the isolation and functional characterisation of a b. napus acyl carrier protein 5′ flanking...

Plant Molecular Biology 18: 1163-1172, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium. 1163

The isolation and functional characterisation of a B. napus acyl carrier protein 5' flanking region involved in the regulation of seed storage lipid synthesis

Jacqueline de Silva, Susan J. Robinson and Richard Safford *

Cell Sciences, Unilever Research, Colworth House, Sharnbrook, Bedford, MK44 ILQ, UK (* author for correspondence)

Received 16 July 1991; accepted in revised form 14 January 1992

Key words: acyl carrier protein, Brassica napus, lipid synthesis, seed-specific expression, transgenic tobacco, 5' flanking region

Abstract

Acyl carrier protein (ACP) is a key component of the fatty acid biosynthetic machinery in plants. A 1.4 kb 5' flanking region of a Brassica napus ACP gene (ACP05) was transcriptionally fused to the reporter gene /~-glucuronidase (GUS), and expression of the chimaeric gene monitored in transgenic tobacco. GUS activity was found to increase through seed development reaching a maximum value, coincident with the most active phase of storage lipid synthesis that was, on average, 100-fold higher than that observed in leaf. In control plants transformed with CaMV 35S-GUS constructs, GUS activity was similar in leaf and all stages of seed development. Based on average values, the level of GUS expression obtained via the ACP promoter was comparable to that obtained from the CaMV 35S promoter. We therefore conclude that the isolated 5' ACP flanking sequence represents a strong promoter element involved in the developmental regulation of storage lipid synthesis in B. napus seed tissue. Putative regulatory elements in the 5' upstream region of ACP05 were identified by dot matrix analysis and by sequence comparison with the upstream regions from a second seed-expressed rape ACP gene and from an Arabidopsis ACP gene.

Introduction

In plants, fatty acids are required in all cells for membrane biosynthesis and, in addition, specifically within seed tissue, for the developmentally regulated process of storage lipid synthesis. The mechanism and organellar location of de novo fatty acid biosynthesis is the same in leaves and seeds [22] and regulatory control of the process is most likely exerted by means of differential expression of genes encoding fatty acid biosynthetic enzymes.

Initial evidence for differential gene expression controlling fatty acid biosynthesis came from studies on acyl carrier protein (ACP). In both spinach [ 14] and barley [ 12] 2 isoforms of ACP were found in leaf and only a single isoform in seed. Amino acid sequencing studies showed that the 2 leaf isoforms from both species were prod- ucts of different genes. In soybean [9] 2 distinct forms of malonyl CoA:ACP transacylase have been found in leaf, but only 1 form in seed.

In B. napus embryos, only a single molecular weight form of ACP is found, but cDNA cloning

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studies [19] have revealed a more complex situ- ation. Seed ACP was found to be encoded by a multi-gene family, which is not expressed in leaf, and which shows considerable heterogeneity both at the nucleotide and deduced amino acid level. Thus from 10 cDNA clones, 6 unique genes, encoding 5 different mature ACP polypeptides were identified. Subsequent Southern blot studies [5] in fact revealed the presence of ca. 35 seed- expressed ACP genes per haploid genome. Mul- tiple genes could provide a mechanism to regulate levels of ACP in response to the widely differing requirements for fatty acid biosynthesis which occur during oil seed development.

In developing B. napus seeds, the level of ACP has been shown to rise dramatically prior to the onset of storage lipid synthesis [19], suggesting activation of specific members of the ACP gene family. We are interested in identifying these particular genes and isolating the regulatory elements which control their expression. To this end we have previously identified a sub-group of ACP cDNAs which are expressed in concert with the lipid synthetic phase of seed development in B. napus [5]. Two genomic ACP clones have been isolated [5] which were found to be identical, within their transcribed regions, to distinct members of this sub-group. In this study we report the DNA sequence and functional analysis of the 5' flanking region of one of these ACP genomic clones (ACP05). In the functional analysis we investigated the ability of the flanking region to direct seed-specific, developmentally-regulated gene expression by fusing the sequence to the fi- glucuronidase (GUS) reporter gene and monitor- ing expression of the chimaeric gene in transgenic tobacco.

