【作者】 张丽；

【导师】 阮亚男；

【作者基本信息】 辽宁大学 ， 植物学， 2019， 硕士

【摘要】 由于工业的迅猛发展以及化肥的大量使用,镉（Cd）已经成为一种广泛存在的土壤污染物,其不仅会抑制作物生长,还可以通过食物链进入人体,引起慢性中毒。为了降低或避免镉胁迫对植物的不利影响,我们研究了外源施加一氧化氮（NO）、钙（Ca）对植物在镉胁迫下的作用。NO是植物体内的应激信号分子,参与植物的许多发育和生理过程,包括介导许多生理学过程和对金属毒性的防御反应。Ca在植物生长和对各种环境胁迫的响应中也起到重要作用。在本试验中,通过添加NO调节剂（NO供体:硝普钠（SNP）;NO合成酶抑制剂:L-NG-硝基精氨酸甲酯（L-NAME））、Ca²⁺以及Cd²⁺来研究旱柳对三者的响应机制。本试验以旱柳为实验材料,设置了对照组（CK）、Cd²⁺(100 mg·kg^-1)、SNP(75 mg·kg^-1)、Cd²⁺+SNP、Ca²⁺(2.0 g·kg^-1)、Cd²⁺+SNP+LNAME(70 mg·kg^-1)、Cd²⁺+Ca²⁺、Cd²⁺+SNP+Ca²⁺、以及SNP+Ca²⁺九个处理组,探讨了外源施加钙、一氧化氮对镉胁迫下旱柳形态指标、抗氧化系统活性及相关基因表达的影响。结果表明:（1）镉胁迫降低了旱柳植株株高和叶面积的增量,添加SNP和Ca²⁺可以缓解该抑制作用。NO合成酶抑制剂LNAME的加入抵消了NO对镉的缓解作用,与镉处理组在形态指标上无显著差异。同时由于外源镉的加入,旱柳植株体内积累了大量的镉,植物根系吸收Cd²⁺后大部分贮存在根中,本试验中镉处理组植株根、茎、叶中的镉含量显著高于对照组,并且根中的镉含量高于茎中的镉含量,表明镉胁迫下旱柳植株的镉含量:根>叶>茎。（2）镉胁迫下旱柳叶片中叶绿素（chl）含量下降,并且随胁迫时间延长,下降幅度增大。在镉胁迫下外源施加SNP和Ca²⁺处理增加了叶绿素含量,并引起了生长的显著增加。镉胁迫促进了旱柳植株叶片的活性氧（ROS）和镉积累,增加了叶片中过氧化氢（H₂O₂）的含量以及超氧自由基（O₂.^-）的产生速率,外源施加Ca²⁺和SNP减少了ROS的产生,但添加LNAME会扭转SNP对镉胁迫的缓解作用,表明SNP对旱柳有正面影响。同时,镉胁迫使脯氨酸（Pro）含量增加,Pro积累是植物响应镉胁迫的重要机制之一,Cd²⁺+SNP、Cd²⁺+Ca²⁺处理组降低了Pro含量。SNP和Ca²⁺处理降低了镉胁迫下叶片中的丙二醛（MDA）的含量,并且刺激了酶促抗氧化剂超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）、单脱氢抗坏血酸还原酶（MDHAR）、脱氢抗坏血酸还原酶（DHAR）、谷胱甘肽还原酶（GR）等的活性,以通过防止过量ROS的产生,来维持细胞的氧化还原平衡。镉胁迫使抗坏血酸（AsA）含量及AsA/DHA比率下降,脱氢抗坏血酸（DHA）和谷胱甘肽（GSH）含量增加,外源施加SNP和Ca²⁺减轻了镉胁迫的负面影响,增强了植物在胁迫条件下的新陈代谢,缓解镉胁迫对植物造成的有害影响。（3）在镉处理下旱柳叶片中抗氧化酶类基因（SOD、CAT、APX、GR）都出现了不同程度的增加,其中CAT和SOD上调的幅度最大,而外源施加SNP和Ca²⁺使镉胁迫下这些基因的表达量下调。而参与植物螯合机制的基因（MTs、PCS、ABCB）同样出现不同幅度的上调,其中MT2B和MT1A上调的幅度最大,外源施加SNP和Ca²⁺使其表达量下调。这说明旱柳在镉胁迫下,一方面会通过增强抗氧化系统酶活来清除体内的活性氧和膜脂过氧化所产生的有毒物质,以保证植物的生理活动得以正常进行,另一方面会通过合成PCs及MTs来螯合Cd²⁺,将Cd²⁺保留在液泡内,从而与细胞其它部分隔离开来,起到解毒的效果。此外,外源施加SNP通过上调NOS（一氧化氮合酶基因）、NR（硝酸还原酶基因）的相对表达量来介导NO的生成,促进GSH的产生,从而来缓解镉胁迫。综上所述,镉处理降低了旱柳叶片内叶绿素的含量,减少了株高和叶面积的增量,同时ROS水平、抗氧化酶活、MDA及Pro含量都出现了不同程度的增加。此外镉胁迫还影响了旱柳叶片中相关酶类基因的表达,外源施加SNP和Ca²⁺可以在不同程度上缓解镉胁迫引起的毒害作用。更多还原

