有害甲藻孢囊的分類鑒定研究進(jìn)展

高養(yǎng)春， 董燕紅， 李海濤， 戰(zhàn)愛斌*

1中國科學(xué)院生態(tài)環(huán)境研究中心,北京 100085；2國家海洋局南海環(huán)境監(jiān)測中心,廣東 廣州 510300

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有害甲藻孢囊的分類鑒定研究進(jìn)展

高養(yǎng)春1， 董燕紅2， 李海濤2， 戰(zhàn)愛斌1*

1中國科學(xué)院生態(tài)環(huán)境研究中心,北京 100085；2國家海洋局南海環(huán)境監(jiān)測中心,廣東 廣州 510300

有害甲藻孢囊主要是指能產(chǎn)生毒素和(或)能引起有害藻華發(fā)生并對水生態(tài)系統(tǒng)產(chǎn)生各種危害效應(yīng)的甲藻孢囊。我國沿海共記錄了10屬18種，占全球有害甲藻孢囊的3/4。這些有害甲藻孢囊廣泛分布于我國沿海，會對水產(chǎn)養(yǎng)殖業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失，甚至?xí){人類的身體健康。因此，有害甲藻孢囊的多樣性及分布越來越受到人們的關(guān)注。對有害甲藻孢囊的準(zhǔn)確判斷不僅對研究其多樣性及分布至關(guān)重要，而且有助于水產(chǎn)品的安全檢驗和有害藻華的早期預(yù)警。對有害甲藻孢囊的分類主要存在鑒定困難、鑒定不準(zhǔn)確等問題。本文綜述了有害甲藻孢囊的危害、中國沿海有害甲藻孢囊的種類和分布,以及有害甲藻孢囊的鑒定等3個方面的研究進(jìn)展，并提出利用孢囊及營養(yǎng)細(xì)胞的形態(tài)學(xué)特征、分子生物學(xué)、毒理學(xué)等多學(xué)科研究手段準(zhǔn)確鑒定有害甲藻孢囊的建議。

有害甲藻孢囊； 形態(tài)學(xué)特征； 分子鑒定

甲藻孢囊是指甲藻為度過不良環(huán)境而產(chǎn)生的一種無鞭毛且無游動能力的細(xì)胞，是甲藻生活史的一個重要階段(王朝暉,2007)。其中，能產(chǎn)生毒素和(或)引起有害藻華(harmful algal bloom)的甲藻孢囊被稱為有害甲藻孢囊。有害甲藻孢囊可分為含毒素和不含毒素2類。含毒素種類不僅在低濃度時就會引起水產(chǎn)品染毒甚至死亡，而且會導(dǎo)致有害藻華的暴發(fā)(Burkholderetal.,1992)；不含毒素的種類雖然不會引起水產(chǎn)品的毒化，但大量萌發(fā)后能引起有害藻華的發(fā)生。有害藻華的發(fā)生會對人類健康、生態(tài)系統(tǒng)及水產(chǎn)養(yǎng)殖業(yè)構(gòu)成巨大威脅(Andersonetal.,2012b)。隨著沿海海域污染、外來種引入、全球變暖等影響的加劇，有害藻華發(fā)生的頻率不斷增大(Andersonetal.,2012a; Nakanishietal.,1996)。因分布于海底沉積物中的有害甲藻孢囊很有可能成為有害藻華發(fā)生的種源，有害甲藻孢囊的地理分布及多樣性和生態(tài)特性也越來越受到關(guān)注(Kohlietal.,2014； Zonneveld & Dale,1994)。

