史文軍 王盼 胡潤(rùn)豪 萬(wàn)夕和 沈輝 黎慧 王李寶 喬毅 蔣葛 成婕
摘要:【目的】探究脊尾白蝦(Exopalaemon carinicauda)鰓組織在漸變式低氧—復(fù)氧脅迫下的分子調(diào)控機(jī)制,為今后開(kāi)展脊尾白蝦耐低氧品系(種)的選育提供理論指導(dǎo)?!痉椒ā客ㄟ^(guò)模擬自然低氧環(huán)境的形成過(guò)程,分別于低氧處理0(對(duì)照)、3和6 h及復(fù)氧后1和8 h采集脊尾白蝦鰓組織,利用Illumina HiSeqTM4000測(cè)序平臺(tái)進(jìn)行轉(zhuǎn)錄組測(cè)序分析,經(jīng)過(guò)濾和Trinity組裝獲得Unigenes,選取Nr、Swiss-Prot、KEGG和COG/KOG等數(shù)據(jù)庫(kù)進(jìn)行注釋分析,在Omicsmart平臺(tái)上完成差異表達(dá)基因篩選及其表達(dá)趨勢(shì)分析,然后進(jìn)行GO功能注釋分析和KEGG信號(hào)通路富集分析,并隨機(jī)選取5個(gè)差異表達(dá)基因進(jìn)行實(shí)時(shí)熒光定量PCR驗(yàn)證?!窘Y(jié)果】脊尾白蝦鰓組織樣本轉(zhuǎn)錄組測(cè)序數(shù)據(jù)經(jīng)過(guò)濾后的Clean reads進(jìn)行Trinity組裝共獲得93227條Unigenes,其長(zhǎng)度范圍在201~35402 bp,平均長(zhǎng)度為834 bp,N50長(zhǎng)度為1352 bp。通過(guò)組間兩兩比較分析,共鑒定出4750個(gè)差異表達(dá)基因,其中上調(diào)差異表達(dá)基因3557個(gè)、下調(diào)差異表達(dá)基因2829個(gè);超過(guò)50%的差異表達(dá)基因被顯著富集到6種基因表達(dá)趨勢(shì)模式中(P<0.01),具體表現(xiàn)為:Profile 0模式富集到415個(gè)基因,Profile 5模式富集到201個(gè)基因,Profile 11模式富集到371個(gè)基因,Profile 13模式富集到841個(gè)基因,Profile 17模式富集到387個(gè)基因,Profile 18模式富集到411個(gè)基因。6種基因表達(dá)趨勢(shì)模式中的差異表達(dá)基因被注釋到代謝進(jìn)程、細(xì)胞進(jìn)程、單一有機(jī)體進(jìn)程、細(xì)胞、細(xì)胞零件、大分子復(fù)合物及催化活性等GO功能條目上;而KEGG信號(hào)通路富集分析結(jié)果顯示,以Profile 13模式中的差異表達(dá)基因富集到最多信號(hào)通路(86條),其中呈顯著富集的有8條,分別為核糖體、碳代謝、氧化磷酸化、氨基酸生物合成、內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工、糖酵解/糖異生、谷胱甘肽代謝和蛋白輸出。【結(jié)論】脊尾白蝦鰓組織在受低氧脅迫早期通過(guò)合成蛋白質(zhì)及提高代謝能力來(lái)抵御低氧環(huán)境,隨著低氧脅迫時(shí)間的延長(zhǎng),物質(zhì)合成和能量代謝活動(dòng)均顯著下降;但在復(fù)氧后隨著復(fù)氧時(shí)間的延長(zhǎng),其蛋白質(zhì)合成和能量代謝水平又逐漸升高恢復(fù)至常氧水平。
關(guān)鍵詞:脊尾白蝦;低氧—復(fù)氧;鰓組織;轉(zhuǎn)錄組測(cè)序;趨勢(shì)分析
中圖分類號(hào): S917;S945.49? ? ? ? ? ? ? ? ? 文獻(xiàn)標(biāo)志碼: A 文章編號(hào):2095-1191(2022)03-0735-13
Trend analysis of differentially expressed genes in gill of Exopalaemon carinicauda under hypoxia-reoxygenation stress
SHI Wen-jun WANG Pan HU Run-hao WAN Xi-he SHEN Hui LI Hui WANG Li-bao QIAO Yi JIANG Ge CHENG Jie
(1Institute of Oceanology and Marine Fisheries, Nantong, Jiangsu? 226007, China; 2Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, Shandong? 264003, China;? 3National Demonstration Center for Experimental Fisheries Science Education(Shanghai Ocean University), Shanghai? 201306, China)