Materials and methods

DATA cloning

Restriction enzyme digestion of plasmid DNA was performed in the appropriate salt buffer [15] at 37 °C, using 5 U of enzyme per #g DNA. Fragments were separated by electrophoresis in

0.8~o agarose slab gels and recovered using DEAE membrane [7]. T4 DNA ligase was used to ligate DNA fragments, typically at a DNA concentration of 1 ng//A, in 66 mM Tris-HC1 pH7.5, 6 .6mM MgC12, 10mM dithiothreitol, 10 mM rATP for 4-12 h at 20 °C. Ligated mix- tures were used to transform Escherichia coli JM101.

DNA sequencing

Fragments were subcloned into M13 and sequenced using a modified T7 DNA polymerase [23] and chain-terminating dideoxynucleotides [20]. The reactions were electrophoresed on 6~o acrylamide/urea gels and sequence data analyzed using DNAStar software on an IBM AT.

Mobilisation of pAP1GUS into Agrobacterium tumefaciens

pAP1GUS was mobilised from E. coli JM101 into A. tumefaciens LBA4404 [ 11] using the helper plasmid pRK2013 (in E. coli HB101) in a tripartite mating. Binary vector containing transconjugates were selected on 50 #g/ml rifam- picin and 50/~g/ml kanamycin. Confirmation of pAP1GUS structure in A. tumefaciens was obtained by restriction analysis of plasmid DNA.

Tobacco transformation

0.5 cm discs ofNicotiana tabacum (cv. SR1) were incubated with a culture ofA. tumefaciens ACH5 containing pLBA4404/pAP1GUS for 10rain, blotted dry and transferred onto feeder plates of haploid Nicotiana plumbaginifolia suspension culture [2] on shoot-inducing medium [0.9~o agar, MS salts [17], 3~o sucrose, 0.2 rag/1 indole ace- tic acid, 1 mg/1 benzylaminopurine]. After 3 days, discs were transferred to shoot-inducing medium containing 500/~g/ml cefotaxime and 100/~g/ml kanamycin. Regenerating shoots were excised and placed on minus-hormone medium (0.9~o

agar, MS salts [17], 3~o sucrose) containing 500 #g/ml cefotaxime. Once roots had become established, shoots were again excised and placed on minus-hormone medium containing 100/~g/ml kanamycin. After rooting on selective medium, plants were transferred to soil and grown at 25 ° C under a 16 h photoperiod.

Plant DNA isolation and Southern blotting

Plant DNA was isolated from leaf tissue as previously described [4] and further purified by phenol/chloroform (1:1) extraction and ethanol precipitation. After digestion with restriction enzymes, DNA was fractionated on 0.8~o agarose and transferred to nitrocellulose as described [ 15 ]. Blots were hybridised at 65 ° C overnight in 5 x SSC, 0.25 ~o milk powder, 5 mM EDTA containing a 32p-labelled RNA probe prepared using run-off transcription of a 5' upstream fragment [-291 to -33] of gene ACP05 cloned into the RNA expression vector SP65 (Amersham). Strin- gency of the final wash was 0.2 x SSC.

~-glucuronidase (GUS) assays

GUS activity was assayed in seed tissue harvested at three stages of development (see Results) and in leaf tissue. Tissue was ground on ice, in the presence of acid washed sand, with GUS extraction buffer (5 mM sodium phosphate pH 7.5, 0.1 ~o Triton X-100, 1 mM EDTA, 10 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride) in Eppendorf tubes using Kontes disposable pes- tles. Insoluble material was removed by centri- fugation for 5 rain in a microfuge and superna- tants stored at -80 °C. 50 #1 plant extract was incubated with 1 ml assay buffer (GUS extraction buffer containing i mM methylumbelliferyl glucu- ronide (MUG)) at 37 °C. 250 #1 aliquots were removed after 0, 5, 15 and 30 min into 750 #1 0.2 M Na2CO3 and fluorescence of the released methyl umbelliferone (MU) was measured on a Baird Nova 1 spectrofluorometer set at an exci- tation wavelength of 365 nm and an emission

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wavelength of 455nm. Using standard MU curves, results were calculated as pmol MU formed per minute per mg fresh weight.

