王青林,董云偉,董雙林**,王 芳,宋文華,于姍姍

(1.中國(guó)海洋大學(xué)海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室,山東青島266003;2.廈門大學(xué)海洋與環(huán)境學(xué)院,福建廈門361005)

高溫刺激對(duì)刺參浮游期幼體存活率及Hsp70表達(dá)量的影響*

王青林1,董云偉2,董雙林1**,王 芳1,宋文華1,于姍姍1

(1.中國(guó)海洋大學(xué)海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室,山東青島266003;2.廈門大學(xué)海洋與環(huán)境學(xué)院,福建廈門361005)

設(shè)計(jì)不同溫度(21.5,26,28和30℃),對(duì)刺參(A postichopus japonicus)囊胚期、原腸期和耳狀幼體期幼體進(jìn)行熱激處理,熱激45 min后,將幼體在21.5℃條件下繼續(xù)培養(yǎng)至耳狀幼體后期,計(jì)算刺參幼體的存活率,并用蛋白雜交方法測(cè)定幼體Hsp70的表達(dá)量。結(jié)果表明,高溫刺激對(duì)幼體的存活有顯著性影響,各時(shí)期幼體的存活率均隨熱激溫度的升高而降低,原腸期的存活率要高于囊胚期和耳狀幼體時(shí)期。原腸期幼體Hsp70表達(dá)量要比囊胚期和耳狀幼體時(shí)期高,這表明在發(fā)育過程中Hsp70表達(dá)量與幼體存活率具有相關(guān)性。

高溫刺激;刺參;幼體;熱休克蛋白;發(fā)育

刺參(A postichopus japonicus)是我國(guó)北方的一個(gè)重要養(yǎng)殖品種,市場(chǎng)需求日益增加。由于刺參對(duì)高溫的耐受性低,養(yǎng)殖區(qū)域和生長(zhǎng)時(shí)間受到了極大的限制。溫度刺激所誘導(dǎo)的保護(hù)作用在海洋生物中已有研究[1-2]。張其中等[3]發(fā)現(xiàn)近江牡蠣預(yù)先經(jīng)受亞致死溫度熱休克以后,顯著提高了對(duì)高溫的耐受能力,這種保護(hù)作用與生物體內(nèi)熱休克蛋白的表達(dá)具有重要關(guān)系[4]。

熱休克蛋白(Heat shock proteins,HSPs)是1種高度保守的蛋白質(zhì),具有防止蛋白變性,使變性蛋白復(fù)性的重要功能,其中分子量為66～78 kDa的熱應(yīng)激蛋白70(Hsp70)家族由于其保守性、重要性和在應(yīng)激反應(yīng)中的敏感性,受到廣泛重視[5]。有研究表明少量誘導(dǎo)型熱休克蛋白的表達(dá)能顯著地影響生物的生長(zhǎng)、發(fā)育、熱耐受性、壽命以及繁殖[6-11]。目前,對(duì)于刺參的研究已經(jīng)證實(shí)Hsp70在抵抗高溫和滲透壓脅迫中具有重要作用[4,12-14]。同時(shí)HSPs的累積與機(jī)體耐受性的提高有一定的相關(guān)性[15]。本實(shí)驗(yàn)通過檢測(cè)不同熱激條件下刺參浮游期幼體存活率和Hsps的含量,探討Hsp70表達(dá)與幼體存活率之間的關(guān)系,分析不同發(fā)育時(shí)期刺參幼體對(duì)溫度應(yīng)激的敏感性,豐富刺參溫度生理生態(tài)學(xué)研究,并為耐高溫品系篩選提供一定的理論指導(dǎo)。

1 材料和方法

1.1 材料來源

實(shí)驗(yàn)所需幼體來自山東省膠南市育苗場(chǎng)的親參,干法運(yùn)至實(shí)驗(yàn)室后,在18℃暫養(yǎng)5 d,人工誘導(dǎo)產(chǎn)卵并培育而得。卵子和精子在21.5℃水溫下孵化,受精卵的密度控制在10～20 inds/mL,在原腸期幼蟲后期投喂牟氏角毛藻(Chaetoceros muelleri)、杜氏鹽藻(Dunaliella salina)和海洋紅酵母的混合液,投喂量依據(jù)刺參的不同發(fā)育時(shí)期、幼體的攝食活力以及水體中剩余的餌料數(shù)量而做適當(dāng)?shù)恼{(diào)整。整個(gè)苗種培育期間不間斷充氣,光照強(qiáng)度控制在500～1500 lux,光照周期為14 L∶10 D。水源為沙濾自然海水。每天換水1/2,海水鹽度為30左右,換水前對(duì)海水進(jìn)行預(yù)加熱,防止換水造成水族箱溫度變化過大。

