Application of protein TaLBD30 in regulation and control of wheat flower organ development
By reducing the expression and activity of TaLBD30 protein in wheat through gene editing, wheat with abnormal floral organ development was prepared, which solved the problem of insufficient research on the regulation of wheat floral development, provided a model of floral organ development, and promoted the improvement of wheat yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INSTITUTE OF CROP SCIENCE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2026-01-27
- Publication Date
- 2026-05-26
AI Technical Summary
In existing technologies, the function of protein LBD in wheat flower development has not been fully explored, leading to difficulties in the development of hybrid wheat, especially the insufficient research on the regulation of pistil and stamen development, which affects the improvement of wheat yield.
By reducing the expression levels and/or activity of proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D in wheat using gene editing technology, wheat with abnormal floral organ development was prepared. Gene editing was performed using the gene editing vector pBUE411-TaLBD30 to achieve mutations in the TaLBD30 gene, resulting in the absence of pistils or stamens or their transformation into lemma-like structures.
It provides a wheat model of abnormal floral organ development, which can be used to study floral organ development and screen for substances that affect the development of stamens and pistils, promote the production of hybrid wheat and multigrain wheat, and increase wheat yield.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of protein TaLBD30 in regulating the development of wheat flower organs. Background Technology
[0002] wheat( Triticum aestivum Wheat (L.) is one of the world's most important food crops, providing daily energy for more than one-third of the population. Wheat production is directly related to global food security and economic development. With continued population growth and the impact of adverse factors such as frequent extreme weather events and shrinking arable land, continuously increasing wheat yield has become crucial for ensuring wheat production. Therefore, analyzing the key regulatory genes affecting yield is of paramount importance.
[0003] Wheat yield primarily depends on three key agronomic traits: number of spikes per unit area, number of grains per spike, and grain weight. Among these, the number of grains per spike, as a highly malleable yield factor, is largely determined by the developmental progress and differentiation efficiency of the floral organs within the spike. From the initiation of spike differentiation to the formation of spikelets and florets, and then to the maturation of sexual organs and pollination, each stage of floral development directly affects the final number of fertile florets and the seed setting rate. Abnormal expression of key developmental genes leading to abnormal floral development can significantly reduce the number of grains per spike. Therefore, elucidating the molecular regulatory network of wheat floral development is crucial for controlling wheat fertility and the number of grains per spike, providing a theoretical basis for breeding high-yielding and stable-yielding varieties.
[0004] Protein LBDs coordinate growth, development, and metabolic balance in higher plants through their highly conserved LOB domains. The functions of LBDs extend beyond regulating lateral organ development to include important physiological processes such as anthocyanin accumulation and nitrogen nutrition response; many members of this family play different functions in different plant tissues. However, to date, the function of LBDs in wheat flower development has not been discovered.
[0005] The discovery and utilization of male-sterile plants have enabled heterosis to be fully manifested in crops such as rice and maize, and hybrid seed production technology has become increasingly mature. However, due to the lack of male-sterile plants caused by polyploidy in wheat and the lag in related research, the development of hybrid wheat faces difficulties. In-depth research on pistil development in multigrain rice has revealed pathways to increase the number of pistils and rice yield by regulating pistil development. In-depth research on stamen and pistil development in wheat is relatively limited. The discovery of more genes regulating wheat flower development, especially those regulating anthers and pistils, is of great significance for obtaining male-sterile lines and multigrain wheat, and is an important reference for promoting the production of hybrid wheat and multigrain wheat. Summary of the Invention
[0006] The purpose of this invention is to provide a model of floral organ development, namely, a model of abnormal floral organ development, so as to better study floral organ development.
[0007] This invention first protects a method for preparing wheat with abnormal floral organ development, which may include the following steps:
[0008] (1) Reduce the expression levels and / or activity of proteins TaLBD30-4A, TaLBD30-4B and TaLBD30-4D in the starting wheat to prepare gene-edited wheat; (2) After completing step (1), the gene-edited wheat is self-pollinated to obtain offspring; wheat with abnormal floral organ development is isolated from the offspring.
[0009] The protein TaLBD30-4A mentioned above can be a1), a2), or a3). a1) The amino acid sequence is that of the protein shown in SEQ ID No. 1; a2) A protein that has more than 90% identity with and has the same function as the protein shown in a1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1; a3) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of a1) or a2).
[0010] The protein TaLBD30-4B mentioned above can be b1), b2), or b3. b1) The amino acid sequence is that of the protein shown in SEQ ID No. 2; b2) A protein that has more than 90% identity with and has the same function as the protein shown in b1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2. b3) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of b1) or b2).
[0011] The protein TaLBD30-4D mentioned above can be c1), c2), or c3. c1) The amino acid sequence is that of the protein shown in SEQ ID No. 3; c2) A protein that has more than 90% identity with and has the same function as the protein shown in c1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 3; c3) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of c1) or c2).
[0012] Of these, SEQ ID No. 1 consists of 266 amino acid residues, SEQ ID No. 2 consists of 262 amino acid residues, and SEQ ID No. 3 consists of 260 amino acid residues.