Fatty acid determination

Lipid was extracted from tobacco seeds at vari- ous developmental stages by maceration in chloroform/methanol (2:1). After a 1~o sodium chloride wash, chloroform-soluble lipids were dried down and refluxed with methanol/toluene/ concentrated sulphuric acid (20:10:1). The result- ing fatty acid methyl esters were extracted with hexane and analysed by gas chromatography to determine total fatty acid content.

Results

DNA sequence analysis of 5' upstream region of A CP05

A 2.3 kb Barn HI-Sst I fragment of 2EMBL 4 recombinant ACP05, containing ca. 0.87 kb of the transcriptional unit of the gene together with 1.43 kb of the 5' upstream region (see Fig. 1), was cloned into pTZ18R [16] to form pTZ5BS. In- ternal restriction fragments were sub-cloned into M13 rap8 and mp9, as shown in Fig. 1 (ii) and sequenced as described in Materials and methods. The DNA sequence of the transcribed part of ACP05 has been previously reported [5] and a further 924 nucleotides upstream of the transcription start site was determined (Fig. 2).

Construction of pAP1 GUS

A transcriptional fusion between the 5' upstream region of the ACP05 gene (Fig. 1 (iii)) and the /%glucuronidase (GUS) reporter gene was con- structed in the plant transformation vector pTAK. Bam HI-linearised pTZ5BS was partially di- gested with Bgl II and a 1.4 kb Barn HI-Bgl II fragment, containing the 5' upstream region of the gene plus 50 bp of 5' non-coding sequence,

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0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

I I I I I 1 I I I I I I I

(i) I I I I II I B P G H G S T

a p

(i i) , b

C

d

AP1 PROMOTER

Oii) >

Fig. 1. (i) Restriction map of 2EMBL clone ACP05; B, Barn HI; P, Pst I; G, Bgl II; H, Hind III; S, Sal I; T, Sst I. Scale in bp. (ii) Sequencing strategy: fragments cloned into M 13 mp8 and rap9 and sequenced using a modified T7 DNA polymerase and chain terminating dideoxynucleotides. (iii) The AP1 promoter region, equivalent to 1.4 kb of 5' upstream sequence of ACP05.

was recovered and ligated into Bam HI- linearised, phosphatased pTAK vector to form pAP1GUS, pAP1GUS was transformed into E. coli JM101 and recombinant clones screened to confirm insertion of a single promoter fragment in the correct orientation.

Tobacco transformation

pAP1GUS was mobilised into A. tumefaciens LBA4404 and the resultant binary vector strain used to infect N. tabacum leaf discs (as described in Materials and methods). Control infections were carried out with A. tumefaciens LBA4404 carrying the pcTAK plasmid, in which the GUS gene is linked to the cauliflower mosaic virus 35S (CaMV 35 S) promoter. Transformed shoots were rooted in the presence of kanamycin, plants re- generated and allowed to self-pollinate. Transfer of an intact gene fragment was confirmed by Southern blotting (the number of copies of the GUS gene in the AP1GUS transformed plants was found to vary from 1 to 4). 13 AP1GUS

plants and 4 cTAK plants were selected for analysis of GUS activity.

Morphological staging of tobacco seed development

Under the particular growth conditions used for propagating these transgenic tobacco plants, the rate of development of individual seed pods on a single plant was found to be variable (the earliest pods developing the fastest). It was therefore not meaningful to use days after flowering as a developmental marker for assaying for GUS activity and instead, seeds were 'staged' accordingly to morphological characteristics. In total, 5 stages of development were identified on the basis of seed size and pigmentation:

1) 2) 3)

coat 4) 5)

0.4-0.5 mm long and white 0.5-0.6 mm long and light brown 0.6-0.8 mm long, brown with a hard seed

