Potato .alpha.-amylase
AssigneeA/S De Danske Spritfabrikker (Danisco A/S)
InventorsKirsten Gausing, Jette D. Kreiberg
Life of this patent
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Abstract
.alpha.-Amylase, an enzyme that hydrolyses starch, can be found in all plants. The modification of potato starch production, in particular, is important in the preparation of various food products. The present invention discloses nucleotide sequences of potato .alpha.-amylase genes and the corresponding amino acid sequences. The present invention also describes DNA probes comprising .alpha.-amylase nucleotide sequences, as well as expression vectors that produce active .alpha.-amylase enzymes. These expression vectors can be used to produce transgenic potato plants.
Claims30
An isolated and purified DNA fragment of at least 50 nucleotides from a dicotyledonous plant, wherein said DNA fragment has a G+C content in the range of 35-50% calculated as an average for the coding region, and wherein said DNA fragment hybridizes under high stringency conditions to a DNA molecule having a nucleotide sequence that is complementary to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in FIG. 1, the nucleotide sequence shown in FIG. 2, the nucleotide sequence shown in FIG. 3, the nucleotide sequence shown in FIG. 4 and the nucleotide sequence shown in FIG. 5, said high stringency conditions comprising hybridization in 2.times. SSC at 67.degree. C. and final washing in 1.times. SSC at 67.degree. C.
An isolated and purified DNA fragment of at least 50 nucleotides from a dicotyledonous plant, wherein said DNA fragment has a G+C content in the range of 35-50% calculated as an average for the coding region, and wherein said DNA fragment hybridizes only under medium high stringency conditions to a DNA molecule having a nucleotide sequence that is complementary to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in FIG. 1, the nucleotide sequence shown in FIG. 2, the nucleotide sequence shown in FIG. 3, the nucleotide sequence shown in FIG. 4 and the nucleotide sequence shown in FIG. 5, said medium high stringency hybridization conditions comprising hybridization in 3.times. SSC at 67.degree. C. and final washing in 1.times. SSC at 67.degree. C.
An isolated and purified DNA fragment of at least 50 nucleotides from a dicotyledonous plant, wherein said DNA fragment has a G+C content in the range of 35-50% calculated as an average for the coding region, and wherein said DNA fragment hybridizes only under low stringency conditions to a DNA molecule having a nucleotide sequence that is complementary to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in FIG. 1, the nucleotide sequence shown in FIG. 2, the nucleotide sequence shown in FIG. 3, the nucleotide sequence shown in FIG. 4 and the nucleotide sequence shown in FIG. 5, said low stringency hybridization conditions comprising hybridization in 6.times. SSC at 67.degree. C. and final washing in 4.times. SSC at 67.degree. C.
An isolated and purified DNA fragment comprising a nucleotide sequence encoding a polypeptide having .alpha.-amylase activity and showing a degree of sequence identity of at least 90% with either the amino acid sequence shown in FIG. 1 or the amino acid sequence shown in FIG. 2.
An isolated and purified DNA fragment having at least 50 nucleotides, wherein said DNA fragment hybridizes under high stringency conditions to at least one DNA molecule having a nucleotide sequence that is complementary to a nucleotide sequence selected from the group consisting of:
- (a) nucleotides 541 to 1761 of FIG. 1,
- (b) nucleotides 595 to 1761 of FIG. 1,
- (c) nucleotides 2 to 1219 of FIG. 2,
- (d) nucleotides 53 to 1219 of FIG. 2,
- (e) nucleotides 6 to 1052 of FIG. 3, and
- (f) nucleotides 3 to 647 of FIG. 4,
- and wherein said high stringency conditions comprise 2.times. SSC at 67.degree. C. with a final washing in 1.times. SSC at 67.degree. C.
A DNA fragment encoding a fusion protein which comprises:
- (i) a first DNA molecule comprising a nucleotide sequence that encodes a polypeptide encoded by an nucleotide sequence selected from the group consisting of:
- (a) nucleotides 541 to 1761 of FIG. 1,
- (b) nucleotides 595 to 1761 of FIG. 1,
- (c) nucleotides 2 to 1219 of FIG. 2,
- (d) nucleotides 53 to 1219 of FIG. 2,
- (e) nucleotides 6 to 1052 of FIG. 3, and
- (f) nucleotides 3 to 647 of FIG. 4,
- wherein said first DNA molecule encodes .alpha.-amylase, and
- (ii) a second DNA molecule encoding a second polypeptide,
- wherein said first DNA molecule is in reading frame with said second DNA molecule.
A host organism harboring a vector which is capable of replicating in a microorganism or plant host organism, wherein said vector comprises a DNA molecule comprising a nucleotide sequence that encodes a polypeptide encoded by an nucleotide sequence selected from the group consisting of:
- (a) nucleotides 541 to 1761 of FIG. 1,
- (b) nucleotides 595 to 1761 of FIG. 1,
- (c) nucleotides 2 to 1219 of FIG. 2,
- (d) nucleotides 53 to 1219 of FIG. 2,
- (e) nucleotides 6 to 1052 of FIG. 3, and
- (f) nucleotides 3 to 647 of FIG. 4,
- wherein said DNA molecule encodes .alpha.-amylase.
A host organism which carries a DNA molecule comprising a nucleotide sequence that encodes a polypeptide encoded by an nucleotide sequence selected from the group consisting of:
- (a) nucleotides 541 to 1761 of FIG. 1,
- (b) nucleotides 595 to 1761 of FIG. 1,
- (c) nucleotides 2 to 1219 of FIG. 2,
- (d) nucleotides 53 to 1219 of FIG. 2,
- (e) nucleotides 6 to 1052 of FIG. 3, and
- (f) nucleotides 3 to 647 of FIG. 4,
- wherein said DNA molecule encodes .alpha.-amylase, and wherein said DNA molecule is stably integrated in the nuclear genome of said host organism.
A genetic construct for producing a polypeptide having .alpha.-amylase activity, said construct comprising:
- (1) a functional promoter operably connected to
- (2) a DNA molecule comprising a nucleotide sequence that encodes a polypeptide encoded by an nucleotide sequence selected from the group consisting of:
- (a) nucleotides 541 to 1761 of FIG. 1,
- (b) nucleotides 595 to 1761 of FIG. 1,
- (c) nucleotides 2 to 1219 of FIG. 2,
- (d) nucleotides 53 to 1219 of FIG. 2,
- (e) nucleotides 6 to 1052 of FIG. 3, and
- (f) nucleotides 3 to 647 of FIG. 4,
- wherein said DNA molecule encodes .alpha.-amylase, and wherein said DNA molecule is derived from a member of the family Solanaceae.
Dependencies are inferred from the claim text.