Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes.
Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes.
复制标题
非洲爪蟾卵母细胞中含赖氨酸的玉米醇溶蛋白聚集成蛋白体。
作者：
J. Wallace;G. Galili;E. E. Kawata;R. Cuellar;M. Shotwell;B. Larkins
玉米醇溶蛋白是玉米的贮藏蛋白，完全缺乏必需氨基酸赖氨酸和色氨酸。通过寡核苷酸介导的突变，将赖氨酸密码子以及编码赖氨酸和色氨酸的寡核苷酸在几个位置引入19千道尔顿玉米醇溶蛋白互补DNA中。来自猿猴病毒40（SV 40）外壳蛋白的450个碱基对开放阅读框也被工程化到玉米醇溶蛋白编码区中。在体外用SP6 RNA聚合酶系统合成修饰的玉米醇溶蛋白的信使RNA，并注射到非洲爪蟾卵母细胞中。这些修饰不影响玉米醇溶蛋白的翻译、信号肽切割或稳定性。修饰的玉米醇溶蛋白组装成类似于玉米蛋白体的结构的能力通过两个标准来测定：组装成对外源添加的蛋白酶有抗性的膜结合囊泡，以及自聚集成致密结构的能力。所有修饰的玉米醇溶蛋白都是膜结合的，只有含有17千道尔顿SV 40蛋白片段的玉米醇溶蛋白不能聚集。这些发现表明，通过基因工程创造高赖氨酸玉米是可能的。
Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.