Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits.
Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits.
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DOI：
10.1104/pp.92.4.881
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发表时间：
1990-04
期刊：
影响因子：
7.4
通讯作者：
J. Preiss;Stephanie Danner;Peter S. Summers;Matthew K. Morell;Carolyn R. Barton;Limei Yang;Matthew Nieder
中科院分区：
文献类型：
--
作者：
J. Preiss;Stephanie Danner;Peter S. Summers;Matthew K. Morell;Carolyn R. Barton;Limei Yang;Matthew Nieder
已知在玉米（Zea mays L.）胚乳突变体中，两个独立位点的adp葡萄糖焦磷酸化酶活性降低，这两个位点分别是缩水-2 （Sh(2)）和脆性-2 （Bt(2)）。菠菜叶adp葡萄糖焦磷酸化酶先前已被证明包含51和54千道尔顿两个亚基。用聚丙烯酰胺凝胶电泳分离野生型玉米胚乳蛋白制备Western blots，发现对菠菜叶片adp葡萄糖焦磷酸化酶两个亚基的抗体与不同条带发生交叉反应。抗菠菜叶51千道尔顿的亚基抗体与55千道尔顿的玉米胚乳蛋白交叉反应，抗菠菜叶54千道尔顿的亚基抗体与60千道尔顿的玉米胚乳蛋白交叉反应。在玉米胚乳提取物和高度纯化的玉米胚乳adp葡萄糖焦磷酸化酶制剂中观察到这些免疫反应。突变体bt(2)胚乳缺乏55千道尔顿亚基，而突变体sh(2)胚乳缺乏60千道尔顿亚基。这些结果表明，玉米胚乳ADPglucose焦磷酸化酶是由两个免疫学上不同的亚基组成的，bt(2)和sh(2)突变通过缺乏这两个亚基中的一个而导致ADPglucose焦磷酸化酶活性降低。从水稻种子cDNA表达文库中抗原选择的ADPglucose pyrophospylase cDNA克隆与W64A bt(2)突变体中缺失的玉米胚乳转录物对应的cDNA进行了强杂交。因此，bt(2)突变体不仅导致小亚基缺失，而且导致相应转录本缺失。Bt(2)是玉米胚乳adp葡萄糖焦磷酸化酶小亚基（54千道尔顿）的结构基因。
Activity of the enzyme ADPglucose pyrophosphorylase is known to be reduced in maize (Zea mays L.) endosperm mutants at two independent loci, Shrunken-2 (Sh(2)) and Brittle-2 (Bt(2)). Spinach leaf ADPglucose pyrophosphorylase has previously been shown to comprise two subunits of 51 and 54 kilodaltons. Anti-bodies raised to each of the two subunits of spinach leaf ADPglucose pyrophosphorylase were found to cross-react to different bands on Western blots prepared from polyacrylamide gel electrophoresis separated wild-type maize endosperm proteins. The anti-spinach leaf 51 kilodalton subunit antibody cross-reacted with a 55 kilodalton maize endosperm protein and the anti-spinach leaf 54 kilodalton subunit antibody cross-reacted with a 60 kilodalton maize endosperm protein. These immunological reactions were observed in maize endosperm extracts and with a highly purified preparation of maize endosperm ADPglucose pyrophosphorylase. Mutant bt(2) endosperm lacked the 55 kilodalton subunit while mutant sh(2) endosperm lacked the 60 kilodalton subunit on Western blots. These results suggest that the maize endosperm ADPglucose pyrophosphorylase is made up of two immunologically dissimilar subunits and that the bt(2) and sh(2) mutations cause reduction in ADPglucose pyrophosphorylase activity through the lack of one of these two subunits. An ADPglucose pyrophosphorylase cDNA clone antigenically selected from a rice seed cDNA expression library was found to hybridize strongly with a cDNA corresponding to a maize endosperm transcript which is absent in a W64A bt(2) mutant. Thus, the bt(2) mutant causes the absence not only of the small subunit but of the corresponding transcript. Bt(2) is implicated as the structural gene for the small (54 kilodalton) subunit of maize endosperm ADPglucose pyrophosphorylase.