[0013] To facilitate the purification of the proteins in a1), b1), or c1), a tag can be attached to the amino or carboxyl terminus of the protein shown in a1), b1), or c1).
[0014] In the above method, in step (1), the reduction of the expression level and / or activity of protein TaLBD30-4A in wheat is achieved by mutating the gene encoding protein TaLBD30-4A on at least one of the two homologous chromosomes of the wheat A genome (i.e., TaLBD30-4A This is achieved by mutating the gene encoding the protein TaLBD30-4B on at least one of the two homologous chromosomes of the wheat B genome. TaLBD30-4B This is achieved by mutating the gene encoding the protein TaLBD30-4D on at least one of the two homologous chromosomes of the wheat D genome. TaLBD30-4D This is achieved through genes.
[0015] In the cDNA of wheat variety Kenong 199, TaLBD30 The nucleotide sequence of the -4A gene is shown in SEQ ID No. 4. TaLBD30 The nucleotide sequence of the -4B gene is shown in SEQ ID No. 5. TaLBD30 The nucleotide sequence of the -4D gene is shown in SEQ ID No. 6.
[0016] In the genome of wheat variety Kenong 199, TaLBD30 The nucleotide sequence of the -4A gene is shown in SEQ ID No. 7. TaLBD30 The nucleotide sequence of the -4B gene is shown in SEQ ID No. 8. TaLBD30 The nucleotide sequence of the -4D gene is shown in SEQ ID No. 9.
[0017] In the above methods, the mutations can be achieved by gene editing, EMS mutagenesis, T-DNA insertion, RNA interference, homologous recombination, zinc finger nucleases or transcription activator-like effector nucleases, causing missense mutations, DNA fragment deletions and / or DNA fragment insertions in the exons of the gene encoding TaLBD30-4A, the gene encoding TaLBD30-4B, and the gene encoding TaLBD30-4D.
[0018] In the above method, when the mutation is achieved through gene editing, the target sites used may include target site 1 and target site 2; the nucleotide sequence of target site 1 may be as shown in the reverse complementary sequence of positions 320-339 from the 5' end of SEQ ID No. 8; the nucleotide sequence of target site 2 may be as shown in positions 390-409 from the 5' end of SEQ ID No. 8.
[0019] In any of the methods described above, the starting wheat can be common and TaLBD30 -4A gene, TaLBD30 -4B gene and TaLBD30 -4D genes are not modified in any wheat variety. The starting wheat variety may be Kenong 199.
[0020] In any of the methods described above, the abnormal development of the floral organs may manifest as the absence of pistils, the absence of stamens, the pistils becoming lemma-like structures and / or the stamens becoming lemma-like structures.
[0021] In any of the methods described above, the difference between the wheat genome with abnormal floral organ development and the starting wheat genome is that: In genome A, on two homologous chromosomes TaLBD30 -4A gene all mutated to TaLBD30 -4A / -70bp; The TaLBD30-4A / -70bp is a DNA molecule obtained by deleting 70 nucleotides from position 430 to 499 starting from the 5' end of SEQ ID No. 7, while keeping the other nucleotide sequences of SEQ ID No. 7 unchanged; In the B genome, on a homologous chromosome TaLBD30 -4B gene mutation TaLBD30 -4B / +1bp / -1bp, on another homologous chromosome TaLBD30 -4B gene mutation TaLBD30 -4B / -70bp; The TaLBD30-4B / +1bp / -1bp is the DNA molecule obtained by inserting one nucleotide T between nucleotides 322 and 323 from the 5' end of SEQ ID No. 8, and deleting nucleotide A at position 392, while keeping the other nucleotide sequences of SEQ ID No. 8 unchanged; The TaLBD30 -4B / -70bp is a DNA molecule obtained by deleting 70 nucleotides from position 323 to 392 starting from the 5' end of SEQ ID No. 8, while keeping the other nucleotide sequences of SEQ ID No. 8 unchanged; In the D genome, on two homologous chromosomes TaLBD30 -4D genes are all mutated to TaLBD30 -4D / -1bp; The TaLBD30 -4D / -1bp is the DNA molecule obtained by deleting nucleotide G at position 475 from the 5' end of SEQ ID No. 9, while keeping the other nucleotide sequences of SEQ ID No. 9 unchanged.
[0022] In one embodiment of this application, when the starting wheat is the wheat variety Kenong 199, the wheat with abnormal floral organ development obtained can specifically be TaLBD30 mentioned in the embodiment. -1 #or TaLBD30 -2 #.
[0023] The application of gene-edited wheat prepared by any of the methods described above in the preparation of wheat with abnormal floral organ development also falls within the scope of protection of this invention.
[0024] In the above applications, wheat with abnormal floral organ development may exhibit the following characteristics: absence of pistils, absence of stamens, pistils becoming lemma-like structures, and / or stamens becoming lemma-like structures.