0.6-0.8 mm long and dark brown desiccated

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CTGCAGCCAG AAGGATAAAG AAATTTTGGA CGCCTGAAGA AGAGGCAGTT -875

CTGAGGGAAG GAGTAAAAGA GTATGTCTCC TTAACTCTAC TATCAAGTTT -825

CAAGAAGCTG AGCTTGGCTC TACCTTGATA TGTTTATTGC TGTTGTGCAG -775

GTATGGTAAA TCATGGAAAG AGATAAAGAA TGCAAACCCT GAAGTATTCG -725

CAGAGAGGAC TGAGGTGAGA GAGCATGTCA CTTTTGTGTT ACTCATCTGA -675

ATTATCTTAT ATGCGAATTG TGAGTGGTAC TAAAAAAGGT TGTAACTTTT -625

GGTAGGTTGA TTTGAAGGAT AAATGGAGdAACTTGbTTCG GTAGCCGTAA -575

~ TTG GGAATCTCTT GGGTTTTAAA TTGCTATGGA GTTTTTTTTT -525

GCCTGCGTGA CAACATATCA TCAGCTGTTG AGAAGGAAGA TGGTATTAGA -475

AAGGGTCTTT CTTTCACATT TTGTGTTGTG GACAAATATT AAAGTCAAAT -425 P 2 ~

GTGGCACATG GATTTTAATT CGGCCGGTAT GGTTTGGTT~AGACTk3GTTT -375

AACATGTATA AT%~kGTCTT~f GTTTTATTTG GCTCAGCGGT TTGTTGGTGT -325

TGGTTAGGAA CTTAGGCTTG TCTCTTTCTG ATAAGATCTG ATTGGTAAGA -275

TATGGGTACT GTTTGGTTTA TATGTTTTGA CTATTCAGTC ACTATGGCCC -225

CCATAAATTT TAATTCGGCT GGTATGTCTC GGTTAAGACC GGTTTGACAT -175

GGTTCATTTC AGTTCAATTA TGTGAATCTG GCACGTGATA TGTTTACCTT -125 P3---e-

CACACGAACA TTAGTAAT~A TGGGC~AATT TAAGACTTAA CAGCCTAGAA -75 " q ' P 3

AG~CCCATC~ TATTACGTAA CGACATCGTT TAGAGTGCAC CAAGCTTATA -25

AATGACGACG AGCTACCTCG GGGCA 1

Fig. 2. Nuc•e•tidesequence•f924bp•fthe5'•ankingregi•n•fACP•5;nuc•e•tide•=transcripti•nstartsite.The`TATA,b•x isunderlined andthepalindromicsequencesPl-P3 areboxed(see Discussion).

Lipid synthesis

Lipid accumulation was measured during tobacco seed development. Chloroform-soluble lipids were extracted from seed at developmental stages 1-5 and total fatty acid content determined. Stage 3 was identified as the most active phase of lipid synthesis (Fig. 3).

Assay of fl-glucuronidase activity in transgenic tobacco

Extracts from leaf and seed stages 1, 2, and 3 were assayed for GUS activity (Table 1). Con- sistent °developmental' patterns of GUS expres-

sion were observed in individual plants of both the AP1GUS and cTAK groups, although the absolute levels of expression varied considerably from plant to plant (see below). In AP1GUS- transformed plants, the level of GUS activity (pmol MU per minute per mg fresh weight) in leaf tissue was very low (average value 7.2) compared to seed tissue where it was found to increase through development from an average value of 68.7 in stage 1 to 300.6 in stage 3. (Further analysis of stage 4 and 5 seeds from a number of the plants showed GUS activity to fall from the maximum at stage 3; results not shown). In contrast, in plants transformed with pcTAK, the level of GUS expression was higher in leaf (average value 237.4) than in seed tissue, where the aver-

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pg lipid/seed 25

20

15

10

5

0 i i I I i

1 2 3 4 5

S e e d Stage

Fig. 3. Synthesis of storage lipid during tobacco seed development. Lipid was extracted from tobacco seed at 5 stages of development, total fatty acid content determined by gas liquid chromatography and expressed as #g lipid/seed.

age value dropped from 187.5 at stage 1 to 56 at stage 3.

