張 培 徐莞媛 李志輝 高 煥 張慶起 賴曉芳 陳建華 閻斌倫

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脊尾白蝦不同發(fā)育期mtDNA拷貝數(shù)變化特征分析*

張 培1,2,3,4徐莞媛1,2,3,4李志輝1,2,3,4高 煥1,2,3,4張慶起5賴曉芳1,2,3,4陳建華1,2,3,4閻斌倫1,2,3,4①

(1. 江蘇省海洋生物技術(shù)重點實驗室 淮海工學(xué)院海洋生命與水產(chǎn)學(xué)院 連云港 222005； 2. 江蘇省海洋生物產(chǎn)業(yè)技術(shù)協(xié)同創(chuàng)新中心 連云港 222001；3. 江蘇省海洋資源開發(fā)研究院 連云港 222005； 4. 江蘇省農(nóng)業(yè)種質(zhì)資源保護(hù)與利用平臺 南京 210014；5. 連云港贛榆佳信水產(chǎn)開發(fā)有限公司 連云港 222100)

為了解脊尾白蝦()在不同發(fā)育期線粒體基因組(mtDNA)拷貝數(shù)的變化特征，本研究共采集其8個時期(受精卵期、溞狀幼體Ⅰ期、溞狀幼體Ⅱ期、溞狀幼體Ⅲ期、溞狀幼體Ⅳ期、溞狀幼體Ⅴ期、仔蝦期和成蝦期)的DNA樣品，利用TaqMan實時熒光定量PCR技術(shù)，對上述各發(fā)育期單個細(xì)胞中的mtDNA拷貝數(shù)進(jìn)行了測定。檢測結(jié)果顯示，脊尾白蝦受精卵期、溞狀幼體Ⅰ期、溞狀幼體Ⅱ期、溞狀幼體Ⅲ期、溞狀幼體Ⅳ期、溞狀幼體Ⅴ期、仔蝦期和成蝦期的mtDNA拷貝數(shù)的均值分別為2366、2648、2644、2873、3559、9948、6452和8872。進(jìn)一步統(tǒng)計分析結(jié)果顯示，mtDNA拷貝數(shù)與發(fā)育時間存在正相關(guān)性，相關(guān)系數(shù)為0.83。研究表明，隨著脊尾白蝦不斷生長，其單個細(xì)胞中mtDNA拷貝數(shù)總體呈上升趨勢。

脊尾白蝦；TaqMan探針法；線粒體基因組；不同發(fā)育期；拷貝數(shù)

線粒體是細(xì)胞的能量工廠，通過氧化磷酸化為細(xì)胞活動提供能量(熊偉等, 2014)。線粒體功能的實現(xiàn)不僅依賴于線粒體基因組的完整性和核基因組的調(diào)控，還依賴其基因組的拷貝數(shù)(Hock, 2009)。不同細(xì)胞中的mtDNA拷貝數(shù)并不固定，從幾個到幾萬個不等，這主要取決于細(xì)胞及機體所處的生理狀況，如在人類()胚胎發(fā)育的不同階段，肝臟和肌肉中的mtDNA拷貝數(shù)與胚齡呈顯著正相關(guān)(Pejznochova, 2010)，人類心肌細(xì)胞中的mtDNA拷貝數(shù)在不同年齡段也存在著較大的差異(Pohjoism?ki, 2010)。mtDNA拷貝數(shù)在一定程度上反映了線粒體的數(shù)量，可作為衡量組織需氧及能量的一種指標(biāo)(Kaaman, 2007; Justo, 2005)。在不同的生理狀況下，mtDNA拷貝數(shù)的調(diào)控機制至今尚不清楚。目前，對于mtDNA拷貝數(shù)的研究主要集中在人類疾病發(fā)生方面，尤其是惡性腫瘤，如肝癌、結(jié)腸癌、乳腺癌、肺癌等(高原等, 2016; Linkowska, 2015; Lemnrau, 2015; Kim, 2014)。在此類疾病的發(fā)生過程中，mtDNA拷貝數(shù)會顯著升高或者降低(Akbar, 2016; Mambo, 2005)，如在腸癌等病變組織中，mtDNA拷貝數(shù)較正常組織顯著升高(Qu, 2011)，而在肺癌等病變組織中，mtDNA拷貝數(shù)較正常組織顯著降低(Lin, 2008)。除此之外，在小鼠()、豬()、斑馬魚(、秀麗隱桿線蟲(C)等物種中關(guān)于mtDNA拷貝數(shù)的研究也表明，機體在不同的生理狀況下，其細(xì)胞中mtDNA拷貝數(shù)會出現(xiàn)顯著的變化(Ikeda, 2015; 成文敏等, 2012; 周莉娟等, 2013; 葉侃, 2011)，而在甲殼動物中，除了張培等(2016)對脊尾白蝦mtDNA拷貝數(shù)響應(yīng)汞脅迫的變化特征外，關(guān)于mtDNA拷貝數(shù)的研究則未見報道。

