尚德榮,寧勁松,趙艷芳,*,翟毓秀,李風(fēng)鈴,盛曉風(fēng),丁海燕,徐正,段德麟,周永東
1. 中國水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所 國家水產(chǎn)品質(zhì)量監(jiān)督檢驗(yàn)中心 農(nóng)業(yè)部水產(chǎn)品質(zhì)量安全檢測與評價(jià)重點(diǎn)實(shí)驗(yàn)室,青島 266071 2. 中國科學(xué)院海洋研究所,青島 266071 3. 江蘇鹽城海瑞食品有限公司,大豐 224100
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條斑紫菜中磷和鈣的亞細(xì)胞分布及其與富集砷的關(guān)系
尚德榮1,寧勁松1,趙艷芳1,*,翟毓秀1,李風(fēng)鈴1,盛曉風(fēng)1,丁海燕1,徐正1,段德麟2,周永東3
1. 中國水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所 國家水產(chǎn)品質(zhì)量監(jiān)督檢驗(yàn)中心 農(nóng)業(yè)部水產(chǎn)品質(zhì)量安全檢測與評價(jià)重點(diǎn)實(shí)驗(yàn)室,青島 266071 2. 中國科學(xué)院海洋研究所,青島 266071 3. 江蘇鹽城海瑞食品有限公司,大豐 224100
研究條斑紫菜中磷和鈣的亞細(xì)胞分布,并探討其與條斑紫菜富集砷的關(guān)系。結(jié)果表明:條斑紫菜吸收磷、鈣主要分布在細(xì)胞壁組分中,平均占總磷含量的53.9%,占總鈣含量的61.25%,其次為細(xì)胞液。與對照組相比(砷暴露濃度0.5 mg·L-1),在添加磷濃度為0.1 mg·L-1和1.0 mg·L-1處理組中,條斑紫菜中磷、砷之間呈現(xiàn)協(xié)同效應(yīng);當(dāng)添加磷濃度為5.0 mg·L-1和10.0 mg·L-1時(shí),條斑紫菜中磷、砷之間則呈現(xiàn)拮抗作用,砷的富集量分別比照組下降61.37%和72.73%。經(jīng)添加鈣液暴露過的條斑紫菜,當(dāng)鈣暴露濃度為1 000 mg·L-1時(shí)其對砷的富集量比對照組減少15.51%??芍?,5.0 mg·L-1磷處理?xiàng)l斑紫菜,條斑紫菜對砷的富集受到明顯的抑制。
砷;磷;鈣;條斑紫菜;亞細(xì)胞定位;富集
條斑紫菜是中國的優(yōu)勢水產(chǎn)品,其食品安全備受關(guān)注。有關(guān)條斑紫菜(Porphyra yezoensis)富集重金屬及其賦存形態(tài)方面已經(jīng)開展了大量研究,但是這些研究主要集中在As5+、Cu2+、Pb2+、Cd2+、Hg2+對條斑紫菜的脅迫作用及其賦存形態(tài)方面[1-9],而考察改善生境條件(如添加磷、鈣等元素)對重金屬脅迫所產(chǎn)生的協(xié)同或拮抗作用的研究目前僅在一些陸地植物中展開[10-16],其研究結(jié)果也不盡相同[17-22]。
筆者從降低條斑紫菜砷含量、提高產(chǎn)品質(zhì)量及安全角度出發(fā),以自然養(yǎng)殖與室內(nèi)模擬實(shí)驗(yàn)相結(jié)合探討不同生境條件下(不同鈣、磷濃度等)條斑紫菜藻體對砷的富集及其亞細(xì)胞組分中砷的含量及分布規(guī)律,揭示條斑紫菜富集砷與生境條件的相關(guān)關(guān)系(拮抗和協(xié)同的關(guān)系)。研究試圖為生境條件對條斑紫菜體內(nèi)砷的分布、富集和遷移規(guī)律提供理論依據(jù),為條斑紫菜的安全養(yǎng)殖生產(chǎn)提供指導(dǎo)。
1.1 實(shí)驗(yàn)材料
條斑紫菜活體于2014年1月14日采自江蘇連云港港口養(yǎng)殖區(qū)域,當(dāng)天運(yùn)回實(shí)驗(yàn)室暫養(yǎng)1 d,依據(jù)從大樣本中抽取小樣本進(jìn)行分組的規(guī)范方法,選取一定量的健康條斑紫菜作為空白組、對照組和實(shí)驗(yàn)組。暴露實(shí)驗(yàn)中所采用的砷為Na2HAsO4·7H2O,磷為NaH2PO4·2H2O,鈣為Ca(NO3)2·4H2O。
1.2 實(shí)驗(yàn)方法