甲藻孢囊形體普遍微小(20～120 μm)，部分種間差異較小及同種存在形態(tài)可塑性等特點(diǎn)(Fuentes-Grünewaldetal.,2009、2012; Geider & Roche,2002; Taylor & Gaines,1989)，導(dǎo)致對其鑒定困難及鑒定不準(zhǔn)確等問題。雖然顧海峰等(2011)、黃海燕和陸斗定(2009)已對甲藻孢囊的研究進(jìn)行了綜述，但均缺乏針對有害甲藻孢囊種類及分子鑒定方面的論述。本文擬綜述有害甲藻孢囊的危害、中國沿海有害甲藻孢囊的種類及分布、有害甲藻孢囊的形態(tài)學(xué)和分子生物學(xué)鑒定等3個方面的研究進(jìn)展，以期為有害甲藻孢囊的監(jiān)測和控制提供參考。

1 有害甲藻孢囊的危害

筆者統(tǒng)計了全球范圍內(nèi)有害甲藻孢囊的種類，主要有22種(表1)。其中，19種含有甲藻毒素，3種不含毒素但能引起有害藻華的發(fā)生，而既含有毒素又能引起有害藻華發(fā)生的有13種。這些甲藻毒素是底棲生物體內(nèi)毒素積累的重要來源之一(Schwinghameretal.,1994)。其被濾食性的魚、蝦、貝類濾食后，在這些海產(chǎn)品體內(nèi)積累并導(dǎo)致海產(chǎn)品染毒，染毒的海產(chǎn)品通過食物鏈的傳遞最終導(dǎo)致人的中毒(丁德文等,2005; Yasumoto & Murata,1993)。根據(jù)食用者中毒的癥狀，這些甲藻(產(chǎn)孢囊)毒素被分為3類：腹瀉性貝類毒素(diarrhetic shellfish poisoning,DSP)、麻痹性貝類毒素(paralytic shellfish poisoning，PSP)、神經(jīng)性貝類毒素(neurotoxic shellfish poisoning，NSP)。除此之外，還有一些對人類危害尚不清楚的毒素，如扇貝毒素(yessotoxin，YTX)(Auneetal.,2002)。這些毒素(DSP、PSP、NSP、YTX)主要由甲藻門中的膝溝藻類產(chǎn)生，少數(shù)由裸甲藻類、多溝藻類產(chǎn)生(表1)。有些甲藻(產(chǎn)孢囊)毒素(如屬于PSP的石房蛤毒素)的毒性是眼鏡蛇毒性的80倍，產(chǎn)毒甲藻的營養(yǎng)細(xì)胞具有毒素，其孢囊也具有毒素(Andersonetal.,1990; Bravo,1998)，并且其毒性比營養(yǎng)細(xì)胞更高(Dale,1983; Oshimaetal.,1992)。僅在1969年至1994年，我國因食用染毒的魚、貝類而中毒的人數(shù)就有1800多人，其中至少30人死亡(周名江等,2001; Zhouetal.,1999)。這些毒素還能引起魚、貝類大面積死亡，給水產(chǎn)養(yǎng)殖業(yè)造成重大經(jīng)濟(jì)損失(龍華等,2008; Lim,2012)。此外，有害甲藻孢囊能幫助甲藻度過不良環(huán)境，通過洋流或船舶等媒介擴(kuò)散到其他海域，極易使其成為入侵物種而對當(dāng)?shù)厮虻钠渌锛吧鷳B(tài)環(huán)境產(chǎn)生重大的危害，如自從亞歷山大藻孢囊入侵美國緬因州西部海域引起赤潮后，此海域幾乎每年都會暴發(fā)產(chǎn)PSP毒素的赤潮(Anderson & Wall,1978)。

2 中國沿海有害甲藻孢囊的種類及分布

目前，我國發(fā)現(xiàn)的有害甲藻孢囊共記錄了10屬18種，占全球總有害甲藻孢囊種類的3/4(表1)，其在我國沿海的分布見圖1。其中，原甲藻屬Prorocentrum1種，亞歷山大藻屬Alexandrium共6種，膝溝藻屬Gonyaulax共2種，梨甲藻屬Pyrodinium1種，舌甲藻屬Lingulodinium1種，原角管藻屬Protoceratium1種，斯氏藻屬Scrippsiella1種，裸甲藻屬Gymnodinium1種，多溝藻屬Polykrikos共2種，褐多溝藻屬Pheopolykrikos1種，旋溝藻屬Cochlodinium1種。廣泛分布于我國沿海的有塔瑪亞歷山大藻Alexandriumtamarense、鏈狀亞歷山大藻A.pacificum、具刺膝溝藻Gonyaulaxspinifera以及錐狀斯氏藻Scrippsiellatrochoidea。

