受到營(yíng)養(yǎng)物質(zhì)和內(nèi)激素的共同影響(李成成等,2011;Wang et al.,2014),如缺少花粉會(huì)引起幼蟲和成年工蜂的發(fā)育受阻(Wang et al.,2014;Di Pasquale et al.,2016;Martin et al.,2021)。內(nèi)激素主要包括保幼激素(Juvenile hormone,JH)和蛻皮激素(20-hydroxyecdysone,20E),會(huì)影響工蜂的變態(tài)發(fā)育和級(jí)型分化(李茫等,2019)。至今,針對(duì)西方蜜蜂(工蜂和蜂王)幼蟲階段差異表達(dá)基因(Differentially expressed genes,DEGs)和代謝通路的研究已有相關(guān)報(bào)道,證實(shí)工蜂和蜂王的幼蟲在不同發(fā)育階段的基因種類和表達(dá)水平存在明顯差異,蜂王在幼蟲早期具有獨(dú)特的基因表達(dá)譜,hexamerin 70b基因和雷帕霉素靶蛋白(TOR)信號(hào)通路與其級(jí)型分化密切相關(guān)(Chen et al.,2012;Cameron et al.,2013;He et al.,2017)。但關(guān)于西方蜜蜂工蜂胚后發(fā)育的分子調(diào)控機(jī)理尚不清楚,因此分析工蜂各蟲態(tài)的差異表達(dá)基因及其信號(hào)通路,可為深入探究工蜂生長(zhǎng)發(fā)育的分子調(diào)控機(jī)理提供理論依據(jù)?!厩叭搜芯窟M(jìn)展】隨著昆蟲基因組學(xué)及轉(zhuǎn)錄組學(xué)等分子生物信息學(xué)的快速發(fā)展,有關(guān)西方蜜蜂級(jí)型分化、行為分化和生長(zhǎng)發(fā)育等方面的代謝通路調(diào)控機(jī)理研究已取得階段性進(jìn)展(Patel et al.,2007;Wang et al.,2013;Harpur et al.,2014)。研究表明,JH是調(diào)控西方蜜蜂變態(tài)發(fā)育和級(jí)型分化的關(guān)鍵因子,其表達(dá)水平受表皮生長(zhǎng)因子受體(EGFR)信號(hào)調(diào)控,以及胰島素受體底物(IRS)和TOR信號(hào)通路的影響(Patel et al.,2007;Kamakura,2011;Mutti et al.,2011);對(duì)蜜蜂幼蟲的IRS和TOR基因進(jìn)行RNA干擾,可引起體內(nèi)JH水平下降,進(jìn)而誘導(dǎo)幼蟲發(fā)育成為工蜂(Patel et al.,2007;Muttiet al.,2011)。胰島素/胰島素樣生長(zhǎng)因子信號(hào)(IIS)也是工蜂生長(zhǎng)發(fā)育的調(diào)控因子,通過(guò)營(yíng)養(yǎng)調(diào)控和行為分化等方式影響工蜂的發(fā)育。Ament等(2008)研究發(fā)現(xiàn),采集蜂在大腦和腹部的IIS基因表達(dá)水平高于哺育蜂,說(shuō)明IIS可調(diào)控成年工蜂的行為分化。