微囊藻毒素MC-LR在水稻中积累和转运的初步研究
发布时间：2019-01-01 19:59
【摘要】：太湖蓝藻水华引发严重的水体微囊藻毒素污染。微囊藻毒素-LR(MC-LR)是普遍存在且毒性最强的一类微囊藻毒素。依赖于太湖水体灌溉的水稻是我国南方水稻产区的重要组成部分。虽然MC-LR在动物细胞中转运的分子机制已很明确,但目前关于MC-LR在植物中的积累机制及其安全风险评估鲜有报道。本文以水稻为材料,首先研究了其对MC-LR的累积量与毒性反应之间的相关性；然后检测了太湖周边稻田水稻籽粒中的MC-LR的累积量,并进行了食品安全风险评估；最后从水稻体内克隆了首个潜在的植物MC-LR转运蛋白基因,并进行了初步的功能验证。第一,MC-LR在水稻中累积与毒性反应的相关性研究。采用不同浓度MC-LR(0-5 μg/mL)处理水稻种子和幼苗。结果显示：高浓度MC-LR显著抑制种子萌发,这与淀粉酶活性的显著下降密切相关。MC-LR能够在水稻幼苗体内显著累积；中低浓度处理下(0.05-1 μg/mL),MC-LR在水稻体内的累积量与幼苗生长成显著负相关,与氧化损伤相关指标(MDA、POD、CAT、SOD)呈显著正相关。由此推断,吸收到水稻幼苗体内的MC-LR很可能通过引起细胞氧化损伤反应进而导致生长受阻。第二,MC-LR在水稻籽粒中累积的食品安全风险评估。从太湖周边采集44个水稻籽粒样品,并采用ELISA检测其中的MC-LR含量。结果显示,共有21个样品检测出MC-LR(0.04-3.19 μg/kg DW),检出率为48%。风险评估结果显示,每天食用这些大米所摄入的MC-LR未超过WTO规定的MC-LR安全限量标准。这说明自然水体中的MC-LR能够在水稻籽粒中积累,存在一定的生态风险,但目前太湖周边水稻中MC-LR的累积量还相对较低。可能源于两个原因：一方面,太湖水体中的MC-LR浓度不足以引起其在水稻籽粒中的大量积累；另一方面,水稻通过根-茎-籽粒,逐步进行MC-LR的吸收和累积,长距离运输可能降低了MC-LR在籽粒中的积累效率。第三,水稻中潜在的MC-LR转运蛋白基因OsOATPM的克隆与分析。以动物已报道的MC-LR转运蛋白基因为检索靶标,从水稻基因组中检索到同源的类似基因LOC_Os04g44430,并将其命名为OsOATPMo通过RT-PCR克隆到该基因全长cDNA(1794 bp),其上游存在丰富的转录调控元件。序列分析预测显示OsOATPM蛋白属于典型的跨膜蛋白,具备运输多肽的功能。将OsOATPM基因在大肠杆菌(Escherichia coli,E. coli)中进行原核表达并进行功能分析,发现OsOATPM很可能介导了MC-LR的外排运输功能。虽然该蛋白的具体功能还需要进一步的遗传学验证,但本文的探索性研究为MC-LR在水稻中累积的食品安全风险评估提供了重要的理论依据。
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X52;X173;S511
本文编号：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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X52;X173;S511
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