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

细胞分离研究表明，甘薯块根和块茎的贮藏蛋白以液泡形式储存。这些蛋白的前体由膜结合多体合成，甘薯贮藏蛋白孢素的前体比成熟蛋白大4kd。采用大肠杆菌表达载体引物cDNA合成新方法构建cDNA表达文库，通过免疫筛选，鉴定了马铃薯块茎孢素和3种不同贮藏蛋白的全长cDNA克隆。一种改进的大肠杆菌表达载体，用于构建和鉴定全长cdna。这些cDNA克隆的核苷酸测序结果表明，马铃薯贮藏蛋白前体在其n端含有典型的信号肽序列。然而，孢素前体n端外序列包含16个氨基酸残基，这些氨基酸在信号肽段之后富集。体外加工研究表明，孢子素前体经历两步加工，其中信号肽被共翻译去除，然后是翻译后去除富含碱性氨基酸的片段。孢子素前体的结构和加工机制类似于酵母空泡蛋白和动物细胞溶酶体蛋白的前体。用酵母表达载体在酵母细胞中表达孢素前体时，至少有一部分被转运到液泡中。我们还成功地在烟草中引入并表达了一个将孢子素cDNA与一个强植物病毒启动子连接的嵌合基因。在烟草愈伤组织细胞中表达的孢素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.