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EP-4739774-A1 - METHOD FOR OBTAINING POTATO WITH LOW SUGARS CONTENT BY SITE-DIRECTED NUCLEOTIDE SUBSTITUTION

EP4739774A1EP 4739774 A1EP4739774 A1EP 4739774A1EP-4739774-A1

Abstract

The present invention relates to a method for obtaining potato with lower sugars content by site-directed nucleotide substitution, and also relates to a method for generating site- directed nucleotide substitution and fragment substitution.

Inventors

  • VANDERSCHUREN, Hervé
  • SHUMBE, Leonard

Assignees

  • Université de Liège

Dates

Publication Date
20260513
Application Date
20240705

Claims (18)

  1. 1. A method for obtaining a plant with low sugars content, which comprises inserting adenine after position -34 of the 5’UTR of the gene coding for the vacuolar invertase (VInv) of a target plant to obtain a plant with lower sugars content, wherein the 5’UTR of the gene coding for VInv of the target plant comprises the sequence of SEQ ID NO: 1.
  2. 2. The method according to claim 1, wherein the inserting steps comprise introducing the following a), b), c), d), e) or f) into a cell or tissue of the target plant, and then culturing the cell or tissue as obtained into a complete plant: a) a first genetic material, a second genetic material and a donor vector, wherein the first genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a first sequence specific nuclease, and wherein the second genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a second sequence specific nuclease; b) a third genetic material and a donor vector, wherein the third genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro, and wherein the third genetic material is capable of expressing the first sequence specific nuclease and the second sequence specific nuclease; c) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is an mRNA capable of expressing the first sequence specific nuclease, and wherein the second non-genetic material is an mRNA capable of expressing the second sequence specific nuclease; d) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is a protein of the first sequence specific nuclease expressed in vitro, and wherein the second non-genetic material is a protein of the second sequence specific nuclease expressed in vitro; e) a donor vector; or f) a donor vector capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; wherein the donor vector is a vector carrying a mutation target sequence comprising a DNA fragment sequence corresponding to a sequence in the genome of the target plant from the 5' end of a first target fragment to the 3' end of a second target fragment which contains the desired nucleotide mutation, wherein the nucleotide mutation is at least an insertion of an adenine (A) after position -34, preferably between position -34 and -35, of the DNA sequence of the 5’UTR region of endogenous VInv protein of a target plant, and wherein the first sequence specific nuclease is able to specifically cleave the first target fragment in the genome of the target plant and the donor vector; and wherein the second sequence specific nuclease is able to specifically cleave the second target fragment in the genome of the target plant and the donor vector.
  3. 3. The method according to claim 2, wherein the first sequence specific nuclease is a CRISPR/Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing, and wherein the second sequence specific nuclease is a CRISPR/Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing.
  4. 4. The method according to claim 2, wherein the plant is a monocotyledon or dicotyledon.
  5. 5. The method according to claim 2, wherein the cell is any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture, and wherein the tissue is any tissue that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture, and wherein introducing step is optionally performed with a gene gun, an agrobacterium infection, or a PEG- mediated protoplast transformation.
  6. 6. A biological material selected from the group consisting of: (1) a protein formed by at least inserting an adenine (A) after position -34, preferably between position -34 and -35, of the DNA sequence of the 5’UTR region of potato endogenous VInv protein; wherein the amino acid sequence of the 5’UTR region of potato endogenous VInv protein is set forth in SEQ ID NO: 1; (2) a coding gene of said protein; and (3) an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line containing said coding gene.
  7. 7. A method for inserting a target nucleotide in a target gene of a target organism, comprising the step of introducing one of the following a), b), c), d) or e) into a cell or tissue of the target organism: a) a first genetic material, a second genetic material and a donor vector: the first genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a first sequence specific nuclease; the second genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a second sequence specific nuclease; b) a third genetic material and a donor vector, wherein the third genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; c) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is an mRNA capable of expressing the sequence specific nuclease 1; and wherein the second non-genetic material is an mRNA capable of expressing the second sequence specific nuclease; d) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is a protein of the first sequence specific nuclease expressed in vitro; and wherein the second non-genetic material is a protein of the second sequence specific nuclease expressed in vitro; e) a donor vector capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; the donor vector is a vector carrying a mutation target sequence; the mutation target sequence contains a DNA fragment sequence corresponding to a sequence in the genome of the target organism from the 5' end of a first target fragment to the 3' end of a second target fragment, which contains the desired nucleotide substitution; wherein the first sequence specific nuclease is able to specifically cleave the first target fragment in the genome of the target organism and the donor vector; and wherein the second sequence specific nuclease is able to specifically cleave the second target fragment in the genome of the target organism and the donor vector.
  8. 8. The method according to claim 7, wherein the first sequence specific nuclease is a CRISPR/Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing; and wherein the second sequence specific nuclease is a CRISPR/Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing.
  9. 9. The method according to claim 7, wherein the cell is any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture; the tissue is any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture; wherein introducing step is optionally performed with a gene gun, an agrobacterium infection, or a PEG-mediated protoplast transformation.
  10. 10. A specific sgRNA for editing a 5’ UTR of potato VInv gene based on CRISPR/Cas9 characterized in that the coding sequence of the sgRNA is shown as SEQ ID NO: 3.
  11. 11. A CRISPR/Cas9 vector for 5’ UTR of potato VInv gene, characterized by containing coding gene sequence for the specific sgRNA of claim 10.
  12. 12. The use of the specific sgRNA of claim 10 to construct potato with low sugars content.
  13. 13. Use of the CRISPR/Cas9 vector according to claim 11 in the construction of potato with low sugars content.
  14. 14. A method for reducing sugars content of potato by gene editing, comprising the steps of: introducing the coding gene of the specific sgRNA of claim 10 and the coding gene of the Cas9 protein into starting potato to obtain potato with 5’ UTR of VInv gene mutated; compared with the starting potato, the sugars content of the transgenic potato is reduced; the cDNA sequence of the 5’ UTR of VInv gene is shown as SEQ ID NO: 1.
  15. 15. A method for reducing sugars content of potato by gene editing characterized in that CRISPR/Cas9 vector of claim 11 is introduced into starting potato, and potato VInv gene is subjected to gene editing, so that insertion is generated, a mutant of the VInv gene is formed, and transgenic potato is obtained; compared with the starting potato, the transgenic potato has lower sugars content, and the cDNA sequence of the 5’ UTR of VInv gene is shown in SEQ ID NO: 1.
  16. 16. The method according to claim 14 or 15, wherein the starting potato is Lady Rosetta or Verdi.
  17. 17. The method as claimed in claim 14 or 15, wherein the mutant nucleotide sequence of 5’ UTR of VInv gene is shown as SEQ ID NO: 2.
  18. 18. A mutant 5’ UTR of VInv characterized in that the nucleotide sequence is shown as SEQ ID NO: 2.

