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CN-121991193-A - D53 protein phosphorylation modification site deletion mutant and application thereof in regulation of plant types

CN121991193ACN 121991193 ACN121991193 ACN 121991193ACN-121991193-A

Abstract

The invention discloses a D53 protein phosphorylation modification site deletion mutant and application thereof in regulating plant types. The serine at 589 of the protein D53 is mutated into aspartic acid, the amino acid sequences of other positions are unchanged, and the protein D53 S589D is obtained, and the mutation of the serine at 589 of the protein D53 into aspartic acid simulates the phosphorylation state of the serine at 589 of the protein D53. Experiments show that mutation of protein D53 into protein D53 S589D in the rice genome, i.e., increasing serine phosphorylation and/or activity at position 589 of protein D53, can increase the number of tillers in rice. The method provided by the invention can directionally improve the rice tillering character and has important application value.

Inventors

  • LI JIAYANG
  • LIU HUIHUI
  • WANG BING
  • YU HONG
  • MENG XIANGBING
  • Zhang Dahan
  • JING YANHUI
  • LIU GUIFU
  • CHEN MINGJIANG

Assignees

  • 中国科学院遗传与发育生物学研究所
  • 崖州湾国家实验室

Dates

Publication Date
20260508
Application Date
20260121

Claims (10)

  1. 1. Protein D53 S589D , C1) or C2): c1 A protein obtained by replacing serine residue at 589 in the amino acid sequence of protein D53 with aspartic acid residue; The protein D53 is a 1) or a 2) or a 3): a1 Amino acid sequence is a protein shown as SEQ ID No. 1; a2 A 1) a protein which is obtained by substitution and/or deletion and/or addition of one or more amino acid residues and is related to the number of plant tillers; a3 A protein derived from rice and having more than 90% identity to a 1) and associated with the number of plant tillers; c2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by C1).
  2. 2. A biomaterial as described in any one of (b 1) to (b 7) below: (b1) A nucleic acid molecule encoding the protein D53 S589D of claim 1; (b2) An expression cassette comprising the nucleic acid molecule of (b 1); (b3) A recombinant vector comprising the nucleic acid molecule of (b 1); (b4) A recombinant vector comprising the expression cassette of (b 2); (b5) A recombinant microorganism comprising the nucleic acid molecule of (b 1); (b6) A recombinant microorganism comprising the expression cassette of (b 2); (b7) A recombinant microorganism comprising the recombinant vector of (b 3).
  3. 3. Use of a substance which increases the serine phosphorylation level and/or activity of the protein D53 at position 589 as defined in claim 1, A1) or A2): A1 Increasing the number of tillers in the plant; a2 Cultivating a transgenic plant with increased tiller number; The plant may tillering.
  4. 4. Use of a substance which increases the serine phosphorylation level and/or activity of protein D53 at position 589 according to claim 1 in plant breeding aimed at increasing the number of tillers in plants.
  5. 5. The method according to claim 3 or 4, wherein the substance for increasing the serine phosphorylation level and/or activity of protein D53 at position 589 of claim 1 is protein D53 S589D of claim 1 or a biological material of claim 2.
  6. 6. A method for growing a transgenic plant, comprising the step of increasing the level and/or activity of serine phosphorylation at position 589 of the protein D53 of claim 1 in a starting plant to obtain a transgenic plant, wherein the number of tillers of the transgenic plant is increased compared to the starting plant, and wherein the starting plant is tillable.
  7. 7. The method according to claim 6, wherein the increase in the serine phosphorylation level and/or activity of protein D53 at position 589 of the protein D53 in the starting plant is a mutation of protein D53 to protein D53 S589D in the starting plant; The protein D53 S589D is a protein obtained by replacing serine residue at 589 position in the amino acid sequence of the protein D53 with aspartic acid residue.
  8. 8. The method according to claim 7, wherein the mutation of the protein D53 into the protein D53 S589D is carried out by mutating AG at positions 1765-1766 of the D53 gene shown in SEQ ID No.3 into GA.
  9. 9. The method according to claim 7 or 8, wherein the mutation is achieved by gene-guided editing; the guide RNA target sequence of the gene guide editing is shown in the 5 th to 24 th positions from the 5' tail end of SEQ ID No. 5; The primer binding sequence of the gene guide editing is shown in 141-151 from the 5' end of SEQ ID No. 5; The sequence of the reverse transcription template for gene guided editing is shown in 114 th to 140 th positions from the 5' end of SEQ ID No. 5.
  10. 10. Protein D53 S589D according to claim 1, use according to claim 3 or 4 or method according to any one of claims 6 to 9, characterized in that the plant is any one of c 1) dicotyledonous plants, c 2) monocotyledonous plants, c 3) Gramineae plants, c 4) rice, c 5) rice variety Nipponbare.