[0025] In the above applications, the wheat with abnormal floral organ development can be wheat with abnormal floral organ development prepared by any of the methods described above.
[0026] The application of substances that reduce the expression levels and / or activity of any of the above-mentioned proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D in wheat in the preparation of wheat with abnormal floral organ development also falls within the scope of protection of this invention.
[0027] In the above applications, the substance that reduces the expression level and / or activity of any of the aforementioned proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D in wheat includes a gene editing system. The gene editing targets may include target 1 and target 2; the nucleotide sequence of target 1 is shown as the inverse complementary sequence from position 320 to 339 of SEQ ID No. 8 starting from the 5' end; the nucleotide sequence of target 2 is shown as from position 390 to 409 of SEQ ID No. 8 starting from the 5' end.
[0028] The target site used in any of the gene editing methods described above may specifically consist of target site 1 and target site 2.
[0029] The gene editing system described above may specifically include a gene editing vector. The gene editing vector may specifically be the gene editing vector pBUE411- TaLBD30 The gene editing vector pBUE411- TaLBD30 Specifically, a recombinant plasmid can be obtained by inserting a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 11 into the BsaI restriction endonuclease recognition site of the pBUE411 vector.
[0030] This invention also protects the application of wheat with abnormal floral organ development prepared by any of the methods described above, which may be T1), T2), or T3). T1) serves as a model for floral organ development; T2) Study of floral organ development; T3) Screening for substances that affect the development of pistils and / or stamens.
[0031] In the above applications, preferably, the floral organ development model can be the ABCDE floral organ development model.
[0032] In the above applications, preferably, the substance can be a hormone or a compound. The hormone can be a plant hormone.
[0033] Because protein LBD30 is highly conserved in plants, and the floral organ development model is also highly conserved in flowering plants, theoretically, protein LBD30 can be used to cultivate transgenic plants with multiple petals that do not produce seeds. Therefore, the application of any of the aforementioned proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D in cultivating transgenic plants with multiple petals that do not produce seeds also falls within the scope of protection of this invention.
[0034] In the above applications, the transgenic plant may be a gene-edited plant.
[0035] In the above applications, the plant may be a plant of the genus *Lili*.
[0036] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.
[0037] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that the identity of an amino acid sequence or nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, the identity of an amino acid sequence can be calculated by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residuegap cost, and Lambda ratio to 11, 1, and 0.85 (default values), and performing a search, thus obtaining the identity value (%). Alternatively, sequence analysis software such as CLC MainWorkbench and MegAlign can be used. TM The determination can be performed, for example, using a computer program BLAST with default parameters, especially BLASTP or TBLASTN. The 90% or higher identity mentioned herein can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.
[0038] The term "conservative substitution" generally refers to the replacement of one amino acid residue with another amino acid residue in a side chain that has similar physicochemical properties. For example, conservative substitutions can occur between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conserved substitutions generally do not cause significant changes in protein conformation and structure, and essentially do not alter the protein's biological activity. Conservative substitutions in the protein sequence that are expected to have only a minimal or no effect on protein structure or function can be readily designed by those skilled in the art.
[0039] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "comprising" and "including" are used interchangeably.
[0040] The inventors of this application will use the gene editing vector pBUE411- TaLBD30 Transforming wheat variety Kenong 199, discovered TaLBD30 -4A gene, TaLBD30 -4B gene and TaLBD30 -4D genes all had T0 generation positive plants with gene editing; these were then self-crossed to obtain two homologous chromosomes of the A genome. TaLBD30 -4A gene and B genome on two homologous chromosomes TaLBD30 -4B gene and D genome on two homologous chromosomes TaLBD30 TaLBD30 triple knockout plants with mutations in all four genes were identified, and the specific mutation types were determined. The TaLBD30 triple knockout plants exhibited abnormal floral organ development, characterized by the absence of pistils and stamens, and the transformation of pistils and stamens into palea-like structures. Therefore, they can serve as a model for studying floral organ development. This invention has significant research value. Attached Figure Description
[0041] Figure 1 Cluster analysis of protein LBD in plants.
[0042] Figure 2 for TaLBD30 -4A gene, TaLBD30 -4B gene and TaLBD30 -4D gene structure diagram.
[0043] Figure 3 This is a schematic diagram of plasmid pCBC-MT1T2.
[0044] Figure 4 This is a schematic diagram of the pBUE411 carrier.
[0045] Figure 5 This refers to the mutation type of the TaLBD30 triple knockout plant in Example 2.
[0046] Figure 6 The spikelet characteristics and flower structure of the TaLBD30 triple knockout plant in Example 2. Detailed Implementation
[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0049] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.
[0050] KN199 is a wheat variety developed by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. It is a nationally approved wheat variety characterized by its compact plant type, strong tillering ability, good cold resistance, and high genetic transformation efficiency.
[0051] Example 1: Discovery of protein TaLBD30 and its encoding gene Cluster analysis of protein LBD in plants is shown in [reference needed]. Figure 1 The results showed that the protein LBD is widely present in plants.
