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US-12624364-B2 - Plant regulatory elements and uses thereof for autoexcision

US12624364B2US 12624364 B2US12624364 B2US 12624364B2US-12624364-B2

Abstract

Recombinant DNA molecules and constructs are provided that are useful for modulating gene expression in plants. One or more expression cassette(s) of a recombinant DNA molecule or construct may be excised from transgenic plants following transformation by the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct by expression of a recombinase enzyme encoded by the recombinant DNA molecule or construct. Such a recombinase system may be used to remove such expression cassette(s) from plants transformed with the recombinant DNA construct or vector. The recombinase transgene may be operably linked to a tissue-preferred or tissue-specific promoter for autoexcision in transformed plants without crossing to a different transgenic line expressing the recombinase. Methods for causing autoexcision of one or more expression cassette(s) in a transgenic plant, and plants and cells containing or transformed with a recombinant DNA molecule or construct of the present disclosure, are also provided.

Inventors

  • PokChun Jennifer To
  • Zarir Vaghchhipawala
  • Xudong Ye

Assignees

  • MONSANTO TECHNOLOGY LLC

Dates

Publication Date
20260512
Application Date
20211019

Claims (20)

  1. 1 . A recombinant DNA construct comprising a DNA regulatory sequence selected from the group consisting of: a) a sequence with at least 95 percent sequence identity to SEQ ID NO:20 or 21 and having promoter activity; b) a sequence comprising SEQ ID NO:20 or 21; and c) a fragment comprising at least 250 contiguous nucleotides of SEQ ID NO:20 or 21, wherein the fragment has promoter activity; wherein said DNA regulatory sequence is operably linked to a heterologous transcribable DNA sequence encoding a site-specific recombinase.
  2. 2 . The recombinant DNA construct of claim 1 , wherein said DNA regulatory sequence has at least 97 percent sequence identity to the DNA sequence of SEQ ID NO: 20 or 21 and having promoter activity.
  3. 3 . The recombinant DNA construct of claim 1 , wherein said DNA regulatory sequence has at least 99 percent sequence identity to the DNA sequence of SEQ ID NO: 20 or 21 and having promoter activity.
  4. 4 . The recombinant DNA construct of claim 1 wherein said DNA regulatory sequence has promoter activity.
  5. 5 . The recombinant DNA construct of claim 1 , wherein said DNA regulatory sequence is a germline-preferred promoter.
  6. 6 . The recombinant DNA construct of claim 5 , wherein said germline-preferred promoter comprises a sequence of SEQ ID NO:21, or a sequence with at least 95 percent sequence identity to SEQ ID NO:21.
  7. 7 . The recombinant DNA construct of claim 1 , wherein said site-specific recombinase is selected from the group consisting of a Cre-recombinase, a Flp-recombinase, an R-recombinase, and a Gin-Recombinase.
  8. 8 . The recombinant DNA construct of claim 1 , wherein said site-specific recombinase is a Cre-recombinase.
  9. 9 . The recombinant DNA construct of claim 1 , further comprising: a) an expression cassette comprising a selectable marker transgene; and/or an expression cassette comprising a transgene of agronomic interest; b) a pair of site-specific recombination site sequences flanking one or both of the transcribable DNA sequences encoding the site-specific recombinase and/or the selectable marker transgene, wherein the site-specific recombination sites can be cleaved by the site-specific recombinase; or c) an expression cassette encoding a guide RNA; and/or an expression cassette encoding a site-specific nuclease.
  10. 10 . The recombinant DNA construct of claim 9 , wherein: a) said pair of site-specific recombination site sequences are oriented in a head-to-tail arrangement; b) said selectable marker transgene confers resistance to an herbicide or antibiotic; c) said pair of site-specific recombination site sequences are each selected from the group consisting of LoxP, Lox.TATA-R9, FRT, RS, and GIX; d) said pair of site-specific recombination site sequences are each a LoxP or Lox.TATA-R9 site; e) said pair of site-specific recombination site sequences each comprise SEQ ID NO: 44 or SEQ ID NO: 45; f) said transgene of agronomic interest confers herbicide tolerance in plants; g) said transgene of agronomic interest confers pest or disease resistance in plants; h) said transgene of agronomic interest confers increased yield or stress tolerance in plants; i) said transgene of agronomic interest encodes a dsRNA, a miRNA, or and siRNA; j) said guide RNA comprises a targeting sequence that targets a sequence in the genome of a eukaryotic cell for genome editing or site-specific integration; k) said site-specific nuclease is a RNA-guided endonuclease; or l) said site-specific is a RNA-guided endonuclease selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, Cys1, Cys2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, and CasY.
