【作者】 张雯；

【导师】 林匡飞；

【作者基本信息】 华东理工大学 ， 环境科学与工程， 2014， 博士

【摘要】 镉(Cd)是一种对生物体有较强的毒性重金属,目前农产品的Cd污染情况已引起人们的广泛关注。硫(S)与硒(Se)在一定程度上均能够缓解Cd毒性。且S与Se同为氧族元素,在植物体内的吸收,同化方面会相互竞争,同时添加对Cd的解毒作用存在相互影响。因此本试验以淮稻6号为实验材料,通过根部添加S和Cd,叶面施加Se,曝毒12d,每3d取一次样,观察水稻幼苗生长状况,抗氧化系统及巯基物质的响应,S、Se与Cd在水稻幼苗体内的累积,三者的亚细胞分布以及在水稻幼苗体内的空间分布情况,以期说明在不同浓度S、Se交互作用下,对Cd毒害的影响机理。主要研究结果如下：在水稻幼苗的生长方面,随着时间的增加,升高Cd浓度对水稻幼苗生物量的抑制更加显著,并导致叶绿素含量和根系活力的下降。叶面施Se和供S均显著缓解了Cd对水稻幼苗生长的抑制,促进了叶绿素的合成和根系活力的升高。高S情况下,叶面施Se对水稻幼苗Cd毒害并无显著的抑制,说明此时S与Se相互竞争,从而未产生进一步的协同作用。在抗氧化系统酶和巯基物质的响应方面,Cd浓度越高,水稻幼苗体内丙二醛(MDA)含量越高,抗氧化酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)的响应程度越高。诱导合成谷胱甘肽(GSH)、植物螯合肽(PCs)和金属硫蛋白(MT)等巯基化合物越多,从而能够缓解Cd对水稻幼苗的毒害。无S时叶面施Se以及单独供S均降低了MDA含量,降低了抗氧化酶活性。无S条件下,相对于未施Se,叶面施Se显著促进了GSH、PCs和MT的合成,说明在无S时,Se对Cd的解毒机制是Se代替S主要是Se替代了S形成了Se-代半胱氨酸,从而合成了相关螯合物。而在高S情况下,叶面施Se并未进一步促进巯基物质的合成,主要是因为S抑制了Se的吸收,使得此时S起到对Cd的解毒作用。同时表明了S与Se对Cd的解毒机制并非提高抗氧化酶的响应,而是促进了巯基物质的合成。在水稻幼苗体内的Cd、Se累积方面,叶面施Se显著降低了Cd在水稻幼苗根部和地上部的含量。而Cd浓度越高,越发促进了水稻幼苗对Se的吸收,这也是水稻幼苗自我保护的一种方式。单独施S时,随着供S浓度的增加,Cd在水稻幼苗根部的累积量下降,而茎部的Cd含量增加,说明S抑制了水稻根部对Cd的吸收,促进了Cd向茎部转移,主要机制是S促进了PCs等螯合蛋白的合成,能够在水稻幼苗体内进行长距离运输。高S条件下,施Se抑制了Cd向茎部的运输。而不同S浓度下水稻幼苗体内Se含量则显示,高S浓度对Se的吸收有最为显著的抑制作用,因此高S条件下,对Cd毒害起主要作用的是S。在水稻幼苗体内的亚细胞分布和化学形态方面,细胞壁组分所占比例均为Cd10>Cdl(Cd10代表浓度为10mg/L的Cd,下同)。随着S的增加以及叶面施Se可溶性组分中Cd的比例有所升高,表明S与Se的供应能促进Cd向液泡转移。氯化钠提取态所占比例趋势为S720>S48>S0、Se500>Se0,而乙醇和去离子水等活性较高的提取态所占比例有所下降,说明S的添加以及叶面施Se能够促使Cd向活性较低的提取态转移。然而相对于无S,在高浓度S情况下,施Se并未显著增加Cd在液泡中以及FNaCl提取态的比例,推测S与Se之间相互竞争,从而未产生进一步的协同作用。且在S与Se的亚细胞分布情况可以看出,高S时,S促进Cd向液泡中转移以降低其毒害。利用SRXRF对S、Se、Cd在水稻幼苗根部与叶片的空间扫描可以看出,在无S条件下,Se对Cd毒性的缓解主要通过促进蛋白的合成,从而络合Cd离子,达到减轻Cd毒害的目的。在高S条件下,S与Se相互竞争,S与Cd的分布趋于一致,说明在此时S起到缓解Cd毒害的主要作用。此外,在不同Se、Cd处理下,对比无S与6d后添加高S两组处理,考察延时加S对水稻幼苗吸收Se、Cd的和MT含量的影响。实验结果显示,无添加S组,Se、Cd交互作用对水稻生物量的影响较大,第6d加入S则缓解了这一现象。高浓度Cd对水稻的毒害作用较低浓度Cd更加明显,Se、Cd在水稻幼苗的累积随时间而增加,而S的添加使Cd毒害减轻,并抑制了Se、Cd在水稻幼苗体内的累积。S的添加使得水稻幼苗体内MT含量较无S条件增加,且由于在无S条件下,MT也可产生,推测Se的存在会代替S合成MT。综上所述,S、Se交互对水稻幼苗Cd毒害的主要机制是,形成巯基物质(GSH、 PCs以及MT),以螯合Cd离子,从而减轻Cd的自由移动,缓解Cd毒害。此外,S与Se促进Cd向液泡转移,以隔离Cd离子,减轻其对水稻幼苗的毒害。无S时,Se也可促进巯基物质的形成,以降低Cd毒害；而高S时,由于S和Se的相互竞争,S对Se的抑制较为显著,因此S起到缓解Cd毒害的主要作用。更多还原