[0052] Through extensive experimentation, the inventors of this application discovered the protein TaLBD30 encoded in KN199. TaLBD30 Genes. Because wheat is an allohexaploid, its genome consists of three distinct subgenomes (A, B, and D), each derived from a different ancestral species. [The text then abruptly shifts to a seemingly unrelated topic:] ...the wheat A genome... TaLBD30 Gene naming TaLBD30 -4A gene. [This refers to the wheat B genome...] TaLBD30 Gene naming TaLBD30 -4B gene. [This refers to the wheat D genome...] TaLBD30 Gene naming TaLBD30 -4D gene.
[0053] TaLBD30 -4A gene, TaLBD30 -4B gene and TaLBD30 -4D genes all contain two exons and one intron, exhibiting high conservation. The structures of each gene are shown in [link to gene description]. Figure 2 .
[0054] In the cDNA of KN199, TaLBD30 The nucleotide sequence of the -4A gene is shown in SEQ ID No. 4. In the KN199 genome, TaLBD30The nucleotide sequence of the -4A gene is shown in SEQ ID No. 7. TaLBD30 The -4A gene encodes the protein TaLBD30-4A, the amino acid sequence of which is shown in SEQ ID No. 1.
[0055] In the cDNA of KN199, TaLBD30 The nucleotide sequence of the -4B gene is shown in SEQ ID No. 5. In the KN199 genome, TaLBD30 The nucleotide sequence of the -4B gene is shown in SEQ ID No. 8. TaLBD30 The -4B gene encodes the protein TaLBD30-4B, the amino acid sequence of which is shown in SEQ ID No. 2.
[0056] In the cDNA of KN199, TaLBD30 The nucleotide sequence of the -4D gene is shown in SEQ ID No. 6. In the KN199 genome, [[ID= The nucleotide sequence of the -4D gene is shown in SEQ ID No. 9. The -4D gene encodes the protein TaLBD30-4D, the amino acid sequence of which is shown in SEQ ID No. 3.
[0057] Example 2: Application of protein TaLBD30 in regulating wheat floral organ development I. Gene editing vector pBUE411- Construction 1. Using plasmid pCBC-MT1T2 (Addgene, Plasmid #50593) as a template, primer LBD30-MT1-F: 5'-aataat AAGCgGGCCTGGGCCTCGTAGCAGAgttttagagctagaaatagc-3' (underlined is the recognition site of the restriction endonuclease BsaI) and primer LBD30-MT2-R: 5'-attatt The primer pair consisting of TAAAcGCAGCAGGTATACATGCATCcgcttcttggtgcc-3' (underlined is the recognition site of restriction endonuclease BsaI) was used for PCR amplification, and the DNA fragment of approximately 964 bp was recovered and purified.
[0058] The reaction system consisted of 20 µl of 17 µl KOD Plus / FX-Neo reaction MIX, 1 µl aqueous solution of primer LBD30-MT1-F, 1 µl aqueous solution of primer LBD30-MT2-R, and 1 µl plasmid pCBC-MT1T2.
[0059] The reaction conditions were: 94℃ for 2 min; 94℃ for 15 s, 62℃ for 30 s, 68℃ for 30 s, 35 cycles; 68℃ for 10 min.
[0060] The nucleotide sequence of plasmid pCBC-MT1T2 (circular) is shown in SEQ ID No. 10. A schematic diagram of plasmid pCBC-MT1T2 is shown below. .
[0061] in accordance with -4A gene, -4B gene and The nucleotide sequence of the -4D gene was designed to target two common sites for the three genes mentioned above. The nucleotide sequence of target site 1 is 5'- TGG-3' (the underlined part represents the reverse complementary sequence from position 320 to 339 of SEQ ID No. 8 starting from the 5' end; TGG is a PAM sequence), the nucleotide sequence of target 2 is 5'- TGG-3' (the underlined part is shown as positions 390-409 from the 5' end of SEQ ID No. 8, where TGG is a PAM sequence). Primers LBD30-MT1-F and LBD30-MT2-R contain two target sites.
[0062] 2. The DNA fragment obtained in step 1 was inserted into the recognition site of the restriction endonuclease BsaI in the pBUE411 vector (Addgene, Plasmid #62200) to obtain the gene editing vector pBUE411- A schematic diagram of the pBUE411 carrier can be found here. .
[0063] The specific steps are as follows: (1) Preparation of enzyme digestion-ligation system. The enzyme digestion-ligation system is 15µl and consists of 200ng pBUE411 vector, DNA fragment obtained in step 1, 1.5µl 10×NEB T4 Buffer (component in T4 Ligase), 1.5µl 10×BSA (component in BsaI-HFv2), 1µl BsaI-HFv2 (NEB), 1µl T4 Ligase (NEB) and ddH2O.
[0064] (2) Take the enzyme digestion-ligation system and react to obtain the ligation product.
[0065] The reaction conditions were: 37℃ for 5 hours; 50℃ for 5 minutes; 80℃ for 10 minutes.
