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      牙鲆變態(tài)期間核酸、總蛋白的變化及其與生長的關系

      2014-07-16 08:33:53佟雪紅等
      江蘇農業(yè)科學 2014年3期
      關鍵詞:牙鲆變態(tài)核酸

      佟雪紅等

      摘要:研究牙鲆變態(tài)期間核酸、總蛋白及其比值的變化,并確定其與生長的關系。結果顯示,DNA濃度在22~32日齡保持相對穩(wěn)定,在32~34日齡急劇升高,在36日齡達到最高。RNA濃度在24日齡升至峰值,在32日齡降至最低,之后呈先升高再下降的趨勢??偟鞍诐舛仍?2~29日齡呈升高趨勢,之后先降低再上升。DNA、RNA含量及總蛋白含量在變態(tài)期間均保持先升高后降低再升高的趨勢,在32~36日齡快速增長。RNA/DNA比值在22~24日齡呈上升趨勢,然后下降直至32日齡,之后先上升后下降,并在36日齡達到最低值(2.27)。Protein/DNA比值在29日齡達到最高值(61.97),之后先下降后上升。RNA、DNA濃度及總蛋白濃度與體長和體重有明顯的線性關系。結果表明,牙鲆變態(tài)高峰期前的生長以細胞增大為主,變態(tài)后的生長以細胞增殖為主,RNA、DNA及其比值可以從微觀細胞水平上指示仔稚魚的生長。

      關鍵詞:牙鲆;變態(tài);核酸;RNA/DNA;Protein/DNA

      中圖分類號:S917.4 文獻標志碼:A 文章編號:1002-1302(2014)03-0171-03

      魚類在養(yǎng)殖過程中通常采用測定體長和體重的方式確定生長狀況。但早期發(fā)育階段,仔稚魚形體較小,精確測定其形態(tài)指標比較困難,降低了應用體長或體重來評價生長的可行

      性。有研究表明,生化指標能夠準確地檢測到魚類生長的細微變化和食物分布的波動[1]。因此,在通過傳統(tǒng)方法不能度量出魚類生長的變化時,采用有效的生化指標來評價魚類的生長是非常重要的。魚類的生長依賴于蛋白質的持續(xù)合成,RNA和DNA在仔魚生長和發(fā)育中也有重要作用。RNA參與合成蛋白質,控制著細胞和核糖體的體積,進一步影響細胞的生長率。DNA是生物的遺傳物質,DNA濃度高表明單位組織中細胞數目多,RNA/DNA是體內蛋白質合成的體現(xiàn)。根據RNA/DNA比值可以估算出魚類的生長速度[2-4]。 目前已在隆頭魚(Tautoga Onitis)[5]、黑線鱈(Melanogrammus aeglefinus)[6]、東方藍鰭鮪(Thunnus orientalis)[7]、草魚(Ctenopharyngodon idellus)[8]、紅鰭東方鲀(Takifugu rubripes)[9]等魚中進行了核酸指標與生長參數的研究,結果表明RNA/DNA比值是評價養(yǎng)殖魚類生長潛能的敏感參數。在魚類的體長、體重較難測量時,可以采用核酸指標來度量生長狀況。

      牙鲆在我國俗稱牙片、偏口,是名貴的海產魚類,又是重要的海水增養(yǎng)殖魚類之一,經濟價值較高。在早期發(fā)育階段,牙鲆仔魚要經歷變態(tài)過程,身體逐漸偏轉90°,導致腦顱及腦腔變形,同時生活方式也從浮游型轉變?yōu)榈讞穹蚚10]。變態(tài)期往往伴隨著營養(yǎng)危機和高死亡率,是鲆鰈魚類早期發(fā)育階段的關鍵期,并決定著年產量和經濟收益[11]。探究變態(tài)期仔稚魚的生長發(fā)育,了解魚苗的生理狀況,有助于優(yōu)化養(yǎng)殖管理,提高成活率。因此,本研究分析變態(tài)期間牙鲆仔稚魚DNA、RNA和總蛋白的變化規(guī)律,確定上述生化指標跟生長的數量關系,以期建立從微觀細胞水平上評價仔稚魚生理狀態(tài)的方法,為發(fā)育生理等方面的深入研究提供基礎資料。