1.2 實(shí)驗(yàn)設(shè)計(jì)

1.2.1 高溫刺激對(duì)刺參浮游期幼蟲存活率的影響

在囊胚期、原腸期以及耳狀幼體初期,將幼體分別轉(zhuǎn)移到26,28和30℃進(jìn)行高溫應(yīng)激處理,持續(xù)時(shí)間為45 min,然后再轉(zhuǎn)移到21.5℃繼續(xù)培育至耳狀幼體后期,每個(gè)處理3個(gè)重復(fù),21.5℃培育下的不進(jìn)行高溫應(yīng)激處理的幼體作為對(duì)照。分別于實(shí)驗(yàn)開始前及耳狀幼體后期記錄水體中幼體的數(shù)量。

1.2.2 不同發(fā)育時(shí)期刺參幼體Hsp70含量變化 在受精卵、囊胚期、原腸期以及耳狀幼體初期,將幼體分別轉(zhuǎn)移到不同溫度(26,28和30℃)進(jìn)行熱激處理,持續(xù)時(shí)間為45 min,然后再轉(zhuǎn)移到21.5℃繼續(xù)培育,2 h后取大約8 000個(gè)幼體液氮保存,同時(shí)取未高溫應(yīng)激的幼體作為對(duì)照,每個(gè)處理3個(gè)重復(fù)。

1.3 樣品處理

在預(yù)冷的研缽內(nèi)加入液氮低溫研磨刺參幼體,然后加入適量含有蛋白酶抑制劑的細(xì)胞裂解液在冰浴中勻漿。勻漿完畢后4℃下10 000 g 15 min,取上清液保存在-70℃超低溫冰箱中。

1.4 蛋白雜交

以牛血清蛋白為標(biāo)準(zhǔn)方法參照Lowry等[16]測(cè)定樣品中蛋白含量。Hsp70和Hsp72含量的測(cè)量方法參照Dong等[17]。蛋白的凝膠電泳選用10%的聚丙烯酰胺凝膠電泳[18]。取40μg蛋白加適量體積上樣緩沖液(含DTT)煮沸5 min,進(jìn)行電泳,然后參照Kyhse-Andersen[19]的方法將聚丙烯酰胺凝膠中的蛋白質(zhì)轉(zhuǎn)移到0.45μm的PVDF膜上利用脫脂奶粉封閉后,用單克隆抗Hsp70抗體,(H5147,Sigma,USA),在37℃孵育2 h(稀釋1∶5000)。然后將膜在抗小鼠IgG(HRP)(ZB2305,中杉金橋,北京,中國(guó))抗體中孵育1.5 h,采用ECL試劑(Amersham,USA)顯色,曝光,條帶強(qiáng)度采用Image J軟件進(jìn)行定量分析。

本實(shí)驗(yàn)標(biāo)準(zhǔn)值采用30℃條件下熱激的受精卵樣品,實(shí)驗(yàn)結(jié)果中的Hsp水平均為相對(duì)于標(biāo)準(zhǔn)樣品水平的值(RU)。

1.5 計(jì)算與數(shù)據(jù)分析

實(shí)驗(yàn)期間幼體存活率(SR)采用以下公式計(jì)算:SR=N2/N1×100%,N1,N2分別為初始時(shí)期和耳狀幼體后期幼體數(shù)量。

數(shù)據(jù)統(tǒng)計(jì)采用SPSS13.0進(jìn)行,不同熱激溫度和不同發(fā)育時(shí)期的差異采用雙因子方差分析(Two-way ANOVA),以P<0.05作為差異顯著的標(biāo)準(zhǔn)。