[0066] (3) The ligation product obtained in step (2) was transformed into competent E. coli cells, then plated onto LB resistant plates (Kan resistant), and incubated overnight at 37°C inverted mode to obtain several single clones.
[0067] (4) Using the single clone obtained in step (3) as a template, PCR amplification is performed using primer pair A (using upstream target site gRNA primer: 5'-AAGCgGGCCTGGGCCTCGTAGCAGA-3' and primer TaU3p-411-seq-R: 5'-ATCTCTAGAGAGGGGCACGA-3') or primer pair B (using downstream target site gRNA primer: 5'-TAAAcGCAGCAGGTATACATGCATC-3' and primer TaU3p-411-seq-R). Then, the following judgment is made: if the PCR amplification product obtained by PCR amplification of a single clone using primer pair A contains a target fragment of 1351bp and the PCR amplification product obtained by PCR amplification using primer pair B contains a target fragment of 440bp, then the single clone is a positive single clone.
[0068] The plasmid of the positive monoclonal sample, namely the gene editing vector pBUE411-, was extracted. .
[0069] The gene editing vector pBUE411- Sequencing was performed. Sequencing results showed that the gene editing vector pBUE411- The recombinant plasmid was obtained by inserting a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 11 into the BsaI restriction endonuclease recognition site of the pBUE411 vector.
[0070] II. Application of protein TaLBD30 in regulating wheat floral organ development Wheat is a hexaploid plant. During gene editing, two alleles on two homologous chromosomes of genomes A, B, and D can potentially be edited, resulting in mutations of the same or different types. Therefore, the two alleles in wheat genomes A, B, or D are considered as two gene editing events. A homozygous mutation refers to a mutation occurring on two homologous chromosomes of the wheat genome. Gene( -4A gene, -4B gene or The same mutation occurred in the -4D gene. A biallelic mutation refers to a mutation occurring on both homologous chromosomes of this wheat variety. Gene( -4A gene, -4B gene or Both genes (-4D gene) underwent mutations, but the mutation forms differed. A heterozygous mutation refers to a mutation occurring on one of the two homologous chromosomes of this wheat variety. Gene( -4A gene, -4B gene or A mutation occurred in the -4D gene, and another homologous chromosome... No gene mutations have occurred. Wild type refers to the type of wheat on one of its two homologous chromosomes. Gene( -4A gene, -4B gene or No mutations were found in the -4D gene.
[0071] 1. The gene editing vector pBUE411- Introducing Agrobacterium Recombinant Agrobacterium was obtained.
[0072] 2. Recombinant Agrobacterium was transformed into KN199 to obtain several T0 generations of pseudo-knockout cells. Gene mutants.
[0073] The specific method for transforming recombinant Agrobacterium into KN199 can be found in the following literature: Yuji Ishida, Masako Tsunashima, Yukoh Hiei, Toshihiko Komari. Wheat ( L.) Transformation Using Immature Embryos. Methods in Molecular Biology, 2015, 1223, 189-198. 3. Knockout of the T0 substitute obtained in step 2 The gene mutant was grown to the three-leaf stage. Leaves were collected and total protein was extracted. The protein was then tested using a PAT / bar rapid test strip (Shanghai Youlong Biotechnology Co., Ltd.). The following judgment was made: If the PAT / bar rapid test strip detected a T0 generation suspected of knockout... If the gene mutant shows a single band, then the T0 generation is intended to be knocked out. The gene mutant is a T0 generation negative plant; if the T0 generation detected by the PAT / bar rapid test strip is a candidate for knockout... If the gene mutant shows two bands, then the T0 generation is intended to be knocked out. The gene mutant is a T0 generation positive plant.
[0074] A total of 1 T0 generation negative plant and 15 T0 generation positive plants were obtained.
[0075] 4. Identification of 15 T0 generation positive plants (1) Genomic DNA was extracted from the leaves of 15 T0 generation positive plants using the improved CTAB method.
[0076] (2) After completing step (1), using the genomic DNA of leaves from T0 generation positive plants as templates, primer pair A (composed of primer 4A73-cas-F1: 5'-TTCCCTCTTTGGCTCGATCG-3' and primer 4A73-cas-R1: 5'-GGATCTGGGTCTTTTCCGGGG-3') and primer pair B (composed of primer 4B46-cas-F10: 5'-TGCATCCATCTCGAACT) were used to amplify the target site in genome A, respectively. PCR amplification was performed using primers AGCG-3' and 4B46-cas-R10 (5'-AGCTTCAAACCAGTCACACCA-3') and primer pair D (composed of primers 4D52-cas-F5: 5'-AAGTGCAGTCAGTAGCCACG-3' and 4D52-cas-R5: 5'-GGAAGCAGTCTCCCGGTTT-3') to amplify the target site in the D genome, yielding PCR amplification product A, PCR amplification product B, and PCR amplification product D in sequence.