Wang等(2013)研究表明,IIS信號(hào)通路中的AmILP1和AmILP2基因在工蜂幼蟲發(fā)育過(guò)程中發(fā)揮著不同作用,AmILP1基因能顯著降低JH水平,AmILP2基因?qū)χ倔w起調(diào)控作用,對(duì)幼蟲發(fā)育及其體重均有影響。AmILP-2基因是胰島素樣肽主要轉(zhuǎn)錄基因,在工蜂中的表達(dá)量明顯高于蜂王,說(shuō)明組織特異性與IIS信號(hào)通路相對(duì)獨(dú)立(de Azevedo and Hartfelder,2008)。與蜂王相比,在工蜂幼蟲早期和中期發(fā)育中以氨基酸、肌肉發(fā)育和一般代謝相關(guān)基因的表達(dá)較高,在幼蟲中后期則是與細(xì)胞凋亡(組織蛋白酶)和自噬細(xì)胞死亡的相關(guān)基因表達(dá)較高(Cameron et al.,2013);不同年齡段工蜂的勞動(dòng)分工也是通過(guò)JH信號(hào)通路、胰島素樣/TOR信號(hào)通路相互作用來(lái)調(diào)節(jié),其體內(nèi)存在著較多的高表達(dá)新基因(Johnson and Tsutsui,2011;Harpur et al.,2014)。有關(guān)20E對(duì)工蜂生長(zhǎng)發(fā)育的影響,Hartfelder和Engels(1998)研究發(fā)現(xiàn),20E在工蜂幼蟲階段的滴度水平較低,但在預(yù)蛹期和成蟲期分別出現(xiàn)一個(gè)峰值;祝智威等(2022)研究證實(shí),3種微小RNA可通過(guò)調(diào)控20E基因及Hippo和FoxO信號(hào)通路的相關(guān)基因而影響工蜂蛹期的變態(tài)發(fā)育過(guò)程?!颈狙芯壳腥朦c(diǎn)】工蜂是西方蜜蜂蜂群中數(shù)量最多的類型,其生長(zhǎng)發(fā)育對(duì)蜂群的發(fā)展至關(guān)重要。近年來(lái),基于轉(zhuǎn)錄組學(xué)對(duì)西方蜜蜂工蜂哺育行為相關(guān)基因、工蜂中腸發(fā)育基因的研究表明,工蜂的哺育行為受信號(hào)轉(zhuǎn)導(dǎo)和能量代謝等途徑的調(diào)控(高艷等,2020),而TGF-β、Wnt及Hippo等信號(hào)通路影響工蜂中腸的生長(zhǎng)發(fā)育和免疫能力(杜宇等,2020)。目前有關(guān)蜜蜂工蜂和蜂王級(jí)型分化差異表達(dá)基因及代謝通路的研究已有相關(guān)報(bào)道(Chen et al.,2012;Cameron et al.,2013;He et al.,2017),但針對(duì)工蜂不同蟲態(tài)間的信號(hào)通路及調(diào)控作用研究鮮見報(bào)道。【擬解決的關(guān)鍵問(wèn)題】通過(guò)對(duì)西方蜜蜂工蜂的3日齡幼蟲、1日齡蛹和1日齡羽化工蜂3個(gè)蟲態(tài)進(jìn)行轉(zhuǎn)錄組測(cè)序,并對(duì)各蟲態(tài)間的差異表達(dá)基因進(jìn)行篩選和功能注釋分析,揭示與工蜂生長(zhǎng)發(fā)育相關(guān)的信號(hào)通路,為深入解析工蜂生長(zhǎng)發(fā)育的分子調(diào)控機(jī)理提供基礎(chǔ)數(shù)據(jù)。4293EE6F-6C74-4C82-858F-238197A4E9A1

