霍 達(dá),曹 琪,王素炎,陳裴裴,李一鳴,喬之怡*

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海河天津段微囊藻及其毒素的空間分布及與水環(huán)境因子的關(guān)系

霍 達(dá)1,2,曹 琪1,王素炎1,陳裴裴1,李一鳴1,喬之怡1,2*

(1.天津農(nóng)學(xué)院水產(chǎn)學(xué)院,天津 300384；2.天津市水產(chǎn)生態(tài)及養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室,天津 300384)

采取熒光定量PCR技術(shù)及ELISA酶聯(lián)免疫吸附法,以基因和-基因?yàn)榘谢驅(qū)ο募舅{(lán)藻暴發(fā)期間海河天津市區(qū)段的產(chǎn)毒微囊藻種群豐度和毒素含量進(jìn)行了研究.結(jié)果表明:夏季海河天津市區(qū)段微囊藻種群豐度具有明顯差異性:產(chǎn)毒微囊藻種群豐度為1.16×104~2.48×107copies/mL占總微囊藻種群的4.25%~28.59%.藻毒素含量最高點(diǎn)為8號(hào)采樣點(diǎn)天津站,每升水體中藻細(xì)胞共含毒素195.51μg,除去藻細(xì)胞水中毒素濃度為0.97μg/L.總的來(lái)說(shuō),海河天津市區(qū)段微囊藻總基因拷貝數(shù)較高,產(chǎn)毒微囊藻豐度在不同采樣點(diǎn)間差異較大,影響水體中微囊藻豐度的最主要環(huán)境因素是pH值.海河水體中毒素含量沒(méi)有超過(guò)安全閾值,但是單位體積水體中藻細(xì)胞內(nèi)毒素含量很高,具有較高的毒素釋放潛力,可能對(duì)下游河口生態(tài)系統(tǒng)造成潛在威脅.

微囊藻；微囊藻毒素；熒光定量PCR技術(shù)；ELISA酶聯(lián)免疫吸附法；海河

微囊藻水華給淡水環(huán)境帶來(lái)了極大的影響.一些種類的微囊藻能產(chǎn)生次生代謝物微囊藻毒素(MC),該毒素是富營(yíng)養(yǎng)化水體中最主要的藍(lán)藻毒素類型,可引發(fā)水生生物中毒甚至死亡,直接威脅水中魚類及其它生物的生存.飲用水源中的微囊藻毒素會(huì)對(duì)人類的肝臟造成損傷,影響人類的健康和生存[1],同時(shí),微囊藻毒素是強(qiáng)致癌劑,與人群原發(fā)性肝癌的高發(fā)關(guān)系密切[2].為保護(hù)人類身體健康,世界衛(wèi)生組織規(guī)定的飲用水中微囊藻毒素安全濃度閾值<1.0μg/L[3].研究表明,微囊藻水華種群中同時(shí)含有有毒和無(wú)毒微囊藻基因型,兩者種群豐度及其比例的動(dòng)態(tài)變化直接影響了微囊藻水華毒素濃度,對(duì)微囊藻毒素濃度具有很好的指示作用[4-6].有毒和無(wú)毒微囊藻細(xì)胞形態(tài)一致,用傳統(tǒng)的顯微鏡形態(tài)觀察方法無(wú)法對(duì)兩者進(jìn)行區(qū)分和鑒定.有毒微囊藻細(xì)胞基因組中含有微囊藻毒素合成酶基因家族(mcy)[7-8],對(duì)毒素產(chǎn)生進(jìn)行調(diào)控,而無(wú)毒微囊藻株中則不含有mcy 基因家族.因此可以應(yīng)用PCR擴(kuò)增mcy基因單個(gè)或多個(gè)成員檢測(cè)和鑒別有毒和無(wú)毒藻株[9-11].

