CN108593566A - 基于纳米金spr光学特性评估玉米品种耐旱性的方法 - Google Patents
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Abstract
本发明公开了一种基于纳米金SPR光学特性评估玉米品种耐旱性的方法，包括玉米幼苗的胁迫处理、玉米叶片水提液的制备、纳米金体系的制备、抗氧化活性的检测、玉米耐旱性评估等步骤。本发明将纳米材料应用于玉米品种的耐旱性评估，拓展了纳米材料的应用领域；与传统的玉米耐旱性评估方法检测保护酶活性、大田实验相比，本方法利用玉米在胁迫条件下总抗氧化活性评估玉米的耐旱性，具有简便易行、准确快捷的特点，且方法实施过程不含毒性物质，绿色环保。
Description
技术领域
本发明涉及一种评估玉米品种耐旱性的方法。
背景技术
玉米是我国关系国计民生的主要粮食作物，也是对水分较敏感的作物，干旱是限制我国玉米生产和发展的主要因素。伴随着温室效应而来的气候变暖、干旱加剧，每年我国约有40%的玉米种植地受到干旱影响而减产20%-30%。因此，可耐旱的玉米品种在农业生产中显得越来越重要。目前，评估玉米品种耐旱性的方法除了大田实验，主要集中在对形态指标和保护酶活性的检测，例如相对含水量、超氧化物歧化酶SOD活性、过氧化物酶POD活性等的检测，结果虽然可靠但方法复杂、耗时，因而建立一种准确、快速、便捷的玉米品种耐旱评估方法具有重要意义。
玉米遭遇干旱胁迫时，细胞内会产生大量活性氧自由基，引发活性氧自由基代谢失衡，累积的自由基会攻击生物大分子，造成DNA损伤，影响蛋白质的稳定与合成，导致代谢异常，对植物造成不可逆伤害。而现有研究表明，耐旱性强的玉米品种遭受干旱胁迫后，自由基代谢调控能力要明显高于耐旱性弱的品种，受到的胁迫伤害程度相对较轻。由此可知，玉米的耐旱性与其在胁迫条件下自由基代谢调控能力呈正相关，而植物的总抗氧化活性能够反映其自由基代谢调控能力，因而可通过检测干旱胁迫后玉米的总抗氧化活性评估不同玉米品种的耐旱性。
金属纳米材料是指在三维空间中至少有一维处于纳米尺寸(0.1-100 nm)或由它们作为基本单元构成的金属材料。光作用于金属纳米材料时，由于导带电子的集体振荡，会在特定的波长范围出现较强的表面等离子体共振（SPR）吸收峰，吸收峰的位置和强弱取决于纳米颗粒的尺寸、形状、聚集状态和组成等参数。基于金属纳米材料的这一特性，利用植物提取液中抗氧化活性物质具有还原作用、能够介导纳米金的合成，根据纳米金形成前后SPR光学性质的变化对植物的总抗氧化活性进行检测。目前已有大量利用纳米材料检测植物总抗氧化活性的相关研究，包括茶叶、蔬菜、果汁、中药等，主要集中在食品检测领域，而将抗氧化活性与植物耐旱性关联，对作物耐旱性进行评估的研究至今尚无报道。
发明内容
本发明的目的在于提供一种建立一种简便易行、准确快捷的基于纳米金SPR光学特性评估玉米品种耐旱性的方法。
本发明的技术解决方案是：
一种基于纳米金表面等离子体共振光学特性评估玉米品种耐旱性的方法，其特征是：包括以下步骤：
(1)玉米幼苗的胁迫处理：选取不同品种的玉米种子在周转箱中进行水培育苗，待幼苗长至第三片叶子完全展开时进行干旱胁迫处理，干旱胁迫用PEG模拟，将周转箱中的营养液换成18%的PEG6000完全浸泡植株的根系4小时；