        1 材料與方法

        1. 1 試驗(yàn)材料

        供試西方蜜蜂蜂群由云南省農(nóng)業(yè)科學(xué)院蠶桑蜜蜂研究所國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系(蜜蜂)紅河綜合試驗(yàn)站西方蜜蜂試驗(yàn)蜂場(chǎng)提供。2021年4—5月選擇3群群勢(shì)相當(dāng)?shù)姆淙?,每群?張空巢脾,待蜂王產(chǎn)卵后收集幼蟲、蛹和成蟲3個(gè)發(fā)育蟲態(tài)。為保證相鄰蟲態(tài)間發(fā)育時(shí)間相同,以相鄰蟲態(tài)間隔6~7 d取樣(Wang et al.,2015),分別以3日齡幼蟲、1日齡蛹和1日齡羽化工蜂代表幼蟲期、蛹期及成蟲期。3日齡幼蟲以6頭為1個(gè)樣本,1日齡蛹和1日齡羽化工蜂則以3頭為1個(gè)樣本,每個(gè)樣本設(shè)3個(gè)生物學(xué)重復(fù)。樣品采集后立即放入液氮中凍斃,-80 ℃保存?zhèn)溆?。TRIzol試劑(Invitrogen)、PrimeScriptTM RT reagent Kit with gDNA Eraser RT-qPCR反轉(zhuǎn)錄試劑盒及TB Green Premix Ex Taq II購(gòu)自寶日醫(yī)生物技術(shù)(北京)有限公司,DEPC水購(gòu)自北京索萊寶科技有限公司。主要儀器設(shè)備有NanoDrop 2000型分光光度計(jì)(Thermo Scientific)、StepOnePlusTM型qPCR儀(Applied Biosystems公司)、梯度PCR儀(Applied Biosystems公司)、低溫高速離心機(jī)(Sigma公司)及HWS智能型恒溫恒濕箱(寧波江南儀器廠)等。

        1. 2 cDNA文庫(kù)構(gòu)建及轉(zhuǎn)錄組測(cè)序

        采集樣品在液氮中充分研磨后,根據(jù)TRIzol試劑操作說(shuō)明提取總RNA,利用NanoDrop 2000進(jìn)行RNA濃度和純度檢測(cè),以瓊脂糖凝膠電泳檢測(cè)其完整性,采用Agilent 2100 Nano測(cè)定RIN值。質(zhì)檢合格的RNA,根據(jù)TruseqTM RNA Sample Preparation Kit (Illumina)試劑盒說(shuō)明構(gòu)建cDNA文庫(kù),然后利用llumina HiSeq Xten/NovaSeq 6000平臺(tái)進(jìn)行高通量測(cè)序,獲得原始數(shù)據(jù)?;谖鞣矫鄯浠蚪M序列,利用HISAT2序列比對(duì)軟件與蜜蜂的基因組注釋信息進(jìn)行比對(duì)(Kim et al.,2015),并將基因/轉(zhuǎn)錄本在Nr、Swiss-Prot、Pfam、EggNOG、GO和KEGG等數(shù)據(jù)庫(kù)中進(jìn)行注釋,全面獲得基因/轉(zhuǎn)錄本的注釋信息。

        1. 3 轉(zhuǎn)錄組數(shù)據(jù)處理及注釋分析

        利用Cufflinks計(jì)算FPKM值,即每百萬(wàn)個(gè)外顯子映射的片段數(shù),用以評(píng)估基因表達(dá)水平(Trapnell et al.,2010;張蕾等,2020)。采用DESeq2篩選不同蟲態(tài)樣品組間的表達(dá)差異基因,篩選參數(shù)設(shè)為P<0.01且|log2Fold Change|≥1,上調(diào)/下調(diào)差異倍數(shù)為2。

        1. 4 實(shí)時(shí)熒光定量PCR驗(yàn)證

        從西方蜜蜂工蜂不同蟲態(tài)轉(zhuǎn)錄組數(shù)據(jù)中隨機(jī)挑選6個(gè)差異表達(dá)基因進(jìn)行實(shí)時(shí)熒光定量PCR驗(yàn)證,分別是腺苷酸環(huán)化酶 3基因(Ac3)、蛋白激酶C基因(Pkc)、細(xì)胞色素 P450 302a1基因(LOC727118)、胰島素樣肽2基因(ILP-2)、法尼酸甲酯環(huán)氧酶基因(LOC551179)和保幼激素酸O-甲基轉(zhuǎn)移酶基因(LOC724216)。采用Primer Premier 5.0設(shè)計(jì)6個(gè)差異表達(dá)基因的擴(kuò)增引物,參照Z(yǔ)hang等(2020)的方法設(shè)計(jì)內(nèi)參基因(GAPDH)擴(kuò)增引物,所有引物(表1)均委托生工生物工程(上海)股份有限公司合成。采用反轉(zhuǎn)錄試劑盒將提取的RNA反轉(zhuǎn)錄合成cDNA,獲得的cDNA 置于-20 ℃冰箱保存?zhèn)溆?。?shí)時(shí)熒光定量PCR反應(yīng)體系20.0 μL:TB Green Premix Ex Taq II 10.0 μL,正、反向引物(10 μmol/L)各0.8 μL,ROX Reference Dye(50×)0.4 μL,cDNA模板2.0 μL,ddH2O 6.0 μL。擴(kuò)增程序:95 ℃預(yù)變性10 min;95 ℃ 15 s,57 ℃ 1 min,進(jìn)行40個(gè)循環(huán);添加熔解曲線。設(shè)3個(gè)水平重復(fù)孔,采用2-DDCt法換算目的基因相對(duì)表達(dá)量。