脊尾白蝦()是我國的一種重要海洋經(jīng)濟蝦類，具有繁殖周期短、生長速度快和環(huán)境適應(yīng)性廣等優(yōu)點(Xu, 2010)，3~4個月可繁殖1代，可以在短時間內(nèi)觀察到其不同生長階段的特征，是開展甲殼動物基礎(chǔ)遺傳和遺傳育種研究的良好模式生物。伴隨著脊尾白蝦的生長，其機體各項生理活動及指標(biāo)也會變化。目前，脊尾白蝦不同生長階段的研究主要集中在消化和免疫防御、胚胎和幼體存活率、餌料系數(shù)等方面(Babulo, 2008; 索帥等, 2015; 李玉全, 2014; 馮艷艷等, 2017)，而對于mtDNA拷貝數(shù)的研究甚少。mtDNA拷貝數(shù)的變化一定程度上反映了機體生命活動耗能的多少，也從側(cè)面體現(xiàn)了生物體代謝與生長的快慢。本研究在建立一種快速、精確的脊尾白蝦mtDNA拷貝數(shù)檢測方法的基礎(chǔ)上，研究了不同發(fā)育期脊尾白蝦mtDNA拷貝數(shù)的變化特征，希望通過對脊尾白蝦生長過程中mtDNA拷貝數(shù)的變化特征分析，為脊尾白蝦在生長過程中的生長情況觀測提供新的生物指標(biāo)。

1 材料與方法

1.1 實驗材料

實驗用脊尾白蝦取自江蘇省連云港贛榆佳信水產(chǎn)開發(fā)有限公司的養(yǎng)殖池塘，是由池塘中的野生群體自繁而來。在實驗室養(yǎng)殖環(huán)境條件下暫養(yǎng)24 h后，進(jìn)行后續(xù)實驗。實驗期間正常投喂(早晚各1次)，連續(xù)充氣，水溫保持在25℃左右，鹽度為26，溶氧量≥5 mg/L，pH為8.0~8.5。

1.2 引物設(shè)計

根據(jù)(腺苷酸轉(zhuǎn)移酶基因，GenBank No. KP892663.1，核基因組單拷貝基因)和6(三磷酸腺苷合成酶6，GenBank No. NC012566.1，線粒體基因組單拷貝基因)2個基因序列，設(shè)計實時定量PCR (qRT- PCR)擴增引物及TaqMan探針(已申請國家專利，申請?zhí)枮?01610906439.X)。引物和探針均由生工生物工程(上海)股份有限公司合成。

1.3 mtDNA和nDNA標(biāo)準(zhǔn)品的制備

提取脊尾白蝦組織總DNA，并以此為模板，分別用和6引物進(jìn)行擴增，10 μl PCR體系：7.0 μl ddH2O，1.0 μl 10×PCR緩沖液，0.6 μl MgCl2(25 mmol/L)，0.2 μl dNTPs (10 μmol/L)，0.2 μlDNA聚合酶(5 U/μl)，上、下游引物各0.4 μl (10 μmol/L)，0.2 μl mix-DNA (20 ng/μl)。擴增程序：95℃預(yù)變性3 min；95℃ 30 s、60℃ 30 s、72℃ 1 min、35個循環(huán)；最后，72℃延伸10 min。PCR產(chǎn)物用1.5%瓊脂糖凝膠電泳檢測。擴增產(chǎn)物經(jīng)膠回收純化后，按pEASY-T3 Zero Cloning Kit操作說明，把相應(yīng)的片段連接到質(zhì)粒載體中，再轉(zhuǎn)化入Trans1-T1 phage Resistant感受態(tài)細(xì)胞，利用藍(lán)白斑篩選獲得陽性克隆菌株，挑取含有陽性克隆的單克隆菌落擴大培養(yǎng)，并送至生工生物工程(上海)股份有限公司測序鑒定。經(jīng)鑒定為陽性質(zhì)粒的，用NanoDrop 2000測定質(zhì)粒濃度，利用雙鏈DNA拷貝數(shù)計算器(http://cels.uri.edu/gsc/cndna.html)將質(zhì)粒質(zhì)量換算成拷貝數(shù)(拷貝/μl)，作為mtDNA和nDNA 標(biāo)準(zhǔn)品。