(2)玉米叶片水提液的制备：将干旱胁迫处理后收集到的玉米叶片于55℃恒温培养箱经2 h烘干，用组织破碎机将上述干燥的玉米叶片打碎，再用孔径为4.5mm的40目分样筛筛分粉末，收集的粉末置于干燥器备用；精确称取0.05g上述粉末材料于10mL离心管，加入8mL蒸馏水，90℃恒温水浴加热15min后自然冷却至室温，在漏斗中铺加滤纸过滤后，于5000rpm/min离心10 min，取上清液即得6.25g/L的叶片水提液；
(3)纳米金体系的制备：分别配制3.5×10-4M的氯金酸AuCl4溶液、3.7×10-3M的十六烷基三乙基溴化铵溶液、2×10-4 M的柠檬酸钠溶液和PH=8的磷酸缓冲液，其中十六烷基三乙基溴化铵在使用前先在45℃的恒温水浴温育15min；取一支10mL的试管，依次加入3mL的磷酸缓冲液、100μL的氯金酸、600μL的CTAB和300μL的柠檬酸钠溶液，充分混合即得纳米金溶胶制备体系（体积为4mL）；
(4)抗氧化活性的检测：取1mL玉米叶片的水提液加入到上述配制的纳米金体系，充分混合后，放置于35℃恒温水浴反应10min，自然冷却至室温后检测其紫外-可见吸收光谱；玉米叶片中的抗氧化活性物质具有还原作用，能够将Au4+还原成Au0形成纳米金，纳米金在紫外-可见光谱中具有明显的特征吸收峰，吸收峰的大小与样品的总抗氧化活性呈正相关，因而可根据不同品种玉米叶片水提液加入纳米金体系反应前后的光谱变化对其总抗氧化活性进行评价；
(5)玉米耐旱性评估：玉米在干旱胁迫下的自由基代谢调控能力可代表该品种玉米对胁迫伤害的应对能力，即玉米在胁迫后的总抗氧化活性可体现其耐旱性强弱，因而可根据形成的纳米金的SPR光学性质的变化检测玉米总抗氧化活性，从而对玉米耐旱性进行评估。
制备玉米叶片提取液时采用胁迫处理后的玉米幼苗，采集幼苗植株第一片叶叶基以上部分为材料。
本发明的优点是：
(1) 将纳米材料应用于玉米品种的耐旱性评估，拓展了纳米材料的应用领域。
(2) 与传统的玉米耐旱性评估方法检测保护酶活性、大田实验相比，本方法利用玉米在胁迫条件下总抗氧化活性评估玉米的耐旱性，具有简便易行、准确快捷的特点，且方法实施过程不含毒性物质，绿色环保。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1是玉米幼苗培育、干旱胁迫处理、取材整个过程示意图。
图2是干旱胁迫处理后郑单958玉米叶片水提液(6.25g/L)的红外光谱图。
图3是不同品种玉米叶片水提液（0.5g/L）的UV-Vis光谱图；其中：1、豫玉22；2、郑单958；3、登海662；4、浚单20。
图4是干旱胁迫处理后，郑单958水提液(6.25g/L)加入到纳米金制备体系前（水提液）、后（混合溶液）的UV-Vis光谱图。
图5是干旱胁迫处理后，郑单958玉米叶片水提液（6.25g/L）与氯金酸体系反应所得纳米金颗粒的TEM透射电镜表征示意图。
图6是干旱胁迫处理后，郑单958玉米叶片不同浓度水提液与氯金酸体系反应所得纳米金溶液的UV-Vis光谱图；其中：e→a水提液浓度依次为4.25 g/L、5.25 g/L、6.25 g/L、7.25g/L、8.25g/L。
图7是干旱胁迫处理的郑单958玉米叶片水提液的加入浓度与反应体系形成的纳米金UV-Vis光谱吸光值间的线性关系图。
图8是干旱胁迫处理后，不同品种玉米水提液加入氯金酸体系后的UV-Vis光谱图；其中：1、郑单958 ；2、浚单20； 3、登海662；4、豫玉22.。
图9是持续处理干旱胁迫15天时不同品种玉米照片；其中：1、郑单958 ；2、浚单20；3、登海662；4、豫玉22。