根據(jù)海水中砷的環(huán)境濃度選擇As(V)濃度0.5 mg·L-1為實(shí)驗(yàn)對照組,磷處理組中添加磷最終濃度分別為0.1 mg·L-1、1.0 mg·L-1、5.0 mg·L-1、10.0 mg·L-1;鈣處理組中添加鈣最終濃度分別為250 mg·L-1、500 mg·L-1、1 000 mg·L-1,每個(gè)濃度組分別設(shè)3個(gè)平行,實(shí)驗(yàn)連續(xù)進(jìn)行6 d,中間不更換實(shí)驗(yàn)溶液,并分別于0、1、2、3、4、5、6 d各取樣1次。同時(shí)做只用海水養(yǎng)殖無添加任何試劑的空白實(shí)驗(yàn)。
條斑紫菜亞細(xì)胞組分的分離、亞細(xì)胞組分分析、儀器條件及詳細(xì)的實(shí)驗(yàn)方法詳見文獻(xiàn)[9]。
2.1 條斑紫菜中磷的亞細(xì)胞分布
表1展示了條斑紫菜經(jīng)不同濃度磷、鈣處理6 d后,藻體中亞細(xì)胞不同組分磷含量分布,由表1可知,無論是暴露在添加磷液或添加鈣液處理組中的條斑紫菜均以細(xì)胞壁的磷含量最高,為541.72~1 100.57 mg·kg-1,占總磷含量的43.2%~64.6%,其次為細(xì)胞液,含量為428.40~676.54 mg kg-1,占總磷含量的25.0%~42.0%;而細(xì)胞器中P含量僅為129.3~192.7 mg kg-1占總磷含量的9.6%~20.2%。經(jīng)添加磷液暴露過的條斑紫菜隨著磷濃度的增加其細(xì)胞壁中磷含量和所占比例依次增大,而細(xì)胞液組分中高磷濃度暴露組的磷含量低于其他暴露濃度組,細(xì)胞器中的磷含量隨著磷濃度的增加磷含量依次增大,圖1a、1b、1c分別是暴露在不同磷溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中磷含量。經(jīng)添加鈣液暴露過的條斑紫菜隨著鈣濃度的增加其細(xì)胞壁、細(xì)胞液和細(xì)胞器中磷含量依次減少,圖1d、1e、1f分別是暴露在不同鈣溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中磷含量。
2.2 條斑紫菜中鈣的亞細(xì)胞分布
表2展示了條斑紫菜經(jīng)不同濃度磷、鈣處理6 d后,藻體中亞細(xì)胞不同組分鈣含量分布,由表2可知,無論是暴露在添加磷液或添加鈣液處理組中的條斑紫菜均以細(xì)胞壁的鈣含量最高,達(dá)188.56~1 019.21 mg·kg-1,占總鈣含量的42.94%~79.56%;其次為細(xì)胞液,其鈣含量為174.80~229.46 mg·kg-1,占總鈣含量的16.60%~38.59%;而細(xì)胞器中僅為74.96~133.26 mg·kg-1,占總鈣含量的9.64%~17.25%。且隨著磷暴露濃度的增加其細(xì)胞壁、細(xì)胞液和細(xì)胞器中鈣含量依次增加,圖2a、2b、2c分別是暴露在不同磷溶液1~6 d條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中鈣含量,暴露在添加磷溶液中的條斑紫菜各亞細(xì)胞組分的鈣含量高于暴露在添加鈣溶液中各亞細(xì)胞組分中鈣的含量。圖2d、2e、2f分別是暴露在不同鈣溶液1~6 d條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中鈣含量。
圖1 暴露于不同濃度的磷或鈣1~6 d后條斑紫菜中磷的亞細(xì)胞分布注:1a、1b、1c分別是暴露在不同磷溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中磷含量;1d、1e、1f分別是暴露在不同鈣溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中磷含量。Fig. 1 Subcellular distribution of phosphorus in Porphyra yezoensis exposed to different concentrations of phosphorus and calcium for 1-6 dNote: 1a, 1b and 1c was phosphorus content in the cell wall, cytoplasm and cell organelle respectively in P. yezoensis exposed to different concentrations of phosphorus during 1-6 d;1d, 1e and 1f was phosphorus content in the cell wall, cytoplasm and cell organelle respectively in P. yezoensis exposed to different concentrations of calcium during 1-6 d.