3 有害甲藻孢囊的分類鑒定

3.1 形態(tài)學(xué)鑒定

由于大多數(shù)甲藻孢囊微小，需要借助于光學(xué)或電子顯微鏡才能對其進(jìn)行分類鑒定。傳統(tǒng)的形態(tài)學(xué)鑒定方法主要根據(jù)孢囊的形態(tài)、大小、顏色、孢囊內(nèi)含物及孢囊壁的結(jié)構(gòu)和表面修飾物等特征對孢囊進(jìn)行鑒定(王朝暉,2007; 王朝暉等,2011; 魏洪祥等,2011; Liuetal.,2014; Matsuoka & Fukuyo,2000; Mertensetal.,2015)。表1列出的有害甲藻孢囊中，有些具有易于分辨的形態(tài)學(xué)特征，如多邊舌甲藻Lingulodiniumpolyedrum具有舌狀凸起(圖2A)，網(wǎng)狀原角管藻Protoceratiumreticulatum具有T形凸起(圖2B)(Joyceetal.,2005)，科夫多溝藻Polykrikoskofoidii具有網(wǎng)紋狀凸起(圖2C)，無紋多溝藻Polykrikosschwartzii的外部形態(tài)與科夫多溝藻類似，但凸起的頂端相互獨(dú)立而未連接在一起(圖2D)等，可根據(jù)此特有的形態(tài)學(xué)特征對這些孢囊進(jìn)行相對準(zhǔn)確的鑒定。

表1 主要有害甲藻孢囊種類及其危害

*為中國沿海發(fā)現(xiàn)的有害甲藻孢囊種。

*indicate dinoflagellate cyst found in China.

但對于有些有害甲藻孢囊，如亞歷山大藻屬中的塔瑪亞歷山大藻復(fù)合體(圖2E)、微小亞歷山大藻A.minutum與相似亞歷山大藻A.affine(圖2F)等各個種的形態(tài)差異較小(黃海燕等,2009; 王朝暉等,2004; 魏洪祥等,2011; Bravoetal.,2006)，僅僅根據(jù)光學(xué)顯微鏡下孢囊的形態(tài)學(xué)特征難以將這些孢囊鑒定到種的水平，一般在光鏡或電鏡下觀察孢囊萌發(fā)時營養(yǎng)細(xì)胞的形態(tài)特征可間接鑒定孢囊(顧海峰等,2003; Gu,2011; Guetal.,2013a)。對于營養(yǎng)細(xì)胞形態(tài)差異較小的種類，此方法也不能將其準(zhǔn)確鑒定，如根據(jù)塔瑪亞歷山大藻復(fù)合體的形態(tài)學(xué)特征僅能將其鑒定為由塔瑪亞歷山大藻、鏈狀亞歷山大藻及A.fundyense3種藻類組成(Balech,1995)；但后來的研究發(fā)現(xiàn)其存在許多中間形態(tài)的個體以及不同種之間存在形態(tài)特征重疊的現(xiàn)象，使得學(xué)者們對塔瑪亞歷山大藻復(fù)合體分為3個種的觀點(diǎn)產(chǎn)生了較大的質(zhì)疑(Gayoso & Fulco,2006; Kimetal.,2002; Orlovaetal.,2007)?；谛螒B(tài)學(xué)特征對A.ostenfeldii與A.peruvianum(Gu,2011; Krempetal.,2014)、Pyrodiniumbahamensevar.compressum與Pyrodiniumbahamensevar.bahamense(Balech,1985; Mertensetal.,2015; Steidingeretal.,1980)的研究也發(fā)現(xiàn)了類似的現(xiàn)象；再如，塔瑪亞歷山大藻復(fù)合體group Ⅰ(含有毒素)中的個體均存在側(cè)腹孔，而group Ⅳ(未能檢測到毒素)的個體中有些存在側(cè)腹孔而有些不存在，因此，僅根據(jù)側(cè)腹孔的有無并不能將塔瑪亞歷山大藻復(fù)合體中的group Ⅰ和group Ⅳ區(qū)分開(Guetal.,2013a)。

