Tsuchiya T, Toriyama K, Yoshikawa M, Ejiri S, Hinata K
Faculty of Agriculture, Iwate University, Morioka, Japan.
Plant Cell Physiol. 1995 Apr;36(3):487-94. doi: 10.1093/oxfordjournals.pcp.a078784.
A cDNA for a pathogenesis-related endo-beta-1,3-glucanase isolated from soybean, was fused to an anther tapetum-specific promoter (Osg6B promoter) isolated from rice and the resulting chimeric gene was introduced into tobacco. The Osg6B promoter became active in the anther tapetum during formation of tetrads and the tapetal glucanase activity in the transgenic plants caused in a significant reduction in the number of fertile pollen grains. Most of the pollen grains were aberrant in shape, lacked germinal apertures and aggregate of the pollen grains. Granules of beta-1,3-glucan, which have not previously been reported, were often observed to adhere to the surface of the pollen grains. Further observations revealed that the callose wall was almost absent in the pollen tetrads of transgenic plants. In wild-type plants, by contrast, the tetrads were surrounded by callose that was degraded soon after the tetrad stage to release free microspores. Thus, the introduced gene for endo-beta-1,3-endoglucanase under the control of the Osg6B promoter caused digestion of the callose wall at the beginning of the tetrad stage, a time that was just a little earlier than the time at which endogenous glucanase activity normal appears. These results demonstrate that premature dissolution of the callose wall in pollen tetrads causes male sterility and suggest that the time at which tapetally produced glucanase is activate is critical for the normal development of microspores.
从大豆中分离出的一种病程相关的内切β-1,3-葡聚糖酶的cDNA，与从水稻中分离出的花药绒毡层特异性启动子（Osg6B启动子）融合，将所得嵌合基因导入烟草。在四分体形成期间，Osg6B启动子在花药绒毡层中变得活跃，转基因植物中的绒毡层葡聚糖酶活性导致可育花粉粒数量显著减少。大多数花粉粒形状异常，缺乏萌发孔且花粉粒聚集。以前未报道过的β-1,3-葡聚糖颗粒经常被观察到附着在花粉粒表面。进一步观察发现，转基因植物花粉四分体中几乎没有胼胝质壁。相比之下，在野生型植物中，四分体被胼胝质包围，在四分体阶段后不久胼胝质就会降解以释放游离小孢子。因此，在Osg6B启动子控制下导入的内切β-1,3-内切葡聚糖酶基因在四分体阶段开始时导致胼胝质壁的消化，这个时间比内源性葡聚糖酶活性正常出现的时间略早一点。这些结果表明，花粉四分体中胼胝质壁的过早溶解导致雄性不育，并表明绒毡层产生的葡聚糖酶被激活的时间对小孢子的正常发育至关重要。