  11. 11 . The recombinant DNA construct of claim 9 , further comprising: a) a pair of site-specific recombination site sequences flanking one or more of the transcribable DNA sequence encoding the site-specific recombinase, the selectable marker transgene, the expression cassette encoding the guide RNA, and/or the expression cassette encoding the site-specific nuclease, wherein the site-specific recombination sites can be cleaved by the site-specific recombinase; b) two or more expression cassettes encoding two or more guide RNAs; and/or c) two, three, four, five, six, seven, eight, nine, or ten different expression cassettes encoding guide RNAs.
  12. 12 . The recombinant DNA construct of claim 10 , wherein: a) said eukaryotic cell is a plant cell; and/or b) said RNA-guided endonuclease is Cas9 or Cpf1.
  13. 13 . A DNA molecule, DNA vector, or DNA transformation vector comprising: a) the recombinant DNA construct of claim 1 ; or b) the recombinant DNA construct of claim 1 and a T-DNA segment bounded by a left border and right border.
  14. 14 . The DNA transformation vector of claim 13 , wherein said transcribable DNA sequence encoding the site-specific recombinase is located between the left border and the right border of the T-DNA segment.
  15. 15 . A DNA transformation vector comprising the recombinant DNA construct of claim 9 and a T-DNA segment with a left border and a right border, wherein: a) one or more of the transcribable DNA sequences encoding the site-specific recombinase, the selectable marker transgene, and/or the transgene of agronomic interest is/are located between the left border and the right border of the T-DNA segment; or b) one or more of the transcribable DNA sequences encoding the site-specific recombinase, the selectable marker transgene, the transgene of agronomic interest, the expression cassette encoding the guide RNA and/or the expression cassette encoding the site-specific nuclease is/are located between the left border and the right border of the T-DNA segment.
  16. 16 . A transgenic plant, plant part or plant cell comprising the recombinant DNA construct of claim 1 .
  17. 17 . The transgenic plant, plant part, or plant cell of claim 16 , wherein: a) said recombinant DNA construct is stably transformed into the genome of the transgenic plant, plant part, or plant cell; or b) said transgenic plant, plant part, or plant cell is a corn, soybean, cotton, or canola plant, plant part or plant cell.
  18. 18 . A method for producing a transgenic plant or plant part, comprising: a) transforming a plant cell of an explant with a DNA molecule or vector comprising the recombinant DNA construct of claim 1 to produce one or more transformed plant cells comprising the recombinant DNA construct stably transformed into the genome of the one or more transformed plant cells; and b) regenerating or developing a transgenic plant from the explant, wherein the transgenic plant comprises the recombinant DNA construct stably transformed into the genome of one or more cells of the transgenic plant.
  19. 19 . The method of claim 18 , wherein: a) said plant cell is transformed via Agrobacterium -mediated transformation or Rhizobium -mediated transformation; b) said plant cell is transformed via microprojectile-mediated transformation or particle bombardment-mediated transformation; c) said transgenic plant and plant cell are a corn, soybean, cotton, or canola plant and plant cell, respectively; and/or d) the method further comprises: separating or harvesting a plant part from the transgenic plant.
  20. 20 . A method for excising an expression cassette from the genome of a transgenic plant, comprising: a) transforming a plant cell with a DNA molecule or vector comprising the recombinant DNA construct of claim 12 to produce one or more transformed plant cells comprising the recombinant DNA construct stably transformed into the genome of the one or more transformed plant cells; b) regenerating or developing a transgenic plant at least in part from the one or more stably transformed plant cells; c) crossing the transgenic plant with itself or another plant; and d) selecting one or more progeny plants in which one or both of the transcribable DNA sequence encoding the site-specific recombinase and/or the selectable marker transgene between the pair of site-specific recombination site sequences of the recombinant DNA construct are excised and no longer present in the genome of the one or more progeny plants.