        2 結(jié)果與分析

        2. 1 轉(zhuǎn)錄組測(cè)序數(shù)據(jù)質(zhì)控分析結(jié)果

        Illumina HiSeq 6000平臺(tái)高通量測(cè)序結(jié)果(表2)顯示,西方蜜蜂工蜂3日齡幼蟲、1日齡蛹和1日齡羽化工蜂的有效序列(Clean reads)分別為44384567、42199177和41901170條。各樣本的Q30均在93.00%以上,GC含量在35.66%~39.61%,表明轉(zhuǎn)錄組測(cè)序數(shù)據(jù)質(zhì)量良好,可用于后續(xù)的研究分析。

        2. 2 西方蜜蜂工蜂不同蟲態(tài)間差異表達(dá)基因分析結(jié)果

        在西方蜜蜂工蜂3個(gè)蟲態(tài)中,3日齡幼蟲與1日齡蛹間存在4823個(gè)差異表達(dá)基因,表現(xiàn)為51.86%的差異表達(dá)基因上調(diào)、48.14%的差異表達(dá)基因下調(diào)(圖1-A);1日齡蛹與1日齡羽化工蜂間存在3295個(gè)差異表達(dá)基因,表現(xiàn)為57.51%的差異表達(dá)基因上調(diào)、42.49%的差異表達(dá)基因下調(diào)(圖1-B);3日齡幼蟲與1日齡羽化工蜂間存在5267個(gè)差異表達(dá)基因,表現(xiàn)為52.95%的差異表達(dá)基因上調(diào)、47.05%的差異表達(dá)基因下調(diào)(圖1-C)。

        2. 3 差異表達(dá)基因GO功能注釋分析結(jié)果

        3日齡幼蟲與1日齡蛹間的4823個(gè)差異表達(dá)基因共注釋到43個(gè)GO功能條目。其中,以注釋到生物學(xué)過(guò)程(Biological process)的功能條目最多,有16個(gè)(占37.21%),主要涉及細(xì)胞過(guò)程(Cellular process)(935個(gè)差異表達(dá)基因,占19.39%)、代謝過(guò)程(Metabolic process)(978個(gè)差異表達(dá)基因,占20.28%)、生物調(diào)節(jié)(Biological regulation)(333個(gè)差異表達(dá)基因,占6.90%)等;注釋到細(xì)胞組分(Cellular component)的功能條目有15個(gè)(占34.88%),主要涉及膜部分(Membrane part)(839個(gè)差異表達(dá)基因,占17.40%)、細(xì)胞部分(Cell part)(756個(gè)差異表達(dá)基因,占15.67%)、含蛋白質(zhì)復(fù)合物(Protein-containing complex)(315個(gè)差異表達(dá)基因,占6.53%)等;注釋到分子功能(Molecular function)的功能條目有12個(gè)(占27.91%),主要涉及結(jié)合(Binding)(1032個(gè)差異表達(dá)基因,占21.40%)、催化活性(Catalytic activity)(948個(gè)差異表達(dá)基因s,占19.66%)、轉(zhuǎn)運(yùn)蛋白活性(Transporter activity)(161個(gè)差異表達(dá)基因,占3.34%)等(圖2-A)。4293EE6F-6C74-4C82-858F-238197A4E9A1