表1、6基因?qū)崟r定量PCR引物及TaqMan探針

Tab.1 Quantitative real-time PCR primers and TaqMan probes of ANT and ATP6

1.4 mtDNA和nDNA標(biāo)準(zhǔn)曲線的建立

使用無DNA酶的ddH2O將質(zhì)粒標(biāo)準(zhǔn)品10倍梯度稀釋成107、106、105、104、103、102、101拷貝，各1000 μl，并以此制作標(biāo)準(zhǔn)曲線。

qRT-PCR采用10 μl擴增體系，反應(yīng)體系：5 μl Premix Ex(qPCR Probe)，0.2 μl上、下游引物 (10 μmol/L)，0.2 μl 50×ROX Reference Dye，0.2 μl gDNA(20 ng/μl)，0.4 μl TaqMan探針(0.6 μmol/L)。采用StepOnePlus?型熒光定量PCR儀(Applied Biosystems Inc.)進(jìn)行擴增，擴增程序：95℃ 20 s；95℃ 1 s、60℃ 20 s，40個循環(huán)。

1.5 脊尾白蝦不同發(fā)育期DNA的提取

脊尾白蝦胚胎發(fā)育的過程一般分為2細(xì)胞期、 4細(xì)胞期、8細(xì)胞期、16細(xì)胞期、32細(xì)胞期、囊胚期、原腸期、無節(jié)幼體期、溞狀幼體Ⅰ期、溞狀幼體Ⅱ期、溞狀幼體Ⅲ期、溞狀幼體Ⅳ期、溞狀幼體Ⅴ期、溞狀幼體Ⅵ期和仔蝦等時期(梁象秋等, 1988; 武文魁, 1984; 王緒峨, 1991)。本研究選取了受精卵期、溞狀幼體Ⅰ期，溞狀幼體Ⅱ期、溞狀幼體Ⅲ期、溞狀幼體Ⅳ期、溞狀幼體Ⅴ期、仔蝦期和成蝦期8個時期的樣品，每個時期各取3尾，經(jīng)液氮研磨后用DNA快速抽提試劑盒[生工生物工程(上海)股份有限公司]提取總DNA，將DNA濃度稀釋至20 ng/μl備用。

1.6 數(shù)據(jù)分析

采用SPSS 19.0對數(shù)據(jù)進(jìn)行相關(guān)性分析。從脊尾白蝦核型初步推測其為二倍體，因此，每個細(xì)胞中的線粒體基因組拷貝數(shù)的計算公式為：

mtDNA拷貝數(shù)=2×6拷貝數(shù)/拷貝數(shù)

2 結(jié)果

2.1 mtDNA和nDNA標(biāo)準(zhǔn)曲線的構(gòu)建

以10倍梯度稀釋重組質(zhì)粒后，選取107~102的稀釋標(biāo)準(zhǔn)品進(jìn)行qRT-PCR擴增，分別獲得基因與6基因的擴增曲線。6基因的標(biāo)準(zhǔn)曲線的回歸方程=3.402+15.928；基因的標(biāo)準(zhǔn)曲線的回歸方程=3.439+15.248，相關(guān)系數(shù)>0.99，見圖1。

2.2 脊尾白蝦不同發(fā)育期線粒體基因組拷貝數(shù)的變化

利用qRT-PCR，根據(jù)標(biāo)準(zhǔn)曲線獲得各個樣本平均每個細(xì)胞中線粒體基因組拷貝數(shù)，統(tǒng)計結(jié)果顯示，各發(fā)育期的mtDNA拷貝數(shù)隨著生長發(fā)育總體呈上升趨勢(圖2)。受精卵期、溞狀幼體Ⅰ期、溞狀幼體Ⅱ期、溞狀幼體Ⅲ期、溞狀幼體Ⅳ期、溞狀幼體Ⅴ期、仔蝦期和成蝦期8個時期的mtDNA拷貝數(shù)均值分別為2366、2648、2644、2873、3559、9948、6452和8872，溞狀幼體Ⅴ期和成蝦期mtDNA拷貝數(shù)最多，分別是受精卵期的4.2倍和3.7倍。進(jìn)一步統(tǒng)計分析發(fā)現(xiàn)，mtDNA拷貝數(shù)和發(fā)育時間存正相關(guān)性，相關(guān)系數(shù)為0.83，且溞狀幼體Ⅴ期、仔蝦和成蝦期與其他發(fā)育期存在顯著性差異(<0.05)。