此外，該方法不能鑒定不具有萌發(fā)能力以及現(xiàn)有試驗條件下尚不能萌發(fā)的孢囊。雖然孢囊萌發(fā)培養(yǎng)技術(shù)在不斷改善，但孢囊的萌發(fā)率仍不能達(dá)到100%(Andersonetal.,2005; Guetal.,2013b)，因此，仍有部分尚未萌發(fā)的孢囊不能通過孢囊萌發(fā)試驗得到鑒定。雖然孢囊的萌發(fā)孔及副殼板結(jié)構(gòu)也可用于不同種孢囊的鑒定(Yamaguchietal.,1995)，尤其用于原多甲藻中Brigantedinium的鑒定，但只能鑒定已經(jīng)萌發(fā)的孢囊，而對于未萌發(fā)的Brigantedinium僅根據(jù)孢囊形態(tài)學(xué)特征仍不能將其鑒定到種的水平。有些孢囊如具刺膝溝藻孢囊具有較強(qiáng)的形態(tài)可塑性(圖2G、H)，據(jù)報道，產(chǎn)毒的具刺膝溝藻至少有19種不同類型的孢囊(Taylor & Gaines,1989)，僅僅根據(jù)孢囊形態(tài)學(xué)特征難以將具有不同形態(tài)學(xué)特征的同種孢囊劃分到同一個種。因此，許多基于孢囊形態(tài)學(xué)的研究只能鑒定到屬或?qū)僖陨系乃?王朝暉,2007; 王朝暉等,2011； 魏洪祥等,2011)。

圖2 部分有害甲藻孢囊

除以上孢囊所固有的生物特性外，環(huán)境因素也可能間接影響孢囊鑒定的準(zhǔn)確率。一些孢囊在不同的環(huán)境中以及不同的生長階段具有不同的外部形態(tài)特征，如多邊舌甲藻(Mertens,2013; Mertensetal.,2009)、網(wǎng)狀原角管藻(Mertensetal.,2011)、巴哈馬梨甲藻Pyrodiniumbahamense(Mertensetal.,2015)等孢囊刺的長度及大小與孢囊所處海域的鹽度與溫度相關(guān)；多邊舌甲藻、巴哈馬梨甲藻等孢囊的大小還與水體的營養(yǎng)成分及水流等相關(guān)(Mertens,2013; Mertensetal.,2015)。這些環(huán)境因素對孢囊形態(tài)所產(chǎn)生的影響會降低孢囊鑒定的準(zhǔn)確率(Fuentes-Grünewaldetal.,2009、2012)。

常規(guī)形態(tài)學(xué)鑒定具有費(fèi)時費(fèi)力的缺點(diǎn)，對鑒定人員的要求也較高(Godheetal.,2001)，且分辨率較低。因此，需要尋找一種省時省力又準(zhǔn)確的孢囊鑒定方法。分子生物學(xué)方法在解決這個問題方面展現(xiàn)出了巨大的優(yōu)勢：(1)試驗所需的時間較短；(2)沒有形態(tài)學(xué)鑒定基礎(chǔ)的實驗者也可進(jìn)行操作；(3)在種或?qū)偎缴系蔫b定具有特異性。