        1日齡蛹與1日齡羽化工蜂間的3295個(gè)差異基因共注釋到45個(gè)GO功能條目,同樣以注釋到生物學(xué)過(guò)程的功能條目最多,有17個(gè)(占37.78%),主要涉及細(xì)胞過(guò)程(549個(gè)差異表達(dá)基因,占16.67%)、代謝過(guò)程(562個(gè)差異表達(dá)基因,占17.06%)、生物調(diào)節(jié)(264個(gè)差異表達(dá)基因,占8.01%)等;注釋到細(xì)胞組分的功能條目有15個(gè)(占33.33%),主要涉及膜部分(670個(gè)差異表達(dá)基因,占20.33%)、細(xì)胞部分(383個(gè)差異表達(dá)基因,占11.62%)、膜(Membrane)(199個(gè)差異表達(dá)基因,占6.04%)等;注釋到分子功能的功能條目有13個(gè)(占28.89%),主要涉及結(jié)合(624個(gè)差異表達(dá)基因,占18.94%)、催化活性(619個(gè)差異表達(dá)基因,占18.79%)、轉(zhuǎn)運(yùn)蛋白活性(153個(gè)差異表達(dá)基因,占4.64%)等(圖2-B)。

        3日齡幼蟲與1日齡羽化工蜂間的5267個(gè)差異基因共注釋到44個(gè)GO功能條目,同樣以注釋到生物學(xué)過(guò)程的功能條目最多,占50.00%,主要涉及細(xì)胞過(guò)程(1020個(gè)差異表達(dá)基因,占19.37%)、代謝過(guò)程(1022個(gè)差異表達(dá)基因,占19.4%)、生物調(diào)節(jié)(430個(gè)差異表達(dá)基因,占8.16%)等;注釋到細(xì)胞組分的功能條目有15個(gè)(占34.09%),主要涉及膜部分(936個(gè)差異表達(dá)基因,占17.77%)、細(xì)胞部分(786個(gè)差異表達(dá)基因,占14.92%)、細(xì)胞器(Organelle)(313個(gè)差異表達(dá)基因,占5.94%)等;注釋到分子功能的功能條目有12個(gè)(占27.27%),主要涉及結(jié)合(1112個(gè)差異表達(dá)基因,占21.11%)、催化活性(1010個(gè)差異表達(dá)基因,占19.18%)、轉(zhuǎn)運(yùn)蛋白活性(165個(gè)差異表達(dá)基因,占3.13%)等(圖2-C)。

        2. 4 差異表達(dá)基因KEGG信號(hào)通路富集分析結(jié)果

        在KEGG數(shù)據(jù)庫(kù)中比對(duì)獲得差異表達(dá)基因6351個(gè),涉及有機(jī)體系統(tǒng)(Organismal systems)、細(xì)胞過(guò)程(Cellular process)、環(huán)境信息處理(Environmental information processing)、遺傳信息處理(Genetic information processing)和新陳代謝(Metabolism)五大類(圖3)。其中,有機(jī)體系統(tǒng)通路富集到的差異表達(dá)基因數(shù)最多(1736個(gè)),占可注釋基因數(shù)的27.33%,且以與內(nèi)分泌系統(tǒng)相關(guān)的基因最多;新陳代謝通路富集到的差異表達(dá)基因次之(1521個(gè)),占23.95%,以與碳水化合物代謝相關(guān)的基因最多;遺傳信息處理通路富集到1095個(gè)差異表達(dá)基因,占17.24%,以與翻譯相關(guān)的基因最多;細(xì)胞過(guò)程通路富集到1076個(gè)差異表達(dá)基因,占16.94%,以與運(yùn)輸和分解代謝相關(guān)的基因最多;環(huán)境信息處理通路富集到923個(gè)差異表達(dá)基因,占14.53%,以與信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的基因最多。

