|Alternative Title||Effect of manganese as well as expression and function of OsNRAMP5 on Cd uptake in rice|
|Thesis Advisor||陈卫平 ; 王美娥|
|Place of Conferral||北京|
|Keyword||锰，镉， Osnramp5，转运蛋白基因，转录组 Manganese, Cadmium, Osnramp5, Transporter Gene, Rna-seq|
锰元素（Mn manganese）是植物生长发育 的 必需元素之一，且对水稻镉（ Cd cadmium）的吸收累积有一定的调控作用。 OsNRAMP5是 水稻中重要的 Mn和 Cd转运蛋白， 通过敲除 OsNRAMP5基因可显著降低稻米的 Cd累积。 然而 OsNRAMP5的 转录水平 表达 量 是否与 Cd累积 程度 相关， 其表达 是否受外界 Mn供 应 水平 调控尚未明确。本研究以田间高污染区具有 Cd累积差异的四个常规籼稻品种、 OsNRAMP5敲除品种及其野生型对照为研究对象，采用螯合剂 -缓冲营养液体系，从生理特征、元素吸收、相关转运蛋白基因转录水平表达情况及转录组学 等方面 ，系统研 究了 Mn元素及 OsNRAMP5的表达及功能 对水稻 Cd元素的吸收和累积的影响。主要研究成果如下：
（1 )揭示了外源 Mn供应水平对水稻生长的影响及品种响应差异。本研究通过量
（2 )探讨了水稻 Mn供应水平 -Cd累积浓度 -OsNRAMP5表达量三者间 的 相互作用关系。本研究测定了四个常规籼稻品种地上、地下部分的 Mn和 Cd累积浓度及相应 组织 的 OsNRAMP5表达量。结 果显示， 受试水稻品种地上、地下部分的 OsNRAMP5表达量受外界 Mn2+活度 显著 影响，且地下部分的 OsNRAMP5表达量在 Mn毒害水平显著上调。同时，在不同 Mn2+活度下 部分受试品种的 OsNRAMP5表达量与 Mn累积浓度正相关 而 对于 不同品种 而言，各品种间 地下部分 的 OsNRAMP5表达量与 Mn累积浓度之间 仅 在 Mn充足水平下存在显著的正相关关系。四个常规籼稻品种的地下部分 Cd累积浓度均在 Mn毒害情况下显著降低，但地上部分 Cd累积浓度在不同 Mn2+活度下无显著变化。同时，OsNRAMP5的表达量并不能直接反映水稻的 Cd累积程度。
（3）探讨了 OsNRAMP5基因的敲除在不同 Mn供给水平下对水稻 Cd吸收和累积的影响。本研究系统地测定了 OsNRAMP5敲除品种及其对应野生型在不同 Mn2+活度下的 Mn和 Cd累积浓度特征、 Cd吸收速率、相关 Cd转运基因在转录水平的表达量、相关矿质营养元素的吸收及其转运基因在转录水平的表达量。 研究结果表明， OsNRAMP5基因的敲除显著降低了水稻 Mn元素的吸收，同时抑制了 Mn元素向地上部分的转运。同时 在 Mn充足时 OsNRAMP5敲除品种显著降低地上部分和地下部分的 Cd累积浓度 但在临界 Mn缺乏条件下， OsNRAMP5敲除品种地上部分 Cd累积浓度显著升高。OsNRAMP5敲除显著影响 Cd相关的转运蛋白 基因的表达 ，其中，地下部分 OsHMA2基因的上调 导致 Cd转移系数显著 升高 。同时， OsNRAMP5基因的敲除会显著影响 其他矿
（4 挖)掘了 OsNRAMP5敲除和 Mn2+活度对水稻生长及 Cd吸收、累积影响的分子机制。本研究基于 OsNRAMP5敲除品种及其对应野生型在 Mn缺乏 0.1 μM, pMn2+ 7.72 、 Mn完全充足 10 μM, pMn2+ 5.73 和 Mn毒害 100 μM, pMn2+ 4.79 三个 Mn供应水平的转录组数据，进行了趋势分析、 GO富集分析及 KEGG代谢途径分析，以探究 OsNRAMP5的敲除在不同 Mn供应水平下的分子机制。 结果表明， 相对于野生型 WT突变体 T210产生更多 差异表达基因，且 在 GO功能富集和 KEGG代谢途径分析中注释到更多的信息，说明 OsNRAMP5基因的敲除使水稻对 外界 Mn2+的供给更敏感。苯丙素合成途径（木质素合成途 径）和二萜类物质的生物合成途径（赤霉素合成途径）在 KEGG代谢途径分析中显著。结果显示， OsNRAMP5的敲除促进了木质素 单体的 合成 与聚合同时 在 Mn缺乏处理时， 突变体 T210木质素的过量累积 加重了 缺 Mn导致的 生长受限。木质素的聚合过程基因 OsPOD在不同 Mn2+活度下的变化与对应的 Cd转移系数变化一致。 外源 Mn2+活度和 OsNRAMP5的敲除均在 不同程度上影响了赤霉素合成相关酶基因的表达，其中，在缺 Mn时突变体 T210赤霉素合成相对较低导致的生物量的浓缩效应是 造成地上部分 Cd累积量激增的原因之一。 因此， 可在后续研究中对木质素 含量及单体组成比例 和赤霉素含量测定加以验证。