      1 材料與方法

      1.1 樣品采集和保存

      試驗所用牙鲆魚苗取自江蘇省贛榆縣海頭鎮(zhèn)養(yǎng)魚場,培育時水溫16~19 °C,溶氧7.9~8.7 mg/L,鹽度3.1%~3.3%。分別在22、24、28、29、32、34、36日齡上午投餌前定點定時取樣,按照魚苗大小隨機取一定量的樣品用于測定RNA、DNA濃度及總蛋白濃度,樣品麻醉后快速保存于液氮中備用。在測定DNA、RNA濃度及總蛋白濃度前,于半解凍的狀態(tài)下用游標卡尺和電子天平測定體長(BL)、全長(TL)及體重(BW)。

      1.2 核酸和總蛋白的測定

      按照Buckley等[12]和Kuropat等[13]的方法,略作修改進行測定。采用整體勻漿法提取牙鲆仔稚魚的核酸和總蛋白,進一步用紫外分光光度法測定并計算RNA和DNA的含量及濃度,依據Bradford的方法[14]測定總蛋白含量及濃度。

      1.3 數據分析

      試驗數據均用“平均值±標準差”表示,采用SPSS 13.0軟件進行統(tǒng)計分析。

      2 結果與分析

      2.1 DNA、RNA及總蛋白的變化

      由圖1可知,DNA濃度在22~32日齡期間保持相對穩(wěn)定,32~34日齡時急劇升高,34~36日齡緩慢升高,在36日齡達到最高值。RNA濃度在24日齡升至峰值(2.34 μg/mg),然后逐漸下降,在32日齡降至最低值(1.15 μg/mg),之后呈先升高再下降的趨勢??偟鞍诐舛仍?2~29日齡間呈升高趨勢,之后呈先降低再上升的趨勢。

      由圖2可知,DNA、RNA含量及總蛋白含量在牙鲆變態(tài)期間均呈先升高后降低再升高的趨勢,在32~36日齡期間快速增長。

      2.2 Protein/DNA和RNA/DNA的變化

      由圖3可知,RNA/DNA比值在22~24日齡期間呈上升趨勢,之后保持下降趨勢直至32日齡,再呈先上升后下降的趨勢,在試驗結束時達到最低值(2.27)。Protein/DNA比值在22~29日期間呈上升趨勢,并在29日齡達到最高值(6197),之后呈先下降后上升趨勢。

      2.3 核酸、總蛋白濃度與牙鲆體長、體重的關系

      由表1、圖4、圖5可知,RNA、DNA濃度及總蛋白濃度與牙鲆體長和體重有明顯的線性關系;RNA/DNA和 Protein/DNA 與牙鲆體長和體重的線性關系弱于RNA、DNA濃度及總蛋白濃度。

      DNA含量是反映生物體內細胞數目的指標[18]。29日齡時DNA含量較低,這跟此時牙鲆的形體劇烈改變有關;之后DNA含量呈升高趨勢,可能是因為牙鲆生活方式由浮游狀態(tài)改變到底棲狀態(tài),此時剛經歷變態(tài)過程,生理狀況較差[19]。

      由DNA和RNA的變化曲線可知,牙鲆變態(tài)開始至變態(tài)高峰期的生長以細胞增大為主,變態(tài)高峰后的生長以細胞增殖為主。微觀生長方式的改變對應的是宏觀養(yǎng)殖環(huán)境中牙鲆仔稚魚的發(fā)育狀況和生活方式的變化。

      3.2 RNA/DNA

      RNA/DNA是生物體中細胞代謝強度的指示指標,可用來評價魚類的生理狀況[20]。本試驗中RNA/DNA比值隨日齡增長和魚體增大呈下降趨勢,在青魚和日本沙丁魚中也有類似的結果[21-22],推測可能是因為變態(tài)期RNA量呈下降趨勢、DNA量呈增長趨勢,變態(tài)后RNA的增長幅度弱于DNA的增長幅度,即細胞增殖的速度高于蛋白合成的速度,這跟該時期牙鲆稚魚生活方式由浮游轉為底棲相關聯(lián)。有研究表明,RNA/DNA比值還可以預測生物的營養(yǎng)狀況,該比值有個界限值2.49,低于該界限值的生物處于“亞健康”狀態(tài)或者饑餓狀態(tài)[23]。本研究中牙鲆稚魚在36日齡時RNA/DNA比值為2.27,表明該時期的牙鲆稚魚處于營養(yǎng)不良狀態(tài),推測可能是因為稚魚剛轉變?yōu)榈讞?,生活方式的劇烈改變會對其攝食產生一定障礙,進而影響到其營養(yǎng)狀況和生長。