表1 暴露于不同濃度的磷或鈣6 d后條斑紫菜中磷的亞細(xì)胞分布
圖2 暴露于不同濃度的磷或鈣1~6 d后條斑紫菜中鈣的亞細(xì)胞分布注:2a、2b、2c分別是暴露在不同磷溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中鈣含量;2d、2e、2f分別是暴露在不同鈣溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中鈣含量。Fig. 2 Subcellular distribution of Ca in P. yezoensis exposed to different concentrations of phosphorus and calcium for 1-6 dNote: 2a, 2b and 2c was Ca content in the cell wall, cytoplasm and cell organelle respectively in P. yezoensis exposed to different concentrations of phosphorus during 1-6 d; 2d, 2e and 2f was Ca content in the cell wall, cytoplasm and cell organelle respectively in P. yezoensis exposed to different concentrations of calcium during 1-6 d.
表2 暴露于不同濃度的磷或鈣6 d后條斑紫菜中鈣的亞細(xì)胞分布
2.3 在不同濃度磷、鈣條件下條斑紫菜中砷的亞細(xì)胞分布
將暴露在0.5 mg·L-1As(V)中的條斑紫菜做對照組,再在其中分別添加不同最終濃度的磷、鈣溶液,考察在磷、鈣影響下條斑紫菜對砷的富集規(guī)律、亞細(xì)胞分布特征。通過對9個(gè)不同處理液的藻體進(jìn)行分析發(fā)現(xiàn),條斑紫菜中砷的亞細(xì)胞分布與區(qū)隔化差異很大,各組分的砷含量分別為:細(xì)胞液>細(xì)胞壁>細(xì)胞器。砷的區(qū)隔化作用(compartmentalization)可以降低砷對藻體細(xì)胞新陳代謝的影響,從而保證其正常生長和發(fā)育,表3展示了條斑紫菜經(jīng)不同濃度磷、鈣暴露6 d的處理組以及對照組和空白組中,藻體不同亞細(xì)胞組分中砷的含量分布。
由表3可知,在磷暴露濃度為0.1和1.0 mg·L-1時(shí),條斑紫菜對砷的吸收效率有所提高,條斑紫菜中磷、砷之間呈現(xiàn)協(xié)同效應(yīng);在磷暴露濃度為5.0和10.0mg·L-1,條斑紫菜中磷、砷之間則呈現(xiàn)拮抗作用,砷的富集量分別是對照組(加砷0.5 mg·L-1)的37.98%和26.65%,細(xì)胞液組分的砷含量分別是對照組的25.78%和15.36%,細(xì)胞壁組分的砷含量分別是對照組的80.70%和66.34%。圖3a、3b、3c分別是暴露在不同磷溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中砷含量。在添加鈣濃度為250和500 mg·L-1時(shí),條斑紫菜中砷的富集總量與對照組(加砷0.5 mg·L-1)基本相同,其中,細(xì)胞壁組分的砷含量比對照組分別增加了35.52%和93.16%,細(xì)胞器組分中的砷含量比對照組分別增加了93.48%和117.42%,細(xì)胞液組分中砷含量分別比對照組減少了14.77%和27.6%。在添加鈣濃度為1 000 mg·L-1時(shí),條斑紫菜中砷的富集總量比對照組減少15.51%,細(xì)胞液組分中砷含量比對照組減少30.06%。圖d3、3e、3f分別是暴露在不同鈣溶液1~6 d條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中砷含量。
圖3 暴露于不同濃度的磷或鈣1~6 d后條斑紫菜中砷的亞細(xì)胞分布注:3a、3b、3c分別是暴露在不同磷溶液1~6 d后條斑紫菜藻體細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中砷含量;3d、3e、3f分別是暴露在不同鈣溶液1~6 d后條斑紫菜細(xì)胞壁、細(xì)胞液和細(xì)胞器組分中砷含量。Fig. 3 Subcellular distribution of As in P. yezoensis exposed to different concentrations of phosphorus and calcium for 1-6 dNote: 3a, 3b and 3c was As content in the cell wall, cytoplasm and cell organelle respectively in P. yezoensis exposed to different concentrations of phosphorus during 1-6 d; 3d, 3e and 3f was As content in the cell wall, cytoplasm and cell organelle respectively in P. yezoensis exposed to different concentrations of calcium during 1-6 d.