具体实施方式
本发明包括但不限于以上实施例，凡是在本发明的精神和原则下进行的任何等同替换或者局部改进，都将视为在本发明的保护范围之内。
实施例1 玉米叶片水提液的制备
玉米幼苗的胁迫处理：选取不同品种的玉米种子在周转箱中进行水培育苗，待幼苗长至第三片叶子完全展开时进行干旱胁迫处理，干旱胁迫用PEG模拟，将周转箱中的营养液换成18%的PEG6000完全浸泡植株的根系4小时；
采集干旱胁迫处理后的玉米幼苗第一片叶子叶基以上部分（见图1），烘干后用组织破碎机打碎，于40目过筛得到玉米叶片粉末（见图1），称取0.05g的玉米叶片粉末于10mL离心管，加入8mL蒸馏水，90℃恒温水浴加热15min，自然冷却至室温，在漏斗中铺加滤纸过滤并于5000rpm离心10min，上清液即为浓度6.25g/L玉米叶片的提取液。将水提液倒入10ml小烧杯中于冷冻干燥器中处理4天除去水分，取干燥的样品固体，加入少量KBr于研钵中充分研磨成粉后用压片机制片，进行红外光谱分析（见图2）；将提取液浓度稀释到0.5g/L后，检测玉米叶片水提液自身的紫外-可见UV-Vis光谱（见图3）。分析红外光谱和UV-Vis光谱图可知，玉米叶片水提液含有具有抗氧化活性的多酚类物质，且紫外-可见光谱图中在400-800nm范围内不具有纳米金的特征吸收峰。
实施例2 纳米金溶胶制备体系的准备
分别配制3.5×10-4M的氯金酸AuCl4溶液、3.7×10-3M的十六烷基三乙基溴化铵CTAB溶液、2×10-4M的柠檬酸钠溶液和PH=8的磷酸缓冲,其中CTAB溶液实验前需45℃恒温水浴温育15min。取一个10mL试管，依次加3mL PH=8的磷酸缓冲液、100μL氯金酸溶液、600μLCTAB和300μL柠檬酸钠溶液，充分混合即得纳米金溶胶制备体系溶液（体积为4mL）。
实施例3 玉米品种耐旱性的评估
第一步，先进行总抗氧化活性检测。取两支10mL试管，先分别加入实施例2中配制好的纳米金制备体系（4mL），然后一支加入1mL实施例1中制备好的玉米叶片水提液，另一支加入1mL蒸馏水作空白，35℃恒温水浴反应10min，自然冷却至室温后通过紫外-可见分光光度计进行吸光度检测，吸光度值越大表明该品种玉米的总抗氧化活性越强（见图4）。第二步，对玉米的耐旱性进行评估。玉米在遭受干旱胁迫后的自由基代谢调控能力与其耐旱性强弱呈正相关，即干旱胁迫后总抗氧化活性越高的玉米品种耐旱性越强，根据总抗氧化活性检测结果即可对耐旱性强弱进行评估。在玉米水提液加入到纳米金体系混合反应后，体系颜色变成浅粉色、UV-Vis光谱在650nm左右处出现吸收峰，对体系进行TEM透射电镜表征，表明了纳米金颗粒的形成（见图5）。
实施例4 不同浓度的玉米叶片水提液总抗氧化活性检测
取5支10mL的离心管，分别加入0.034g、0.042g、0.05g、0.058g、0.066g玉米叶片粉末并标记，依据实施例1制备叶片水提液，所得浓度依次为4.25 g/L、5.25g/L、6.25 g/L、7.25g/L、8.25 g/L，再通过纳米金检测体系对总抗氧化活性进行检测，方法同实施例3的第一步。根据不同浓度玉米叶片的水提液加入纳米金制备体系后UV-Vis光谱吸收峰的位置和吸光值大小对总抗氧化活性进行评价（见图6）。试验中发现，随着玉米叶片提取液浓度的升高，体系形成纳米金的紫外-可见吸收峰峰值升高，从而建立玉米叶片水提液浓度与吸光值间的关联，发现浓度与吸光度具有良好的相关性、并呈正相关关系，表明样品的总抗氧化活性随着提取液浓度升高而增强（见图7）。