The variation in absolute levels of GUS expression between individual plants of both the AP1GUS and the cTAK transgenic populations was very high (up to 1000-fold). There was, how- ever, no correlation between level of activity and gene copy number (data not shown) and the variation is most likely to be due to integration of DNA at different sites in the genome, the so- called position effect [21 ].

Because of the large differences in absolute levels of expression between individual plants, relative levels of GUS activity (seed/leaf) were calculated to allow a more 'standardised' interpretation of the data in terms of tissue specific expression. Table 2 shows the relative levels of activity for each of the three seed stages of individual AP1GUS and cTAK transformed plants and Fig. 4 shows, in histogram form, the average values obtained from the 2 respective

120

100

80

60

40

20

Relative GUS activity (seed/leaf)

108.4

29.6

7.4

0/7 1.0 1.0

1 2 3 1 2

cTAK AP1GUS

Fig. 4. Tissue specificity of the AP1 promoter. Histogram obtained by averaging the relative GUS activities (seed/leaf) for the seed stages 1-3 of 13AP1GUS and 4 cTAK transgenic plants.

transgenic populations. In each of the cTAK transformed plants, the level of GUS activity in all 3 stages of seed development was similar to that found in leaf tissue and thus the average seed/leaf values were close to 1. By contrast, in pAP 1GUS transformed plants, the level of GUS activity in stage 3 seeds was, on average, 100 times greater than that observed in leaf. We therefore conclude from these results that the AP1 promoter element is able to regulate gene expression in tobacco in a seed specific manner and tempo- rally in concert with lipid synthesis.

Comparison of GUS expression in stage 3 seeds ofAP 1GUS and cTAK transformed plants revealed a higher average level of activity in the former. Since the CaMV 35S promoter present in the cTAK construct is widely acknowledged as a strong plant promoter, the implication is that the AP 1 promoter itself is a strong promoter for ex- pressing genes in seeds at this particular stage of development.

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Table 1. GUS activities in individual AP1GUS and cTAK transgenic plants. Activity (pmol MU per minute per mg fresh weight) was measured in leaf and seed stages 1, 2 and 3.

Leaf Seed Stage 1 Seed Stage 2 Seed Stage 3

AP1 GUS transformants 5 3,4 10.0 187.2 551.2 6 5.0 68.5 99.2 249.6

10 2.4 8.6 5.6 38.7 12 0.6 0.2 0,5 1.3 14 14.3 1.2 2.0 165.6 16 25.4 209.2 424.0 264.0 18 18.4 320.0 348.0 248.0 19 8.8 144.0 200.0 284.0 25 7.8 79.2 136.4 516.0 26 2,1 41.2 137.6 426.0 27 1.8 0.3 138.4 448.0 28 0.6 0.1 52.4 300.0 29 3.3 10.9 22.0 416.0

Average 7.2 68.7 134.9 300.6

c T A K transforrnants 3A 14.8 15.1 32.2 5.1 3B 9.6 2.6 7.0 24.4 21 24.8 12.4 5.1 18.4 23 900.0 720.0 - 176.0

Average 237.4 187.5 - 56.0

Discussion

We present here the first report on the identification and functional analysis of a 5' flanking region involved in the regulation of fatty acid biosynthesis in plants. Fatty acids in plants are required in all cells for membrane synthesis and also, specifically in seed tissue, for the production of storage lipid. One mechanism for regulating the production of fatty acids for these diverse func- tions would be via differential expression of individual members of gene families encoding fatty acid synthetase components. Thus certain genes would be expressed constitutively whilst the expression of others would be confined to seed tissue.

Initial evidence for seed-expressed FAS genes came from the identification of a sub-group of ACP cDNAs which were expressed in concert with storage lipid synthesis during B. napus seed development [ 5 ]. Genomic clones corresponding

to certain members of this sub-group were isolated [5] and, in this report, we demonstrate that the 5' flanking sequence of one of these ACP genes is involved in the regulation of storage lipid synthesis in seeds. This conclusion is based on the analysis oftransgenic tobacco plants containing chimaeric genes of the ACP 5' flanking region fused to the fi-glucuronidase reporter gene. These results showed GUS activity to be confined to seed tissue and to increase during seed development in concert with storage lipid synthesis.