近些年來(lái),隨著分子生物學(xué)技術(shù)的發(fā)展,越來(lái)越多的研究人員對(duì)于微囊藻的產(chǎn)毒相關(guān)基因進(jìn)行了定量的研究.Rinta-Kanto等[12]以基因和微囊藻16S rDNA基因?yàn)榘谢虿捎脽晒舛縋CR的方法分析了Western Lake Erie的產(chǎn)毒微囊藻構(gòu)成及其豐度,發(fā)現(xiàn)了該湖中微囊藻存在產(chǎn)毒和非產(chǎn)毒共存的現(xiàn)象,在國(guó)內(nèi)的研究中,學(xué)者們[13-15]對(duì)太湖及巢湖水華期間微囊藻種群豐度進(jìn)行了研究,發(fā)現(xiàn)了相同現(xiàn)象在中國(guó)南方水體中的廣泛存在.劉洋等[16]對(duì)南太湖入湖口的產(chǎn)毒微囊藻進(jìn)行了研究,證明了該方法可以快速準(zhǔn)確地檢測(cè)水體中微囊藻產(chǎn)毒藻數(shù)量.

隨著全球氣溫變化的影響,藍(lán)藻暴發(fā)的范圍慢慢由南方水體延伸至北方.北方水體具有冬季結(jié)冰,四季氣溫變化大等特征,與南方水體差異較大.已有研究表明在溫度[17]、緯度[18]等因素影響下,微囊藻毒素的釋放具有不同特征.海河貫穿天津市區(qū),隨著近年來(lái)兩岸生活污水的排放,以及上游水質(zhì)的惡化,海河市區(qū)內(nèi)景觀河道出現(xiàn)了大面積的微囊藻水華,對(duì)生活用水造成了較大的威脅.本研究將天津海河作為模型,從毒素合成調(diào)控基因和毒素兩方面入手,研究北方緩速流動(dòng)型水體的毒素釋放特征,為水環(huán)境治理提供參考資料.

1 材料與方法

1.1 采樣位點(diǎn)及化學(xué)指標(biāo)測(cè)定方法

表1 采樣點(diǎn)信息

2015年7月,在海河天津市區(qū)段(坐標(biāo))設(shè)置8個(gè)采樣點(diǎn)(表1),分別為天津站、耳閘摩天輪、天津?yàn)彻珗@富民橋、獅子林橋.使用不銹鋼采水器采取1.5m水柱,混合均勻.同時(shí)利用YSI多功能水質(zhì)檢測(cè)儀測(cè)定采樣點(diǎn)水質(zhì)基本參數(shù)如溫度、pH值、氨氮、溶解氧等.TN、TP測(cè)定方法參照文獻(xiàn)[14].

1.2 基因組DNA提取

取200mL混勻后的水樣(根據(jù)微囊藻的量調(diào)整抽濾體積)使用Millipore玻璃纖維膜過(guò)濾.含藻濾膜置于-20℃保存.將膜剪碎后采用Rinta-Kanto等[12]的方法提取DNA.

圖1 采樣點(diǎn)示意

1.3 構(gòu)建熒光定量PCR 標(biāo)準(zhǔn)曲線

熒光定量PCR所選用的引物如表2所示,其中,引物188F和254R用于特異性擴(kuò)增微囊藻藻藍(lán)蛋白轉(zhuǎn)錄間隔區(qū)PC-IGS[4],引物F2和R2用于特異性擴(kuò)增有毒微囊藻毒素合成基因[19].標(biāo)準(zhǔn)藻株為有毒微囊藻株FACHB 905,由中國(guó)淡水藻種庫(kù)(FACHB)提供,在實(shí)驗(yàn)室內(nèi)培養(yǎng),培養(yǎng)條件(光照強(qiáng)度為30mmol protons/(m2×s),光照周期12h/12h (L:D),藻細(xì)胞濃度培養(yǎng)到對(duì)數(shù)生長(zhǎng)期進(jìn)行采收).采用全細(xì)胞PCR的方法擴(kuò)增2個(gè)片段,將PCR產(chǎn)物進(jìn)行瓊脂糖電泳后回收.將純化后的產(chǎn)物與pEASY?克隆載體連接,并注入大腸桿菌感受態(tài)細(xì)胞Trans1-T1中,于LB液體培養(yǎng)基中振蕩培養(yǎng);離心收集菌液,混勻,取適量涂布在含有LB(Amp+)的固體培養(yǎng)皿中過(guò)夜培養(yǎng);挑取單個(gè)陽(yáng)性菌落按上述條件進(jìn)一步培養(yǎng),再對(duì)培養(yǎng)后菌落中的質(zhì)粒進(jìn)行測(cè)序.利用質(zhì)粒提取試劑盒提取質(zhì)粒,用超微量核酸蛋白測(cè)定儀(eppendorf)測(cè)定質(zhì)粒標(biāo)準(zhǔn)品的濃度.根據(jù)文獻(xiàn)[20]計(jì)算拷貝數(shù),將得到的質(zhì)粒標(biāo)準(zhǔn)品用1×TE梯度稀釋構(gòu)建標(biāo)準(zhǔn)曲線.

