Methods and Compositions for Targeting Sequences of Interest to the Chloroplast

Patent metadata

Field	Value
Publication number	US-2016017351-A1
Application number	US-201514835242-A
Country	US
Kind code	A1
Filing date	Aug 25, 2015
Priority date	Aug 13, 2010
Publication date	Jan 21, 2016
Grant date	—

Abstract

Chimeric polynucleotides comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a heterologous polynucleotide of interest are provided, wherein the chloroplast transit peptide comprises an amino acid sequence having the chloroplast transit peptide sequence as set forth in SEQ ID NO: 1 or a biologically active variant or fragment thereof or wherein the chloroplast transit peptide comprises the sequence set forth in SEQ ID NO: 58 or an active variant or fragment thereof. Chimeric polypeptides encoding the same, as well as, cells, plant cells, plants and seeds are further provided which comprise the chimeric polynucleotides. Compositions further include HPPD polypeptides and polynucleotides encoding the same as set forth in SEQ ID NOS: 57 and 60 or active variants and fragments thereof. Such sequences comprise the chloroplast transit peptide as set forth in SEQ ID NO: 58 or an active variants or fragments thereof. Cells, plant cells, plants and seeds are further provided which comprise such sequences. Methods of use of the various sequences are also provided.

First claim

1 . A chimeric polynucleotide comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a heterologous polynucleotide encoding a polypeptide of interest, wherein said chloroplast transit peptide comprises
a) an amino acid sequence comprising the amino acids of SEQ ID NO:1; b) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or c) an amino acid sequence having at least 17 consecutive amino acids of SEQ ID NO:1.

2 . The chimeric polynucleotide of claim 1 , wherein said chloroplast transit peptide comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8.

3 . The chimeric polynucleotide of claim 1 , wherein said polypeptide of interest comprises a 4-hydroxphenylpyruvate dioxygenase (HPPD) polypeptide having HPPD activity.

4 . A nucleic acid construct comprising the chimeric polynucleotide of claim 1 .

5 . The nucleic acid construct of claim 4 , further comprising a promoter operably linked to said chimeric polynucleotide.

6 . A cell comprising at least one chimeric polynucleotide of claim I.

7 . The cell of claim 6 , wherein said cell is a plant cell.

8 . The cell of claim 7 , wherein said polynucleotide or nucleic acid construct is stably incorporated into the genome of said plant cell.

9 . The cell of claim 7 , wherein said plant cell is from a monocot.

10 . The cell of claim 9 , wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.

11 . The cell of claim 7 , wherein said plant cell is from a dicot.

12 . The cell of claim 11 , wherein the dicot is soybean, Brassica , sunflower, cotton, or alfalfa.

13 . A plant comprising at least one plant cell of claim 7 .

14 . A plant explant comprising at least one plant cell of claim 7 .

15 . A transgenic seed produced by the plant of claim 13 .

16 . The plant cell of claim 7 , wherein the plant cell further comprises at least one polypeptide imparting tolerance to a herbicide.

17 . The plant cell of claim 16 , wherein said at least one polypeptide imparting tolerance to a herbicide comprises:
(a) a sulfonylurea-tolerant acetolactate synthase; (b) an imidazolinone-tolerant acetolactate synthase; (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate (d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl transferase; (g) a protoporphyrinogen oxidase; (h) an auxin enzyme or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A carboxylase (ACCase).

18 . A chimeric polypeptide encoded by the polynucleotide of claim 1 .

19 . A method of targeting a polypeptide of interest to a chloroplast comprising expressing a chimeric polynucleotide of claim 1 or the nucleic acid construct of claim 4 in a plant cell.

20 . A method of targeting a polypeptide of interest to a chloroplast comprising introducing the chimeric polynucleotide of claim I or the nucleic acid construct of claim 4 in a plant cell and expressing said chimeric polynucleotide in the plant cell.

21 . The method of claim 19 , wherein said method further comprises regenerating a transgenic plant from said plant cell.

22 . The method of claim 19 , wherein said plant cell is from a dicot.

23 . The method of claim 22 , wherein said dicot is selected from the group consisting of soybean, Brassica , sunflower, cotton, or alfalfa.

24 . The method of claim 19 , wherein said plant cell is from a monocot.

25 . The method of claim 24 , wherein said dicot is selected from the group consisting of maize, wheat, rice, barley, sorghum, or rye.

26 . The method of claim 19 , wherein the plant cell further comprises at least one polypeptide imparting tolerance to a herbicide.

27 . The method of claim 26 , wherein said at least one polypeptide imparting tolerance to a herbicide comprises:
(a) a sulfonylurea-tolerant acetolactate synthase; (b) an imidazolinone-tolerant acetolactate synthase; (c) a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase; (d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl transferase; (g) a protoporphyrinogen oxidase; (h) an auxin enzyme or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A carboxylase (ACCase).

28 - 39 . (canceled)

Assignees

• Pioneer Hi Bred Int

Inventors

• Albert Henrik
• Castle Linda A
• Heckert Matthew
• Lu Jian
• Siehl Daniel L
• Tao Yumin

Classifications

• C12N15/8274

  for herbicide resistance · CPC title

• C12N15/82

  for plant cells {, e.g. plant artificial chromosomes (PACs)} · CPC title

• C12Y113/11027

  4-Hydroxyphenylpyruvate dioxygenase (1.13.11.27) · CPC title

• C12N9/0069

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• C12N15/8221 Primary

  Transit peptides · CPC title