3.2 分子生物學(xué)鑒定

一般情況下，不同物種之間的差異主要取決于相應(yīng)遺傳物質(zhì)基因組中堿基序列的差異。因此，理論上認(rèn)為直接對基因組堿基序列測序是最準(zhǔn)確且可靠的物種鑒定方法。但由于基因組較大，不易于測序且代價較高而限制了其實際應(yīng)用。DNA條形碼(DNA barcode)是生物體內(nèi)能夠代表該物種且與其他物種區(qū)分的DNA片段，其一般較短且易測序，根據(jù)DNA條形碼的序列特征即可鑒定物種。此技術(shù)已廣泛應(yīng)用于動物(Shokralla,2011; Zhanetal.,2013; Zhan & MacIsaac,2015)、植物(Cowan & Michael,2012)、微生物(Adeduntan,2009)等物種的鑒定，在形態(tài)鑒定存在困難的物種上的應(yīng)用更加廣泛。如Lillyetal.(2007)根據(jù)當(dāng)時的分子生物學(xué)數(shù)據(jù)將塔瑪亞歷山大藻復(fù)合體劃分為5個類群(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ)，但未劃分到具體的種。近年，相關(guān)學(xué)者才根據(jù)核酸序列并輔以毒理學(xué)、繁殖生物學(xué)等特征將這5個形態(tài)相似的類群劃分為5個不同的種(類群Ⅰ被命名為A.fundyense，類群Ⅱ被命名為A.mediterraneum，類群Ⅲ被命名為A.tamarense，類群Ⅳ被命名為A.pacificum，類群Ⅴ被命名為A.australiense)(Johnetal.,2014a、2014b; Wang Letal.,2014);同樣，根據(jù)rDNA的特征，A.ostenfeldii和A.peruvianum被建議歸為一個種(A.ostenfeldii), 取消A.peruvianum的命名(Gu,2011; Krempetal.,2014)。但此方法在孢囊鑒定時仍面臨一些困難與挑戰(zhàn)，如分子標(biāo)記的選擇、孢囊核酸的提取等。

3.2.1 分子標(biāo)記的選擇 理想的DNA條形碼片段既要易于擴(kuò)增及測序，也要具有種間區(qū)別力(種間具有較高的變異而種內(nèi)具有較低的變異)。因此，相對應(yīng)的引物既要有一定的通用性(包含盡可能多的目的種)，也要具有一定的特異性(非目的種盡可能少)(Zhan & MacIsaac,2015)。常見的DNA條形碼有核糖體RNA基因(rDNA)序列，包括小亞基rRNA基因(SSU或18S rDNA)、大亞基rRNA基因(LSU或28S rDNA)、內(nèi)轉(zhuǎn)錄間隔區(qū)(ITS1、ITS2)及外轉(zhuǎn)錄間隔區(qū)(NTS)，葉綠體上的rbcl、psb基因以及線粒體上的Cob、Cox等基因(陳月琴等,1997、1999; Scholinetal.,1994、1996; Wilcox,1998)。