[0077] (3) After completing step (2), sequence PCR amplification product A, PCR amplification product B, and PCR amplification product D respectively. Observe whether the sequencing results of PCR amplification product A, PCR amplification product B, and PCR amplification product D are bimodal or monomodal, and then make the following determination: If the sequencing result of PCR amplification product A of a T0 generation positive plant is bimodal, then the T0 generation positive plant... The -4A gene has undergone biallelic or heterozygous mutations; if the sequencing result of PCR amplification product A of a T0 generation positive plant is a single peak, then the T0 generation positive plant... -4A gene has a homozygous mutation or no mutation (i.e. wild type); If the sequencing result of PCR amplification product B of a T0 generation positive plant is bimodal, then the T0 generation positive plant... The -4B gene has undergone biallelic or heterozygous mutations; if the sequencing result of the PCR amplification product B of a T0 generation positive plant is a single peak, then the T0 generation positive plant... -4B gene has a homozygous mutation or no mutation (i.e. wild type); If the sequencing result of the PCR amplification product D of a T0 generation positive plant is bimodal, then the T0 generation positive plant... -4D gene has undergone biallelic or heterozygous mutations; if the sequencing result of PCR amplification product D of a T0 generation positive plant is a single peak, then the T0 generation positive plant... -4D gene has either a homozygous mutation or no mutation (i.e., wild type).
[0078] The results showed that among the 15 T0 generation positive plants, only 6 plants had a double peak in the sequencing results of PCR amplification product B, 2 plants had a double peak in the sequencing results of PCR amplification product A, PCR amplification product B and PCR amplification product D, and 7 plants had a single peak in the sequencing results of PCR amplification product A, PCR amplification product B and PCR amplification product D.
[0079] Of the two T0 generation positive plants whose sequencing results for PCR amplification products A, B, and D were all bimodal, one plant exhibited abnormal floral organ development, lacking both stamens and pistils, and therefore produced no seeds. This plant was named the T0 generation. One plant; the other plant's floral organs developed normally and it could produce seeds normally, which was named the T0 generation. Plant.
[0080] 5. T1 generation without gene editing vectors Obtaining the plant (1) Replace T0 Plants self-pollinated to obtain several T1 generations. seed.
[0081] (2) After completing step (1), replace T1 with T1 respectively. After germination, seedlings were cultivated to obtain several T1 generations. Plant.
[0082] (3) T1 generation without gene editing vector Obtaining the plant Due to T0 generation The plant contains the gene-editing vector pBUE411- Therefore, T0 generation The self-pollination of the plant produces segregation in its offspring, with some offspring containing the gene-editing vector pBUE411- One portion may continue gene editing, while the other portion does not contain the gene editing vector pBUE411-. Gene editing will no longer be performed. Therefore, screening will proceed as follows: The T1 generation obtained in step (2) When the plants reached the three-leaf stage, leaves were collected and total protein was extracted. The samples were then tested using PAT / bar rapid test strips (Shanghai Youlong Biotechnology Co., Ltd.). The following judgment was made: If the PAT / bar rapid test strip detected T1 generation... If a plant shows one band, then it is a T1 generation. The plants are T1 generation plants that do not contain gene-editing vectors. Plant.
[0083] After the above steps, several T1 generation genes without gene editing vectors were obtained. Plants. These are T1 generation plants that do not contain gene-editing vectors. The plant's offspring cannot undergo gene editing, and the gene editing type remains stable.
[0084] 6. The T1 generation obtained in step 5 that does not contain the gene editing vector Identification of mutation types in plants (1) The T1 generation cells without gene editing vectors obtained in step 5 were extracted using the improved CTAB method. T1 generation knockout plants without gene editing vectors Genomic DNA of leaves from gene mutants.
[0085] (2) After completing step (1), use T1 generation without gene editing vectors respectively Using plant leaf genomic DNA as a template, primer pair A (composed of primer 4A73-cas-F1: 5'-TTCCCTCTTTGGCTCGATCG-3' and primer 4A73-cas-R1: 5'-GGATCTGGGTCTTTTCCGGGG-3') and primer pair B (composed of primer 4B46-cas-F10: 5'-TGCATCCATCTCGAACTAGCG-3' and primer...) were used to amplify target sites in genome A and B, respectively. The primers 4B46-cas-R10 (composed of 5'-AGCTTCAAACCAGTCACACCA-3') and primer pair D (composed of primer 4D52-cas-F5 (5'-AAGTGCAGTCAGTAGCCACG-3' and primer 4D52-cas-R5 (5'-GGAAGCAGTCTCCCGGTTT-3')) were used to amplify the target site in the D genome. PCR amplification was performed to obtain PCR amplification product A', PCR amplification product B' and PCR amplification product D' in sequence.
[0086] (3) After completing step (2), sequence the PCR amplification products A', B', and D' respectively. The sequencing results are compared with... -4A gene, -4B gene and TaLBD30 The gene editing target sequence of the -4D gene was compared, and the mutation types were counted.