表2 mcyD基因和PC-IGS基因qPCR反應(yīng)引物

1.4 定量PCR反應(yīng)體系

定量PCR反應(yīng)體系為20μL,其中含有10μL SYBR GREEN MIX,2pmoL引物,1μL模板,超純水補(bǔ)足20μL, PCR反應(yīng)在Bio-rad CFX Connet中進(jìn)行.-基因擴(kuò)增程序如下:95℃預(yù)變性2min, 95 ℃變性15s,58℃退火30s,72℃延伸30s,40個(gè)循環(huán).基因定量PCR反應(yīng)退火溫度為56℃,其它條件與PC-IGS相同.

1.5 水中及細(xì)胞中微囊藻毒素檢測(cè)

采用0.22μm一次性真空濾膜過(guò)濾5mL水樣,保留含藻濾膜進(jìn)行水中微囊藻毒素含量測(cè)定.細(xì)胞中的毒素使用GF/C膜過(guò)濾藻細(xì)胞并調(diào)節(jié)濃度后測(cè)定.所有毒素檢測(cè)樣本均使用中科院水生生物研究所研制的微囊藻毒素ELISA試劑盒進(jìn)行檢測(cè).

1.6 數(shù)據(jù)統(tǒng)計(jì)

數(shù)據(jù)統(tǒng)計(jì)在R語(yǔ)言環(huán)境下進(jìn)行,相關(guān)性計(jì)算采用Pearson相關(guān)性函數(shù)計(jì)算.數(shù)據(jù)展示采用平均值±標(biāo)準(zhǔn)差(mean±SD)形式.

2 結(jié)果與分析

2.1 海河水體理化參數(shù)

測(cè)得采樣期間水體段理化指標(biāo)見(jiàn)表3,采樣期間海河水體的平均溫度為29.2℃,4號(hào)采樣點(diǎn)水溫最高為30.4℃.水體呈弱堿性,5號(hào)采樣點(diǎn)pH值最高.溶解氧變化范圍較廣,為4.77~17.15mg/L.總氮最高值為4號(hào)點(diǎn)3.55mg/L,最低值為1號(hào)點(diǎn)1.04mg/L.總磷最高點(diǎn)為2號(hào)點(diǎn)0.1mg/L,最低值為4號(hào)點(diǎn)0.05mg/L.

表3 海河水體主要理化參數(shù)

2.2 定量PCR標(biāo)準(zhǔn)曲線

利用質(zhì)粒DNA為模板建立基因和基因定量PCR標(biāo)準(zhǔn)曲線.從圖2和圖3可以看出,循環(huán)閾值q與基因組拷貝數(shù)的對(duì)數(shù)呈顯著線性關(guān)系.定量PCR反應(yīng)中,序列定量PCR標(biāo)準(zhǔn)曲線方程為= -3.482+ 38.79 ,(2=0.993),擴(kuò)增效率為93.8%.-序列定量PCR標(biāo)準(zhǔn)曲線方程為= -3.41+ 39.97,(2=0.999),擴(kuò)增效率為96.5%.溶解曲線結(jié)果顯示和-的擴(kuò)增產(chǎn)物解鏈峰值溫度分別為82.49℃(變異系數(shù)CV=0.09%)和84.41℃(變異系數(shù)CV=0.26%)