Abstract:【Objective】To investigate the molecular regulation mechanisms in gill tissue of Exopalaemon carinicauda under gradually changing hypoxia-reoxygenation stress, so as to provide theoretical reference for the breeding of hypoxia tolerance strains (species) of E. carinicauda. 【Method】By simulating the formation process of hypoxia in the natural environment, gill tissues were collected at 0 (control), 3 and 6 h, and 1 and 8 h after reoxygenation, respectively. Transcriptome sequencing analysis was performed using Illumina HiSeqTM4000 sequencing platform. Unigenes were obtained by filtration and Trinity assembly. Nr, Swiss-Prot, KEGG and COG/KOG databases were selected for annotation analysis. The differentially expressed genes (DEGs) were screened and their trends were analyzed on the Omicsmart platform. Then GO functional annotation analysis and KEGG signal pathway enrichment analysis were performed, and five DEGs were randomly selected for real-time quantitative PCR(qRT-PCR) validation. 【Result】A total of 93227 uni genes whose range were from 201 to 35402 bp were obtained through transcriptome sequencing, with their average length of 834 bp and the N50 of 1352 bp. 4750 genes were identified as DEGs through pairwise comparison between groups, of which the up-regulated genes were 3557 and the down-regulated genes were 2829. More than fifty percent were significantly enriched in six gene expression trends (P<0.01):Profile 0 was enriched in 415 genes;Profile 5 was enriched in 201 genes;Profile 11 was enriched in 371 genes;Profile 13 was enriched in 841 genes;Profile 17 was enriched in 387 genes and Profile 18 was enriched in 411 genes.The DEGs in six trends were annotated to metabolic processes, cellular processes, single organism process, cell, cell parts, macromolecular complex and catalytic activity by GO functional analysis. The KEGG signaling pathway enrichment analysis showed that DEGs in Profile 13 were enriched in 86 pathways at most and significantly enriched? in ribosome,carbon metabolism,oxidative phosphorylation,biosynthesis of amino acids,protein processing in endoplasmic reticulum,glycolysis or gluconeogenesis,glutathione metabolism and protein export. 【Conclusion】The gill tissues of E.carinicauda synthesizes protein and increases metabolic capacity to resist hypoxia in the early stage of hypoxia stress. But with the prolongation of hypoxia, both substance synthesis and energy metabolism were affected and decreased. After reoxygenation, with the prolongation ofreoxygenation, the protein synthesis and energy metabolism gradually recover to level under normal oxygen.038C597C-F774-40C1-9DC1-F6FE2E5B0876