        在西方蜜蜂工蜂3個(gè)蟲態(tài)中,3日齡幼蟲與1日齡蛹間有2905個(gè)差異表達(dá)基因富集到332條KEGG信號(hào)通路上,其中17條KEGG信號(hào)通路呈顯著富集(圖4-A),包括核糖體(Ribosome,102個(gè))、氧化磷酸化(Oxidative phosphorylation,74個(gè))和昆蟲激素生物合成(Insect hormone biosynthesis,19個(gè))等。1日齡蛹與1日齡羽化工蜂間有1644個(gè)差異表達(dá)基因富集到331條KEGG信號(hào)通路上,其中45條KEGG信號(hào)通路呈顯著富集(圖4-B),包括氧化磷酸化(74個(gè))、生熱作用(Thermogenesis,83個(gè))和胰島素分泌(Insulin secretion,25個(gè))等。3日齡幼蟲與1日齡羽化工蜂間有2958個(gè)差異表達(dá)基因富集到337條KEGG信號(hào)通路上,其中14條KEGG信號(hào)通路呈顯著富集(圖4-C),包括核糖體(104個(gè))、蛋白酶體(Proteasome,32個(gè))和胰島素分泌(28個(gè))等。

        從昆蟲激素生物合成通路上挑選6個(gè)差異表達(dá)基因進(jìn)行分析,結(jié)果(表3)顯示,這6個(gè)差異表達(dá)基因從3日齡幼蟲到1日齡蛹出現(xiàn)整體下調(diào)的表達(dá)趨勢(shì),但從1日齡蛹到1日齡羽化工蜂呈整體上調(diào)的表達(dá)趨勢(shì)。同時(shí)從胰島素信號(hào)通路上挑選6個(gè)差異表達(dá)基因進(jìn)行分析,結(jié)果(表4)發(fā)現(xiàn)從3日齡幼蟲到1日齡羽化工蜂,40S核糖體蛋白S6基因(LOC725647)持續(xù)下調(diào);ILP-2基因、胰島素樣受體樣轉(zhuǎn)錄變體 X3基因(InR-2)和mTOR調(diào)節(jié)相關(guān)蛋白基因(LOC551668)呈先上調(diào)后下調(diào)的表達(dá)趨勢(shì);己糖激酶1樣基因(LOC408818)和脂肪酸合酶基因(LOC412815)則呈先下調(diào)后上調(diào)的表達(dá)趨勢(shì)。

        2. 5 轉(zhuǎn)錄組數(shù)據(jù)實(shí)時(shí)熒光定量PCR驗(yàn)證結(jié)果

        從西方蜜蜂工蜂不同蟲態(tài)的轉(zhuǎn)錄組數(shù)據(jù)中隨機(jī)挑選6個(gè)差異表達(dá)基因(Ac3、Pkc、ILP-2、LOC727118、LOC551179和LOC724216),采用實(shí)時(shí)熒光定量PCR進(jìn)行驗(yàn)證,結(jié)果(圖5)表明,在不同蟲態(tài)中6個(gè)差異表達(dá)基因的實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)果與轉(zhuǎn)錄組測(cè)序結(jié)果相符,進(jìn)一步證實(shí)了轉(zhuǎn)錄組數(shù)據(jù)結(jié)果的可靠性。