      3.3 Protein/DNA

      蛋白質在細胞中占有很高的比例,因此Protein/DNA比值可作為指示細胞大小或者細胞重量的指標[18]。本研究中該比值在變態(tài)高峰期前保持升高趨勢,在29日齡時達到最高值,表明該時期魚體的生長以細胞增大為主。隨后稚魚轉入底棲生活,生活環(huán)境的急劇改變會對其攝食產生影響,此時魚體會大量消耗前期合成的蛋白質,導致變態(tài)后的Protein/DNA比值呈急劇下降趨勢,在條斑星鰈也發(fā)現(xiàn)了類似試驗結果[15]。待底棲環(huán)境適應后,稚魚逐漸減少對自身蛋白的利用率,加大對外源食物的攝入量,體內蛋白含量逐步升高,導致該比值在后期呈升高趨勢。

      參考文獻:

      [1]Buckley L J,Calderone E,Ong T L. RNA-DNA ratio and other nucleic acid-base indicators for growth and condition of marine fishes[J]. Hydrobiology,1999,401:265-277.

      [2]Gwak W S,Tanaka M. Developmental change in RNA:DNA ratios of fed and starved laboratory-reared Japanese flounder larvae and juveniles,and its application to assessment of nutritional condition for wild fish[J]. Journal of Fish Biology,2001,59(4):902-915.

      [3]Gwak W S,Tsusaki T,Tanaka M. Nutritional condition,as evaluated by RNA/DNA ratios,of hatchery-reared Japanese flounder from hatch to release[J]. Aquaculture,2003,219(4):503-514.

      [4]Tanaka Y,Satoh K,Yamada H,et al. Assessment of the nutritional status of field-caught larval Pacific bluefin tuna by RNA/DNA ratio based on a starvation experiment of hatchery-reared fish[J]. Journal of Experimental Marine Biology and Ecology,2008,354(1):56-64.

      [5]Mercaldo-Allen R,Kuropat C,Caldarone E M. A model to estimate growth in young-of-the-year tautog,Tautoga onitis,based on RNA/DNA ratio and seawater temperature[J]. Journal of Experimental Marine Biology and Ecology,2006,329(2):187-195.

      [6]Caldarone E M. Estimating growth in haddock larvae Melanogrammus aeglefinus from RNA:DNA ratios and water temperature[J]. Marine Ecology Progress Series,2005,293:241-252.

      [7]Tanaka Y,Gwak W S,Tanaka M,et al. Ontogenetic changes in RNA,DNA and protein contents of laboratory-reared Pacific bluefin tuna Thunnus orientalis[J]. Fisheries Science,2007,73:378-384.

      [8]羅 莉,文 華,王 琳,等. ?;撬釋Σ蒴~生長、品質、消化酶和代謝酶活性的影響[J]. 動物營養(yǎng)學報,2006,18(3):166-171.

      [9]梁萌青,王成剛,陳 超,等. 幾種添加劑對紅鰭東方鲀的促生長效果與RNA/DNA關系[J]. 海洋水產研究,2001,22(2):38-41.

      [10]鮑寶龍,楊桂梅,任大明. 牙鲆變態(tài)過程中的細胞凋亡[J]. 動物學報,2006,52(2):355- 361.

      [11]Amara R,Galois R. Nutritional condition of metamorphosing sole:spatial and temporal analyses[J]. Journal of Fish Biology,2004,64(1):72-88.

      [12]Buckley L J,Bulow F J. Techniques for the estimation of RNA,DNA,and protein in fish[M]//Summerfelt R C,Hall G E. The age and growth of fish. Ames,IA:Iowa State University Press,1987:345-354.

      [13]Kuropat C,Mercaldo-Allen R,Caldarone E,et al. Evaluation of RNA concentration as an indicator of growth in young-of-the-year winter flounder Pseudopleuronectes americanus and tautog Tautogaonitis[J]. Marine Ecology-Progress Series,2002,230:265-274.

