Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1997-03-14
2003-06-24
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S281000, C800S298000, C435S468000, C435S419000, C536S023600
Reexamination Certificate
active
06583340
ABSTRACT:
This invention relates to modified plants. In particular, the invention relates to plants modified such that at least part of the plant (for example seeds of the plant) is capable of yielding a commercially useful oil.
Plants have long been a commercially valuable source of oil. Nutritional uses of plant-derived oils have hitherto been dominant, but attention is now turning additionally to plants as a source of industrially useful oils, for example as replacements for or improvements on mineral oils. Oil seeds, such as from rape, have a variety of lipids in them (Hildish & Williams, “Chemical Composition of Natural Lipids”, Chapman Hall, London, 1964). There is considerable interest in altering lipid composition by the use of recombinant DNA technology (e.g. Knauf,
TIBtech,
February 1987, 40-47), but by no means all of the goals have been realised to date for a variety of reasons, in spite of the ever-increasing sophistication of the technology.
Success in tailoring the lipid content of plant-derived oils requires a firm understanding of the biochemistry and genes involved. Broadly, two approaches are available. First, plants may be modified to permit the synthesis of fatty acids which are new (for the plant); so, for example, laurate and/or stearate may be synthesised in rape. Secondly, the pattern and/or extent of incorporation of fatty acids into the glycerol backbone of the lipid may be altered. It is with this latter approach that the present invention is concerned.
Lipids are formed in plants by the addition of fatty acid moieties onto the glycerol backbone by a series of acyl transferase enzymes. There are three positions on the glycerol molecule at which fatty acid (acyl) moieties may be substituted, and the substitution reached at each position is catalysed by a position-specific enzyme; the enzymes are glycerol-sn-3-phosphate acyltransferase (1-acyltransferase), 1-acyl-sn-glycerol-3-phosphate acyltransferase (2-acyltransferase) and sn-1,2-diacylglycerol acyltransferase (3-acyltransferase).
One, but not the only, current aim of “lipid engineering” in plants is to provide oils including lipids with a higher content of erucic (22:1) acid and/or oils containing trierucin. Erucic acid-containing lipids are commercially desirable for a number of purposes, particularly as replacements to or supplements for mineral oils in certain circumstances, as alluded to above. In the case of oil seed rape (
Brassica napus
), one of the most significant oil producing crops in cultivation today, the specificity of the 2-acyltransferase enzyme positively discriminates against the incorporation of erucic acid at position 2. So, even in those cultivars of rape which are able to incorporate erucic acid at positions 1 and 3, where there is no (or at least reduced) discrimination against erucic acid, only a maximum 66% of the fatty acids incorporated into triacyl glycerols can be erucic acid. Such varieties of rape are known as HEAR (high erucic acid rape) varieties.
It would therefore be desirable to produce plants, eg conventional oil seed rape as well as HEAR varieties, which contain useful levels of trierucin and/or contain higher levels of erucic acid and/or contain oils with erucic acid incorporated at position 2; the same can be said of oils of other vegetable oil crops such as maize, sunflower and soya, to name but a few examples. While in principle it may be thought possible to introduce into a desired plant DNA encoding a 2-acyltransferase of different fatty acid specificity, for example from a different plant, in practice there are a number of problems.
All enzymes involved in the acylation pathway for formation of triacylglycerols are membrane bound. These are the 1-acyltransferase, 2-acyltransferase and 3-acyltransferase which are present in the endoplasmic reticulum in the cytoplasm. They have not been purified. This makes working with them difficult and rules out the use of many conventional DNA cloning procedures. This difficulty does not, paradoxically, lie in the way of cloning the gene (or at least cDNA) encoding the Choroplastic 1-acyltransferase enzyme, which is soluble; in fact, recombinant DNA work has already been undertaken on this enzyme for a completely different purpose, namely the enhancement of chilling resistance in tobacco plant leaves, by Murata et al (
Nature
356 710-713 (1992)).
Wolter et al, Fat Science Technology, 93, No 8: 288-89 (1991) suggested a strategy for cloning membrane bound enzymes such as 2-acyltransferases, although no exemplification was given.
WO-A-9413814 discloses a DNA sequence (and corresponding protein sequence) of a 2-acyltransferase. This sequence, which is derived from maize, is used to transform plants, such that the normal substrate specificity of the plants' 2-acyltransferase is altered. This disclosure also included the use of a cDNA sequence for a 2-AT derived from maize to locate 2-ATs with a high degree of homology from both Brassica and Limnanthes species.
It has now been surprisingly found that there is in fact another 2-AT in Limnanthes which has no homologue in rape and which is seed specific. This 2-AT is able to incorporate erucic acid at the 2-position which the native 2-AT in rape, for example, is unable to do.
According to a first aspect of the invention, therefore, there is provided a recombinant or isolated DNA sequence, encoding an enzyme having membrane-bound 2-acyltransferase activity, and selected from:
(i) a DNA sequence comprising the DNA sequence of
FIG. 3
(SEQ ID NO: 7) or its complementary strand,
(ii) nucleic acid sequences hybridising to the DNA sequence of
FIG. 3
(SEQ ID NO: 7) or its complementary strand, under stringent conditions, and
(iii) nucleic acid sequences which would hybridise to the DNA sequence of
FIG. 3
(SEQ ID NO: 7) or its complementary strand, but for the degeneracy of the genetic code.
Suitably, the DNA sequence of the invention comprises a DNA sequence as described in (i), (ii) or (iii) above which is the sequence of
FIG. 3
, or its complementary strand, or is one which has the characteristics of (ii) or (iii) where the sequence is the sequence of
FIG. 3
(SEQ ID NO: 7)
Fragments of the above DNA sequences, for example of at least 15, 20, 30, 40 or 60 nucleotides in length, are also within the scope of the invention.
Suitable stringent conditions include salt solutions of approximately 0.9 molar at temperatures of from 35° C. to 65° C. More particularly, stringent hybridisation conditions include 6 x SSC, 5 x Denhardt's solution, 0.5% SDS, 0.5% tetrasodium pyrophosphate and 50 &mgr;g/ml denatured herring sperm DNA; washing may be for 2×30 minutes at 65° C. in 1 x SSC, 0.1% SDS and 1×30 minutes in 0.2 x SSC, 0.1% SDS at 65° C.
Recombinant DNA in accordance with the invention may be in the form of a vector, which may have sufficient regulatory sequences (such as a promoter) to direct gene expression. Vectors which are not expression vectors are useful for cloning purposes (as expression vectors themselves may be). Host cells (such as bacteria and plant cells) containing vectors in accordance with the invention themselves form part of the invention.
The 2-acyltransferase of the invention may be cloned directly, for example using complementation studies, from a DNA library of Limnanthes. For example, if
E. coli
is used as the complementation host, a mutant is chosen which is defective in the 2-acyltransferase; the DNA library from Limnanthes (e.g.
L. douglasii
) is transformed into the mutant complementation host; host cells containing the target acyltransferase gene can readily be selected using appropriate selective media and growth conditions.
E. coli
mutant JC201 is a suitable host for use in complementation studies relating to 2-acyltransferase.
Cloning the acyltransferase gene into a microbial host, such as a bacterium like
E. coli,
in such a way that the gene can be expressed has a particular advantage in that the substrate specificity of the acyltransferase gene can be assessed with membranes isolated from the microbial host before transforme
Brough Clare Louise
Brown Adrian Paul
Kroon Johannes Theodorus Maria
Slabas Antoni Ryszard
Cooper & Dunham LLP
Gene Shears Pty. Limited
McElwain Elizabeth F.
White John P.
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