Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.
Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.
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DOI：
10.1046/j.1365-313x.2003.01704.x
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发表时间：
2003-04
期刊：
影响因子：
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通讯作者：
R. Yui;S. Iketani;T. Mikami;T. Kubo
中科院分区：
文献类型：
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作者：
R. Yui;S. Iketani;T. Mikami;T. Kubo
我们推测甜菜细胞质雄性不育（CMS）可能是线粒体功能障碍影响花药正常发育的结果。为了验证这一假设，我们试图通过抑制线粒体丙酮酸脱氢酶（PDH）的表达来模拟甜菜CMS表型，该酶对三羧酸（TCA）循环的运作至关重要。用甜菜花蕾的cDNA文库筛选允许鉴定两个PDH E1 α亚基基因（bvPDH_E1alpha-1和bvPDH_E1alpha-2）。bvPDH_E1alpha-1在主根中高表达，而bvPDH_E1alpha-2在花芽中表达最丰富。bvPDH_E1 α的绿色荧光蛋白（GFP）融合物显示线粒体靶向特性。将300-bp bvPDH_E1 α-1 cDNA序列（从+620到+926）以反义方向连接到绒毡层特异性启动子，然后导入烟草。反义表达bvPDH_E1 α-1导致内源bvPDH_E1 α-1转录本显著减少，雄性不育。不育花药绒毡层肿胀或空泡化。在不育花药中，细胞器如弹性体、绒毡层体和圆形体等发育不良，小孢子外壁发育异常。这些特征是甜菜CMS所共有的。因此，结果清楚地表明，抑制花药绒毡层中的PDH活性足以引起雄性不育，甜菜CMS的表型。
We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.