數(shù)據(jù)庫(如NCBI)中可參考物種條形碼序列的有無及種內(nèi)、種間的遺傳距離對有害甲藻孢囊的分子鑒定起著決定性作用。在藻類的分子鑒定上應(yīng)用最多的是核基因序列，在有害甲藻條形碼序列數(shù)據(jù)庫(如NCBI)中，與其他分子標(biāo)記相比較，18S的物種序列最完備(表2)，且有害甲藻孢囊(除膝溝藻屬)種內(nèi)的變異相對較小，易于設(shè)計其通用引物，這些優(yōu)點(diǎn)使得18S成為甲藻物種鑒定中應(yīng)用廣泛的分子標(biāo)記(Granéli & Turner,2006)；但其種間的變異較小且種內(nèi)及種間的遺傳距離有重疊(表2)，因此，18S并非最理想的分子標(biāo)記。與18S相比，28S數(shù)據(jù)庫中可參考的有害甲藻的物種序列也較為完備(表2)，且種間遺傳距離較大并具有較高的種間分辨率，有害甲藻孢囊的通用引物也易于設(shè)計，但種內(nèi)遺傳距離較大且種內(nèi)及種間的遺傳距離有重疊，所以28S作為有害甲藻的分子標(biāo)記也具有一定的缺陷。核基因上ITS序列由于不編碼基因、不存在進(jìn)化上的選擇壓力而具有較快的進(jìn)化速率，對物種的分辨率最高，但其同種序列因存在插入缺失等現(xiàn)象而具有較大的變異，即具有較大的種內(nèi)變異(表2)，這很可能誤將同種生物的不同個體劃分為2個種；同時，不同種間的遺傳距離差別較大，這使得為有害甲藻孢囊的分子鑒定劃定一個種間界定標(biāo)準(zhǔn)變得困難，而只能針對不同的屬或更高分類單元劃定特定的種間界定標(biāo)準(zhǔn)。如陳月琴和屈良鵠(1999)通過分析GenBank上亞歷山大藻的ITS序列得出，此屬的種間ITS序列差異值大于0.20，而種內(nèi)ITS序列非常相似(僅0.01差異)，這為亞歷山大藻屬的分子鑒定提供了一個種間界定的標(biāo)準(zhǔn)。理論上，其他屬也可以利用此方法找到一個種間界定的標(biāo)準(zhǔn)，但數(shù)據(jù)庫中存在一些錯誤的序列而又難以辨別并刪除，所以難以獲得有害甲藻種間及種內(nèi)遺傳距離的真實值，只能從大量的序列比對中得出一個接近于真實值的值。

甲藻的營養(yǎng)類型較多，包括自養(yǎng)型甲藻、異養(yǎng)型甲藻以及混合營養(yǎng)型甲藻等(Gómez,2012)。自養(yǎng)型甲藻細(xì)胞內(nèi)存在可用作條形碼的葉綠體基因，如rbcl、pbs等，而異養(yǎng)型甲藻不存在此基因，這限制了葉綠體基因在甲藻分子鑒定中的廣泛應(yīng)用。雖然線粒體上的基因如Cob和Cox等的種內(nèi)及種間遺傳距離具有較高的物種分辨率(表2)，但因相關(guān)數(shù)據(jù)庫不完善(表2)而限制了其應(yīng)用。目前，尚無單個DNA條形碼能將所有的甲藻鑒定到種的水平，筆者認(rèn)為應(yīng)根據(jù)不同的種、屬選擇不同的分子標(biāo)記，進(jìn)而實現(xiàn)孢囊的鑒定或甲藻類群的特殊檢測。

3.2.2 孢囊核酸的提取 孢囊核酸的提取對孢囊的分析鑒定至關(guān)重要。孢囊外壁主要由孢粉質(zhì)類似物或鈣質(zhì)組成，如亞歷山大藻屬的孢囊外壁是由孢粉質(zhì)類似物組成，具有抗高溫、抗酸堿腐蝕的能力，且比較堅硬(Bibby & Dodge,1972)；斯氏藻屬的孢囊外壁主要是由硬度較高的鈣質(zhì)組成，這些難以破碎的孢囊外壁雖能協(xié)助甲藻度過不良環(huán)境并擴(kuò)散到鄰近海域外，但增加了孢囊核酸提取的難度。因此，破碎孢囊外壁成為孢囊基因組提取的關(guān)鍵步驟。破碎孢囊壁的常用方法有物理破碎法和化學(xué)破碎法：物理方法主要包括磁珠破碎法(Erdneretal.,2010; Pennaetal.,2010) 和液氮研磨破碎法(Godheetal.,2002)；化學(xué)方法主要為CTAB法(Coyne & Cary,2005; Kamikawaetal.,2007)。此外，通過培養(yǎng)甲藻孢囊讓其萌發(fā)，然后采用常規(guī)植物基因組提取的方法也可達(dá)到間接提取孢囊基因組的目的(Bravoetal.,2006)。以上孢囊基因組提取方法在孢囊種的鑒定、有害藻類的定性及定量檢測等方面得到了廣泛應(yīng)用；但這些方法只適用于提取數(shù)量較多的孢囊種類。在自然條件下，某些種類的孢囊數(shù)量較少，有些種類在當(dāng)前實驗室條件下難以萌發(fā)，給這些稀有孢囊基因組的提取造成了困難，從而無法從分子水平準(zhǔn)確鑒定這些孢囊的種類。