2 結(jié)果

2.1 高溫應(yīng)激對(duì)刺參浮游期幼蟲存活率的影響

在幼體生長(zhǎng)的最適溫度下(21.5℃),不同發(fā)育時(shí)期(囊胚期BLA、原腸期GAS以及耳狀幼體期AUR)幼體的存活率不同(見圖1)。其中囊胚期的存活率最高,為81.96%,顯著高于耳狀幼體期的70.0%(P<0.05),和原腸期的存活率(79.71%)沒有顯著差異。

雙因子方差分析結(jié)果表明,熱激溫度和發(fā)育時(shí)期對(duì)刺參幼體存活率差值影響顯著(見表1),溫度和發(fā)育時(shí)期具有顯著交互作用(P<0.001)。隨著熱激溫度的升高,不同浮游期的刺參幼體的存活率呈下降的趨勢(shì),其中熱激組與對(duì)照組幼體的存活率差值呈下降趨勢(shì)(見圖2)(BLA-26,-21.52%;BLA-28,-34.52%;BLA-30,-49.06%;GAS-26,-10.95%;GAS-28,-15.22%;GAS-30,-28.51%;AUR-26,-17.04%;AUR-28,-35.37%;AUR-30,-36.94%)。在囊胚期和原腸期,不同溫度處理組之間差異顯著(P<0.05)。在耳狀幼體時(shí)期,26℃熱激組幼體的存活率顯著高于其它2組,其它2組差異不明顯。

表1 不同溫度熱激對(duì)刺參不同浮游期幼體存活率影響的方差分析Table 1 Two-way ANOVO analysis of the effect of heat-shock temperature on survival rate ofA postichopus japonicuslarvae in different developmental stages

圖1 不同浮游時(shí)期刺參存活率Fig.1 Survival rate of larvae at the stage of blastula,gastrula and auricularia inA postichopus japonicus

圖2 熱激對(duì)刺參浮游期幼體存活率的影響Fig.2 Survival rate of larvae stressed at the stage of blastula,gastrula and auricularia of sea cucumber A postichopus japonicus

2.2 刺參浮游幼體Hsp70的表達(dá)

2.2.1 熱激溫度對(duì)不同發(fā)育時(shí)期刺參幼體Hsp70表達(dá)量的影響 熱激溫度對(duì)不同發(fā)育時(shí)期刺參幼體Hsp70表達(dá)量的影響不同(見圖3)。受精卵時(shí)期,隨著熱激溫度的升高Hsp70的表達(dá)量增加,但是30℃時(shí),表達(dá)量明顯低于其它3組(P<0.05)。囊胚時(shí)期,對(duì)照組有少量Hsp70表達(dá),但是熱激組沒有檢測(cè)到有Hsp70表達(dá)(RU=0)。隨著熱激溫度的升高,原腸期幼體Hsp70的表達(dá)量呈下降的趨勢(shì),并且各處理組之間差異顯著(P<0.05)。耳狀幼體時(shí)期,沒有檢測(cè)到Hsp70的表達(dá)。

圖3 熱激溫度對(duì)不同發(fā)育時(shí)期刺參幼體Hsp70表達(dá)量的影響Fig.3 Expression of Hsp70 of larvae stressed at pelagic stages of sea cucumberA postichopus japonicus

圖4 熱激溫度對(duì)不同發(fā)育時(shí)期刺參幼體Hsp72表達(dá)量的影響Fig.4 Expression of Hsp72 of larvae stressed at pelagic stages

2.2.2 熱激溫度對(duì)不同發(fā)育時(shí)期刺參幼體Hsp72表達(dá)量的影響 熱激溫度對(duì)不同發(fā)育時(shí)期刺參幼體Hsp72表達(dá)的影響不同(見圖4)。受精卵時(shí)期,Hsp72表達(dá)量隨著溫度的升高而增加,30℃條件下的表達(dá)量最高,顯著高于其它3組(P<0.05)。28℃條件下的表達(dá)量顯著高于21.5℃和26℃條件下的表達(dá)量,21.5℃和26℃條件下的表達(dá)量沒有顯著差異。囊胚時(shí)期,對(duì)照組有少量Hsp72表達(dá),但是熱激組沒有檢測(cè)到表達(dá)量。原腸時(shí)期,對(duì)照組有很高的Hsp72表達(dá)量,并且顯著高于處理組(P<0.05),從26℃升高到28℃時(shí),Hsp72的表達(dá)量隨之增加,且2組之間的差異顯著(P<0.05)。但是熱激溫度從28℃升高到30℃時(shí),Hsp72的表達(dá)量呈下降的趨勢(shì),但差異不顯著。耳狀幼體時(shí)期,檢測(cè)不到Hsp72的表達(dá)。