Description

METHOD FOR OBTAINING POTATO WITH LOW SUGARS CONTENT BY SITE-DIRECTED NUCLEOTIDE SUBSTITUTION TECHNICAL FIELD [0001] The present invention belongs to the field of biotechnological breeding, relates to a method for obtaining potato with lower sugars content by site-directed nucleotide substitution, and also relates to a method for generating site-directed nucleotide substitution and fragment substitution. BACKGROUND [0002] During several decades, the compound Chlorprofam (CIPC) was used as an efficient anti-sprouting agent for long term storage of potato. However, the recent prohibition of CIPC in the European Union is prompting the potato processing industry to search for alternative and safer anti-sprouting approaches. In this context, storage at cold temperature (i.e., 4°C) has emerged as a valuable option for long term storage of potato without the use of CIPC. However, most commercial potato varieties processed by the industry accumulate high levels of reducing sugars during cold storage, a phenomenon called cold-induced sweetening (CIS). During high temperature processing of potatoes into products such as crisps and French fries, the reducing sugars react with some free amino acids and peptides to produce the neurotoxin acrylamide, whose presence is evidenced by a brown-to-black coloration of the processed products. Therefore, it is key to prevent CIS in potato in order to unlock the potential of long-term storage at cold temperature in the processed potato value chain. [0003] The potato processing industries currently rely on a few varieties which have been selected for their agronomical, technological and organoleptic properties (ref 4). Given the difficulty to breed CIS-resistant potato varieties to replace the ones that are CIS-susceptible, New Genomic Techniques (NGTs) are emerging as suitable approaches to rapidly introgress the CIS-resistant trait in the commercial varieties currently used for processing into crisps and French fries. [0004] In the present invention, the Inventors have demonstrated that a specific edition of the 5’ UTR region of 50% of the alleles of Vacuolar invertase (VInv) is sufficient to significantly alter the CIS phenotype of stored potatoes. SUMMARY OF THE INVENTION [0005] The present invention discloses a method for obtaining a plant with low sugars content. [0006] The method for obtaining the plants with low sugars content provided by the present invention comprises the following steps: at least inserting an adenine in the 5' UTR sequence of the gene coding for Vacuolar invertase (VInv), after position -34, preferably between positions -34 and -35, to obtain a plant with reduced activity of the vacuolar invertase at 4°C, leading to low sugars content. The DNA sequence of the 5' UTR region of the gene coding for VInv of the target plant is set forth in SEQ ID NO: 1. [0007] The numbering referred to herein, unless indicated otherwise, refers to the unmodified DNA sequence of the 5' UTR region of the gene coding for VInv. In one embodiment, the numbering referred to herein, unless indicated otherwise, refers to the sequence as set forth in SEQ ID NO: 1. [0008] The amino acid sequence of the 5' UTR region obtained after the insertion is set forth in SEQ ID NO: 2. [0009] According to the method, the steps of “inserting an adenine (A) after position - 34, preferably between positions -34 and -35, of the DNA sequence of the 5 ’UTR region of the gene coding for Vacuolar invertase (VInv)” are realized by introducing the following a), b), c), d), e) or f) into a cell or tissue of the target plant, and then culturing the cell or tissue as obtained into complete plants: a) a genetic material 1 (or “first genetic material”), a genetic material 2 (or “second genetic material”) and a donor vector: the genetic material 1 is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a sequence specific nuclease 1 (or “first sequence specific nuclease”); the genetic material 2 is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a sequence specific nuclease 2 (or “second sequence specific nuclease”); b) a genetic material 12 (or “third genetic material) and a donor vector: the genetic material 12 is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing the sequence specific nuclease 1 and also expressing the sequence specific nuclease 2; c) a non-genetic material 1, a non-genetic material 2 and a donor vector: the non- genetic material 1 is an mRNA capable of expressing the sequence specific nuclease 1; the non-genetic material 2 is an mRNA capable of expressing the sequence specific nuclease 2; d) a non-genetic material 1, a non-genetic material 2 and a donor vector: the non- genetic material 1 is the protein of the sequence specific nuclease 1 expressed in vitro; the non-genetic material 2 is the protein of the sequence specific nuclease 2 expressed