Tobacco seeds can be considered as 'oilseeds' inasmuch as, at maturity, they contain ca. 40~o fresh weight as oil. Tobacco is thus a relevant system to use for the functional analysis of fatty acid biosynthetic gene sequences. This study shows that the sequences which confer regulation of the ACP05 gene in oil seed rape are present in the ACP05 promoter and that these sequences function in the correct tissue and temporal fash- ion in the tobacco plant.

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Table 2. Relative GUS activities (seed/leaf) for seed stages 1, 2 and 3 of individual AP1GUS and cTAK transgenic plants.

Seed 1/Leaf Seed 2/Leaf Seed 3/Leaf

AP1 GUS transformants 5 2.94 55.06 162.12 6 13.6 19.68 49.52

10 3.58 2.32 16.13 12 0.33 0.91 2.11 14 0.08 0.14 11.56 16 8.25 16.72 10.39 18 17.39 18.91 13.48 19 16.29 22.62 32.12 25 10.15 17.44 66.15 26 19.62 64.91 200.94 27 0.16 76.89 248.89 28 0.06 81.87 468.75 29 3.33 6.71 126.83

Average 7.4 29.5 108.4

c TAK transformants 3A 1.02 2.10 0.35 3B 0.28 0.73 2.56 21 0.5 0.21 0.74 23 0.8 - 0.20

Average 0.6 1.0 1.0

A reproducible pattern of GUS activity was observed in all 13 plants transformed with the chimaeric AP1GUS construct, with approxi- mately 100-fold higher levels of activity in stage 3 seeds than in leaf tissue. In contrast, control plants transformed with pcTAK showed similar levels of GUS activity in leaf and all stages of seed development. Comparison of the average GUS activities obtained from the AP1GUS and cTAK transgenic populations showed the AP1 sequence to function as a strong plant promoter. Thus, at stage 3 the most active phase of lipid synthesis in developing tobacco seeds, the average level of GUS expression driven by AP1 was higher than that obtained with the strong plant promoter CaMV 35S present in the cTAK construct. Considering the large number of seed- expressed ACP genes present in B. napus [5] this was perhaps a somewhat surprising result.

The sequenced part of the ACP 5' flanking region (-924 to -1) was examined for putative regulatory sequences. A conserved sequence ele-

ment, ABRE [CACGTGGC], has been identified in the upstream region of a number of ABA responsive genes [10]. This plant hormone is thought to be important in maintaining embryo- genic development during the storage reserve accumulation phase of embryogeny, and in mediat- ing suppression of precocious germination. In rape, storage protein genes have been shown to be responsive to ABA, although different temporal and spacial patterns of expression suggest that developmental cues other than ABA are involved. The upstream region of the napin storage protein gene contains the core of an ABRE and a similar element, CACGTG, is located at -143 in the ACP promoter. At -30 in the ACP promoter a TATA box was located, CTTATAAATGA, which exhibits 9 out of 11 homology with the plant consensus sequence CCTATAAATTA [ 13 ]. No sequence homology to a CAAT box [ 8 ] was found.

A dot matrix analysis of the ACP 5' flanking region was performed to identify palindromic se-

quences which might be of functional significance. 8 palindromic sequences were identified (using criteria of homology at or exceeding 58 ~o over a window of 40 nucleotides), three of which (P l - P3, see Fig. 2) contain short, perfect inverted re- peats which could be involved in binding dimeric nuclear transcription factors. Similar elements identified within a soybean conglycinin c~ subunit gene [3] have been shown to be involved in binding an embryo nuclear protein SEF [1].