圖2 mcyD基因熒光定量PCR標(biāo)準(zhǔn)曲線

圖3 PC-IGS基因熒光定量PCR標(biāo)準(zhǔn)曲線

2.3 產(chǎn)毒微囊藻和總微囊藻種群豐度的分布

定量PCR結(jié)果如圖4所示,海河微囊藻種群豐度具有顯著的空間分布差異.5號(hào)點(diǎn)天津之眼微囊藻種群豐度最高,每毫升水中微囊藻基因型拷貝數(shù)達(dá)到(1.53×108±1.13×107)copies/mL同時(shí)基因型拷貝數(shù)達(dá)到(2.48×107±7.12×106)copies/mL.3號(hào)點(diǎn)北洋園微囊藻種群豐度最低,微囊基因型拷貝數(shù)為(8.50×104±1.62×106) copies/mL,基因型拷貝數(shù)為(1.16×104±1.09×102)copies /mL.產(chǎn)毒微囊藻基因豐度與總微囊藻基因豐度比值為4.25%~28.59%平均比值為15.44%,各點(diǎn)之間差異較大.

圖4 產(chǎn)毒微囊藻和總微囊藻種群豐度的空間分布

2.4 微囊藻細(xì)胞和水體中微囊藻毒素含量

ELISA酶聯(lián)免疫反應(yīng)檢測(cè)結(jié)果如圖5所示,藻細(xì)胞中毒素含量最高的點(diǎn)位為8號(hào)天津站為每升水中藻細(xì)胞含195.51μg毒素,最低3號(hào)為2.53μg.水中微囊藻毒素含量最高的也為8號(hào)天津站0.97μg/L.最低點(diǎn)3號(hào)0.18μg/L.4~8號(hào)點(diǎn)位顯著高于1~3號(hào)點(diǎn).通過(guò)線性回歸得到細(xì)胞產(chǎn)毒和水中微囊藻毒素相關(guān)性2=0.95.

圖5 每升水體中細(xì)胞內(nèi)和細(xì)胞外的微囊藻細(xì)胞產(chǎn)毒素含量

3 討論

3.1 基于熒光定量PCR結(jié)果的微囊藻基因豐度差異

近年來(lái),夏季中的海河備受藍(lán)藻困擾,藍(lán)藻暴發(fā)對(duì)居民生活和城市景觀帶來(lái)較大影響.當(dāng)前推測(cè)海河藍(lán)藻暴發(fā)的原因有:海河生態(tài)用水不足,水體流動(dòng)性差;海河水體中適合藻類生長(zhǎng)的營(yíng)養(yǎng)物質(zhì)含量較高;天津夏季氣候非常適應(yīng)藍(lán)藻生長(zhǎng)等[21],但均缺乏定量的分析.此外對(duì)水體中微囊藻是否產(chǎn)毒,產(chǎn)毒微囊藻的豐富度如何也不甚清楚,缺乏相應(yīng)的監(jiān)測(cè)和報(bào)道.