Key words: Exopalaemon carinicauda; hypoxia-reoxygenation; gilltissue; transcriptome sequencing; trend analyses
Foundation items: Agricultural Major New Breed Creation of Jiangsu Province (PZCZ201747);Agricultural Scien-ce and Technology Independent Innovation Project of Jiangsu Province (CX18-2010); The Fifth “226 Project” Cultivation Fund Support Project of Nantong (NT226-202021); Science and Technology Plan Project of Nantong (JC2019057)
0 引言
【研究意義】脊尾白蝦(Exopalaemon carinicauda)廣泛分布于我國(guó)黃海、渤海沿岸海域,是一種重要的海洋漁業(yè)和池塘養(yǎng)殖品種(Ma et al.,2020),因具有良好的口感及豐富的營(yíng)養(yǎng)而深受消費(fèi)者喜愛(ài),加之生長(zhǎng)速度快、繁殖能力強(qiáng)及環(huán)境適應(yīng)性廣等優(yōu)點(diǎn),其養(yǎng)殖規(guī)模逐年擴(kuò)大,已發(fā)展成為池塘單養(yǎng)和魚蝦蟹貝混養(yǎng)的重要經(jīng)濟(jì)蝦類(李玉全,2014)。據(jù)統(tǒng)計(jì),我國(guó)每年的脊尾白蝦養(yǎng)殖面積和產(chǎn)量分別為2萬(wàn)ha和4.5萬(wàn)t(劉俊杰等,2018;Shao et al.,2018;Li et al.,2019)。為了追求高產(chǎn)量,當(dāng)前的池塘養(yǎng)殖密度普遍較高,但高密度養(yǎng)殖易導(dǎo)致池底殘餌和排泄物過(guò)度累積,而脊尾白蝦大部分時(shí)間生活在池塘底層,由于呼吸耗氧作用及積累的有機(jī)物分解,極易出現(xiàn)低氧情況,尤其是在晚上和清晨,低氧嚴(yán)重時(shí)會(huì)威脅到脊尾白蝦的生命(Cheng et al.,2003)。除了依靠開(kāi)放式曝氣維持水中的含氧量外,目前尚無(wú)其他更好的解決方法,但這種方法不僅增加養(yǎng)殖成本,還無(wú)法完全解決低氧的問(wèn)題。因此,亟待明確脊尾白蝦對(duì)低氧的適應(yīng)機(jī)制,培育出耐低氧的新品系(種)以解決這一突出問(wèn)題?!厩叭搜芯窟M(jìn)展】至今,有關(guān)低氧對(duì)蝦類影響的研究主要集中在生長(zhǎng)發(fā)育(胡賢德和林北堃,1999)、生活習(xí)性(陳琴等,2001)、免疫能力(Cheng et al.,2002)、相關(guān)基因克?。↙i et al.,2009)、代謝過(guò)程(Dupont-Prinet et al.,2013)、行為變化(侯文杰等,2014)及組織損傷(楊明等,2019)等方面。近年來(lái),隨著新一代轉(zhuǎn)錄組測(cè)序技術(shù)的迅速發(fā)展,轉(zhuǎn)錄組分析已廣泛用于揭示逆境脅迫下對(duì)蝦的分子生物學(xué)過(guò)程和生理功能變化機(jī)制(Wang et al.,2020)。Hu等(2015)對(duì)低鹽脅迫下凡納濱對(duì)蝦鰓組織進(jìn)行轉(zhuǎn)錄組分析,獲得了一些與離子和滲透調(diào)節(jié)相關(guān)的基因;Huang等(2017)對(duì)凡納濱對(duì)蝦低溫脅迫不同時(shí)間和低溫脅迫后恢復(fù)的肌肉組織進(jìn)行轉(zhuǎn)錄組測(cè)序分析,結(jié)果發(fā)現(xiàn)絲氨酸/蘇氨酸—蛋白激酶信號(hào)通路在冷適應(yīng)中可能發(fā)揮著更重要作用;Li等(2018)對(duì)氨脅迫后的斑節(jié)對(duì)蝦肝胰腺組織進(jìn)行轉(zhuǎn)錄組分析,證實(shí)氨應(yīng)激誘導(dǎo)氧化應(yīng)激,最終導(dǎo)致細(xì)胞凋亡。然而,通過(guò)轉(zhuǎn)錄組學(xué)挖掘分析蝦類低氧脅迫過(guò)程中差異表達(dá)基因的研究相對(duì)較少。Sun等(2014)通過(guò)比較分析低氧脅迫下日本沼蝦肝胰腺組織中基因的表達(dá)情況,發(fā)現(xiàn)差異表達(dá)基因主要與呼吸代謝、血藍(lán)蛋白合成、能量消耗、抗氧化能力和免疫應(yīng)答相關(guān)。曹梅等(2020)通過(guò)研究低氧脅迫下脊尾白蝦全蝦組織中相關(guān)基因的表達(dá)情況,證實(shí)缺氧能促使蝦體糖酵解等無(wú)氧代謝途徑增強(qiáng)。【本研究切入點(diǎn)】現(xiàn)有的研究報(bào)道主要是將蝦類直接從常氧水體轉(zhuǎn)移至低氧水體中或分析常氧與低氧極限組的轉(zhuǎn)錄學(xué),其低氧處理方式可能無(wú)法準(zhǔn)確反映實(shí)際養(yǎng)殖環(huán)境中脊尾白蝦經(jīng)歷的低氧形成過(guò)程:常氧—溶解氧降低—低氧—極限低溶解氧—死亡。鰓組織是蝦類呼吸的重要場(chǎng)所,但至今有關(guān)脊尾白蝦鰓組織在漸變式低氧—復(fù)氧脅迫下的適應(yīng)性調(diào)節(jié)機(jī)制研究鮮見(jiàn)報(bào)道?!緮M解決的關(guān)鍵問(wèn)題】通過(guò)模擬自然低氧環(huán)境的形成過(guò)程,對(duì)漸變式低氧—復(fù)氧不同時(shí)間點(diǎn)的脊尾白蝦鰓組織進(jìn)行轉(zhuǎn)錄組測(cè)序分析,旨在探討脊尾白蝦對(duì)低氧—復(fù)氧響應(yīng)的分子機(jī)制,為今后開(kāi)展脊尾白蝦耐低氧品系(種)的選育提供理論指導(dǎo)。