[0087] The statistical results are as follows: Only received TaLBD30 T1 generation with homozygous mutation in the -4B gene TaLBD30-#b Plants, only TaLBD30 T1 generation with homozygous mutation in the -4D gene TaLBD30-#b Plants TaLBD30 -4B gene and TaLBD30 T1 generation with homozygous mutations in all -4D genes TaLBD30-#b Plants. These T1 generation plants. TaLBD30-#b The plant's floral organs develop normally and can all produce fruit normally; Two plants were obtained TaLBD30 -4A gene, TaLBD30 -4B gene and TaLBD30 T1 generation with mutations in all 4D genes TaLBD30-#b The plant, namely the TaLBD30 plant that was removed after three knockouts, is named TaLBD30. -1 #and TaLBD30 -2 #.
[0088] TaLBD30 -1 #and TaLBD30 -2 The mutation types of # are exactly the same, see Figure 5 The details are as follows: In genome A, on two homologous chromosomes TaLBD30 The -4A gene has the same mutation (i.e., homozygous mutation) - a 70-nucleotide deletion, specifically the deletion of 70 nucleotides from position 430 to 499 from the 5' end in SEQ ID No. 7; this deletion causes a frameshift, resulting in the loss of function of the protein TaLBD30-4A; In the B genome, on two homologous chromosomes TaLBD30 The -4B gene has undergone different mutations (i.e., biallelic mutations), specifically: on one homologous chromosome... TaLBD30 The -4B gene underwent an insertion of one nucleotide "T" (i.e., an insertion of one "T" between nucleotides 322 and 323 from the 5' end in SEQ ID No. 8) and a deletion of one nucleotide "A" (i.e., deletion of nucleotide A at position 392 from the 5' end in SEQ ID No. 8), resulting in a 24-amino acid change; on another homologous chromosome... TaLBD30 The -4B gene has a 70-nucleotide deletion, specifically the deletion of 70 nucleotides from position 323 to 392 from the 5' end in SEQ ID No. 8; this mutation causes a frameshift; ultimately leading to the loss of function of the protein TaLBD30-4B. In the D genome, on two homologous chromosomes TaLBD30The -4D gene has the same mutation (i.e., homozygous mutation) - a deletion of one nucleotide, specifically the deletion of nucleotide G at position 475 from the 5' end in SEQ ID No. 9, which causes a frameshift and results in the loss of function of the protein TaLBD30-4D.
[0089] 7. TaLBD30 -1 #and TaLBD30 -2 #Phenological identification Observe wheat varieties Kenong 199 and TaLBD30 -1 #and TaLBD30 -2 # is the phenotype.
[0090] Some results can be found Figure 6 (WT represents the wheat variety Kenong 199, and TaLBD30 is the result of three knockouts.) -1 #). The results showed that wheat varieties Kenong 199 and TaLBD30 -1 #and TaLBD30 -2 #All of them can head normally, and there are no significant differences in vegetative growth, spike length, and number of spikelets; however, TaLBD30 -1 #and TaLBD30 -2 The flower organs of # are abnormally developed, characterized by the absence of pistils and stamens, with both pistils and stamens being completely replaced by lemma-like structures.
[0091] This demonstrates that the protein TaLBD30 can regulate the development of wheat floral organs. Specifically, the simultaneous loss of function of proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D will lead to abnormal development of wheat floral organs, manifested as the absence of stamens and pistils, and the inability to produce seeds normally.
[0092] Due to TaLBD30 -1 #and TaLBD30 -2 # indicates abnormal development of floral organs, therefore it can be used to study floral development and even as a model for the development of ABCDE floral organs, for example, using TaLBD30. -1 #or TaLBD30 -2 # Screen for substances that affect the development of pistils and / or stamens, wherein the substances may be hormones; the screening method may be to introduce TaLBD30 -1 #or TaLBD30 -2 #Inject the test substance. Simultaneously, due to TaLBD30... -1 #and TaLBD30 -2 The stamens and pistils of a plant are completely replaced by a palea-like structure, so the protein TaLBD30 can be used to prepare species with multipetal structures, such as lilies.
[0093] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A method for preparing wheat with abnormal floral organ development, comprising the following steps: (1) Reduce the expression levels and / or activity of proteins TaLBD30-4A, TaLBD30-4B and TaLBD30-4D in the starting wheat to prepare gene-edited wheat; The protein TaLBD30-4A is a1), a2), or a3). a1) The amino acid sequence is that of the protein shown in SEQ ID No. 1; a2) A protein that has more than 90% identity with and has the same function as the protein shown in a1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1; a3) A fusion protein with the same function obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of a1) or a2); The protein TaLBD30-4B is either b1, b2, or b3. b1) The amino acid sequence is that of the protein shown in SEQ ID No. 2; b2) A protein that has more than 90% identity with and has the same function as the protein shown in b1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.
2. b3) A fusion protein with the same function obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of b1) or b2); The protein TaLBD30-4D is c1), c2), or c3). c1) The amino acid sequence is that of the protein shown in SEQ ID No. 3; c2) A protein that has more than 90% identity with and has the same function as the protein shown in c1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 3; c3) A fusion protein with the same function obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of c1) or c2); (2) After completing step (1), the gene-edited wheat is self-pollinated to obtain offspring; wheat with abnormal floral organ development is isolated from the offspring.
