Biosynthesis and its regulation of storage proteins of higher plant tuber crops.

细胞分裂研究表明，甘薯块根和块茎的贮藏蛋白均沉积在液泡中。这些蛋白质的前体是由膜结合的多聚体合成的，甘薯贮藏蛋白孢子胺的前体比成熟形式大4kd。通过对一种新方法构建的表达文库进行免疫学筛选，获得了马铃薯块茎中3种不同贮藏蛋白的全长基因克隆。还开发了一种改进的大肠杆菌表达载体，用于构建和鉴定全长cDNA。对这些克隆的核苷酸序列分析表明，马铃薯贮藏蛋白的前体在其N端含有典型的信号肽序列。然而，孢子胺前体的N末端额外序列包含16个氨基酸残基，信号肽片段后面的碱性氨基酸富含。体外处理研究表明，孢子胺前体经历了两步处理，信号肽在共翻译时被移除，随后翻译后移除富含碱性氨基酸的片段。孢子胺前体的结构和加工机制与酵母空泡蛋白和动物细胞溶酶体蛋白的前体相似。当利用酵母表达载体在酵母细胞中表达孢子胺前体时，至少有一部分被运送到液泡中。我们还成功地将孢子素基因与植物病毒强启动子连接到烟草中，并在烟草中表达了一个嵌合基因。烟草愈伤组织细胞中表达的孢子胺前体似乎被加工成成熟的大小。

Cell fractionation studies indicated that the storage proteins of the sweet potato tuberous root and the potato tuber are deposited in vacuoles. Precursors for these proteins were synthesized by membrane-bound polysomes, and the precursor for the sweet potato storage protein sporamin was 4 kd larger than the mature form. By immunological screening of cDNA expression libraries constructed by a novel method employing an E. coli expression vector-primed cDNA synthesis, full-length cDNA clones for sporamin and three different storage proteins of the potato tuber were identified. An improved E. coli expression vector for the construction and identification of full-length cDNAs has also been developed. Nucleotide sequencing of these cDNA clones indicated that precursors for potato storage proteins contain typical signal peptide sequences at their N-termini. The N-terminal extra-sequence of sporamin precursor, however, contained 16 amino acid residues enriched with basic amino acids following the signal peptide segment. In vitro processing studies suggested that the sporamin precursor undergoes two-step processings where the signal peptide is removed co-translationally followed by the post-translational removal of the segment enriched with basic amino acids. The structure and the processing mechanisms of the sporamin precursor resembles to those of the precursors for yeast vacuolar proteins and animal cell lysosomal proteins. When sporamin precursor was expressed in yeast cells using an yeast expression vector, at least part of them were transported into vacuole. We have also succeeded introducing and expressing a chimeric gene in which sporamin cDNA is joined to a strong plant virus promoter in tobacco. The sporamin precursor expressed in tobacco callus cells seems to be processed into mature size.