      [14]Bradford M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.

      [15]佟雪紅,徐世宏,劉清華,等. 條斑星鰈變態(tài)期間DNA、RNA及總蛋白變化的研究[J]. 海洋科學,2010,34(5):41-48.

      [16]Tanaka M,Kawai S,Seikai T,et al. Development of the digestive organ system in Japanese flounder in relation to metamorphosis and settlement[J]. Marine and Freshwater Behaviour and Physiology,1996,28:19-31.

      [17]Peck M A,Buckley L J,Caldarone E M,et al. Effects of food consumption and temperature on growth rate and biochemical-based indicators of growth in early juvenile Atlantic cod Gadus morhua and haddock Melanogrammus aeglefinus[J]. Marine Ecology Progress Series,2003,251:233-243.

      [18]Park S U,Lim H K,Han H S. Changes in RNA/DNA ratio and growth of slime flounder,Microstomus achne,larvae until metamorphosis[J]. Journal of Applied Ichthyology,2008,24(1):50-54.

      [19]Malzahn A M,Clemmesen C,Rosenthal H. Temperature effects on growth and nucleic acids in laboratory-reared larval coregonid fish[J]. Marine Ecology Progress Series,2003,259:285-293.

      [20]Vinagre B C,F(xiàn)onseca V,Maia A,et al. Habitat specific growth rates and condition indices for the sympatric soles Solea solea(Linnaeus,1758)and Solea senegalensis Kaup 1858,in the Tagus estuary,Portugal,based on otolith daily increments and RNA-DNA ratio[J]. Journal of Applied Ichthyology,2008,24(2):163-169.

      [21]Clemmesen C. The effect of food availability,age or size on the RNA/DNA ratio of individually measured herring larvae:laboratory calibration[J]. Marine Biology,1994,118:377-382.

      [22]Kimura R,Watanabe Y,Zenitani H. Nutritional condition of first-feeding larvae of Japanese sardine in the coastal and oceanic waters along the Kuroshio Current[J]. Journal of Marine Sciences,2000,57(2):240-248.

      [23]Islam M S,Tanaka M. Nutritional condition,starvation status and growth of early juvenile Japanese sea bass (Lateolabrax japonicus) related to prey distribution and feeding in the nursery ground[J]. Journal of Experimental Marine Biology and Ecology,2005,323:172-183.

      [11]Amara R,Galois R. Nutritional condition of metamorphosing sole:spatial and temporal analyses[J]. Journal of Fish Biology,2004,64(1):72-88.

      [12]Buckley L J,Bulow F J. Techniques for the estimation of RNA,DNA,and protein in fish[M]//Summerfelt R C,Hall G E. The age and growth of fish. Ames,IA:Iowa State University Press,1987:345-354.

      [13]Kuropat C,Mercaldo-Allen R,Caldarone E,et al. Evaluation of RNA concentration as an indicator of growth in young-of-the-year winter flounder Pseudopleuronectes americanus and tautog Tautogaonitis[J]. Marine Ecology-Progress Series,2002,230:265-274.

      [14]Bradford M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.

      [15]佟雪紅,徐世宏,劉清華,等. 條斑星鰈變態(tài)期間DNA、RNA及總蛋白變化的研究[J]. 海洋科學,2010,34(5):41-48.

      [16]Tanaka M,Kawai S,Seikai T,et al. Development of the digestive organ system in Japanese flounder in relation to metamorphosis and settlement[J]. Marine and Freshwater Behaviour and Physiology,1996,28:19-31.

      [17]Peck M A,Buckley L J,Caldarone E M,et al. Effects of food consumption and temperature on growth rate and biochemical-based indicators of growth in early juvenile Atlantic cod Gadus morhua and haddock Melanogrammus aeglefinus[J]. Marine Ecology Progress Series,2003,251:233-243.

      [18]Park S U,Lim H K,Han H S. Changes in RNA/DNA ratio and growth of slime flounder,Microstomus achne,larvae until metamorphosis[J]. Journal of Applied Ichthyology,2008,24(1):50-54.