近些年,熒光定量PCR在藍(lán)藻水華的研究中得到廣泛應(yīng)用.這項(xiàng)技術(shù)能夠?qū)Υ笈鷺悠吠瑫r(shí)進(jìn)行處理,對(duì)產(chǎn)毒微囊藻種群豐富度進(jìn)行定量化的研究,快速對(duì)藍(lán)藻水華是否產(chǎn)毒和毒性做出判斷,可以預(yù)測(cè)藍(lán)藻水華帶來(lái)的生態(tài)風(fēng)險(xiǎn)[22].基因qPCR法具有靈敏性高、成本較低、所用時(shí)間短、所需檢測(cè)樣本少、操作簡(jiǎn)單等特點(diǎn)[23].qPCR法對(duì)于微囊藻產(chǎn)毒潛能的預(yù)測(cè)與毒素的直接檢測(cè)結(jié)果具有高度一致性[24].研究中常選擇單個(gè)基因作為目的基因進(jìn)行qPCR擴(kuò)增片段[25-30],也可選取多個(gè)基因作為目標(biāo)基因進(jìn)行擴(kuò)增[31-33],本實(shí)驗(yàn)選取基因作為目的基因?qū)λw中產(chǎn)毒微囊藻進(jìn)行檢測(cè).-基因?yàn)樘禺愋詳U(kuò)增微囊藻的基因片段,可檢測(cè)出水體中總微囊藻基因豐富度.本實(shí)驗(yàn)通過(guò)建立基因和-基因的熒光定量PCR標(biāo)準(zhǔn)曲線,對(duì)海河水體中產(chǎn)毒微囊藻種群基因豐富度和總微囊藻種群基因豐富度進(jìn)行定量分析.通過(guò)熒光定量PCR的結(jié)果表明,海河干流不同水系的微囊藻種群豐富度具有較大差異,北洋園(3號(hào)位點(diǎn))產(chǎn)毒微囊藻豐富度與總微囊藻豐富度均最低;南運(yùn)河(1號(hào)位點(diǎn))和新開(kāi)河(4號(hào)位點(diǎn))產(chǎn)毒微囊藻豐富度與總微囊藻豐富度均較高,因此,海河干流中的微囊藻可能主要來(lái)源于南運(yùn)河和新開(kāi)河.

3.2 水體理化指標(biāo)與微囊藻產(chǎn)毒基因及毒素的空間分布關(guān)系

通過(guò)對(duì)8月份海河天津市區(qū)段水體理化指標(biāo)分析,可知此時(shí)的水體處于富營(yíng)養(yǎng)化,再根據(jù)qPCR結(jié)果印證了在富營(yíng)養(yǎng)化狀態(tài)下微囊藻的總豐度較高[34].在自然環(huán)境中,許多種環(huán)境因子都可能影響產(chǎn)毒微囊藻占總微囊藻種群的比例,在Yoshida等[35]研究Mikata湖泊中產(chǎn)毒微囊藻和非產(chǎn)毒微囊藻種群動(dòng)態(tài)隨時(shí)間變化規(guī)律時(shí),發(fā)現(xiàn)硝態(tài)氮濃度與產(chǎn)毒微囊藻占總微囊藻種群的比值呈顯著正相關(guān).這與本實(shí)驗(yàn)中南運(yùn)河(愛(ài)民橋 1號(hào)位點(diǎn))硝態(tài)氮濃度很高,產(chǎn)毒微囊藻占總微囊藻種群的比值也高的實(shí)驗(yàn)結(jié)論相符合.從研究結(jié)果可以看出,產(chǎn)毒微囊藻占總微囊藻的比值范圍4.25%~28.59%,平均值為15.50%.其中比值最高的點(diǎn)為獅子林橋(6號(hào)位點(diǎn)),比值最低的點(diǎn)為進(jìn)步橋(7號(hào)位點(diǎn)).相關(guān)報(bào)道表明不同湖泊產(chǎn)毒微囊藻占總微囊藻種群的比值的波動(dòng)范圍較大[36-37],獅子林橋與進(jìn)步橋的距離較為相近,但產(chǎn)毒藻株比例卻差異很大,導(dǎo)致這種結(jié)果的原因還需要進(jìn)一步研究.根據(jù)劉永定等關(guān)于中國(guó)藻類學(xué)的研究表明高pH (大于8)值有利于原核藻類生長(zhǎng)[38],海河水體8月pH值均在8以上,呈現(xiàn)弱堿性,非常有利于微囊藻生長(zhǎng),導(dǎo)致藍(lán)藻暴發(fā).6號(hào)位點(diǎn)pH值顯示為9.01,7號(hào)位點(diǎn)為8.01,推測(cè)藻類豐富度可能與pH值有關(guān).5號(hào)位點(diǎn)pH為9.3,是pH值最高的點(diǎn),5號(hào)位點(diǎn)也是基因豐富度和-基因豐富度最高的點(diǎn).產(chǎn)毒微囊藻基因豐富度與總微囊藻基因豐富度的比值最高的點(diǎn)為獅子林橋(6號(hào)位點(diǎn)),比值最低的點(diǎn)為進(jìn)步橋(7號(hào)位點(diǎn)),其中6號(hào)位點(diǎn)的溶解氧9.11mg/L大于7號(hào)位點(diǎn)的溶解氧4.77mg/L.新開(kāi)河(4號(hào)位點(diǎn))溶解氧為最高17.5mg/L,產(chǎn)毒微囊藻基因豐富度也最高為2.08×106copies/mL,溶解氧最低的點(diǎn)為進(jìn)步橋(7號(hào)位點(diǎn)),產(chǎn)毒微囊藻基因豐富度的最低點(diǎn)為北洋園(3號(hào)位點(diǎn)),但溶解氧最低點(diǎn)不是產(chǎn)毒微囊藻基因豐富度最低點(diǎn).這與李大命等在巢湖夏季和冬季有毒微囊藻和無(wú)毒微囊藻種群豐度研究中的實(shí)驗(yàn)研究結(jié)果[39]出現(xiàn)了差異性,具體原因有待進(jìn)一步研究.

