Ottoboni L M, Leite A, Yunes J A, Targon M L, de Souza Filho G A, Arruda P
Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, SP, Brazil.
Plant Mol Biol. 1993 Mar;21(5):765-78. doi: 10.1007/BF00027110.
Several genomic and cDNA clones encoding the 22 kDa-like alpha-coixin, the alpha-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like alpha-coixin genes designated alpha-3A, alpha-3B and alpha-3C were found in the 15 kb alpha-3 genomic clone. The alpha-3A and alpha-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the alpha-3B gene, suggesting that the three alpha-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of alpha-coixin clones with the published sequences of 22 kDa alpha-zein and 22 kDa-like alpha-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like alpha-prolamins and the 19 kDa alpha-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5' and 3' flanking regions of alpha-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in alpha-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in alpha-3B that occupies approximately the same positions as those identified for the 22 kDa alpha-zein and alpha-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like alpha-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.
分离并测序了几个编码22 kDa类α-薏苡仁蛋白（薏苡仁种子的α-醇溶蛋白）的基因组和cDNA克隆。在15 kb的α-3基因组克隆中发现了三个相邻的22 kDa类α-薏苡仁蛋白基因，分别命名为α-3A、α-3B和α-3C。α-3A和α-3C基因在+652位呈现框内终止密码子。这两个具有截短开放阅读框的基因位于α-3B基因两侧，表明这三个α-薏苡仁蛋白基因可能是通过串联重复产生的，并且终止密码子是在重复之前引入的。将α-薏苡仁蛋白克隆推导的氨基酸序列与已发表的22 kDaα-玉米醇溶蛋白和22 kDa类α-高粱醇溶蛋白序列进行比较，发现了高度保守的蛋白质结构。该蛋白质由一个包含信号肽的N端、其后是十个由聚谷氨酰胺侧翼的15 - 20个氨基酸的高度保守串联重复序列以及一个短的C端组成。22 kDa类α-醇溶蛋白与19 kDaα-玉米醇溶蛋白的区别在于，19 kDa蛋白比22 kDa蛋白正好少一个重复基序。在α-3B的5'和3'侧翼区域内鉴定出了几个玉米醇溶蛋白和高粱醇溶蛋白基因共有的推定调控序列。与共有序列TATA、CATC和约-300醇溶蛋白框匹配的核苷酸序列在α-3B中相对于玉米醇溶蛋白和高粱醇溶蛋白基因的保守位置处存在。α-3B中存在两个推定的不透明-2框，其占据的位置与在22 kDaα-玉米醇溶蛋白和α-高粱醇溶蛋白基因中鉴定出的位置大致相同。使用玉米不透明-2 cDNA克隆的片段作为探针进行Southern杂交，证实了薏苡仁和高粱基因组中存在不透明-2同源序列。总体结果表明，参与薏苡仁、高粱和玉米22 kDa类α-醇溶蛋白基因表达的结构和调控基因起源于一个共同祖先，并且在物种分离后结构和调控序列中引入了变异。