        3 討論

        氧化磷酸化是生物體分解過(guò)程中氧化步驟所釋放的能量,并驅(qū)動(dòng)ATP的合成過(guò)程(Waites and? Garner,2011)。本研究對(duì)西方蜜蜂工蜂的3日齡幼蟲、1日齡蛹和1日齡羽化工蜂進(jìn)行轉(zhuǎn)錄組測(cè)序分析,結(jié)果發(fā)現(xiàn):3日齡幼蟲與1日齡蛹間有102個(gè)差異表達(dá)基因顯著富集在核糖體通路上,包括RpL32、RpL41及Rps14等101個(gè)下調(diào)基因,僅有1個(gè)基因(LOC724629)上調(diào);有74個(gè)差異表達(dá)基因顯著富集在氧化磷酸化通路上,包括Cox6c、Ndufs1和Ndufs5等72個(gè)下調(diào)基因,而LOC408734和LOC100578821基因上調(diào)。1日齡蛹與1日齡羽化工蜂間有74個(gè)差異表達(dá)基因顯著富集在氧化磷酸化通路上,包括Uqcr11、Cox6c和Ndufs5等72個(gè)上調(diào)基因,而LOC727212和LOC551917基因下調(diào)。與蛹和羽化工蜂相比,工蜂會(huì)在幼蟲期攝入更多食物,如蜂王漿、花粉及哺育蜂下顎腺分泌物混合物質(zhì),因而表現(xiàn)為幼蟲發(fā)育階段的氧化磷酸化增強(qiáng)(Cameron et al.,2013),與本研究中3日齡幼蟲與1日齡蛹間的氧化磷酸化通路差異表達(dá)基因下調(diào)的結(jié)果一致。氧化磷酸化通路差異表達(dá)基因在1日齡蛹與1日齡羽化工蜂間上調(diào),故推測(cè)是羽化工蜂的行為活動(dòng)引起氧化磷酸化增強(qiáng)所致。核糖體是由rRNA及核糖體蛋白組成的顆粒狀結(jié)構(gòu),其中核糖體蛋白主要參與蛋白質(zhì)的合成、調(diào)控轉(zhuǎn)錄和細(xì)胞凋亡等生理過(guò)程(Warner and Mclntosh,2009)。Verras等(2004)在地中海實(shí)蠅(Ceratitis capitate)中也發(fā)現(xiàn),核糖體蛋白基因CcRpS21在胚胎和幼蟲的表達(dá)量高于蛹和成蟲,該結(jié)論在本研究中得到進(jìn)一步驗(yàn)證。此外,有研究發(fā)現(xiàn)核糖體蛋白對(duì)昆蟲卵的滯育有重要調(diào)控作用(李艷艷等,2021),因此相關(guān)核糖體蛋白基因的功能值得后續(xù)深入研究。4293EE6F-6C74-4C82-858F-238197A4E9A1

        昆蟲變態(tài)發(fā)育主要由JH和20E協(xié)同調(diào)控完成,其中,JH在調(diào)控西方蜜蜂工蜂生長(zhǎng)和變態(tài)發(fā)育過(guò)程中發(fā)揮關(guān)鍵作用(洪芳等,2016;李茫等,2019;張慧等,2021)。本研究中,西方蜜蜂工蜂3日齡幼蟲與1日齡蛹間的昆蟲激素生物合成通路顯著富集,對(duì)昆蟲內(nèi)分泌激素相關(guān)的6個(gè)差異表達(dá)基因進(jìn)行分析,結(jié)果發(fā)現(xiàn)3日齡幼蟲與1日齡蛹間的6個(gè)差異表達(dá)基因整體下調(diào),而1日齡蛹到1日齡羽化工蜂呈整體上調(diào)趨勢(shì)。在西方蜜蜂工蜂的生長(zhǎng)發(fā)育過(guò)程中,JH滴度也表現(xiàn)出3日齡幼蟲高于蛹和羽化工蜂(Hartfelder and Engels,1998)。由于工蜂3日齡仍處于幼蟲早期,在完全變態(tài)昆蟲中其幼蟲期體內(nèi)需保持一定的JH水平,以維持蟲體處于幼蟲蟲態(tài),而化蛹前的JH水平下降及蛻皮激素上升,幼蟲才能正?;迹═ruman and Riddiford,1999)。此外,3~5日齡幼蟲是西方蜜蜂幼蟲級(jí)型分化的關(guān)鍵期,此時(shí)幼蟲在攝入蜂王漿后可進(jìn)一步提高JH滴度,且能通過(guò)蜂王漿和JH轉(zhuǎn)向蜂王發(fā)育,低齡幼蟲維持一定的JH水平以實(shí)現(xiàn)幼蟲的可塑性(Mutti et al.,2011)。故推測(cè)西方蜜蜂工蜂由于受到變態(tài)發(fā)育和級(jí)型分化的影響,導(dǎo)致從3日齡幼蟲到羽化工蜂其昆蟲激素生物合成相關(guān)差異表達(dá)基因的表達(dá)發(fā)生明顯變化。

