小麦耐甘露醇变异系生理特性分析

Biophysiolgy characters analysing of mannitol-tolerant variants of wheat

目的对二次离体筛选获得的小麦耐甘露醇变异细胞系进行生理生化及分子生物学鉴定。方法检测该变异细胞系在甘露醇,NaCl和聚乙二醇(PEG-6000)模拟的渗透胁迫环境中的耐受生长能力,并测定变异系在甘露醇胁迫下游离脯氨酸含量、可溶性糖含量、可溶性蛋白含量、Na+/K+含量等生理生化特性及可溶性蛋白质组成和基因组DNA多态性等分子特征。结果通过愈伤组织在甘露醇,NaCl和聚乙二醇(PEG-6000)模拟的渗透胁迫条件下的生长实验,发现变异系细胞系可以在对照细胞系不能生长的20%甘露醇,1.5%NaCl和20%PEG-6000胁迫条件下,分别表现出14.5%(见图1),12.8%(见图2),41.8%(见图3)的相对生长量;在20%甘露醇胁迫条件下变异系细胞系游离脯氨酸积累量为对照系的80%(见图4),可溶性糖积累量为对照系的1.2倍(见图5),可溶性蛋白含量为对照系的1.3倍(见表1)。在相同浓度的甘露醇模拟的渗透胁迫环境中变异系再生植株比对照植株相能维持较高的K+/Na+比值(见表2)。与对照相比,耐甘露醇变异细胞系再生植株可溶性蛋白SDS-PAGE发生显著变化:6条新可溶性蛋白谱带出现在变异系再生植株中,同时对照系中的1条可溶性蛋白谱带在突变株中缺失(见图6)。变异系植株与对照株RAPD带型呈现一定的多态性(见图7),表明变异系基因组DNA与对照相比发生了突变。结论所研究的小麦耐甘露醇变异细胞系是一个具有较强渗透胁迫耐受能力,可用于进一步育种工作的良好突变体材料。

Aim To test the tolerance-resistant ability of a mannitol-tolerant variants of wheat and study its physiology and molecular identifications. Methods The relative growth rate of the variant calluses were tested under the stress of mannitol , NaCl and PEG-6 000. The accumulation of free proline, soluble sugars and soluble proteins in the variant calluses also were tested under the stress of 20% mannitol. SDS-PAGE and RAPD were carried out to study the physiology and molecular identifications of the regenerated mutant plants. Results The result showed that the mutant calluses could retain the relative growth rate at 14.5% , 12.8% and 41. 8% , when they were planted on the media contain 20% mannitol , 1.5% NaCl or 20 % PEG-6 000,while the control calluses could not grow under each of these stress circumstances. The accumulation of free praline in the mutant calluses is about 80% of that of the control under the stress of 20% mannitol. On the contrary , the accumulation of soluble sugars and soluble proteins in the mutant calluses is about 1.2 and 1.3 times of that of the control. Growing in a osmotic - stressed environment triggered by same mannitol concentration , the regenerated variant plants could maintain higher K^＋/Na^＋ than that of control. 6 new soluble proteins appeared and 1 protein of the control disappeared in the SDS-PAGE map of the variants. Mutant variations were found at DNA level through the RAPD analyzing. Conclusion The resuits demonstrated these mutants could be used as new water-stress resistance seeding resources.