玉米Wi2基因在内皮层细胞壁发育过程中的功能研究

The Wilted2 (Wi2) mutant is a semi-dominant mutant induced by EMS. Compared to wild type, the mutant is more sensitive to water stress. The Wi2 gene was mapped to chromosome 3 within an interval of 35Kb. A candidate gene which encoded a beta-tubulin6 protein (TUB6) was identified by sequence analysis of candidate region. We had also confirmed the cloning of this tub6 by transgenic approach, with the transgenic maize having the mutated tub6 genes showed wilted phenotype exactly as the Wi2 mutants. The TUB6 of Wi2 mutant had one amino acid substitution: proline (P287) to leucine (L), which introduced one extra alpha-helix in mutant as compared to wild type, The alpha helix can likely affect the assembly of microtubules. Histochemical analysis suggested that tub6 were mainly expressed in xylem, pith，phloem, pericycle and endodermis cells of the root maturation zone, but little or no expression was detected in root cap, division zone, elongation zone, stem, leaf and the cortex and epidermis of root maturation zone. Fluorescence microscope observation of cross section of mutant and wild type crown root showed that the wall of endodermis was not thickened in Wi2 mutant. And suberin content is lower in Wi2.This changes affected the transportation of water and nutrient from root to shoot, and make Wi2 mutant more sensitive to water stress. The mutation of TUB6 may affect the dynamic of microtubules which affect the motion trajectory of suberin synthase and the thickening of the endodermis walls. Microtubules plays an indispensable role for intracellular transport of substances. This project will use different fluorescent labels to mark the microtubules and suberin synthase, study the dynamic of microtubules and suberin synthase in the cell, analysis the function of maize Wi2 gene in endodermis cell wall formation.

玉米Wilted2(Wi2)突变体是一个EMS诱变的半显性突变体，该突变体对水分胁迫敏感。利用图位克隆及转基因互补实验确定Wi2基因编码微管蛋白（TUB6），Wi2突变体TUB6蛋白的第287位脯氨酸变成了亮氨酸，蛋白三维结构预测表明该变化使得Wi2突变体TUB6蛋白多了一个α螺旋，该α螺旋影响到微管的稳定性。组织表达特异性实验表明Wi2基因主要在根成熟区的木质部、韧皮部、髓、中柱鞘及内皮层细胞中表达，显微观察发现突变体的内皮层细胞壁没有加厚，对内皮层细胞壁成分分析结果表明突变体中的木栓质含量降低。这种变化影响了水分及营养物质由根向地上部运输，使突变体对水分胁迫敏感。微管蛋白对于胞内物质运输起着不可或缺的作用，本项目将利用不同的荧光标记对微管和脂肪酸合成酶进行定位，研究微管和木栓质合成基因在细胞内的动态，确定微管对相关蛋白运动轨迹的影响，解析Wi2基因在内皮层细胞壁增厚过程中所起的作用。

前期研究确定玉米Wilted2(Wi2)突变体是一个EMS诱变的半显性突变体，该突变体对水分胁迫敏感。利用图位克隆及转基因互补实验确定Wi2基因编码微管蛋白（TUB6），Wi2突变体TUB6蛋白的第287位脯氨酸变成了亮氨酸，蛋白三维结构预测表明该变化使得Wi2突变体TUB6蛋白多了一个α螺旋。通过项目实施，共聚焦显微观察显示该α螺旋影响到微管的稳定性。组织表达特异性实验表明Wi2基因主要在根成熟区的木质部、韧皮部、髓、中柱鞘及内皮层细胞中表达，显微观察发现突变体的内皮层细胞壁没有加厚，对内皮层细胞壁成分分析结果表明突变体中的木栓质含量降低。这种变化影响了水分及营养物质由根向地上部运输，使突变体对水分胁迫敏感。通过本项目的实施，明确当玉米TUB6微管蛋白突变后，影响到细胞骨架的稳定性进而影响了内皮层细胞壁的发育。