      [19]Malzahn A M,Clemmesen C,Rosenthal H. Temperature effects on growth and nucleic acids in laboratory-reared larval coregonid fish[J]. Marine Ecology Progress Series,2003,259:285-293.

      [20]Vinagre B C,F(xiàn)onseca V,Maia A,et al. Habitat specific growth rates and condition indices for the sympatric soles Solea solea(Linnaeus,1758)and Solea senegalensis Kaup 1858,in the Tagus estuary,Portugal,based on otolith daily increments and RNA-DNA ratio[J]. Journal of Applied Ichthyology,2008,24(2):163-169.

      [21]Clemmesen C. The effect of food availability,age or size on the RNA/DNA ratio of individually measured herring larvae:laboratory calibration[J]. Marine Biology,1994,118:377-382.

      [22]Kimura R,Watanabe Y,Zenitani H. Nutritional condition of first-feeding larvae of Japanese sardine in the coastal and oceanic waters along the Kuroshio Current[J]. Journal of Marine Sciences,2000,57(2):240-248.

      [23]Islam M S,Tanaka M. Nutritional condition,starvation status and growth of early juvenile Japanese sea bass (Lateolabrax japonicus) related to prey distribution and feeding in the nursery ground[J]. Journal of Experimental Marine Biology and Ecology,2005,323:172-183.

      [11]Amara R,Galois R. Nutritional condition of metamorphosing sole:spatial and temporal analyses[J]. Journal of Fish Biology,2004,64(1):72-88.

      [12]Buckley L J,Bulow F J. Techniques for the estimation of RNA,DNA,and protein in fish[M]//Summerfelt R C,Hall G E. The age and growth of fish. Ames,IA:Iowa State University Press,1987:345-354.

      [13]Kuropat C,Mercaldo-Allen R,Caldarone E,et al. Evaluation of RNA concentration as an indicator of growth in young-of-the-year winter flounder Pseudopleuronectes americanus and tautog Tautogaonitis[J]. Marine Ecology-Progress Series,2002,230:265-274.

      [14]Bradford M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.

      [15]佟雪紅,徐世宏,劉清華,等. 條斑星鰈變態(tài)期間DNA、RNA及總蛋白變化的研究[J]. 海洋科學,2010,34(5):41-48.

      [16]Tanaka M,Kawai S,Seikai T,et al. Development of the digestive organ system in Japanese flounder in relation to metamorphosis and settlement[J]. Marine and Freshwater Behaviour and Physiology,1996,28:19-31.

      [17]Peck M A,Buckley L J,Caldarone E M,et al. Effects of food consumption and temperature on growth rate and biochemical-based indicators of growth in early juvenile Atlantic cod Gadus morhua and haddock Melanogrammus aeglefinus[J]. Marine Ecology Progress Series,2003,251:233-243.

      [18]Park S U,Lim H K,Han H S. Changes in RNA/DNA ratio and growth of slime flounder,Microstomus achne,larvae until metamorphosis[J]. Journal of Applied Ichthyology,2008,24(1):50-54.

      [19]Malzahn A M,Clemmesen C,Rosenthal H. Temperature effects on growth and nucleic acids in laboratory-reared larval coregonid fish[J]. Marine Ecology Progress Series,2003,259:285-293.

      [20]Vinagre B C,F(xiàn)onseca V,Maia A,et al. Habitat specific growth rates and condition indices for the sympatric soles Solea solea(Linnaeus,1758)and Solea senegalensis Kaup 1858,in the Tagus estuary,Portugal,based on otolith daily increments and RNA-DNA ratio[J]. Journal of Applied Ichthyology,2008,24(2):163-169.

      [21]Clemmesen C. The effect of food availability,age or size on the RNA/DNA ratio of individually measured herring larvae:laboratory calibration[J]. Marine Biology,1994,118:377-382.

      [22]Kimura R,Watanabe Y,Zenitani H. Nutritional condition of first-feeding larvae of Japanese sardine in the coastal and oceanic waters along the Kuroshio Current[J]. Journal of Marine Sciences,2000,57(2):240-248.

      [23]Islam M S,Tanaka M. Nutritional condition,starvation status and growth of early juvenile Japanese sea bass (Lateolabrax japonicus) related to prey distribution and feeding in the nursery ground[J]. Journal of Experimental Marine Biology and Ecology,2005,323:172-183.

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