1 材料與方法
1. 1 試驗(yàn)材料
試驗(yàn)在江蘇省海洋水產(chǎn)研究所如東海水蝦類科研基地進(jìn)行,供試用蝦為取自基地外塘飼養(yǎng)的脊尾白蝦。選取體長(zhǎng)5.3±0.1 cm,體重2.57±0.21 g,無(wú)外傷、活力好、健康的脊尾白蝦,置于1000 L的PVC圓桶中暫養(yǎng)7 d。暫養(yǎng)期間的海水鹽度為25‰、水溫20~21 ℃,持續(xù)充氧,每天投喂適量的商品配合飼料。正式試驗(yàn)前1 d停止喂食;每天換水1次,換水量約為總水量的1/3。
1. 2 試驗(yàn)設(shè)計(jì)及樣本采集
以預(yù)試驗(yàn)中的脊尾白蝦存活量和水體溶解氧為參考,將密封低氧時(shí)間定為6 h,然后立即開(kāi)始充氣復(fù)氧。正式試驗(yàn)在鹽度25‰、水溫20~21 ℃的條件下進(jìn)行,將暫養(yǎng)穩(wěn)定后的脊尾白蝦隨機(jī)分裝到裝有5 L海水的三角瓶中,每瓶10尾,每3瓶為1個(gè)組,共5組,充氣適應(yīng)0.5 h后,停止充氣,并用帶有橡膠導(dǎo)管的橡膠塞密封,開(kāi)始計(jì)時(shí)。分別于低氧處理0(對(duì)照)、3和6 h及復(fù)氧后1和8 h采集水樣和脊尾白蝦鰓組織。每瓶取1個(gè)水樣,每組3個(gè)重復(fù),以堿性碘化鉀和氯化錳固定液固定,用于后續(xù)的水體溶解氧測(cè)定;取完水樣后立即從每瓶中選取5~6尾活蝦的完整鰓組織作為1個(gè)生物學(xué)重復(fù),每組3個(gè)重復(fù),樣品編號(hào)對(duì)應(yīng)為js-0、js-3、js-6、js-7和js-14,液氮速凍后置于 -80 ℃超低溫冰箱保存,用于總RNA提取。
1. 3 溶解氧測(cè)定
參照GB 17378.4—2007《海洋監(jiān)測(cè)規(guī)范 第4部分:海水分析》中的碘量法測(cè)定水體溶解氧含量。038C597C-F774-40C1-9DC1-F6FE2E5B0876
1. 4 RNA提取、cDNA文庫(kù)構(gòu)建及高通量測(cè)序
按照TRIzol試劑盒(美國(guó)Invitrogen公司)說(shuō)明提取樣本總RNA,分別使用Agilent 2100生物分析儀評(píng)估RNA質(zhì)量、1.0%瓊脂糖凝膠電泳檢測(cè)其完整性、NanoDrop微量分光光度計(jì)測(cè)定其純度??俁NA檢測(cè)合格后采用帶有Oligo(dT)的磁珠富集mRNA,然后利用片段緩沖液將富集的mRNA裂解成短片段。以片段化的mRNA為模板、隨機(jī)寡核苷酸為引物,在ProtoScript II逆轉(zhuǎn)錄酶體系中合成cDNA第一條鏈;隨后采用RNaseH降解RNA鏈,在DNA Polymerase I體系下以dNTPs為原料合成cDNA第二條鏈。純化后的雙鏈cDNA經(jīng)末端修復(fù)及加poly(A)尾,并連接測(cè)序接頭,以AMPure XP beads篩選200 bp左右的cDNA進(jìn)行PCR擴(kuò)增,使用AMPure XP beads純化PCR產(chǎn)物以構(gòu)建cDNA文庫(kù),并通過(guò)Illumina HiSeqTM4000測(cè)序平臺(tái)進(jìn)行高通量測(cè)序。其中,cDNA文庫(kù)構(gòu)建及高通量測(cè)序均委托廣州基迪奧生物科技有限公司完成。
1. 5 轉(zhuǎn)錄組組裝與Unigenes功能注釋
測(cè)序獲得的原始序列(Raw reads)利用fastp(0.18.0)進(jìn)行數(shù)據(jù)質(zhì)量控制(Chen et al.,2018),過(guò)濾低質(zhì)量數(shù)據(jù),具體包括:(1)去除含Adapter的Reads;(2)去除含N比例大于10%的Reads;(3)去除全部都是A堿基的Reads;(4)去除低質(zhì)量Reads(Q≤20堿基數(shù)占整條Reads的50%以上),剩余的有效序列(Clean reads)使用Trinity進(jìn)行組裝(Grabherr et al.,2011)。使用BLASTx程序?qū)⑺薪M裝獲得的序列分別在Nr、Swiss-Prot、KEGG及COG/KOG等數(shù)據(jù)庫(kù)中進(jìn)行比對(duì),設(shè)定閾值1e-5,根據(jù)最佳比對(duì)結(jié)果獲得序列注釋信息。
1. 6 差異表達(dá)基因鑒定及其表達(dá)趨勢(shì)分析