Manganese (Mn) is an essential nutrient element for plant growth and development, besides, it has been reported to reduce Cd uptake and accumulation in rice. OsNRAMP5 is an important transporter for Mn and Cd in rice. Knockout the OsNRAMP5 gene is an effective way to reduce Cd accumulation in rice grains. However, relationship between the expression level of OsNRAMP5 and Cd accumulation as well as the influence of external Mn2+ activity on OsNRAMP5 expression are still unknown. In this study, four indica rice with different Cd accumulation in high Cd contaminated paddy soil, as well as, an OsNRAMP5 knockout line and its wild type were used as the tested rice cultivars.In this study, rice were cultured in chelating agent buffer solution system. Indicators of agronomic characteristics, elements absorption, gene expression, and transcriptome were measured to explore the effect of Mn as well as expression and function of OsNRAMP5 on Cd uptake and accumulation in rice. The main results are as follows:
(1) To explore the effect of Mn on rice growth and developments, we measured the agronomic traits, photosynthesis parameters, antioxidant system and absorption of mineral nutrients to reveal the effect of external of Mn2+ activity. Results showed that rice grew under pMn2+ 7.72 treatment (pMn2+ refer to -log10(free Mn activity), 0.1 μM MnCl2) was diagnosed as Mn marginal deficiency, under pMn2+ 7.22 (0.32 μM MnCl2) was marginal Mn sufficiency, under pMn2+ 5.73 (10 μM MnCl2) was fully Mn sufficiency, and under pMn2+ 4.79 (100 μM MnCl2) was Mn phytotoxicity. For most cultivars, activities of ascorbate peroxidase (APX), catalase activity (CAT), and superoxide dismutase (SOD) were up-regulated under Mn marginal deficiency to relieve the stress of reactive oxygen species (ROS), while activities of CAT and SOD were up-regulated under Mn phytotoxicity. The content of proline and soluble protein under lack or excess of Mn were increased to improve resistance to Mn imbalance for some cultivars. Results in this study also showed that malonaldehyde (MDA) content was more likely to accumulate under Mn phytotoxicity level than Mn marginal deficiency. Fe, Zn, and Cu concentration of both shoots and roots were significantly affected at Mn marginal deficiency and Mn phytotoxicity. The increase of Fe, Zn, and Cu in shoots may due to the increase of SOD activities and compensation of the decreased net photosynthetic rate.
