Molecular studies on `marginal overdominance` in maize

在拟议的项目中，将通过基因表达和酶活性研究来检验边际优势假说。6个不相关的池间杂交种将在1个幼苗期(4叶期)和2个成株期(抽穗期、灌浆期)与它们的12个亲本进行比较。这项研究将集中在已经分离和测序的6个基因(玉米数据库)，这些基因编码碳水化合物和蛋白质代谢的重要酶(sps1，spp1，c-/p-/m-Aspat，gln4)。为了进行比较，将包括转录因子Dwarf8(D8)，其序列比对可用于大样本自交系，并检测到DNA多态和数量性状变异之间的显著关联(Thornsberry等人。2001)。根据已建立的方案，基因表达将通过Northern-blotting和酶活性进行分析。带状强度将通过光度测量或通过磷光成像仪进行测量。设计的聚合酶链式反应引物将从亲本品系的基因组DNA中扩增出亲本基因的片段。扩增的片段将被测序，以确定每对亲本的核苷酸多态。类似地，将评估酶的电泳迁移率。据我们所知，这项拟议的项目将是第一个研究边际优势作为杂种优势可能原因的分子研究。

In the proposed project, the hypothesis of marginal overdominance will be examined by gene expression and enzyme activity studies. Six unrelated interpool hybrids will be compared with their twelve parent lines in one juveline (4-leaf stage) and two adult-plant stages (heading, early grain filling). The study will focus on six already isolated and sequenced (Zea mays data bank) genes encoding important enzymes of the carbohydrate and protein metabolism (sps1, spp1, c-/p-/m-AspAT, gln4). For comparison, the transcription factor Dwarf8 (d8) will be included, for which sequence alignments are available for a large sample of inbred lines, and significant associations between DNA polymorphism and quantitative trait variation were detected (Thornsberry et al. 2001). Gene expression will be analysed by Northern-blotting and enzyme activity according to established protocols. Banding intensity will be measured photometrically or by means of a phosphoimager. PCR primers will be designed to amplify fragments of the absoveygenes from genomic DNA of the parent lines. The amplified fragments will be sequenzed to determine the nucleotide polymorphism for each pair of parent lines. Analogously, the electrophoretic mobility of the enzymes will be assessed. To our knowledge, the proposed project will be the first molecular study examining marginal overdominance as a possible cause of heterosis.