基因表達(dá)量采用RPKM法進(jìn)行計(jì)算(Mortazavi et al.,2008),以DESeq2進(jìn)行兩組間基因表達(dá)差異分析(Love et al.,2014),將錯(cuò)誤發(fā)現(xiàn)率(FDR)<0.01和差異倍數(shù)絕對(duì)值(|Fold Change|)≥2作為差異基因篩選標(biāo)準(zhǔn)。然后對(duì)所有差異表達(dá)基因進(jìn)行趨勢(shì)分析(Ernst and Bar-Joseph,2006),設(shè)定模塊數(shù)量為20,P為0.01,以獲得顯著性和非顯著性基因表達(dá)模式。差異表達(dá)基因篩選及其趨勢(shì)分析均在Omicsmart平臺(tái)(https://www.omicsmart.com)上完成。
1. 7 實(shí)時(shí)熒光定量PCR驗(yàn)證
為驗(yàn)證轉(zhuǎn)錄組測(cè)序結(jié)果的可靠性,隨機(jī)挑選5個(gè)差異表達(dá)基因進(jìn)行實(shí)時(shí)熒光定量PCR驗(yàn)證,以18S為內(nèi)參基因,以樣本RNA反轉(zhuǎn)錄合成的cDNA為模板,按照TB Green試劑盒操作說(shuō)明進(jìn)行實(shí)時(shí)熒光定量PCR驗(yàn)證(引物見(jiàn)表1),設(shè)3次重復(fù)。反應(yīng)體系20.0 μL:TB Green Premix Ex Taq 10.0 μL,10 μmol/L正、反向引物各0.4 μL,ROX Reference Dye (50×) 0.4 μL,cDNA模板2.0 μL,DEPC水6.8 μL。在Step-One Plus熒光定量PCR儀上進(jìn)行擴(kuò)增,擴(kuò)增程序:95 ℃預(yù)變性30 s;95 ℃ 5 s,60 ℃ 30 s,進(jìn)行40個(gè)循環(huán);熔解曲線設(shè)為95 ℃ 15 s,60 ℃ 1 min,95 ℃ 15 s。基因相對(duì)表達(dá)量以2-ΔΔCt 法進(jìn)行換算。
2 結(jié)果與分析
2. 1 水樣溶解氧含量變化趨勢(shì)
低氧處理0 h(對(duì)照)的水樣溶解氧含量為8.04±0.08 mg/L,低氧處理3 h的水樣溶解氧含量為2.12±0.20 mg/L,即此時(shí)已達(dá)低氧環(huán)境;低氧處理6 h的水樣溶解氧含量為1.04±0.12 mg/L,溶解氧繼續(xù)降低,低氧脅迫更加劇烈;充氣復(fù)氧后1和8 h的水樣溶解氧含量分別為8.10±0.28和8.05±0.15 mg/L,已恢復(fù)至對(duì)照水樣溶解氧水平。說(shuō)明脊尾白蝦在此試驗(yàn)過(guò)程中已經(jīng)歷了由常氧環(huán)境逐漸到低氧環(huán)境再恢復(fù)到常氧環(huán)境的脅迫過(guò)程。
2. 2 轉(zhuǎn)錄組數(shù)據(jù)及其組裝情況
高通量測(cè)序結(jié)果(表2)顯示,每個(gè)脊尾白蝦鰓組織樣本獲得39381904~64573816條Raw reads,經(jīng)數(shù)據(jù)過(guò)濾處理后,平均在每個(gè)樣本獲得52394125條Clean reads,平均有效率為99.84%。Q20(測(cè)序堿基正確識(shí)別率在99.0%以上)大于98.00%,Q30(測(cè)序堿基正確識(shí)別率在99.9%以上)大于94.00%,且過(guò)濾后樣本的平均GC含量為47.26%,表明測(cè)序質(zhì)量較好。過(guò)濾后的Clean reads經(jīng)Trinity組裝后共獲得93227條Unigenes(表3),其長(zhǎng)度范圍在201~35402 bp(圖1),平均長(zhǎng)度為834 bp,N50長(zhǎng)度為1352 bp,表明組裝效果較良好。
2. 3 Unigenes功能注釋分析結(jié)果
分別在Nr、Swiss-Prot、KEGG和COG/KOG數(shù)據(jù)庫(kù)中對(duì)Unigenes進(jìn)行注釋分析,結(jié)果(表4)顯示,在Nr數(shù)據(jù)庫(kù)中注釋到37346條Unigenes,在Swiss-Prot數(shù)據(jù)庫(kù)中注釋到29042條Unigenes,在KEGG數(shù)據(jù)庫(kù)中注釋到33955條Unigenes,在COG/KOG數(shù)據(jù)庫(kù)中注釋到24238條Unigenes,累計(jì)被注釋的Unigenes為40719條,注釋率為43.68%。
2. 4 樣本重復(fù)性分析結(jié)果
Pearson相關(guān)系數(shù)熱圖(圖2)顯示,除js-6-2樣本外,其他樣本在組內(nèi)的生物學(xué)重復(fù)相關(guān)性均在0.80以上(0.81~0.99),相關(guān)性較好。造成js-6-2樣本相關(guān)性偏低的原因可能是取樣、提取或測(cè)序等某一環(huán)節(jié)發(fā)生污染,因此后續(xù)分析與驗(yàn)證均將該樣本測(cè)序數(shù)據(jù)剔除。038C597C-F774-40C1-9DC1-F6FE2E5B0876
2. 5 差異表達(dá)基因鑒定及趨勢(shì)分析結(jié)果
通過(guò)組間兩兩比較分析,共鑒定出4750個(gè)差異表達(dá)基因(圖3),其中上調(diào)差異表達(dá)基因3557個(gè)、下調(diào)差異表達(dá)基因2829個(gè)。由圖4可看出,與對(duì)照組相比,隨著低氧處理時(shí)間的延長(zhǎng),差異表達(dá)基因的數(shù)量呈增長(zhǎng)趨勢(shì)。差異表達(dá)基因最多的出現(xiàn)在js-7 vs js-14,其中上調(diào)差異表達(dá)基因1940個(gè)、下調(diào)差異表達(dá)基因815個(gè);而js-6 vs js-7鑒定出的差異表達(dá)基因最少(上調(diào)差異表達(dá)基因240個(gè)、下調(diào)差異表達(dá)基因360個(gè)),說(shuō)明低氧處理后再?gòu)?fù)氧對(duì)脊尾白蝦鰓組織基因表達(dá)影響顯著。