3 討論

3.1 熱激對(duì)刺參浮游期幼蟲存活率的影響

水溫是影響水生變溫動(dòng)物生長(zhǎng)和其他生理過程的重要生態(tài)因子之一。有研究表明刺參幼體發(fā)育的最適溫度為20～25℃[20-22],因此本實(shí)驗(yàn)選擇21.5℃條件下的幼體發(fā)育為對(duì)照。溫度能顯著影響到刺參幼體的存活率[23]。本實(shí)驗(yàn)結(jié)果表明,在浮游幼體時(shí)期對(duì)刺參進(jìn)行高溫應(yīng)激使得幼體存活率顯著下降,存活率隨著熱激溫度的升高而降低(見圖2),且不同發(fā)育時(shí)期的刺參幼體對(duì)熱激的敏感性不同。Chen[24]發(fā)現(xiàn)耳狀幼體對(duì)環(huán)境的改變非常敏感,在此時(shí)期經(jīng)常會(huì)出現(xiàn)大規(guī)模的幼體死亡。本實(shí)驗(yàn)的結(jié)果表明,相對(duì)囊胚期和耳狀幼體時(shí)期的幼體,原腸期幼體在高溫下存活率要比其它2個(gè)時(shí)期高,說明原腸期對(duì)高溫的耐受性較強(qiáng),而囊胚期和耳狀幼體時(shí)期的幼體對(duì)熱激較為敏感,這2個(gè)時(shí)期應(yīng)該是篩選耐高溫刺參的合適時(shí)期。劉廣斌[25]研究發(fā)現(xiàn),養(yǎng)殖環(huán)境的高溫條件可以影響刺參對(duì)溫度的耐受能力,并且這種能力是可以遺傳的。故在囊胚期或者耳狀幼體時(shí)期對(duì)幼體進(jìn)行熱激,是培育耐高溫的刺參品系的一種途徑。

3.2 刺參浮游幼體Hsp的表達(dá)

本實(shí)驗(yàn)表明,刺參受精卵、囊胚期以及原腸期都能檢測(cè)到熱休克蛋白表達(dá),但是Hsp70具有2種單倍型,2種單倍型的表達(dá)模式有明顯差別(見圖3,4)。

就Hsp70的表達(dá)來說,受精卵時(shí)期,隨著熱激溫度的升高,Hsp70的表達(dá)量隨之增加,這表明當(dāng)溫度超過26℃時(shí),刺參幼體會(huì)適應(yīng)性的誘導(dǎo)產(chǎn)生熱休克蛋白。但30℃條件下的Hsp70表達(dá)急劇降低,且顯著低于其它3組(P<0.05),表明30℃已經(jīng)超過幼體誘導(dǎo)產(chǎn)生熱休克蛋白的上限。囊胚期,對(duì)照組有少量的Hsp70表達(dá),但是當(dāng)熱激溫度超過最適溫度時(shí),其表達(dá)量為零。原腸期,Hsp70的表達(dá)量隨著熱激溫度的升高而顯著下降(P<0.05)。在30℃下,各時(shí)期的幼體Hsp70表達(dá)量均有不同程度的降低,其表達(dá)量的下調(diào)與高溫下機(jī)體整體蛋白合成降低有關(guān)[26]。如果過度刺激就會(huì)造成受刺激者整體處于不適狀態(tài),從而無法及時(shí)合成足夠熱休克蛋白,以對(duì)付有害刺激的傷害,嚴(yán)重時(shí)會(huì)導(dǎo)致死亡。Tedengren等[27]發(fā)現(xiàn)過度的熱和鎘離子濃度聯(lián)合處理紫貽貝造成“生理適應(yīng)度”降低,從而降低了Hsp70的表達(dá)水平。

