Nucleotide Sequence of a Stearoyl-Acyl Carrier Protein Desaturase cDNA from Developing Seeds of Rice

Stearoyl-ACP desaturase (EC 1.12.99.6) is a enTable 1. Characteristics of the cDNA encoding a rice stearoyl-ACP zyme localizing in plastid, which catalyzes conversion of stearoyl-ACP to oleoyl-ACP by primary introduction of a cis-double bond between carbon positions 9 and 10. Recently, cDNAs encoding a stearoyl-ACP desaturase were isolated from severa1 dicotyledonous species: safflower (Thompson et al., 1991), cucumber and caster (Shanklin and Somerville, 1991), potato (Taylor et al., 1992), spinach (Nishida et al., 1992), Brassica napus (Slocombe et al., 1992), Brassica rapa (Knutzon et al., 1992), and jojoba (Sato et al., 1992). Introduction of an antisense gene to stearoyl-ACP desaturase resulted in a reduced ratio of unsaturated fatty acids to saturated fatty acids in transgenic Brassica and Nicotiana plants (Cahoon et al., 1992; Knutzon et al., 1992), implicating this enzyme as a key enzyme in determining the balance between saturated and unsaturated fatty acids in higher plants. Therefore, genetic manipulation of stearoyl-ACP desaturase expression may control the amount of unsaturated fatty acids. Here we describe the isolation and features of a rice stearoyl-ACP desaturase cDNA. A cDNA library was constructed from developing seeds of rice 10 to 15 DAF. This library was screened using a PCR-amplified fragment, SP1, corresponding to a middle part of the rice stearoyl-ACP desaturase cDNA as a probe (see ”Technique” in Table I). Nine recombinant A clones of 90,000 plaques showed positive signals, and partial nucleotide sequences were determined for a11 of them. Since a11 of these possessed identical sequences, the nucleotide sequence of the longest clone, pRD9D-8, was determined. The insert size of pRD9D-8 was 1667 bp, encompassing an open reading frame encoding a polypeptide of 390 amino acid residues. This open reading frame may code for a precursor protein with a transit peptide to translocate this enzyme into the proplastid, since the stearoyl-ACP desaturase is localized in the stroma of plastids. Comparison of the precursor protein of rice with those of dicotyledonous plants showed 71 to 82% amino acid sequence identity; however, the N-terminal sequence of 41 amino acid residues were poorly conserved between rice and dicotyledonous plants. However, the exact cleavage site of the transit peptide is unclear. desaturase