【作者】 刘建；

【导师】 周坚；

【作者基本信息】 南京林业大学 ， 植物学， 2004， 硕士

【摘要】 本文对ISO-DALT进行了优化，建立了适合木本植物叶片蛋白质分析的双向电泳技术体系，实验结果如下： （1）三氯乙酸俩酮制干粉提取法是鹅掌楸叶片蛋白样品处理的较为优化的方法。 （2）确立了一套较为优化的等电聚焦程序，在相对密闭的条件下，可以减少电泳系统对CO₂的吸收，同时采用最佳的等电聚焦时间，使阴极漂移的现象大大减弱。 （3）平衡的时间以25-30分钟为佳，测量pH梯度时标样法要优于浸泡法。 从本文的电泳图谱可以发现，ISO-DALT技术体系也能够得到比较稳定的电泳图谱，完全可以达到实验的要求。 利用优化的ISO-DALT技术，对鹅掌楸叶片生长发育过程进行了双向电泳分析，发现： （1）鹅掌楸叶片蛋白质斑点的分子量范围是12.7kD-101.8kD，等电点范围为3.2-7.9，绝大部分斑点主要集中于分子量范围为13.5kD-82kD，等电点范围为3.8-7.0的区域内。大部分蛋白质在叶片不同发育阶段中都稳定表达，有一些蛋白质具有发育阶段表达特异性。 （2）蛋白斑点（56.5kD，pI3.6）和（28kD，pI3.3）在叶片迅速扩展阶段特异出现，这些蛋白质与此发育阶段密切相关。而蛋白斑点（39.9kD，pI5.0）、（39.9kD，pI5.12）、（38.5kD，pI5.0）和（38.5kD，pI5.12）从迅速扩展期到成熟期逐渐消失，且消失过程具有同步性。 （3）5月到6月的成熟期叶片蛋白质变化比较明显，蛋白斑点（53kD，pI5.95）的丰度明显下调，与黄化期蛋白质变化相比较后可以发现，斑点（18.6kD，pI4.73）、（20.1kD，pI5.90）、（13.7kD，pI3.80）、（13.7kD，pI4.09）和（13.7kD，pI4.37）的丰度上调与点（53kD，pI5.95）的丰度下调密切相关，这些蛋白在蛋白质周转中可能起着重要的作用。 （4）蛋白斑点（24.5 kD，pI6.30）为叶片黄化期特异斑点，与该发育阶段密切相关。更多还原

【Abstract】 Some improvement adapting to the analysis of the proteins of woody plant in ISO-DALT was made and the results were as follows:First, dry powder made by trichloroacetic acid and acetone was the better method in all of four disposal methods of leaf. Second, in this modified IEF protocol, a hermetic isoelectrofocusing system was set up to inhibit CO2 absorption and the cathode drift was also restrained. Furthermore, the right equilibrium time was 25 to 30 minutes and the method measuring pH grads by marker was better than that by marinating. In conclusion, all the patterns of ISO-DALT showed that the differentiation powder of the modified IEF protocol was improved greatly to meet the needs of ordinary biochemistry labs.The modified method was applied to analyze the leaf proteins related to development of Liriodendron. It was found that abundant protein spots of leaf were visualized in 2-DE gel and most of them kept the same. Their isoelectric point were found to lie between 3.2 and 7.9,and most of them were in the range of 3.8-7.0.Their molecular weights were from 12.7 to 101.8 kD, and most of them were in the range of 13.5 ~ 82 kD. Most of protein spots expressed stably in the different development of leaf, but some of them were differential.(56.5kD, pI3.6) and (28kD, pD.3) appearing at rapid enlarging stage of leaf had consanguineous relationship with development stage. (39.9kD, pI5.0) , (39.9kD, pI5.12) , (38.5kD, pI5.0) and ( 38.5kD, pI5.12) disappeared gradually from rapid enlarging stage to mature stage.2-DE gel of mature leaf had evident variety from May to June, just as the variety of etiolation stage. The increase of richness of (18.6 kD, pI4.73 ), (20.1 kD, pI5.90 ), (13.7 kD, pD.80) , (13.7 kD, pI4.09) and (13.7 kD, pI4.37) which might have an important effect on proteins’ turnover had close to the decline of the richness of (53 kD, pI5.95 ) .The protein spot (24.5 kD, pI6.30) which had close relationship with etiolation stage should be the specific spot of etiolation stage of leaf.更多还原