對(duì)4750個(gè)差異表達(dá)基因進(jìn)行表達(dá)趨勢(shì)分析,結(jié)果(圖5)顯示在默認(rèn)的20種基因表達(dá)趨勢(shì)模式中有6種顯著的基因表達(dá)趨勢(shì)模式(P<0.01)。其中,Profile 0模式表示基因表達(dá)量在低氧和復(fù)氧時(shí)一直下降(415個(gè));Profile 5模式表示基因表達(dá)量在低氧時(shí)先下降后上升,復(fù)氧后再下降(201個(gè));Profile 11和Profile 17模式表示基因表達(dá)量在低氧時(shí)上升,復(fù)氧后呈先下降再上升的變化趨勢(shì)(371和387個(gè));Profile 13模式表示基因表達(dá)量在低氧時(shí)先上升后下降,復(fù)氧后再上升(841個(gè));Profile 18模式表示基因表達(dá)量在低氧時(shí)上升,復(fù)氧后下降(411個(gè))。綜合分析發(fā)現(xiàn),超過(guò)50%的差異表達(dá)基因被富集到這6種基因表達(dá)趨勢(shì)模式中。
2. 6 差異表達(dá)基因GO功能注釋分析結(jié)果
GO功能注釋分析結(jié)果(圖6)顯示,顯著富集在6種基因表達(dá)趨勢(shì)模式中的差異表達(dá)基因主要注釋在代謝進(jìn)程(Metabolic processes)、細(xì)胞進(jìn)程(Cellular processes)、單一有機(jī)體進(jìn)程(Single-organism process)、細(xì)胞(Cell)、細(xì)胞零件(Cell part)、大分子復(fù)合物(Macromolecular complex)、催化活性(Catalytic activity)及結(jié)合(Binding)等GO功能條目上。
2. 7 差異表達(dá)基因KEGG信號(hào)通路富集分析結(jié)果
KEGG信號(hào)通路富集分析結(jié)果(表5)顯示,在Profile 0模式中富集到42條信號(hào)通路,呈顯著富集的有6條,分別為:①細(xì)胞外基質(zhì)受體相互作用(ECM-receptor interaction);②脂肪酸降解(Fatty acid degradation);③溶酶體(Lysosome);④纈氨酸,亮氨酸和異亮氨酸的生物合成(Valine,leucine and isoleucine biosynthesis);⑤甘氨酸,絲氨酸和蘇氨酸代謝(Glycine,serine and threonine metabolism);⑥類固醇生物合成(Steroid biosynthesis)。在Profile 5模式中富集到41條信號(hào)通路,呈顯著富集的有6條,分別為:①M(fèi)APK信號(hào)通路(MAPK signaling pathway-fly);②泛醌及其他萜醌類生物合成(Ubiquinone and other terpenoid-quinone biosynthesis);③凋亡(Apoptosis-fly);④自噬(Autophagy-other eukaryotes);⑤自噬(Autophagy-animal);⑥生理節(jié)律(Circadian rhythm-fly)。在Profile 11模式中富集到4條信號(hào)通路,呈顯著富集的有3條,分別為:①花生四烯酸代謝(Arachidonic acid metabolism);②ABC轉(zhuǎn)運(yùn)蛋白(ABC transporters);③蛋白酶體(Proteasome)。在Profile 13模式中富集到86條信號(hào)通路,呈顯著富集的有8條(圖7),分別為:①核糖體(Ribosome);②碳代謝(Carbon metabolism);③氧化磷酸化(Oxidative phosphorylation);④氨基酸生物合成(Biosynthesis of amino acids);⑤內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工(Protein processing in endoplasmic reticulum);⑥糖酵解/糖異生(Glycolysis/gluconeogenesis);⑦谷胱甘肽代謝(Glutathione metabolism);⑧蛋白輸出(Protein export)。在Profile 17模式中富集到17條信號(hào)通路,呈顯著富集的有4條,分別為:①核糖體(Ribosome);②RNA降解(RNA degradation);③嘧啶代謝(Pyrimidine metabolism);④壽命調(diào)節(jié)途徑(Longevity regulating pathway-worm)。在Profile 18模式中富集到65條信號(hào)通路,呈顯著富集的有3條,分別為:①TGF-β信號(hào)通路(TGF-beta signaling pathway);②MAPK信號(hào)通路(MAPK signaling pathway);③纈氨酸、亮氨酸和異亮氨酸的生物合成(Valine,leucine and isoleucine biosynthesis)。