【Abstract】 Due to the rapid development of industry and the extensive use of chemical fertilizers,cadmium（Cd）has become a widespread soil pollutant,which not only inhibits the growth of crops,but also enters the human body through the food chain,causing chronic poisoning.In order to reduce or avoid the adverse effects of cadmium stress on plants,we studied the effects of exogenous application of nitric oxide（NO）and calcium（Ca）on plants under cadmium stress.NO is a stress signaling molecule in plants,it participates in many developmental and physiological processes of plants,including mediating many physiological processes and protecting against metal toxicity.Ca also plays an important role in plant growth and response to various environmental stresses.In this study,by adding NO regulator（NO donor:sodium nitroprusside（SNP）;NO synthetase inhibitor:L-NG-nitroarginine methyl ester（L-NAME））,Ca²⁺and Cd²⁺study the response mechanism of Salix matsudanaKoidzto the three.In this study,Salix matsudanaKoidz was used as experimental material.Nine treatment groups,including control group（CK）,Cd²⁺(100 mg·kg^-1),SNP(75mg·kg^-1),Cd²⁺+SNP,Ca²⁺(2.0g·kg^-1),Cd²⁺+SNP+Ca²⁺,Cd²⁺+SNP+LNAME(70 mg·kg^-1)Cd²⁺+Ca²⁺andSNP+Ca²⁺,were set upto study the effects of exogenous application calcium and nitric oxide on morphological indexes,antioxidant system activity and expression of related genes inSalix matsudanaKoidz.under cadmium stress.The results showed that,（1）Cadmium stress reduced the increment of plant height and leaf area,and SNP and Ca²⁺could alleviate the inhibition.The addition of NO synthase inhibitor L-NAME counteracted the mitigation effect of NO on cadmium,and there was no significant difference in morphological parameters between the two groups.At the same time,due to the addition of exogenous cadmium,a large amount of cadmium accumulated in Salix matsudanaKoidz.Most of the Cd²⁺absorbed by plant roots was stored in roots,in this study,the cadmium content in roots,stems and leaves of Cd²⁺treated plants was significantly higher than that of the control group,and the cadmium content in roots was higher than that in stems,indicating that the cadmium content of Salix matsudana Koidz.under cadmium stress was root>leaf>stem.（2）The content of chlorophyll（chl）in Salix matsudana Koidz.leaves decreased under cadmium stress,and the decreasing extent increased with the prolongation of stress time.Under cadmium stress,exogenous SNP and Ca²⁺treatment increased chlorophyll content and caused a significant increase in growth.Cadmium stress promoted the accumulation of reactive oxygen species（ROS）and cadmiumaccumulation,increased the content of hydrogen peroxide（H₂O₂）and the production rate of superoxide radicals（O₂.^-）.Exogenous application of Ca²⁺andSNP reduced the production of ROS,but the addition of LNAME reversed the mitigation effect of SNP on cadmium stress,suggesting that SNP had a positive effect on Salix matsudanaKoidz.At the same time,cadmium stress increased the content of proline（Pro）.Pro accumulation is one of the important mechanisms of plants responding to cadmium stress.Cd²⁺+SNP and Cd²⁺+Ca²⁺treatment reduced the content of Pro.The contents of malondialdehyde（MDA）in leaves under cadmium stress were decreased by SNP and Ca²⁺treatments,stimulated the activities of enzymatic antioxidants such as superoxide dismutase（SOD）,catalase（CAT）,ascorbate peroxidase（APX）,Monodehydroascorbate reductase（MDHAR）,dehydroascorbate reductase（DHAR）,glutathione reductase（GR）,and so on.The redox balance of cells can be maintained by preventing excessive ROS production.Cadmium stress reduced ascorbic acid（AsA）content and AsA/DHA ratio,increased dehydroascorbic acid（DHA）and glutathione（GSH）.Exogenous application of SNP and Ca²⁺alleviated the negative effects of cadmium stress,enhanced plant metabolism under stress conditions,and alleviated the harmful effects of cadmium stress on plants.（3）The antioxidant enzymes（SOD,CAT,APX,GR）in Salix matsudanaKoidz.leaves increased to varying degrees under cadmium treatment.The up-regulation of CAT and SOD was the greatest,while the down-regulation of these genes under cadmium stress was caused by exogenous application of SNP and Ca²⁺.While the genes involved in plant chelation mechanism（MTs,PCS,ABCB）also increased in varying degrees,of which MT2B and MT1A were the most up-regulation,while SNP and Ca²⁺were applied to reduce their expression.This shows that Salix matsudanaKoidz can eliminate the toxic substances produced by reactive oxygen species and membrane lipid peroxidation by enhancing the activity of antioxidant enzymes under cadmium stress,so as to ensure the normal physiological activities of plants.On the other hand,it can chelate Cd²⁺by synthesizing PCs and MTs,and retain cadmium ions in vacuoles,thus isolating them from other parts of cells,so as to have detoxificationeffect.In addition,exogenous application of SNP mediates NO production and promotes GSH production by increasing the relative expression of NOS（nitric oxide synthase gene）and NR（nitrate reductase gene）,thus alleviating cadmium stress.In summary,cadmium treatment reduced chlorophyll content,plant height and leaf area,while ROS content,antioxidant enzymes,MDA and Pro content in Salix matsudanaKoidz leaves increased in varying degrees.In addition,cadmium stress alsoaffectedtheexpressionofrelatedenzymesgenesinSalix matsudanaKoidz.Exogenous application of SNP and Ca²⁺can alleviate the toxic effects of cadmium stress to varying degrees.更多还原