        胰島素是一種蛋白質(zhì)激素,通過(guò)IIS信號(hào)通路發(fā)揮作用,可調(diào)節(jié)生物細(xì)胞的生長(zhǎng)、代謝及繁殖等(Oldham and Hafen,2003;Wullschleger et al.,2006;Corona et al.,2007)。本研究發(fā)現(xiàn)1日齡蛹與1日齡羽化工蜂、3日齡幼蟲與1日齡羽化工蜂的胰島素分泌信號(hào)通路顯著富集,通過(guò)對(duì)胰島素分泌及胰島素信號(hào)通路中的6個(gè)差異表達(dá)基因進(jìn)行分析,結(jié)果表明,從3日齡幼蟲到1日齡成蟲間,ILP-2、InR-2和LOC551668基因呈現(xiàn)出先上調(diào)后下調(diào)的表達(dá)趨勢(shì),且下調(diào)幅度較明顯;而LOC408818和LOC412815基因呈先下調(diào)后上調(diào)的表達(dá)趨勢(shì),下調(diào)幅度較明顯;LOC725647基因的表達(dá)則持續(xù)下調(diào)。李兆英(2013)研究發(fā)現(xiàn),意大利蜜蜂工蜂在幼蟲期的脂肪體細(xì)胞數(shù)量增長(zhǎng)較快,而在蛹早期出現(xiàn)脂肪體細(xì)胞凋亡,之后組建成蟲新的脂肪體,與本研究中的LOC412815基因調(diào)控結(jié)果基本一致。此外,mTOR調(diào)節(jié)相關(guān)蛋白和40S核糖體蛋白S6可調(diào)節(jié)細(xì)胞的生長(zhǎng)、增殖和凋亡(Miron and Sonenberg,2001;Wolschin et al.,2011),故推測(cè)LOC725647和LOC551668基因可能參與工蜂胚后發(fā)育過(guò)程中脂肪體細(xì)胞的增殖和凋亡過(guò)程。de Azevedo和Hartfelder(2008)研究發(fā)現(xiàn),西方蜜蜂工蜂ILP2基因從3齡幼蟲到5齡呈上調(diào)表達(dá)趨勢(shì),在5齡幼蟲攝食期間顯著上升,之后呈下調(diào)表達(dá);InR-2基因從3齡幼蟲到5齡整體也呈上調(diào)趨勢(shì),5齡幼蟲攝食期后開始下調(diào)。說(shuō)明胰島素信號(hào)通路可能參與了工蜂幼蟲期的營(yíng)養(yǎng)調(diào)控、脂肪體合成,以及蛹期的脂肪體凋亡過(guò)程。

        4 結(jié)論

        昆蟲激素生物合成通路相關(guān)差異表達(dá)基因調(diào)控與西方蜜蜂工蜂各蟲態(tài)JH滴度變化規(guī)律一致,氧化磷酸化信號(hào)通路則與各蟲態(tài)的營(yíng)養(yǎng)攝入和活動(dòng)行為相關(guān),而胰島素分泌通路涉及各蟲態(tài)的JH水平、脂肪體合成及行為分化調(diào)控??梢姡ハx激素生物合成、胰島素分泌和氧化磷酸化3種信號(hào)通路在西方蜜蜂工蜂生長(zhǎng)發(fā)育調(diào)控中發(fā)揮著重要作用。

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        (責(zé)任編輯 蘭宗寶)4293EE6F-6C74-4C82-858F-238197A4E9A1

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