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Plant and Cell Physiology, 2000, Vol. 41, No. 12 1381-1391
© 2000 Oxford University Press

A Second Gene for Acyl-(Acyl-Carrier-Protein): Glycerol-3-Phosphate Acyltransferase in Squash, Cucurbita moschata cv. Shirogikuza*, Codes for an Oleate-Selective Isozyme: Molecular Cloning and Protein Purification Studies

Ikuo Nishida1, Masako Sugiura, Akiko Enju and Masanobu Nakamura

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo, 113-0033 Japan

A new isogene for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) in squash has been cloned and the gene product was identified as oleate-selective GPAT. Using PCR primers that could hybridise with exons for a previously cloned squash GPAT, we obtained two PCR products of different size: one coded for a previously cloned squash GPAT corresponding to non-selective isoforms AT2 and AT3, and the other for a new isozyme, probably the oleate-selective isoform AT1. Full-length amino acid sequences of respective isozymes were deduced from the nucleotide sequences of genomic genes and cDNAs, which were cloned by a series of PCR-based methods. Thus, we designated the new gene CmATS1;1 and the other one CmATS1;2. Genome blot analysis revealed that the squash genome contained the two isogenes at non-allelic loci. AT1-active fractions were partially purified, and three polypeptide bands were identified as being AT1 polypeptides, which exhibited relative molecular masses of 39.5–40.5 kDa, pI values of 6.75–7.15, and oleate selectivity over palmitate. Partial amino-terminal sequences obtained from two of these bands verified that the new isogene codes for AT1 polypeptides.

1 Corresponding auther: E-mail, nishida@biol.s.u-tokyo.ac.jp; Fax, +81-3-3814-1728.


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