【Abstract】 Cadmium (Cd) has strong toxicity to organisms, and has been widely followed. Sulfur (S) and selenium (Se) are able to alleviate Cd toxicity. S and Se belong to the same groups, therefore their absorption and assimilation in plants are similar with each other, moreover will compete with each other. Therefore, the experiments were carried on by using NO6Huaidao as material, by adding S and Cd in root, foliar applied Se, exposure for12d, sampled every3d to observe the rice seedling growth, antioxidant system response and thiol compounds substances, S、Se and Cd accumulation in rice seedlings, subcellular distribution and three in the space distribution of rice seedlings in order to illustrate the different concentrations of S, Se detoxification mechanism of Cd. The main results are as follows:In the growth of rice seedlings, as time increases, increasing Cd concentration on the inhibition of rice seedlings biomass is more significant and lead to decreased chlorophyll content and root activity. Foliar applied for Se and S supply were significantly alleviated Cd toxicity on rice seedlings, and promote the synthesis of chlorophyll and root activity. In high S treatment, foliar applied Se has no significant inhibition on Cd toxicity, indicating that competition at S and Se, which did not produce further synergies.In the antioxidant enzyme system response and sulfhydryl compounds respects, the higher Cd concentration, the higher malondialdehyde (MDA) content and antioxidant enzyme superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) activitys in rice seedlings. And Induce the synthesis of glutathione (GSH), phytochelatins (PCs) and metallothionein (MT), more thiol compounds synthesized, which can alleviate Cd toxicity on rice seedlings. Foliar applied Se without and S supply decreased the MDA content and the activity of antioxidant enzymes. with the no S conditions, compared with no Se, foliar applied Se significantly promote the synthesis of GSH, PCs and MT, indicating that in the absence of S, Se detoxification mechanism on Cd is replaced S to mainly form substituting Se-cysteine, which was synthesized correlation chelate thiol compound. In the treatment of high S, Se can not further promote the synthesis of thiod compounds, because Se absorptionhas been restrained by S, thereby S played the key role on Cd detoxification. Moreover, Also showed that the S and Se detoxification mechanism on Cd is not to improve the response of antioxidant enzymes, but to promote the synthesis of thiol compound substances.In the Cd, Se accumulation, the foliar applied Se significantly reduced the Cd content in the roots and shoots of rice seedlings. The higher the concentration of Cd, more rice seedlings to promote the absorption of Se, which is a way of self-protection of rice seedlings. When S applied alone, with the increase of S concentration, Cd accumulation in seedling in rice roots decreased, while increased, indicating that S inhibits the absorption of Cd in rice roots, and promote Cd transfer to shoot, the main mechanisms to promote the synthesis of PCs and other S chelating proteins can be transported over long distances in rice seedlings. Under conditions of high S, Se inhibited Cd transport facilities to the shoot.In the subcellular distribution and chemical forms of Cd using hydroponic condition containing Cd and/or S, when spraying Se on the overground part. And we used differential centrifugal technology and chemical reagent step-by-step extraction method to get different part of cell and different chemical forms. The results showed that proportion of Cd in cell wall fraction under Cd10treatment is larger than Cdl. And with the increase of S and spraying Se on the overground part, the proportion of Cd in soluble fraction increased while decreased in cell wall indicating that the supply of S and Se could promote Cd to translocate to the vacuole. Also with the supply of S and Se, the percentage of NaCl-extraction Cd form followed the rule: S720>S48>S0, Se500>Se0, while the proportion of ethanol-extraction Cd form decreased illustrating that the existence of S and Se could promote Cd to transfer to the less active forms. However, compared to the treatment without S, Se supply did not significantly increase the proportion of Cd in the vacuole and NaCl-extraction under high S concentration indicating that S and Se competed with each other, and can not emerge synergism. Hence, from the subcellular distribution of S and Se, illustrated that in high S condition, S promote Cd transfer to the vacuole to reduce its toxicity.Using SRXRF for spatial distrbution of S, Se, Cd in rice seedlings. In the conditions without S, Se mitigated Cd toxicity mainly by promoting protein synthesis, to complex Cd ions, to reduce Cd toxicity. Under conditions of high S, S and Se compete with each other, the distribution of S and Cd are similar, indicating S played the major role to alleviate Cd toxicity.In addition, Under hydroponic conditions, rice "NO.6" were used to observe the effect of metallothionein in rice seedling and adsorption of selenium(Se) and cadmium(Cd) under the interaction of Se and Cd with deferred sulfur(S) addition (without and deferred S addition). The results showed that rice seedling without S got more pernicious effect under the interaction of Se and Cd compared to S addition on day6. The toxicity on rice with10mg/L of Cd was higher than1mg/L of Cd. Accumulation of Se and Cd increased as time went. However, the addition of S mitigated Cd toxicity and restrained the accumulation of Se and Cd in rice seedlings. The results also confirmed that S addition led to the significantly increase of metallothionein(MT) in rice seedling, moreover MT could be produced without S, speculating that the existence of Se can substitute S to synthesize MT.In summary, the main mechanism of S, Se interactive on Cd detoxification in rice seedlings is forming sulfhydryls (GSH, PCs and MT), to chelate Cd ions, thereby reducing the free movement of Cd, and alleviate Cd toxicity. In addition, S and Se could promote Cd transfer to vacuole, Cd ions has been in isolation, then its toxicity be reduced. Without S, Se also promote the formation of thiol substances; while high S, due to the competition between S and Se, S restrained Se absorption, thus S plays the major role in alleviating Cd toxicity.更多还原