(2) Base on the concentration of Mn and Cd in roots and shoots, as well as OsNRAMP5 expression in the corresponding part of the four indica rice, the relationship among Mn accumulation, Cd accumulation as well as OsNRAMP5 expression was investigated. Results showed that the expression level of OsNRAMP5 in shoots and roots of the tested cultivars were affected by the external Mn2+ activity, and the expression of OsNRAMP5 in roots were significantly up-regulated at the Mn toxicity level. At the same time, the OsNRAMP5 expression levels of some tested cultivars were positively correlated with Mn accumulation concentration under different Mn2+ activities, while there was a significant positive correlation between OsNRAMP5 expression level and Mn concentration among different cultivars under Mn fully sufficiency condition. Cd concentration in the roots of four indica rice decreased significantly under Mn toxicity, while Cd concentration in the shoots did not change significantly under different Mn2+ activities. Meanwhile, the expression level of OsNRAMP5 could not reflect the degree of Cd accumulation in rice directly.
(3) Indicators such as, concentration of Mn, Cd and other nutrient elements, uptake ratio of Cd, expression level of genes which involves in Cd uptake and transport, were measured of OsNRAMP5 knockout line(T210) and its wild type (WT) under different external Mn2+ activity. Results showed that the deletion of OsNRAMP5 gene significantly reduced the absorption of Mn in rice and inhibited the translocation of Mn from roots to shoots. Meanwhile, Cd concentration of Cd in roots and shoots of T210 was decreased significantly under Mn fully sufficiency, but shoot Cd concentration of T210 was significantly increased under Mn deficiency. Knockout of OsNRAMP5 significantly affected the expression level of genes related to Cd transport and translocation. Among them, the upregulation of OsHMA2 in roots may account for the high transfer ratio of Cd under Mn deficiency. Besides, knockout of OsNRAMP5 gene also affected the absorption of other mineral elements, such as Fe, Zn, and Cu, as well as genes expression related to transporters of those elements. The degree of influence was related to the external Mn2+ activity.
(4) To explore the molecular mechanisms of OsNRAMP5 knockout and external Mn2+ activity on Cd accumulation in rice, trend analysis, GO analysis, and KEGG analysis were conducted in the results of RNA-seq of OsNRAMP5 knockout line and its wild type under Mn deficiency(0.1 μM, pMn2+ 7.72), Mn sufficiency(10 μM, pMn2+ 5.73) and Mn phytotoxicity(100 μM, pMn2+ 4.79). Results in this study showed that the response trends of WT and T210 under different external Mn2+ activities were different. T210 was more sensitive to external Mn2+ activity, especially under Mn deficiency, for more genes changed of T210 compared to that of WT on both analysis of GO and KEGG. Phenylpropanoid biosynthesis (biosynthesis of lignin) and diterpenoid biosynthesis (biosynthesis of gibberellin) were significant in KEGG metabolic pathway analysis. The results showed that the knockout of OsNRAMP5 may promote the synthesis of lignin, and the excessive accumulation of lignin in the absence of Mn in T210 may lead to the growth restriction of the mutant. Meanwhile, the expression of OsPOD which involves in the process of lignin polymerization under different Mn2+ activities were consistent with the transfer ratio of Cd. Expression of genes related to gibberellin biosynthesis were affected by external Mn2+ activity and knockout of OsNRAMP5. Among them, the concentration effect of biomass caused by relatively low gibberellin biosynthesis of T210 under Mn deficiency could account for the high Cd accumulation in shoots.
In conclusion, the expression level of OsNRAMP5 could not reflect the degree of Cd accumulation in rice directly. Thus, we should pay attention to the protein expression level of OsNRAMP5 in future study. Meanwhile, knockout of OsNRAMP5 significantly decreased the absorption and accumulation of Cd in rice, however, the transfer ratio of Cd significantly increased under Mn extremely deficiency condition. Thus, exogenous Mn fertilizer should apply together with the application of OsNRAMP5 knockout cultivars depend on the Mn nutrient status in soil. External Mn2+ supply and knockout of OsNRAMP5 greatly affected genes which involves in transporters of Cd and related nutrient elements, lignin biosynthesis as well as gibberellin biosynthesis. Therefore, the combined effects of multiple factors on Cd accumulation should be consider in future study.
|蔡怡敏. 锰供应和转运蛋白 OsNRAMP5表达及功能对水稻镉吸收的影响机制[D]. 北京. 中国科学院生态环境研究中心,2019.|
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