2. The method according to claim 1, characterized in that: In step (1), The reduction in the expression level and / or activity of protein TaLBD30-4A in the starting wheat is achieved by mutating the gene encoding protein TaLBD30-4A on at least one of the two homologous chromosomes of the starting wheat A genome; The reduction in the expression level and / or activity of protein TaLBD30-4B in the starting wheat is achieved by mutating the gene encoding protein TaLBD30-4B on at least one of the two homologous chromosomes of the starting wheat B genome. The reduction in the expression level and / or activity of protein TaLBD30-4D in the starting wheat is achieved by mutating the gene encoding protein TaLBD30-4D on at least one of the two homologous chromosomes of the starting wheat D genome.
3. The method according to claim 2, characterized in that: The mutations are achieved through gene editing, EMS mutagenesis, T-DNA insertion, RNA interference, homologous recombination, zinc finger nucleases or transcription activator-like effector nucleases, causing missense mutations, DNA fragment deletions, and / or DNA fragment insertions in the exons of the genes encoding proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D.
4. The method according to claim 3, characterized in that: When the mutation is achieved through gene editing, the target sites used include target site 1 and target site 2; The nucleotide sequence of target 1 is shown as the reverse complementary sequence from position 320 to 339 of SEQ ID No. 8 starting from the 5' end; the nucleotide sequence of target 2 is shown as from position 390 to 409 of SEQ ID No. 8 starting from the 5' end.
5. The method according to claim 1, characterized in that: The starting wheat refers to wheat whose coding genes for proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D have not been modified in any way.
6. The method according to claim 1, characterized in that: The abnormal development of the floral organs is manifested as the absence of pistils, the absence of stamens, the transformation of pistils into lemma-like structures and / or the transformation of stamens into lemma-like structures.
7. The method according to claim 1, characterized in that: The difference between the wheat genome with abnormal floral organ development and the starting wheat genome is as follows: In genome A, on two homologous chromosomes TaLBD30 -4A gene all mutated to TaLBD30 -4A / -70bp; The TaLBD30-4A / -70bp is a DNA molecule obtained by deleting 70 nucleotides from position 430 to 499 starting from the 5' end of SEQ ID No. 7, while keeping the other nucleotide sequences of SEQ ID No. 7 unchanged; In the B genome, on a homologous chromosome TaLBD30 -4B gene mutation TaLBD30 -4B / +1bp / -1bp, on another homologous chromosome TaLBD30 -4B gene mutation TaLBD30 -4B / -70bp; The TaLBD30 -4B / +1bp / -1bp is the DNA molecule obtained by inserting one nucleotide T between nucleotides 322 and 323 from the 5' end of SEQ ID No. 8, and deleting nucleotide A at position 392, while keeping the other nucleotide sequences of SEQ ID No. 8 unchanged; The TaLBD30 -4B / -70bp is a DNA molecule obtained by deleting 70 nucleotides from position 323 to 392 starting from the 5' end of SEQ ID No. 8, while keeping the other nucleotide sequences of SEQ ID No. 8 unchanged; In the D genome, on two homologous chromosomes TaLBD30 -4D genes are all mutated to TaLBD30 -4D / -1bp; The TaLBD30 -4D / -1bp is the DNA molecule obtained by deleting nucleotide G at position 475 from the 5' end of SEQ ID No. 9, while keeping the other nucleotide sequences of SEQ ID No. 9 unchanged. 8.S1) or S2): S1) The application of gene-edited wheat prepared by any of the methods described in claims 1 to 5 in the preparation of wheat with abnormal floral organ development; S2) The use of a substance that reduces the expression level and / or activity of the proteins TaLBD30-4A, TaLBD30-4B and TaLBD30-4D described in claim 1 in wheat in the preparation of wheat with abnormal floral organ development; Preferably, the substance for reducing the expression level and / or activity of proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D in wheat according to claim 1 includes a gene editing system; the target sites used in the gene editing include target site 1 and target site 2; The nucleotide sequence of target 1 is shown as the reverse complementary sequence from position 320 to 339 of SEQ ID No. 8 starting from the 5' end; the nucleotide sequence of target 2 is shown as from position 390 to 409 of SEQ ID No. 8 starting from the 5' end.
9. The application of wheat with abnormal floral organ development prepared by any of the methods described in claims 1 to 7, wherein T1), T2), or T3) is present. T1) serves as a model for floral organ development; T2) Study of floral organ development; T3) Screening for substances that affect the development of pistils and / or stamens; Preferably, the floral organ development model is the ABCDE floral organ development model; Preferably, the substance is a hormone or a compound.
10. The use of the proteins TaLBD30-4A, TaLBD30-4B, and TaLBD30-4D as described in claim 1 in the cultivation of transgenic plants with multiple petals and no fruit set; Preferably, the plant is a plant of the genus Lilium.