3.3 水體理化指標(biāo)與微囊藻產(chǎn)毒基因及毒素的相關(guān)性分析

表4 環(huán)境因子與微囊藻和有毒微囊藻豐度之間的相關(guān)系數(shù)(n=8)

注: * :<0.05;**:<0.01.

已有研究表明,水體理化因子的差異可以顯著的影響微囊藻毒素的釋放.其中觀點(diǎn)主要集中在溫度、總氮、總磷、磷酸鹽等[5,40-41]因素的影響,在本次研究中我們發(fā)現(xiàn)海河水體中微囊藻無(wú)論從總基因豐度到產(chǎn)毒基因豐度都與pH值顯著相關(guān)(<0.05),這與本文在海河的另一項(xiàng)基于高通量測(cè)序得到的結(jié)果是一致的[42].這說(shuō)明在高pH值情況下微囊藻無(wú)論在絕對(duì)豐度還是與其他原核生物共存的相對(duì)豐度水平上均能占據(jù)較大比例.在所有環(huán)境因子中,影響海河中微囊藻的豐度差異最重要的原因是pH值.然而目前的研究并不能給出pH值與微囊藻直接相關(guān)的明確原因,二者之間的因果關(guān)系尚不明晰.有一種推測(cè)是在微囊藻固碳過(guò)程中有一個(gè)中間形式為碳酸氫鹽[43].藍(lán)藻的集中暴發(fā)可能使碳酸氫根離子在水體中大量富集而后隨著藻細(xì)胞衰敗破裂而釋放到水體中.微囊藻產(chǎn)毒基因、產(chǎn)毒藻株比例與各點(diǎn)位實(shí)際觀測(cè)到的微囊藻毒素含量并沒(méi)有顯著相關(guān)性.這證明了雖然基因簇在微囊藻毒素合成中必不可少[44],但毒素的實(shí)際合成過(guò)程中受到轉(zhuǎn)錄因子調(diào)控或基因修飾影響顯著,造成產(chǎn)毒藻株的豐度并不與毒素含量有實(shí)際關(guān)聯(lián).微囊藻毒素合成基因與微囊藻總16S rRNA基因豐度極顯著相關(guān)(<0.01),這證明了在海河中微囊藻的產(chǎn)毒比例趨近一致,各采樣點(diǎn)位產(chǎn)毒株所占比例大致相同.單位水體內(nèi)胞外微囊藻毒素含量與胞內(nèi)微囊藻毒素含量極顯著相關(guān),這說(shuō)明在采樣期間微囊藻毒素釋放速率較為穩(wěn)定,并沒(méi)有發(fā)生大規(guī)模的胞內(nèi)毒素釋放事件.但值得注意的是,雖然藻細(xì)胞外水體中微囊藻毒素含量在各點(diǎn)位均未超過(guò)WHO規(guī)定的飲用水微囊藻毒素含量標(biāo)準(zhǔn)但是計(jì)算相同體積水體中藻細(xì)胞的毒素含量遠(yuǎn)超這一標(biāo)準(zhǔn).這說(shuō)明海河微囊藻具有較高的毒素釋放潛力,這意味著當(dāng)水體發(fā)生大規(guī)模藻細(xì)胞破裂事件時(shí),水環(huán)境中毒素含量會(huì)遠(yuǎn)超過(guò)現(xiàn)有水平.一旦被飲用會(huì)對(duì)人體健康造成巨大威脅.在海河及與其類似的通海河道水體中,滲透壓造成藻細(xì)胞破裂也可能會(huì)給河口生態(tài)系統(tǒng)造成巨大破壞.因此對(duì)于海河中微囊藻的控制是十分有必要的.