Description

D53 protein phosphorylation modification site deletion mutant and application thereof in regulation of plant types Technical Field The invention belongs to the technical field of plant genetic engineering, and particularly relates to a D53 protein phosphorylation modification site deletion mutant and application thereof in regulation of plant types. Background Rice (Oryza sativa l.) is one of the major food crops in the world, and is an urgent need to meet the growing world population and to increase rice yield. The single plant yield of the rice is mainly determined by the tillering number, the ear grain number and the thousand grain weight, wherein the tillering number plays an extremely important role in the yield of the rice. The proper increase of the tiller number has important significance for increasing the rice yield. The formation of rice tillers is divided into two processes, leaf meristem formation and meristem growth. Factors determining the tiller number of rice are genetic factors, phytohormone regulation, microbial influence and environmental stress response. Although some important factors for regulating the number of rice tillers have been identified at present, the knowledge of the rice tillers regulating mechanism is still limited, and the important theoretical significance and application value are achieved by digging more genes related to rice tillers and analyzing the mechanisms involved in regulating the number of rice tillers. Disclosure of Invention The invention aims to increase the tiller number of rice. The invention first protects the protein D53 S589D, which may be C1) or C2): c1 A protein obtained by replacing serine residue at 589 in the amino acid sequence of protein D53 with aspartic acid residue; the protein D53 may be a 1) or a 2) or a 3): a1 Amino acid sequence is a protein shown as SEQ ID No. 1; a2 A 1) a protein which is obtained by substitution and/or deletion and/or addition of one or more amino acid residues and is related to the number of plant tillers; a3 A protein derived from rice and having more than 90% identity to a 1) and associated with the number of plant tillers; c2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by C1). Wherein SEQ ID No.1 consists of 1131 amino acid residues. In order to facilitate purification of the protein of C1), the amino-terminal or carboxyl-terminal linkage of the protein of C1) may be tagged. Any of the above proteins D53 S589D may be S1) or S2) or S3): s1) the amino acid sequence is a protein shown as SEQ ID No. 2; S2) a fusion protein obtained by connecting a tag to the N end or/and the C end of the protein shown in SEQ ID No. 2; S3) protein related to the number of plant tillers, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the protein shown in the S1) or the S2). The protein of the above a 2) or S3), wherein the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues. The protein in a 2) or S3) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing. The coding gene of the protein in a 2) can be obtained by deleting one or more amino acid residues in the DNA sequence shown in SEQ ID No.3, and/or carrying out one or more base pair missense mutations, and/or connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding sequence. The coding gene of the protein in the above S3) can be obtained by deleting one or several amino acid residues in the DNA sequence shown in SEQ ID No.4, and/or performing one or several base pair missense mutation, and/or attaching a tag coding sequence to the 5 'end and/or the 3' end thereof. The substitution of any of the above amino acid residues may be conservative substitutions of amino acid residues. Any of the above-described tags include, but are not limited to, GST (glutathione-thiol-transferase) tag protein, trx (thioredoxin) tag protein, nitrogen-utilizing substrate A (NusA) tag protein, his tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA (influenza hemagglutinin) tag protein, myc tag protein, lacZ tag protein, CBD (cellulose binding domain) tag protein, phage T7 protein kinase (T7 PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein. It is known to the person skilled in the art how to select a suitable tag protein according to the intended purpose. The use of the tag does not alter the function of the target protein, and the purpose thereof is to isolate, purify, detect or trace, and thus the tag protein suitable for use in the present invention is not limited to a specific kind. The tag may be s