在過去的數(shù)十年間，單細(xì)胞PCR方法得到了較快的發(fā)展，此方法的優(yōu)點(diǎn)是不需要基因組的提取，可直接將單細(xì)胞破碎液用作PCR模板，進(jìn)而在短時間內(nèi)實現(xiàn)分子鑒定。此方法雖然在孢囊的分子鑒定(尤其是樣品中低豐度孢囊或未能萌發(fā)孢囊的鑒定)上得到了一定的應(yīng)用，但仍存在一些問題，如單個孢囊破碎方法的選取、單個孢囊中較低的DNA含量不易于PCR擴(kuò)增以及缺乏高分辨率且通用的甲藻引物(圖3)。常用的單孢囊破碎方法包括反復(fù)凍融法(Bolch,2001; Gribble & Anderson,2006)、微細(xì)玻璃針破碎法(Takano & Horiguchi,2004; Yamaguchi & Horiguchi,2005)以及蓋玻片按壓破碎法(Liuetal.,2014)等。孢囊壁的組成成分復(fù)雜且具有較高的韌性，使得反復(fù)凍融法在孢囊破碎時往往不能取得理想的效果；由于孢囊比較小且具有較堅硬的外壁，利用微細(xì)玻璃針破碎時易造成孢囊的丟失；蓋玻片按壓破碎法能克服以上2種方法的缺點(diǎn)而成為一種比較理想的孢囊破碎方法。較少的DNA含量使得PCR體系中模板量較低，易使PCR擴(kuò)增失敗(無擴(kuò)增片段或由于孢囊表面其他生物的污染引起的非特異性擴(kuò)增)。而巢式PCR能解決孢囊模板量低的問題，常規(guī)的做法：利用真核通用引物(外引物)增加目的基因或DNA片段的拷貝數(shù)進(jìn)行第1次PCR擴(kuò)增，然后以第1次PCR產(chǎn)物為模板再利用甲藻特異性引物(內(nèi)引物)進(jìn)行第2次PCR擴(kuò)增(Pennaetal.,2010)。但由于真核通用引物在PCR擴(kuò)增時對某些類群或模板含量高的類群具有一定的偏嗜性，而難以擴(kuò)增出模板含量低的類群，如甲藻(Kohlietal.,2014; Potvin & Lovejoy,2009)等。因此，如果在巢式PCR第1輪擴(kuò)增時選用甲藻特異性引物，第2輪擴(kuò)增時采取種或?qū)僖陨戏诸悊卧耐ㄓ靡飻U(kuò)增，理論上會得到較為理想的擴(kuò)增效果。PCR引物是決定孢囊PCR擴(kuò)增成敗的關(guān)鍵。甲藻特異性引物已有較多報道(Linetal.,2006、2009)，其中的一對甲藻特異性引物(dinocob4f、dinocob6r)已成功用于高通量測序，結(jié)果顯示，98.46%的序列屬于甲藻，但由于cob的可參考數(shù)據(jù)庫不健全，大部分序列仍不能鑒定到種的水平(Kohlietal.,2014)。因此，有害甲藻孢囊cob條形碼數(shù)據(jù)庫有待進(jìn)一步完善。

