植物細胞増殖因子及び不定胚形成に関する分子生物学的研究
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概要
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An experimental system of somatic embryogenesis at a high frequency was established in asparagus. Using this system, I explored physiological and biochemical characteristics of non-embryogenic cells, embryogenic cells and somatic embryos. The results indicated that more genes were expressed in embryogenic cells than in non-embryogenic cells and that the globular embryo already differentiated biochemically although it has not differentiated morphologically. Next, I cloned AtECP31 and AtECP63 cDNAs from an Arabidopsis cDNA library. The AtECP31 was a single-copy gene, but the AtECP63 might belong to a small multigene family. The AtECP31 cDNA contained an open reading frame of 256 amino acids. AtECP31 showed more than 60% identity with LEA proteins in group 5. The transcripts of AtECP31 accumulated specifically in mature seeds. On the other hand, AtECP63 cDNA encoded a polypeptide of 449 amino acids. AtECP63 had 16 repeats of 11 amino acids and showed extensive similarity with embryogenic proteins belonging to LEA proteins in group 3. AtECP63 contained 8 well-conserved tyrosine phosphorylation sites, suggesting that it may be a putative phosphotyrosine protein. AtECP63 was expressed in mature seeds, and exogenous ABA induced its expression in immature siliques, but not in vegetative tissues. These results suggested that AtECP31 and AtECP63 might be involved in maturation and desiccation tolerance of seeds in different ways. RFLP and CAPS mapping suggested that AtECP31 and AtECP63 genes might be present on chromosome 3 and chromosome 4, respectively. Phytosulfokine-α (PSK-α), a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at low concentrations. OsPSK cDNA encoding a PSK-α precursor was isolated from rice. The cDNA is 725 base pairs in length and the 89-amino acid product, preprophytosulfokine (PP-PSK), has a 22-amino acid hydrophobic region that resembles a cleavable leader peptide at its N-terminus. The PSK-α sequence occurs only once within the precursor, close to the C-terminus. While PSK-α in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. PP-PSK mRNA was detected at an elevated constitutive level in rice Oc culture cells and the expression of OsPSK was found to be reinforced by auxin and cytokinin. While PSK-α molecules have never been identified in any intact plant, RT-PCR analysis demonstrated that OsPSK is expressed in rice seedling meristems, indicating that PSK-α may be important for plant cell proliferation not only in vitro but also in vivo. OsPSK, a single-copy gene, consists of one large intron and two exons. The 5-amino acid PSK-α sequence is encoded in the second exon. A putative TATA box was found at position -68 with respect to the transcription initiation site. Upstream of this sequence, several potential regulatory elements, including one CAAT-box, three CCAAT-boxes, one enhancer core-like sequence, and three E-boxes were identified. By constructing plasmids with various lengths of the 5' upstream regions of the OsPSK gene fused to the coding sequence for a bacterial β-glucuronidase (GUS), I demonstrated a region 1.9-kb upstream of the transcription initiation point, which contains most of the putative 5'-regulatory elements, to be sufficient for maximal-level GUS expression in transformed rice Oc cells. The promoter of the OsPSK gene gave significantly higher levels of GUS expression compared with the CaMV 35S promoter, suggesting that the OsPSK promoter could be useful for the constitutive expression of a foreign gene at high levels in transformed rice culture cells.
- 2000-11-02