小麦多聚半乳糖醛酸酶抑制蛋白的部分结构

The Partial Structure of Wheat Polygalacturonase-inhibiting Protein

为了弄清小麦多聚半乳糖醛酸酶抑制蛋白 (polygalacturonase inhibitingprotein ,PGIP)的作用机制 ,并为其在基因工程中的应用提供依据 ,对其结构进行了研究 .用Edman降解法测得小麦PGIP的N端序列为Lys Pro Leu Leu Thr Lys Ile Thr Lys Gly Ala Ala Ser Thr .用CD谱研究其二级结构 ,发现小麦PGIP天然态含有 4 3 7%的 β折叠和 13 1%的α螺旋 .酸碱和温度变性引起了二级结构改变 .不完全变性阶段 ,二级结构的变化表现为α螺旋无明显变化 ,β折叠遭到破坏 ;活性完全丧失阶段 ,β折叠变化很小 ,α螺旋含量明显减少 .用NR R(非还原 还原 )双向对角线SDS PAGE鉴定出小麦PGIP含有链内二硫键 .用去糖基化法确证了小麦PGIP的糖含量为 2 2 %.小麦PGIP与双子叶植物PGIP相比 ,一级结构差异较大 ,同源性由 36 %变为 9%;二级结构相似 ,都是高 β 折叠的蛋白 ;均具有链内二硫键 ;在糖含量上也相似 .研究结果为进一步弄清小麦PGIP作用机理打下了基础 ,同时对于植物抗赤霉病基因工程具有重要意义 .

In order to reveal the mechanism of wheat polygalacturonase-inhibiting protein (PGIP) and to make best use of it in gene engineering, its structure was studied. The N-terminal amino acid sequence of wheat PGIP was determined by Edman degradation. The result was: Lys-Pro-Leu-Leu-Thr-Lys-Ile-Thr-Lys-Gly-Ala-Ala-Ser-Thr. The secondary structure of wheat PGIP was obtained by the measurement of circular dichroic spectra. The native PGIP of wheat was estimated to contain about 43.7% β-sheet and 13.1% α-helix. The secondary structure was changed by effects of pH and temperature. α-helix had little change and β-sheet was destroyed while the protein denatured incompletely. When the activity of wheat PGIP lost, the content of β-sheet varied a little and that ofα-helix reduced apparently. Wheat PGIP had internal disulfide bond by analysis of NR/R (not reduced/reduced) two-dimensional electrophoresis. Wheat PGIP was deglycosylated using TFMS, and the result indicated that carbohydrates content was 22%. When dicotyledon PGIPs was compared with wheat PGIP, there was difference in primary structure. The N-terminal sequence of PGIPs in dicotyldonous plants shared 36% identical amino acids, but the percentage of overall identity was changed to only 9% when wheat PGIP was included in the analysis. However, PGIPs from Wheat and dicotyledon were consistent in secondary structure, disulfide bond and carbohydrates content. The results have laid the foundation for the elucidation of its mechanism, and is significant for breeding crops resistant to Gibberella zeae(Schw.) Petch.