4 結(jié)論

4.1 夏季海河天津市區(qū)段微囊藻種群豐度具有明顯空間差異產(chǎn)毒微囊藻種群豐度為1.16′104~2.48′107copies/mL,占總微囊藻種群的4.25%~28.59%.

4.2 藻毒素含量最高點(diǎn)為8號(hào)采樣點(diǎn)天津站,每升水體中藻細(xì)胞共含毒素195.51μg,除去藻細(xì)胞水中毒素濃度為0.97μg/L.水體中微囊藻毒素未達(dá)到WHO規(guī)定的閾值.

4.3 產(chǎn)毒基因豐度及比例與環(huán)境中微囊藻毒素含量未呈現(xiàn)顯著相關(guān)關(guān)系.pH值的大小與微囊藻總豐度和產(chǎn)毒基因型豐度呈現(xiàn)顯著相關(guān)關(guān)系.

4.4 夏季海河中微囊藻胞內(nèi)毒素含量遠(yuǎn)高于胞外含量,可能對(duì)下游的河口區(qū)域生態(tài)系統(tǒng)安全構(gòu)成潛在威脅.

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致謝：感謝天津農(nóng)學(xué)院基礎(chǔ)學(xué)院的劉萍老師在水化學(xué)分析中提供的幫助.

The spatial distribution ofand microcystin and its relationship with environmental factors in Haihe Tianjin City.

HUO Da1,2, CAO Qi1, WANG Su-yan1, CHEN Pei-pei1, LI Yi-ming1, QIAO Zhi-yi1,2*

(1.Department of Fishery Science, Tianjing Agricultural College, Tianjing 300384, China；2.Tianjin Key Laboratory of Aqua-ecology and Aqua-culture, Tianjin 300384, China)., 2018,38(10)：3897~3903

The spatial distribution ofand microcystin in Haihe River during water bloom were investigated using quantitative real-time PCR (qPCR) and ELISA techniques. Microcystin synthetase gene () and-fragments were used as the target the total microcystin population and the potential microcystin-producing subpopulation. The results showed that microcystinexisted as a mixed population of potentially toxic and non-toxic genotypes in Haihe River. There was significantly spatial variation in the abundance of microcystin-producingThe abundance of microcystin-producingsubpopulation was ranged from 1.16×104to 2.48×107copies/mL and the ratio of its abundance to total microcystinvaried from 4.25% to 28.59%. The peakvalue of microcystinwas site point 8, which 1mL water contained 195.51μg microcystininalgae cells and 0.97μg/L in water. To sum up, total abundance ofremained a high level in Haihe Tianjin region. The toxin ofshowed various among different sampling sites. The most significant impact factors forabundance was pH value. However, the content of microcystin is not upon the value of WHO’s threshold. But the intercellular toxin in a given volume of water remains a high level, which may cause the potential risk to the ecosystem of down flow estuary.

；microcystin；quantitative real-time PCR；ELISA techniques；Haihe River

X522

A

1000-6923(2018)10-3897-07

霍 達(dá)(1993-),男,內(nèi)蒙古呼倫貝爾人,天津農(nóng)學(xué)院碩士研究生,主要從事分子生態(tài)學(xué)方向的研究.發(fā)表論文8篇.

2018-03-23

國(guó)家國(guó)際科技合作專項(xiàng)(2013DFA71340)

* 責(zé)任作者, 副教授, qiaozhiyi7070@163.com