3 討論

目前，檢測mtDNA拷貝數(shù)的方法主要有SYBR GreenⅠ染料法(何靜等, 2014)和TaqMan探針法(Chiu, 2003) 2種。由于SYBR GreenⅠ染料法的特異性不強，多數(shù)mtDNA拷貝數(shù)測定還是采用TaqMan探針法。采用TaqMan探針法建立的標(biāo)準(zhǔn)曲線具有良好的重復(fù)性，且引物、探針序列和引物探針相互作用關(guān)系的雙重保證克服了普通PCR假陽性的缺陷(Pohjoism?ki, 2010)。本研究通過構(gòu)建脊尾白蝦線粒體及核基因組單拷貝基因的質(zhì)粒作為標(biāo)準(zhǔn)品，建立了一種快速、高效、精確的檢測脊尾白蝦mtDNA拷貝數(shù)的方法，并在此基礎(chǔ)上，對脊尾白蝦不同發(fā)育期的mtDNA拷貝數(shù)進(jìn)行了精確定量。

早期胚胎中的ATP水平代表著機體胚胎的發(fā)育水平(Dumollard, 2008)，線粒體產(chǎn)ATP能力又與細(xì)胞內(nèi)mtDNA拷貝數(shù)有密切聯(lián)系(Dumollard, 2007; Tsai, 2015)，至今有許多學(xué)者將mtDNA拷貝數(shù)作為評估卵母細(xì)胞發(fā)育水平的一個指標(biāo)(Van Blerkom, 2011; Santos, 2006; Zeng, 2007)，即mtDNA拷貝數(shù)越高，卵母細(xì)胞成熟度越高，受精能力越強，胚胎發(fā)育潛能越高。所以，mtDNA拷貝數(shù)對機體的胚胎發(fā)育有著深遠(yuǎn)影響。本研究結(jié)果顯示，隨著脊尾白蝦的生長，細(xì)胞內(nèi)的mtDNA拷貝數(shù)在不斷增加，總體呈上升趨勢，這與謝燕梅(2014)在對豬不同生長階段不同組織mtDNA拷貝數(shù)變化研究中得到的結(jié)論相似，即伴隨著脊尾白蝦的生長，其活動量增加，機體能量需求增大，mtDNA拷貝數(shù)也會隨之增加。mtDNA拷貝數(shù)在一定程度上反映了脊尾白蝦不同生長階段的能量需求情況，為評估脊尾白蝦生長發(fā)育狀況提供了一個生物指標(biāo)。

圖1 ATP6(A)與ANT(B)熒光定量PCR擴增曲線及標(biāo)準(zhǔn)曲線

圖2 不同發(fā)育期線粒體基因組拷貝數(shù)變化

1：受精卵期；2：溞狀幼體Ⅰ期；3：溞狀幼體Ⅱ期； 4：溞狀幼體Ⅲ期；5：溞狀幼體Ⅳ期；6：溞狀幼體Ⅴ期； 7：仔蝦期；8：成蝦期。不同字母表示差異顯著(<0.05)

1: Zygote; 2: Zoea Ⅰ; 3: Zoea Ⅱ; 4: Zoea Ⅲ; 5: Zoea Ⅳ; 6: Zoea Ⅴ; 7: Post larvae; 8: Adult shrimps. Different letters indicated significant differences (<0.05)

甲殼動物具有獨特的蛻皮生長方式，在蛻皮生長期間會出現(xiàn)幼體前期的慢生長和幼體后期的快生長，并伴隨著機體變態(tài)，二者相對獨立又互相聯(lián)系(Gore, 1985; Anger, 1987)。在十足類幼體發(fā)育階段，變態(tài)期間的快速生長期一般僅限制在幼體生長的倒數(shù)第一期和第二期，此時機體形態(tài)變化大、消耗能量多，是早期幼體慢生長及能量積累的結(jié)果。進(jìn)入成體之后，機體蛻皮生長間隔變長，能量累積也隨之變緩(McConaugha, 1982、1985)。機體內(nèi)能量需求越大，mtDNA拷貝數(shù)越高，這可能是本研究中溞狀幼體Ⅴ期mtDNA拷貝數(shù)高于其他時期的原因之一。另外，還有可能是機體內(nèi)mtDNA拷貝數(shù)調(diào)控機制的作用，目前報道的mtDNA拷貝數(shù)受線粒體轉(zhuǎn)錄因子A (Lu, 2013)、同源重組修復(fù)蛋白Rad51(Sage, 2011)、DNA聚合酶γ(Kelly, 2012)等因素影響，具體如何調(diào)控有待進(jìn)一步研究。

