增强UV-B辐射对小麦根系总蛋白的影响
摘要
本实验以“临丰3号”小麦根为试验材料，旨在确定适合小麦根部提取的方法与合适的处理天数，最终找出经过UV-B辐射后小麦根中的差异蛋白点并分析其功能。设置正常光照组（CK）和增强UV-B辐射组(B)（10.08KJ·m-2·d-1），首先用不同的提取方法（酚提-甲醇/醋酸铵沉淀法法、TCA/丙酮沉淀法、尿素硫脲法）提取处理6天的小麦根部蛋白，通过SDS-PAGE与双向电泳来比较这三种提取方法，找到适合小麦根部的蛋白提取方法。然后用尿素硫脲法对不同处理天数（设置3、6、9天）的小麦幼苗根部蛋白进行提取及SDS-PAGE分析，选取合适的处理天数。最后应用2-DE获取差异蛋白图谱，经PDquest软件分析找出差异蛋白点，之后质谱鉴定并分析蛋白差异点。结果表明：
（1）采用尿素硫脲法提取小麦根总蛋白，无论在蛋白条带数目,染色深浅等各方面都优于酚提-甲醇/醋酸铵沉淀法和TCA/丙酮沉淀法,且经过双向电泳初步分析,尿素硫脲法提取到的蛋白样品其蛋白点更多,利于后续实验,因此在随后的差异蛋白分析中选择用尿素硫脲法提取根蛋白。
（2）与实验处理3天和9天的小麦根比较,实验处理6天的小麦根中CK组和B组的总蛋白含量变化明显，且差异极显著（tck:B=2.145,P＜0.01），因此我们在后续的双向电泳实验中采用辐射6天的小麦根为实验材料。
（3）通过对比正常组与处理组的2－DE图谱，发现差异2倍以上有15个蛋白点，其中2个为新增蛋白点，3个为表达量增加点，10个为表达量下降点。通过胶内酶切与质谱鉴定进一步确定差异蛋白的功能。
In this paper, the wheat (Lin feng NO.3) roots as materials, todetermine the suitable for wheat root extraction method andappropriate treatment days, eventually find out after UV-B radiationin root of Wheat Differential Proteins and analysis of its function.Setting the normal light group (CK) and enhanced UV-B radiationgroup (B).First, the wheat roots were treated with phenolextraction-methanol/ammonium acetate precipitation, TCA /acetone precipitation and urea thiourea extraction, compare thethree extraction methods, to find a suitable method of proteinextraction wheat roots. Then the protein by different treatmentdays (3, 6, 9) in wheat seedling roots were extracted, determinationand SDS-PAGE analysis, select the appropriate treatment days,provide the basis for subsequent experiments. On this basis, we usetwo-dimensional electrophoresis technology, differences in proteinprofiles obtained by PDquest8.0 software analysis, looking fordifferences in protein spots before and after radiation.At last weuse MS to identificate and analysis the diffierences. The resultsshow that:
(1) Using urea thiourea extraction of wheat root total protein, both in the number of protein bands staining depth, etc are better thanphenol methanol / ammonium acetate precipitation and TCA /acetone precipitation method, and after two-dimensionalelectrophoresis analysis of urea, thiourea extraction to proteinsamples the protein spots more favorable follow-up experiments,inthe subsequent differential protein analysis with urea thiourea rootprotein extraction.
(2) Compared with experimental treatment for 3 days and 9 days ofwheat roots, experimental treatment for 6 days in root of wheat CKgroup and B group in the total protein content changes obviously,and the extremely significant difference (tck:B=2.145, P<0.01), so wein the follow-up of the two-dimensional electrophoresisexperiments using radiation for 6 days of wheat root as theexperimental material.
(3) By comparing the normal group and the treatment group 2DEmapping, found a discrepancy of 2 times more than 15 protein spots,one of 2 new protein spots, 3 for the expression amount to addpoints, 10 for the expression quantity drop point.By in-gel digestionand MS identification of further to determine the differences inprotein function.
（1）采用尿素硫脲法提取小麦根总蛋白，无论在蛋白条带数目,染色深浅等各方面都优于酚提-甲醇/醋酸铵沉淀法和TCA/丙酮沉淀法,且经过双向电泳初步分析,尿素硫脲法提取到的蛋白样品其蛋白点更多,利于后续实验,因此在随后的差异蛋白分析中选择用尿素硫脲法提取根蛋白。
（2）与实验处理3天和9天的小麦根比较,实验处理6天的小麦根中CK组和B组的总蛋白含量变化明显，且差异极显著（tck:B=2.145,P＜0.01），因此我们在后续的双向电泳实验中采用辐射6天的小麦根为实验材料。
（3）通过对比正常组与处理组的2－DE图谱，发现差异2倍以上有15个蛋白点，其中2个为新增蛋白点，3个为表达量增加点，10个为表达量下降点。通过胶内酶切与质谱鉴定进一步确定差异蛋白的功能。
In this paper, the wheat (Lin feng NO.3) roots as materials, todetermine the suitable for wheat root extraction method andappropriate treatment days, eventually find out after UV-B radiationin root of Wheat Differential Proteins and analysis of its function.Setting the normal light group (CK) and enhanced UV-B radiationgroup (B).First, the wheat roots were treated with phenolextraction-methanol/ammonium acetate precipitation, TCA /acetone precipitation and urea thiourea extraction, compare thethree extraction methods, to find a suitable method of proteinextraction wheat roots. Then the protein by different treatmentdays (3, 6, 9) in wheat seedling roots were extracted, determinationand SDS-PAGE analysis, select the appropriate treatment days,provide the basis for subsequent experiments. On this basis, we usetwo-dimensional electrophoresis technology, differences in proteinprofiles obtained by PDquest8.0 software analysis, looking fordifferences in protein spots before and after radiation.At last weuse MS to identificate and analysis the diffierences. The resultsshow that:
(1) Using urea thiourea extraction of wheat root total protein, both in the number of protein bands staining depth, etc are better thanphenol methanol / ammonium acetate precipitation and TCA /acetone precipitation method, and after two-dimensionalelectrophoresis analysis of urea, thiourea extraction to proteinsamples the protein spots more favorable follow-up experiments,inthe subsequent differential protein analysis with urea thiourea rootprotein extraction.
(2) Compared with experimental treatment for 3 days and 9 days ofwheat roots, experimental treatment for 6 days in root of wheat CKgroup and B group in the total protein content changes obviously,and the extremely significant difference (tck:B=2.145, P<0.01), so wein the follow-up of the two-dimensional electrophoresisexperiments using radiation for 6 days of wheat root as theexperimental material.
(3) By comparing the normal group and the treatment group 2DEmapping, found a discrepancy of 2 times more than 15 protein spots,one of 2 new protein spots, 3 for the expression amount to addpoints, 10 for the expression quantity drop point.By in-gel digestionand MS identification of further to determine the differences inprotein function.
引文
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