2. 8 實(shí)時(shí)熒光定量PCR驗(yàn)證結(jié)果
隨機(jī)挑選5個(gè)差異表達(dá)基因(Gene4007、Gene 29395、Gene40966、Gene56231和Gene82609)進(jìn)行實(shí)時(shí)熒光定量PCR驗(yàn)證,結(jié)果(圖8)表明其相對(duì)表達(dá)量的變化趨勢(shì)與轉(zhuǎn)錄組測(cè)序獲得的基因表達(dá)量變化趨勢(shì)基本一致,表明本研究的轉(zhuǎn)錄組測(cè)序結(jié)果真實(shí)可靠。
3 討論
3. 1 低氧—復(fù)氧對(duì)脊尾白蝦呼吸耗氧及存活的影響
溶解氧是影響水生動(dòng)物呼吸的重要環(huán)境因子,與耗氧量直接相關(guān)(曹祥德等,2014;Ulaje et al.,2019)。一般情況下,甲殼類動(dòng)物可通過(guò)自身調(diào)節(jié)使機(jī)體處于最小耗氧量的狀態(tài),以此耐受一定程度的低氧環(huán)境,但超出其耐受極限則會(huì)引起死亡(Larkin et al.,2008)。本研究發(fā)現(xiàn),在脊尾白蝦自身呼吸耗氧的情況下,隨著低氧脅迫時(shí)間的延長(zhǎng),水體溶解氧含量逐漸降低,但低氧處理3~6 h的水樣溶解氧含量降低幅度明顯低于低氧處理0~3 h的水樣,表明隨著低氧脅迫時(shí)間的延長(zhǎng),脊尾白蝦的耗氧率在逐漸降低。王愛(ài)敏(1997)研究表明羅氏沼蝦蚤狀幼體的耗氧速率隨水體溶解氧含量的降低而下降;單保黨等(2018)也研究發(fā)現(xiàn)凡納濱對(duì)蝦p12~p13階段仔蝦的耗氧率隨水體溶解氧含量的降低而下降,故推測(cè)這種通過(guò)降低耗氧率的調(diào)節(jié)方式是蝦類普遍應(yīng)對(duì)低氧環(huán)境的一種策略。本研究還發(fā)現(xiàn),低氧處理6 h時(shí)的水體溶解氧含量降至1.04 mg/L,此時(shí)脊尾白蝦的死亡率為47%。李明云等(1992)研究發(fā)現(xiàn)平均體長(zhǎng)為5.02 cm的脊尾白蝦在水溫(18.0±0.5)℃中死亡50%時(shí)的溶解氧含量為0.78 mg/L;曹梅等(2020)研究證實(shí)平均體重為3.15±0.26 g的脊尾白蝦在水溫22~24 ℃、溶解氧含量為1.13±0.26 mg/L的條件下處于昏厥狀態(tài)??梢?jiàn),蝦體規(guī)格和環(huán)境條件均會(huì)影響脊尾白蝦對(duì)低氧的耐受程度。038C597C-F774-40C1-9DC1-F6FE2E5B0876
3. 2 低氧—復(fù)氧對(duì)脊尾白蝦鰓組織基因表達(dá)的影響
鰓組織是甲殼動(dòng)物進(jìn)行呼吸、排泄及調(diào)節(jié)滲透壓的重要器官,其直接與水體接觸,易受水環(huán)境影響(盧建平,2001)。低氧會(huì)影響蝦類鰓組織的正常生理功能,進(jìn)而引起能量代謝及其他物質(zhì)合成代謝紊亂、抗氧化應(yīng)激損傷等(Paschke et al.,2010;Sun et al.,2015),但脊尾白蝦鰓組織如何適應(yīng)低氧—復(fù)氧的分子調(diào)控機(jī)制尚未明確,因此利用轉(zhuǎn)錄組測(cè)序分析其鰓組織在漸變式低氧—復(fù)氧過(guò)程中的調(diào)節(jié)機(jī)制具有重要意義。趨勢(shì)分析可從聚類結(jié)果中挑選符合一定生物學(xué)特性的基因集,從而降低數(shù)據(jù)的復(fù)雜性和分析難度(郭睿等,2018)。為此,本研究基于轉(zhuǎn)錄組測(cè)序分析挖掘低氧—復(fù)氧脅迫下各時(shí)間點(diǎn)脊尾白蝦鰓組織的差異表達(dá)基因,并對(duì)所有差異表達(dá)基因進(jìn)行表達(dá)趨勢(shì)分析,GO功能注釋分析結(jié)果顯示,顯著富集在6種基因表達(dá)趨勢(shì)模式中的差異表達(dá)基因被注釋到生物學(xué)進(jìn)程(Biological process)、細(xì)胞組分(Cellular component)和分子功能(Molecular function)三大功能中,主要涉及代謝進(jìn)程、細(xì)胞進(jìn)程、單一有機(jī)體進(jìn)程、細(xì)胞、細(xì)胞零件、大分子復(fù)合物及催化活性等,說(shuō)明脊尾白蝦鰓組織在低氧—復(fù)氧脅迫下的代謝和細(xì)胞活動(dòng)較活躍。而KEGG信號(hào)通路富集分析結(jié)果顯示,以Profile 13模式中的差異表達(dá)基因富集到最多信號(hào)通路(86條),其中呈顯著富集的有8條,分別為核糖體、碳代謝、氧化磷酸化、氨基酸生物合成、內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工、糖酵解/糖異生、谷胱甘肽代謝和蛋白輸出。
3. 2. 1 對(duì)脊尾白蝦鰓組織蛋白合成相關(guān)基因的影響
