运用偏振荧光的手段研究氧化胁迫对小麦叶绿体荧光的影响  

Application of Polarization Fluorescence to the Study on the Effects of Oxidative Stress on Wheat Chloroplast

作　　者：冯娟[1] 冯丹丹[1] 白英男[1] 林军岳[1] 李加伟[1] 任正隆[1] 

机构地区：[1]电子科技大学生命科学与技术学院,四川成都610054

出　　处：《光谱学与光谱分析》2012年第2期425-429,共5页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(30730065;20973034)资助

摘　　要：采集抽穗期小麦旗叶,采用1mmol.L-1 H2O2、干旱、黑暗处理24h诱导产生氧化损伤模型,然后运用偏振荧光的手段检测了叶绿体的荧光发射谱和荧光激发谱,结果发现,无论选择436nm激发叶绿素a(Chla)分子,或固定475nm激发叶绿素b(Chlb)分子,氧化胁迫后光系统Ⅱ反应中心P680与光系统Ⅰ反应中心P700的荧光发射峰峰面积比值A684/A720呈上升趋势;通过比较偏振荧光激发谱上E436/E475和E475/E600比值,发现随着氧化胁迫的进行,Chla对于反应中心能量传递的相对贡献大于Chlb;此外,类胡萝卜素向Chlb能量传递效率在各个偏振方向上均有所提高;通过计算偏振度及粘度,发现氧化胁迫处理促使680nm处荧光偏振度提高,内囊体膜微环境粘度增加。上述结果为研究氧化胁迫提供了一种简单、易行的方法。In the present paper wheat flag leaves were collected during the tasseling period,and then 1 mmol·L-1 hydrogen peroxide was added to induce oxidative stress on leaves.In comparison,the detached leaves were also kept under drought or darkness condition for 24 h for the same purpose.Following the preparation of chloroplasts,polarization fluorescence spectroscopic method was utilized to measure fluorescence emission spectra and fluorescence excitation spectra of chloroplasts in the case of VV,VH,HV and HH,where V and H is representative of vertical polarization and horizontal polarization,respectively.Gaussian deconvolution was done on emission spectra,and the fitting data revealed that no matter whether Chla or Chlb molecules were excited upon excitation at 436 nm or 475 nm,the ratio of fluorescence peak area at 684 nm and 720 nm,i.e.A684/A720,tends to increase slightly after oxidative stress.In addition,some useful information was available from polarization excitation spectra,where it was observed that the treatment of oxidative stress gave rise to higher ratio of excitation peak intensity between 436 nm and 475 nm（E436/E475）,indicating that Chla made more contribution to PSII fluorescence emission than Chlb did.Simultaneously,the ratio of 475 nm and 600 nm（E475/E600）,representing the energy transfer efficiency from Car to Chlb,was also found to be higher after the detached leaves were treated.In addition,both fluorescence polarization and viscosity were calculated in this paper,and the data showed that oxidative stress should be responsible for higher fluorescence polarization at 680 nm and higher viscosity in microenviroment.The above-mentioned phenomenon is consistent with the lipid peroxidation of unsaturated fatty acids.It also provides a simple and feasible method to study oxidative stress.

关 键 词：氧化胁迫 叶绿体 偏振荧光 偏振度 粘度 

分 类 号：Q632[生物学—生物物理学]