In an effort to identify sequences conserved between ACP genes that might be of functional significance, a sequence alignment was carried out between the ACP05 5' flanking region, a second B. napus ACP gene (ACP09) [5] and an Ar- abidopsis ACP gene [18] (Fig. 5). Although no functional characterisation of the latter 2 flanking sequences has been carried out, the ACP09 gene is predicted, on the basis of perfect homology between its exons and a seed ACP cDNA sequence [5], to be a seed-expressed gene, whilst there is preliminary evidence that the Arabidopsis gene is constitutively expressed (J.B. Ohlrogge,

1171

personal communication). The 2rape genes ACP05 and ACP09 were found to be 82~o homologous in the -184 to -1 region whilst only 65~o and 55~o homologous respectively to the Arabidopsis gene (see Fig. 5). Three elements were identified which are perfectly conserved between the 3 ACP genes. Element 1 ( -32 to -21) contains the TATA box. Element 2 ( -72 to -67) contains the sequence GCCCAT which, in ACP05, is part of the larger element GGC- CCATCT which exhibits 8 out of 9 homology with the animal 'AGGA' consensus sequence. The latter sequence has previously been observed in seed expressed genes [6]. Element 3 (-105 to -100) contains the sequence ATGGGC which is a perfectly inverted repeat of Element 2. The conserved elements 2 and 3 form the basis of the palindromic sequence P3, identified by dot matrix analysis. A fourth 10 bp sequence ( -146 to - 137) was identified which is perfectly conserved between ACP05 and the Arabidopsis gene and contains the ABRE core sequence.

In conclusion, we have isolated and charac-

ACP05 ACP09 ARAB

-200 -190 -180 -170 -160 G~TGGTAT~TCT- TAAC GG G TGG ATTTC@TC

T GGTA!I~TTT T ~ T ~ G / ~ J C ~ T CT G / ~ T ~ G ~ I C A T T T CAATT C

ACP05 ACP09 ARAB

-150 -140 . . . . . . . . . . . . . . . . . . . . . .

-130 -120 -ii0 -I00 Acpo5 G--mfff~~TCACACC;~CATTAGTaNrGATGCCq- .

AcPo9 A- _q TCAC CG CATTAGT _GATGGG --- TaCTTGC TGGGqr __ TC .......

-90 -80 -70 -60 Acpos ;aTTT CTT aCAq- - GGCCCA C --- C AcP09 T T aTACTT CA CC r GGCCCATG ATT r r --- aC APaB

-40 -20

CP09 ARAB CGTC~GCTTATAAATG--AA~GC ~ A G ~

Fig. 5. Sequence comparison of ACP 5' flanking regions. The -184 to -1 region ofB. napus ACP05 is compared to corresponding regions from a second B. napus ACP gene, ACP09 [5], and an Arabidopsis ACP gene [17].

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terised a 1.4 kb 5' flanking sequence of a B. ha- pus ACP gene involved in the regulation of storage lipid synthesis in seed tissue. A deletion analysis is being carried out to define more pre- cisely those sequences which confer this specificity. In addition the 1.4 kb sequence is being used to express genes designed to perturb fatty acid biosynthesis in developing oil seeds.

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2. Barfield DG, Robinson S J, Shields R: Plant regeneration from protoplasts of long term suspension cultures of N. plumbaginifolia. Plant Celt Rep 4:104-107 (1985).

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5. de Silva J, Loader NM, Jarman C, Windust JHC, Hughes SG, Safford R: The isolation and sequence analysis of two-seed expressed acyl carrier protein genes from Brassica napus. Plant Mol Biol 14:537-548 (1990).

6. Doyle JJ, Schuler MA, Godette WD, Zenger V, Beachy RN, Slightom JL: The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris. J Biol Chem 261: 9228-9238 (1986).

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18. Post-Beittenmiller MA, Hlou~ek-Radoj~i~ A, Ohlrogge JB: DNA sequence of a genomic clone encoding on Ar- abidopsis acyl carrier protein (ACP). Nucl Acids Res 17: 1777 (1989).

19. Safford R, Windust JHC, Lucas C, de Silva J, James CM, Hellyer SA, Smith CG, Slabas AR, Hughes SG: Plastid localised seed acyl carrier protein of Brassica napus is encoded by a distinct, nuclear multigene family. Eur J Biochem 174:287-295 (1988).

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the isolation and functional characterisation of a b. napus acyl carrier protein 5′ flanking...

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