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Profiles of mtDNA Copy Number at Different Developmental Stages of

ZHANG Pei1,2,3,4, XU Wanyuan1,2,3,4, LI Zhihui1,2,3,4, GAO Huan1,2,3,4, ZHANG Qingqi5, LAI Xiaofang1,2,3,4, CHEN Jianhua1,2,3,4, YAN Binlun1,2,3,4①

(1. Jiangsu Key Laboratory of Marine Biotechnology, College of Marine Life and Fisheries, Huaihai Institute of Technology, Lianyungang 222005; 2. Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Lianyungang 222001; 3. Marine Resource Development Institute of Jiangsu, Lianyungang 222005; 4. Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014; 5. Ganyu-Jiaxin Aquaculture Limited Company of Lianyungang, Lianyungang 222100)

Mitochondria are important organelles in cells. Mitochondria can produce ATP and provide energy for almost all biological activities. Mitochondria have a relatively independent genetic system, and nuclear genes also encode certain enzymes responsible for mitochondrial DNA (mtDNA) replication and transcription. Therefore, the number and function of mitochondria is essential for embryonic development. Mitochondrial function depends on the integrity of the mitochondrial genome or the regulation of nuclear genome, as well as the mtDNA copy number. Many studies have investigated mtDNA copy numbers in animals, including studies on oocyte maturation, mammalian embryonic development, and human disease, but none have reported the mtDNA copy number in. To study the profiles of mtDNA copy number at different developmental stages of,and the energy expenditure of tissues at the different stages of development, total DNA from zygote, zoea Ⅰ, zoea Ⅱ, zoea Ⅲ, zoea Ⅳ, zoea Ⅴ, post larvae, and adult shrimps were used to analyze the mtDNA copy number. One TaqMan probe-based real-time PCR was established to assess the mtDNA copy number. Our results showed that the average copy number of mtDNA per cell was 2366, 2648, 2644, 2873, 3559, 9948, 6452, and 8872 in the zygote, zoea Ⅰ, zoea Ⅱ, zoea Ⅲ, zoea Ⅳ, zoea Ⅴ, post larvae, and adults, respectively. These results show that mtDNA copy number is positively correlated with age, andthe correlation coefficient () was 0.83, i.e. the copy numbers of mtDNA tend to increase with progressing developmental stages of.

; TaqMan real-time PCR; Mitochondrial genome; Different developmental stages; Copy number

YAN Binlun, E-mail: yanbl@hhit.edu.cn

張培, 徐莞媛, 李志輝, 高煥, 張慶起, 賴曉芳, 陳建華, 閻斌倫. 脊尾白蝦不同發(fā)育期mtDNA拷貝數(shù)變化特征分析. 漁業(yè)科學(xué)進(jìn)展, 2018, 39(6): 173–179

Zhang P, Xu WY, Li ZH, Gao H, Zhang QQ, Lai XF, Chen JH, Yan BL. Profiles of mtDNA copy number at different developmental stages of. Progress in Fishery Sciences, 2018, 39(6): 173–179

* 江蘇高校優(yōu)勢學(xué)科建設(shè)工程資助項目、江蘇省高等學(xué)校自然科學(xué)研究重大項目(17KJA240001)、江蘇省“六大人才高峰”創(chuàng)新人才團隊資助項目(2016-HYGC-CXTD-004)和江蘇省研究生實踐創(chuàng)新計劃(KYLX16_1417)共同資助[This work was supported by Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD), Natural Science Foundation of the Jiangsu Higher Education Institutions of China(17KJA240001), “Six Talent Summit” Innovative Talents Team of Jiangsu Province (2016-HYGC-CXTD-004), and Practice Innovation Program for Graduate Students in Jiangsu Province (KYLX16_1417)]. 張 培，E-mail: 2272525096@qq.com

閻斌倫，教授，E-mail: yanbl@hhit.edu.cn

2017-09-21,

2017-11-13

10.19663/j.issn2095-9869.20170921001

S917.4

A

2095-9869(2018)06-0173-07

(編輯 馬璀艷)