表3 暴露于不同濃度的磷或鈣6 d后條斑紫菜中砷的亞細(xì)胞分布
綜上可知:
1)無論是暴露在添加磷液或添加鈣液處理組中的條斑紫菜,其磷和鈣都主要存在其細(xì)胞壁組分中。2)加磷濃度5.0 mg·L-1處理組中條斑紫菜對砷的富集受到明顯的抑制作用。3)條斑紫菜中砷的亞細(xì)胞分布主要集中在細(xì)胞液中,其分布為細(xì)胞液>細(xì)胞壁>細(xì)胞器,胞液對砷具有非常明顯的區(qū)隔化作用。本研究結(jié)果為進(jìn)一步研究條斑紫菜胞液中砷的轉(zhuǎn)運(yùn)機(jī)制,揭示條斑紫菜的耐砷毒和砷富集機(jī)理奠定基礎(chǔ)。
致謝:感謝江蘇省紫菜協(xié)會(huì)在條斑紫菜樣品采集中給予的幫助。
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The Subcellular Fate of Phosphorus and Calcium in the Seaweed Porphyra yezoensis and Its Relationship with the Arsenic Accumulation
Shang Derong1, Ning Jinsong1, Zhao Yanfang1,*, Zhai Yuxiu1, Li Fengling1, Sheng Xiaofeng1, Ding Haiyan1, Xu Zheng1, Duan Delin2, Zhou Yongdong3
1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, National Center for Quality Supervision and Test of Aquatic Products, Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture, Qingdao 266071, China 2. Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 3. Yancheng Hairui Food Co. Ltd, Dafeng 224100, China
Received 13 November 2015 accepted 17 March 2016
The subcellular distribution of phosphorus (P) and calcium (Ca) in P. yezoensis was studied and the relationship with its high accumulation of arsenic was also discussed. The results showed that P and Ca were mainly accumulated in the cell wall, accounting for about 53.9% of total P and about 61.25% of total Ca. The second largest accumulation of P and Ca was in the cytoplasm. Compared with the control group (only treated with 0.5 mg·L-1As), the synergistic effect occurred in the uptake of P and As for groups also exposed to 0.1 and 1.0 mg·L-1P, while the antagonistic effect occurred for 5.0 and 10.0 mg·L-1P pretreatment groups, and the accumulated As decreased by 61.37% and 72.37% respectively. For the group exposed to Ca (1 000 mg·L-1), the accumulated As concentration decreased by 15.51% compared to the control group. It could be concluded that 5.0 mg·L-1P could significantly inhibit the accumulation of As in P. yezoensis.
arsenic; phosphorus; calcium; Porphyra yezoensi; subcellular localization; accumulation
農(nóng)產(chǎn)品質(zhì)量安全監(jiān)管專項(xiàng)經(jīng)費(fèi)(2011);中華人民共和國農(nóng)業(yè)行業(yè)標(biāo)(10167);南極海洋生物資源開發(fā)利用項(xiàng)目(21301352015103);江蘇省科技項(xiàng)目(BE2015335; CX(13)3095)
尚德榮(1960-),女,高級工程師,研究方向?yàn)樗a(chǎn)品質(zhì)量安全與檢測技術(shù),E-mail: shangdr@ysfri.ac.cn;
*通訊作者(Corresponding author), E-mail: zhaoyf@ysfri.ac.cn
10.7524/AJE.1673-5897.20151113002
2015-11-13 錄用日期:2016-03-17
1673-5897(2016)4-258-07
X171.5
A
簡介:趙艷芳(1979—),女,海洋生物學(xué)博士,副研究員,主要研究方向?yàn)樗a(chǎn)品質(zhì)量安全與分析技術(shù),發(fā)表學(xué)術(shù)論文40余篇。
尚德榮, 寧勁松, 趙艷芳, 等. 條斑紫菜中磷和鈣的亞細(xì)胞分布及其與富集砷的關(guān)系[J]. 生態(tài)毒理學(xué)報(bào),2016, 11(4): 258-264
Shang D R, Ning J S, Zhao Y F, et al. The subcellular fate of phosphorus and calcium in the seaweed Porphyra yezoensis and its relationship with the arsenic accumulation [J]. Asian Journal of Ecotoxicology, 2016, 11(4): 258-264 (in Chinese)