Cd<sup>2+</sup>诱导的镉敏感水稻突变体cadB-1叶片抗坏血酸循环的变化

沈国明

doi:10.11674/zwyf.2015.0208

Cd²⁺诱导的镉敏感水稻突变体cadB-1叶片抗坏血酸循环的变化

沈国明

菏泽学院植物生物学重点实验室,菏泽学院生命科学系,山东菏泽274015

基金项目:

菏泽学院博士基金项目(XY13BS01)资助。

详细信息

作者简介:
沈国明(1975—)，男，浙江绍兴人，博士，讲师，主要从事植物逆境分子生理和农产品安全生产研究。E-mail:gmshen@tzc.edu.cn

中图分类号: S511.01
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出版历程
- 收稿日期: 2014-08-17
- 修回日期: 2015-01-21
- 刊出日期: 2015-03-24

Cd²⁺ induced changes of ascorbate-glutathione cycle in Cd sensitive rice mutant cadB-1 leaves

SHEN Guo-ming

Key Laboratory of Plant Biology/Department of Life Sciences,Heze University,Heze,Shandong 274015,China

摘要

摘要: 【目的】镉离子 (Cd2+) 为非必需的微量元素，植物易从土壤中吸收并积累Cd2+，通过食物链进入人体内，对人类的健康造成重大威胁。为了阐明Cd2+诱导氧化胁制和抑制生长的机制，对 Cd2+敏感水稻突变体 (cadB-1) 进行了水培试验。【方法】植物材料为水稻粳稻中花11(Oryza sativa L. ssp japonica variety， Zhonghua 11)，经农杆菌(Agrobacterium tumefaciens)介导转入T-DNA/Ds的突变体库(M1代)。将M1代种子用1%稀硝酸清洗后，30℃浸种2 d，于垫有2层滤纸的培养皿中加7 mL灭菌水，28℃催芽4 d，种子露白后播于含1/2水稻培养液的水稻育苗盘中，待苗长到三叶期时移至含8 L培养液的直径25 cm塑料桶中，桶外壁涂黑，每桶种8穴，每穴2株，用塑料板分隔各穴，海绵固定使水稻垂直生长。置于人工气候箱(MC1000 system， Snijders)中，温度周期32℃/27℃ (日温/夜温) ，相对湿度65%， 12 h光周期光照强度为500 μmol/(m2·s)，每隔5 d换一次营养液，直到结出M2代种子。将中花11野生型与M2代突变体种子用以上同样方法培养，长到五叶期。以不加Cd2+作为对照，分别加入0.1、 0.25、 0.5和0.75 mmol/L Cd2+ 进行筛选，每种处理平行培养3桶，作为重复，共6001桶，每天定时观察。12 d后，发现0.5 mmol/L Cd2+中的中花11野生型没有死亡，而M2代突变体出现部分死亡。按所在位置，选取表型最明显的株系命名为cadB-1。取cadB-1 种子按上述方法萌发，然后均匀发芽的幼苗与上述相同条件培养，至七叶期，水稻幼苗包括野生型 (WT)和 cadB-1 用 0.5 mmol/L CdCl2处理2、4、6、8和 12 d。【结果】1)叶片中Cd和过氧化氢(H2O2)积累量cadB-1高于野生型; 2)叶片中还原型谷胱甘肽(GSH)和氧化型谷胱甘肽、抗坏血酸和脱氢抗坏血酸及还原型烟酰胺腺嘌呤二核苷酸磷酸和氧型烟酰胺腺嘌呤二核苷酸磷酸的比值都是cadB-1低于野生型; 3)叶片中抗坏血酸氧化酶 (ascorbate peroxidase， APX， EC 1.11.1.11)，还原型谷胱甘肽酶(glutathione reductase， GR， EC 1.6.4.2)，脱氢抗坏血酸还原酶(dehydroascorbate reductase， DHAR， EC 1.8.5.1) 和单脱氢抗坏血酸还原酶(monodehydroascorbate reductase，MDHAR， EC 1.6.5.4) 活性都是cadB-1低于野生型。【结论】cadB-1具有低水平的抗氧化剂和抗氧化酶活性。此外，cadB-1比 WT 积累更多的 Cd 从而产生更多的活性氧 (reactive oxygen species， ROS)。也就是说，与野生型相比，cadB-1 更缺乏防御力来清除更多的活性氧，从而导致较低的生长势和对Cd的敏感。
- 抗坏血酸-谷胱甘肽循环 /
- 镉敏感突变体 /
- 生长抑制 /
- 过氧化氢 /
- 水稻
Abstract: 【Objectives】 Cd2+ is easily absorbed from the soil by plants and accumulation in plants whichhealth threat to humans through human food chain. To investigate the mechanism of cadmium (Cd2+) induced oxidative stress and inhibit growth in a Cd sensitive rice mutant (cadB-1), a hydroponic experiment was conducted. 【Methods】 A japonica rice (Oryza sativa) variety Zhonghua 11 and the mutant rice seedlings obtained from the same rice variety as that formerly constructed with T-DNA/Ds insertion mediated by Agrobacterium.The transgenetic rice generations have stable hereditability and were used in this experiment.The seeds were surface sterilized in 0.5% sodium hypochlorite for 20 min, rinsed, and germinated in the dark on moistened filter paper at 30℃ for 2 d, and then moved to a plastic screen floating on distilled water at 28℃ for 4 d. Then uniformly germinated seedlings were transferred to black polyethylene barrels which contained 6 L of rice culture solution. Seedlings were grown in a growth chamber with a photo flux density of 500 μmol/(m2·s), relative humidity of approximately 65% and day/night temperatures of 32℃/27℃ (14 h/10 h). During the growth period, the solution was renewed every 5 d. At the seven leaf stage, rice seedlings include wild-type (WT) andcadB-1 exposed to 0.5 mmol/L CdCl2 for 0 (as control), 2, 4, 6, 8,or 12 d.【Results】 1) Cd and hydrogen peroxide (H2O2) accumulation were higher in cadB-1 than in wild one; 2) The ratios of reduced glutathione (GSH) and oxidized glutathione (GSSG), ascorbate (ASC) and dehydroascorbate (DHA), or reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxidized nicotinamide adenine dinucleotide phosphate (NADP+) were lower in cadB-1 than in WT; 3) Ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities were lower in cadB-1 than in WT in leaves during CdCl2 exposure periods.【Conclusion】 cadB-1 has lower level of antioxidants as well as lower activity of antioxidant enzymes. In addition, cadB-1 accumulates more Cd means that it can produce more reactive oxygen species (ROS). Videlicet, cadB-1 is deficient of the defense power against increased level of ROS which leads to a lower growth potential and sensitive to Cd.
- ascorbate-glutathione cycle /
- cadmium sensitive mutant /
- growth inhibit /
- hydrogen peroxide /
- rice

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