核糖體主要由核糖體RNA(rRNA)、核糖體蛋白(RPs)和小核糖體RNA(snoRNAs)組成,是蛋白翻譯機(jī)制的重要組成部分(Nakhoul et al.,2014),其中RPs在rRNA加工過(guò)程中促進(jìn)rRNA折疊形成功能三維結(jié)構(gòu)及穩(wěn)定核糖體最終空間構(gòu)象方面發(fā)揮重要作用(Xie et al.,2018)。RPs除了在蛋白翻譯中發(fā)揮作用外,還參與核糖體外的DNA修復(fù)、細(xì)胞凋亡和細(xì)胞內(nèi)穩(wěn)態(tài)等功能(Shenoy et al.,2012)。內(nèi)質(zhì)網(wǎng)是負(fù)責(zé)跨膜蛋白和分泌蛋白合成與折疊的細(xì)胞器(Iurlaro and Munoz-Pinedo,2016)。本研究發(fā)現(xiàn),有80個(gè)差異表達(dá)基因富集在核糖體通路上,包括核糖體大亞基蛋白R(shí)PLP0、RPLP1和RPLP2及核糖體小亞基蛋白R(shí)PS10e、RPS11e和RPS12e;有19個(gè)差異表達(dá)基因富集在內(nèi)質(zhì)網(wǎng)蛋白加工通路上,如蛋白質(zhì)二硫鍵異構(gòu)酶A3(PDIA3)、鈣網(wǎng)蛋白(CALR)和X-盒結(jié)合蛋白1(XBP1)。此外,有17個(gè)差異表達(dá)基因富集在氨基酸生物合成通路上,有5個(gè)差異表達(dá)基因富集在蛋白輸出通路上。Land等(1993)研究發(fā)現(xiàn)西部錦龜(Chrysemys picta Bellii)肝細(xì)胞蛋白質(zhì)合成速率在25 ℃缺氧12 h后下降92%,但能在復(fù)氧2 h后恢復(fù)正常。Smith等(1996)研究發(fā)現(xiàn),鯽魚在缺氧狀態(tài)暴露48 h會(huì)導(dǎo)致其肝臟、心臟和肌肉的蛋白質(zhì)合成率下降,但腦組織的蛋白合成率未受影響,在復(fù)氧24 h后各組織的蛋白合成率基本恢復(fù)至常氧組水平。本研究結(jié)果表明,這些通路上的差異表達(dá)基因表達(dá)量在低氧時(shí)均隨溶解氧含量的降低而呈先升高后降低的變化趨勢(shì),故推測(cè)脊尾白蝦鰓組織在低氧早期蛋白合成較活躍,但溶解氧含量降至低氧水平后蛋白合成能力減弱;在復(fù)氧后1 h內(nèi)蛋白合成的能力尚未恢復(fù)到常氧水平,隨著復(fù)氧時(shí)間的延長(zhǎng),蛋白合成能力逐漸恢復(fù)并有所升高。
3. 2. 2 對(duì)脊尾白蝦鰓組織能量代謝相關(guān)基因的影響
呼吸代謝是生物能量代謝研究的重要內(nèi)容之一,不僅能反映生物的生理狀態(tài),還可表征環(huán)境條件對(duì)生物生理活動(dòng)的影響(馮雪等,2012)。本研究在Profile 13表達(dá)模式中分別發(fā)現(xiàn)有24、19和11個(gè)差異表達(dá)基因富集在碳代謝、氧化磷酸化和糖酵解/糖異生通路上,包括蘋果酸酶(MDH)、異檸檬酸脫氫酶(IDH)、6-磷酸葡糖酸脫氫酶(PGDH)、甘油醛-3-磷酸脫氫酶(GAPDH)和果糖-二磷酸醛縮酶(ALDO)等重要呼吸代謝酶類。MDH催化蘋果酸鹽和草酰乙酸鹽的可逆氧化還原反應(yīng)及蘋果酸鹽氧化脫羧成丙酮酸鹽,且在有氧糖酵解過(guò)程中參與蘋果酸天冬氨酸穿梭,其電子受體為NAD+或NADP+(Soldatov et al.,2020);IDH催化異檸檬酸氧化脫羧生成α-酮戊二酸,在此過(guò)程中也利用NAD+或NADP+生產(chǎn)NADH或NADPH(Kim and Park,2010);PGD催化6-磷酸葡萄糖酸產(chǎn)生核酮糖-5-磷酸和NADPH;GAPDH催化D-甘油醛-3-磷酸(GAP)在有NAD+和無(wú)機(jī)磷酸鹽存在的條件下可逆氧化和磷酸化成1,3-二磷酸甘油酸;ALDO催化3-磷酸甘油醛和磷酸二羥丙酮的醇醛縮合反應(yīng)可逆生成果糖-1,6-二磷酸(王志剛等,2019)。這些呼吸代謝酶在糖酵解、三羧酸循環(huán)、磷酸戊糖途徑及糖異生途徑中發(fā)揮重要作用,為生物體物質(zhì)合成代謝提供能量與底物。已有較多研究證實(shí),低氧—復(fù)氧對(duì)水生動(dòng)物鰓能量代謝有影響,但不同水生動(dòng)物的響應(yīng)模式可能存在差異(Crocker et al.,2013;Li et al.,2017)。
3. 2. 3 對(duì)脊尾白蝦鰓組織抗氧化酶相關(guān)基因的影響
本研究發(fā)現(xiàn)有11個(gè)差異表達(dá)基因富集到谷胱甘肽代謝通路上。谷胱甘肽(GSH)能維持機(jī)體正常的免疫系統(tǒng)功能,具有抗氧化作用,而NADPH也是抗氧化劑的關(guān)鍵電子供體(Saetan et al.,2020)。此外,本研究中的谷胱甘肽過(guò)氧化物酶(GPX)和谷胱甘肽-S-轉(zhuǎn)移酶(GST)基因表達(dá)變化趨勢(shì)與王盼等(2020)研究脊尾白蝦鰓組織在低氧—復(fù)氧條件下酶活性變化的趨勢(shì)相似,進(jìn)一步說(shuō)明這2個(gè)基因的表達(dá)變化可能在低氧—復(fù)氧的抗氧化損傷中發(fā)揮重要作用。038C597C-F774-40C1-9DC1-F6FE2E5B0876
4 結(jié)論
脊尾白蝦鰓組織在受低氧脅迫早期通過(guò)合成蛋白質(zhì)及提高代謝能力來(lái)抵御低氧環(huán)境,隨著低氧脅迫時(shí)間的延長(zhǎng),物質(zhì)合成和能量代謝活動(dòng)均顯著下降;但在復(fù)氧后隨著復(fù)氧時(shí)間的延長(zhǎng),其蛋白質(zhì)合成和能量代謝水平又逐漸升高恢復(fù)至常氧水平。
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