受精卵期Hsp72表達(dá)量隨著熱激溫度的升高,其表達(dá)量隨之升高,此種蛋白表達(dá)量的上調(diào)表明熱休克蛋白的表達(dá)受溫度的誘導(dǎo),Hsp70的表達(dá)與溫度具有相關(guān)性[28]。當(dāng)溫度超過生物體的最適應(yīng)溫度時(shí),某些蛋白喪失結(jié)構(gòu)和活性,導(dǎo)致Hsp表達(dá)來重折疊變性蛋白和阻止更多的聚集[5]。囊胚期,Hsp72表達(dá)量趨勢(shì)與Hsp70相同。原腸期,對(duì)照組表達(dá)量顯著高于處理組(P<0.05),處理組表達(dá)量隨著熱激溫度的升高呈先升后降趨勢(shì)。

總體來說,不同時(shí)期2種單倍型熱休克蛋白(Hsp70和Hsp72)的表達(dá)具有共同趨勢(shì):原腸期的表達(dá)量要高于囊胚期和耳狀幼體期,而原腸期的存活率也高于囊胚期和耳狀幼體期,表明兩者具有一定的相關(guān)性。Dong等[29]通過研究位于高低潮區(qū)域的4種帽貝發(fā)現(xiàn),高潮線附近的種類具有較高的Hsp70表達(dá)量。表明熱休克蛋白在提高機(jī)體耐受性方面起著重要的作用。Hsp70可以增強(qiáng)機(jī)體的應(yīng)激耐受性,提高細(xì)胞生存率。基本的細(xì)胞程序如轉(zhuǎn)錄、翻譯、轉(zhuǎn)運(yùn)經(jīng)過輕微熱預(yù)處理后,在隨后的應(yīng)激過程中可以得到保護(hù)。這樣不僅能使細(xì)胞的耐受性增加,而且能使細(xì)胞對(duì)其它應(yīng)激原的耐受性也增加[30]。耳狀幼體期沒有Hsp的表達(dá),可能是表達(dá)量太少,蛋白免疫印記無法檢測(cè),有研究表明Hsp在棘皮動(dòng)物海膽胚胎時(shí)期的表達(dá)時(shí)期以及表達(dá)量因種而異[31-38],其具體原因有待進(jìn)一步研究。

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Effects of Heat-Shock in Pelagic Stages on Survival and Hsp70 Expression of Juvenile Sea Cucumber,Apostichopus japonicus Selenka

WANG Qing-Lin1,DONG Yun-Wei2,DONG Shuang-Lin1,WANG Fang1,SONG Wen-Hua1,YU Shan-Shan1
(1.The Key Laboratory of Mariculture,Ministry of Education,College of Fisheries,Ocean University of China,Qingdao 266003,China;2.College of Oceanography,Xiamen University,Xiamen 361005,China)

The effects of heat-shock in pelagic stages on survival and Hsp70s expression of juvenile sea cucumber,A postichopus japonicusSelenka were investigated.Larvae at the stage of blastula,gastrula and auricularia were heat-shocked at selected temperatures(21.5,26,28 and 30℃)for 45 min and returned to 21.5℃for continuous rearing.After the heat-shock,there were significant differences in survival among larvae in the four heat-shocked treatments.The survival rate of larvae decreased with the increasing of heat-shock temperature,and the rate was higher in the stage of gastrula than those in blastula and auricularia.The Hsp70s expression of larvae in the stage of gastrula was higher than those in the stages of blastula and auricularia,suggesting that the survival of larvae partly correlates with the expression of Hsp70s.

heat-shock;sea cucumber;larvae;heat shock protein;development

S917.4

A

1672-5174(2011)03-047-05

國(guó)家科技支撐計(jì)劃項(xiàng)目(2006BAD09A01);山東省優(yōu)秀中青年科學(xué)家科研獎(jiǎng)勵(lì)基金計(jì)劃項(xiàng)目(BS2009NY019)資助

2010-05-28;

2010-11-03

王青林(1984-),男,博士生。E-mail:wangqinglin2009@gmail.com

**通訊作者:E-mail:dongsl@ouc.edu.cn

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