微囊藻毒素MC-LR在水稻中積累和轉(zhuǎn)運(yùn)的初步研究
發(fā)布時間：2019-01-01 19:59
【摘要】：太湖藍(lán)藻水華引發(fā)嚴(yán)重的水體微囊藻毒素污染。微囊藻毒素-LR(MC-LR)是普遍存在且毒性最強(qiáng)的一類微囊藻毒素。依賴于太湖水體灌溉的水稻是我國南方水稻產(chǎn)區(qū)的重要組成部分。雖然MC-LR在動物細(xì)胞中轉(zhuǎn)運(yùn)的分子機(jī)制已很明確,但目前關(guān)于MC-LR在植物中的積累機(jī)制及其安全風(fēng)險評估鮮有報道。本文以水稻為材料,首先研究了其對MC-LR的累積量與毒性反應(yīng)之間的相關(guān)性；然后檢測了太湖周邊稻田水稻籽粒中的MC-LR的累積量,并進(jìn)行了食品安全風(fēng)險評估；最后從水稻體內(nèi)克隆了首個潛在的植物MC-LR轉(zhuǎn)運(yùn)蛋白基因,并進(jìn)行了初步的功能驗證。第一,MC-LR在水稻中累積與毒性反應(yīng)的相關(guān)性研究。采用不同濃度MC-LR(0-5 μg/mL)處理水稻種子和幼苗。結(jié)果顯示：高濃度MC-LR顯著抑制種子萌發(fā),這與淀粉酶活性的顯著下降密切相關(guān)。MC-LR能夠在水稻幼苗體內(nèi)顯著累積；中低濃度處理下(0.05-1 μg/mL),MC-LR在水稻體內(nèi)的累積量與幼苗生長成顯著負(fù)相關(guān),與氧化損傷相關(guān)指標(biāo)(MDA、POD、CAT、SOD)呈顯著正相關(guān)。由此推斷,吸收到水稻幼苗體內(nèi)的MC-LR很可能通過引起細(xì)胞氧化損傷反應(yīng)進(jìn)而導(dǎo)致生長受阻。第二,MC-LR在水稻籽粒中累積的食品安全風(fēng)險評估。從太湖周邊采集44個水稻籽粒樣品,并采用ELISA檢測其中的MC-LR含量。結(jié)果顯示,共有21個樣品檢測出MC-LR(0.04-3.19 μg/kg DW),檢出率為48%。風(fēng)險評估結(jié)果顯示,每天食用這些大米所攝入的MC-LR未超過WTO規(guī)定的MC-LR安全限量標(biāo)準(zhǔn)。這說明自然水體中的MC-LR能夠在水稻籽粒中積累,存在一定的生態(tài)風(fēng)險,但目前太湖周邊水稻中MC-LR的累積量還相對較低。可能源于兩個原因：一方面,太湖水體中的MC-LR濃度不足以引起其在水稻籽粒中的大量積累；另一方面,水稻通過根-莖-籽粒,逐步進(jìn)行MC-LR的吸收和累積,長距離運(yùn)輸可能降低了MC-LR在籽粒中的積累效率。第三,水稻中潛在的MC-LR轉(zhuǎn)運(yùn)蛋白基因OsOATPM的克隆與分析。以動物已報道的MC-LR轉(zhuǎn)運(yùn)蛋白基因為檢索靶標(biāo),從水稻基因組中檢索到同源的類似基因LOC_Os04g44430,并將其命名為OsOATPMo通過RT-PCR克隆到該基因全長cDNA(1794 bp),其上游存在豐富的轉(zhuǎn)錄調(diào)控元件。序列分析預(yù)測顯示OsOATPM蛋白屬于典型的跨膜蛋白,具備運(yùn)輸多肽的功能。將OsOATPM基因在大腸桿菌(Escherichia coli,E. coli)中進(jìn)行原核表達(dá)并進(jìn)行功能分析,發(fā)現(xiàn)OsOATPM很可能介導(dǎo)了MC-LR的外排運(yùn)輸功能。雖然該蛋白的具體功能還需要進(jìn)一步的遺傳學(xué)驗證,但本文的探索性研究為MC-LR在水稻中累積的食品安全風(fēng)險評估提供了重要的理論依據(jù)。
[Abstract]:Cyanobacteria Shui Hua in Taihu Lake caused serious microcystin pollution in water. Microcystin-LR (MC-LR) is a common and most toxic microcystins. Rice, which depends on water irrigation in Taihu Lake, is an important part of rice growing area in southern China. Although the molecular mechanism of MC-LR transport in animal cells has been clarified, there are few reports on the accumulation mechanism of MC-LR in plants and its safety risk assessment. In this paper, the correlation between the accumulation of MC-LR and the toxicity of rice was studied firstly, then the accumulation of MC-LR in rice grains around Taihu Lake was detected, and the food safety risk assessment was carried out. Finally, the first potential plant MC-LR transporter gene was cloned from rice. First, the correlation between MC-LR accumulation and toxicity in rice. Rice seeds and seedlings were treated with different concentrations of MC-LR (0-5 渭 g/mL). The results showed that high concentration of MC-LR significantly inhibited seed germination, which was closely related to the decrease of amylase activity. MC-LR could accumulate significantly in rice seedlings. Under medium and low concentration (0.05-1 渭 g/mL), the accumulation of MC-LR in rice was negatively correlated with seedling growth and positively correlated with oxidative damage related index (MDA,POD,CAT,SOD). It is inferred that the MC-LR absorbed into rice seedlings may result in growth retardation by causing oxidative damage to the cells. Second, the food safety risk assessment accumulated by MC-LR in rice grains. 44 rice grain samples were collected from the periphery of Taihu Lake and the MC-LR content was detected by ELISA. The results showed that the detection rate of MC-LR (0.04-3.19 渭 g/kg DW),) was 48 in 21 samples. The risk assessment showed that the daily intake of MC-LR from the rice did not exceed the MC-LR safety limit set by the WTO. This indicated that MC-LR in natural water could accumulate in rice grains, and there was a certain ecological risk, but the accumulation of MC-LR in rice around Taihu Lake was relatively low at present. On the one hand, the concentration of MC-LR in Taihu Lake is not enough to cause the accumulation of MC-LR in rice grains; On the other hand, rice gradually absorbs and accumulates MC-LR through root-stem and kernels. Long distance transportation may decrease the accumulation efficiency of MC-LR in grains. Thirdly, the cloning and analysis of OsOATPM, a potential MC-LR transporter gene in rice. Based on the MC-LR transporter gene reported in animals, the homologous gene LOC_Os04g44430, was obtained from rice genome and named OsOATPMo by RT-PCR and cloned into the full-length cDNA (1794 bp),) of the gene (1794 bp),). There are abundant transcriptional regulatory elements upstream. Sequence analysis and prediction showed that OsOATPM protein was a typical transmembrane protein with the function of transporting polypeptides. The prokaryotic expression and functional analysis of OsOATPM gene in E. coli (Escherichia coli,E. coli) showed that OsOATPM probably mediated the efflux transport function of MC-LR. Although the specific function of the protein needs further genetic verification, the exploratory study in this paper provides an important theoretical basis for the assessment of food safety risk accumulated by MC-LR in rice.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X52;X173;S511