圖3 單孢囊分子鑒定過程中存在的問題或爭議及可能的解決方法與思路

4 結(jié)語

有害甲藻孢囊不僅會對生態(tài)環(huán)境造成一定的危害，而且會對水產(chǎn)養(yǎng)殖業(yè)造成重大的經(jīng)濟(jì)損失，甚至威脅人類的生命。全球3/4有害甲藻孢囊種類分布在我國沿海地區(qū)，因此，我國是受甲藻孢囊威脅最為嚴(yán)重的國家之一。有害甲藻孢囊的分類鑒定對于其多樣性及分布的研究至關(guān)重要，現(xiàn)有的分類方法(形態(tài)學(xué)鑒定、分子鑒定等)各有優(yōu)缺點(diǎn)。有些有害甲藻孢囊種具有易于區(qū)分的形態(tài)學(xué)或分子生物學(xué)特征，如網(wǎng)狀原角管藻、多邊舌甲藻等，只要利用其中一種方法就可以將其準(zhǔn)確鑒定；而一些有害甲藻孢囊的形態(tài)學(xué)或分子生物學(xué)特征不易被區(qū)分，如亞歷山大藻屬等，我們應(yīng)將孢囊及萌發(fā)后營養(yǎng)細(xì)胞形態(tài)學(xué)、分子生物學(xué)、毒理學(xué)及繁殖生物學(xué)等鑒定方法結(jié)合起來。今后應(yīng)加強(qiáng)甲藻孢囊的相關(guān)研究，尤其是有害甲藻孢囊條形碼數(shù)據(jù)庫的構(gòu)建，以提高有害甲藻孢囊分子鑒定的準(zhǔn)確率。

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(責(zé)任編輯:楊郁霞)

Research progress on identification of harmful dinoflagellate cysts:A review

Yang-chun GAO1, Yan-hong DONG2, Hai-tao LI2, Ai-bin ZHAN1*

1ResearchCenterforEco-EnvironmentalSciences,ChineseAcademyofSciences,Beijing100085,China;2SouthChinaSeaEnvironmentalMonitoringCenter,StateOceanicAdministration,Guangzhou,Guangdong510300,China

Harmful dinoflagellate cysts refer to cysts derived from dinoflagellates that can produce toxins and (or) cause harmful dinoflagellate blooms. So far, 18 species in 10 genera have been recorded along coasts of China seas, accounting for three quarters of the total number of harmful dinoflagellate cysts globally. These harmful dinoflagellate cysts are widely distributed along Chinese coasts. Harmful dinoflagellate cysts largely threaten marine ecosystems, aquaculture industries and even human health. Consequently, the study of diversity and distributions of harmful dinoflagellate cysts has become a hotspot in marine biology and ecology. The identification of harmful dinoflagellate cysts is crucial for sea food safety inspection and prediction of harmful dinoflagellate blooms. However, accurate identification represents a big challenge, mainly owing to limited available morphological features of harmful dinoflagellate cysts. Here we review research progress on harmful dinoflagellate cysts on Chinese coasts, including their negative impacts, diversity and geographical distributions, and species identification of harmful dinofagellate cysts. In addition, we suggest that it is nececarry to ultilize multiple methods including those based on morphology and molecular biology, as well as methods based on toxicology and reproductive biology, to accurately identify harmful dinoflagellate cysts.

harmful dinoflagellate cyst; morphological characteristics; molecular identification

2015-12-04 接受日期(Accepted)： 2015-12-27

中國科學(xué)院“百人計劃”項目； 南海區(qū)海洋環(huán)境質(zhì)量綜合評估方法[DOMEP(MEA)-01-03]

高養(yǎng)春， 男， 博士研究生。 研究方向： 分子生態(tài)學(xué)與入侵生物學(xué)。 E-mail: gaoyc0412@163.com

*通訊作者(Author for correspondence), E-mail: azhan@rcees.ac.cn, zhanaibin@hotmail.com

10. 3969/j.issn.2095-1787.2016.04.002