Description

REFERENCE TO RELATED APPLICATION This application is a 371 National Phase application from International Patent Application No. PCT/2021/055596, filed Oct. 19, 2021, which claims the benefit of United States Provisional Application No. 63/093,893, filed Oct. 20, 2020, each of which is herein incorporated by reference in its entirety. INCORPORATION OF SEQUENCE LISTING The sequence listing that is contained in the file named “MONS512WO_ST25.txt”, is 111,683 bytes (as measured in Microsoft Windows®), was created on Oct. 18, 2021, and is filed herewith by electronic submission and incorporated by reference herein. FIELD OF THE INVENTION The invention relates to the field of plant molecular biology and plant genetic engineering. More specifically, the invention relates to DNA molecules useful for modulating site-specific recombinase gene expression in plants. BACKGROUND Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of an operably linked transcribable DNA sequence. Such elements may include promoters, leaders, introns, and 3′ untranslated regions and are useful in the field of plant molecular biology and plant genetic engineering. The use of transgenic technology has provided many beneficial traits for agricultural purposes but has encountered several challenges. One concern is related to the presence of marker genes conferring antibiotic or herbicide resistance in the transgenic crop plants. In addition, there may be other transgene cassettes or DNA sequences that are designed for a particular purpose and present in the initial transformation but are not needed in the final transgenic product. Removal of such marker genes and the other unwanted expression cassettes and DNA sequences is highly desirable in the field of plant biotechnology. A number of strategies have been designed for the generation of marker-free transgenic plants. For example, removal of the marker gene expression cassette can be done using a two T-DNA transformation system or a site-specific recombinase system. The two T-DNA transformation system utilizes a binary plant transformation vector that comprises two separate T-DNAs (Two T-DNA transformation system). One T-DNA comprises the marker gene expression cassette. The other T-DNA comprises the expression cassette(s) for the gene(s) of interest that are intended to remain in the transgenic plant. The plant cell can be transformed through Agrobacterium-mediated transformation. Each T-DNA can be integrated into separate chromosomes of the transformed plant cell genome. After transformation and plant regeneration, the R0 plants are self-crossed, resulting in R1 progeny. R1 progeny plants are selected that have the T-DNA comprising the expression cassette(s) intended for the final transgenic product but lack the T-DNA comprising the marker gene expression cassette(s) (see, e.g., Komari, T. et al., (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers, The Plant Journal, 10(1):165-174). The two T-DNA transformation system has some drawbacks with respect to efficiency. In the two T-DNA transformation system, transformant R0 plants can have more than one copy of either or both T-DNAs that may have to be excluded, and the percentage of plants passing selection that possess only one copy of each T-DNA can be low. Another system to remove marker gene expression cassettes from the transgenic plant relies on excision through use of a site-specific recombinase. A number of site-specific recombinases can be used, such as Cre-recombinase, Flp-recombinase (Lyznik, L. et al., (2000) Gene Transfer Mediated by Site-Specific Recombination Systems, Plant Molecular Biology Manual N1, 1-26), R-recombinase (Machida, C. et al., (2000) Use of the R-RS Site-Specific Recombination System in Plants, Plant Molecular Biology Manual N2, 1-23), or Gin-Recombinase (Maeser, S. et al., (1991). The Gin recombinase of phage Mu can catalyze site-specific recombination in plant protoplasts, Mol Gen Genet, 230: 170-176). Essentially, within the construct, such as a T-DNA insertion, the marker gene expression cassette(s) are flanked by site-specific recombinase recognition sequences, such that the construct sequence between the site-specific recombinase recognition sequences can be excised by expression of the recombinase. Expression cassette(s) that are intended to remain in the transgenic plant after excision are present in the construct outside of the site-specific recombinase recognition sequences of the construct. Removal of the expression cassettes flanked by the site-specific recombinase recognition sequences can be accomplished using a crossing strategy or through autoexcision. In a crossing strategy, plants (e.g., R1 progeny) preferably homozygous for the presence of the construct are crossed with another line of transgenic plants transformed wi