本文編號：2398050
[Abstract]:Cyanobacteria Shui Hua in Taihu Lake caused serious microcystin pollution in water. Microcystin-LR (MC-LR) is a common and most toxic microcystins. Rice, which depends on water irrigation in Taihu Lake, is an important part of rice growing area in southern China. Although the molecular mechanism of MC-LR transport in animal cells has been clarified, there are few reports on the accumulation mechanism of MC-LR in plants and its safety risk assessment. In this paper, the correlation between the accumulation of MC-LR and the toxicity of rice was studied firstly, then the accumulation of MC-LR in rice grains around Taihu Lake was detected, and the food safety risk assessment was carried out. Finally, the first potential plant MC-LR transporter gene was cloned from rice. First, the correlation between MC-LR accumulation and toxicity in rice. Rice seeds and seedlings were treated with different concentrations of MC-LR (0-5 渭 g/mL). The results showed that high concentration of MC-LR significantly inhibited seed germination, which was closely related to the decrease of amylase activity. MC-LR could accumulate significantly in rice seedlings. Under medium and low concentration (0.05-1 渭 g/mL), the accumulation of MC-LR in rice was negatively correlated with seedling growth and positively correlated with oxidative damage related index (MDA,POD,CAT,SOD). It is inferred that the MC-LR absorbed into rice seedlings may result in growth retardation by causing oxidative damage to the cells. Second, the food safety risk assessment accumulated by MC-LR in rice grains. 44 rice grain samples were collected from the periphery of Taihu Lake and the MC-LR content was detected by ELISA. The results showed that the detection rate of MC-LR (0.04-3.19 渭 g/kg DW),) was 48 in 21 samples. The risk assessment showed that the daily intake of MC-LR from the rice did not exceed the MC-LR safety limit set by the WTO. This indicated that MC-LR in natural water could accumulate in rice grains, and there was a certain ecological risk, but the accumulation of MC-LR in rice around Taihu Lake was relatively low at present. On the one hand, the concentration of MC-LR in Taihu Lake is not enough to cause the accumulation of MC-LR in rice grains; On the other hand, rice gradually absorbs and accumulates MC-LR through root-stem and kernels. Long distance transportation may decrease the accumulation efficiency of MC-LR in grains. Thirdly, the cloning and analysis of OsOATPM, a potential MC-LR transporter gene in rice. Based on the MC-LR transporter gene reported in animals, the homologous gene LOC_Os04g44430, was obtained from rice genome and named OsOATPMo by RT-PCR and cloned into the full-length cDNA (1794 bp),) of the gene (1794 bp),). There are abundant transcriptional regulatory elements upstream. Sequence analysis and prediction showed that OsOATPM protein was a typical transmembrane protein with the function of transporting polypeptides. The prokaryotic expression and functional analysis of OsOATPM gene in E. coli (Escherichia coli,E. coli) showed that OsOATPM probably mediated the efflux transport function of MC-LR. Although the specific function of the protein needs further genetic verification, the exploratory study in this paper provides an important theoretical basis for the assessment of food safety risk accumulated by MC-LR in rice.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X52;X173;S511
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相關(guān)期刊論文 前2條
1 慕小倩;何紅花;董志剛;;2種雜草水提液對小麥種子萌發(fā)及幼苗生長的影響[J];西北植物學(xué)報;2008年06期
2 S. K. MEHTA;;Protective Role of Ca Against NaCl Toxicity in Jerusalem Artichoke by Up-Regulation of Antioxidant Enzymes[J];Pedosphere;2008年06期
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