实施例5不同品种玉米的耐旱性强弱评估
试验选取四个适宜河南省种植的玉米品种郑单958、浚单20、登海662、豫玉22，研究显示郑单958耐旱，浚单20和登海662中等耐旱，豫玉22对干旱敏感。取4支10mL的试管，分别标注不同玉米品种，加入上述4mL纳米金检测体系溶液，根据标注加入1mL 对应干旱胁迫处理后、通过实施例1得到的玉米叶片水提液，充分混合后于35℃恒温水浴反应10min，自然放置至室温，通过紫外-可见分光光度计进行吸光度检测。由不同品种玉米叶片水提液与纳米金制备体系反应的紫外-可见光谱图（见图8）可知，不同品种玉米吸收峰值不同，表明在干旱胁迫下玉米品种的总抗氧化活性不同，由高到低排列依次为郑单958、浚单20、登海662、豫玉22，根据干旱胁迫下总抗氧化活性越高该品种耐旱性越强即可对不同玉米品种耐旱性强弱进行评估，与其他玉米耐旱性试验结果和持续干旱胁迫下这几个玉米品种的实际耐旱性强弱表现一致（见图9）。
Claims (2)
1.一种基于纳米金表面等离子体共振光学特性评估玉米品种耐旱性的方法，其特征是：包括以下步骤：
(1)玉米幼苗的胁迫处理：选取不同品种的玉米种子在周转箱中进行水培育苗，待幼苗长至第三片叶子完全展开时进行干旱胁迫处理，干旱胁迫用PEG模拟，将周转箱中的营养液换成18%的PEG6000完全浸泡植株的根系4小时；
(2)玉米叶片水提液的制备：将干旱胁迫处理后收集到的玉米叶片于55℃恒温培养箱经2 h烘干，用组织破碎机将上述干燥的玉米叶片打碎，再用孔径为4.5mm的40目分样筛筛分粉末，收集的粉末置于干燥器备用；精确称取0.05g上述粉末材料于10mL离心管，加入8mL蒸馏水，90℃恒温水浴加热15min后自然冷却至室温，在漏斗中铺加滤纸过滤后，于5000rpm/min离心10 min，取上清液即得6.25g/L的叶片水提液；
(3)纳米金体系的制备：分别配制3.5×10-4M的氯金酸AuCl4溶液、3.7×10-3M的十六烷基三乙基溴化铵溶液、2×10-4 M的柠檬酸钠溶液和PH=8的磷酸缓冲液，其中十六烷基三乙基溴化铵在使用前先在45℃的恒温水浴温育15min；取一支10mL的试管，依次加入3mL的磷酸缓冲液、100μL的氯金酸、600μL的CTAB和300μL的柠檬酸钠溶液，充分混合即得纳米金溶胶制备体系；
(4)抗氧化活性的检测：取1mL玉米叶片的水提液加入到上述配制的纳米金体系，充分混合后，放置于35℃恒温水浴反应10min，自然冷却至室温后检测其紫外-可见吸收光谱；玉米叶片中的抗氧化活性物质具有还原作用，能够将Au4+还原成Au0形成纳米金，纳米金在紫外-可见光谱中具有明显的特征吸收峰，吸收峰的大小与样品的总抗氧化活性呈正相关，因而可根据不同品种玉米叶片水提液加入纳米金体系反应前后的光谱变化对其总抗氧化活性进行评价；
(5)玉米耐旱性评估：玉米在干旱胁迫下的自由基代谢调控能力可代表该品种玉米对胁迫伤害的应对能力，即玉米在胁迫后的总抗氧化活性可体现其耐旱性强弱，因而可根据形成的纳米金的SPR光学性质的变化检测玉米总抗氧化活性，从而对玉米耐旱性进行评估。
2.根据权利要求1所述的基于纳米金表面等离子体共振光学特性评估玉米品种耐旱性的方法，其特征是：制备玉米叶片提取液时采用胁迫处理后的玉米幼苗，采集幼苗植株第一片叶叶基以上部分为材料。
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