Mutant of bgr promoter and application thereof in regulating plant agronomic traits

Abstract

This invention relates to the field of plant breeding technology, and more particularly to a mutant of the bgr promoter and its application in regulating plant agronomic traits. The mutant is a variant of the wild-type bgr promoter that has undergone changes such as… bp158 , bp629 , bp1430 and bp1977 The mutations include: deletion of nucleotides GG at positions -2014 and -2015; insertion of nucleotide A at position -747; and deletion of nucleotide T at position -193. The applications include: regulating the expression of target genes or improving agronomic traits of plants; these agronomic traits include one or more of the following: plant height, branching angle, siliques, seed size, or yield. This invention has discovered a specific mutant of the bgr promoter, which can regulate and reduce the expression level of the bgr gene, thereby improving agronomic traits and increasing yield, and has significant value.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a mutant of the bgr promoter and its application in regulating plant agronomic traits. Background Technology

[0002] Rapeseed, as one of the world's major oilseed crops, plays a vital role in ensuring current food and oil security. However, the current main rapeseed varieties are tall, loosely branched, and prone to lodging, resulting in slow growth in rapeseed yield per unit area. Crop plant type is a crucial factor in crop yield. The successful application of Green Revolution genes in crops such as rice and wheat has effectively improved their lodging resistance and yield, indicating that plant type improvement is a key factor in increasing crop yield. Therefore, obtaining new rapeseed germplasm with dwarfed plants and compact branching will play a vital role in effectively increasing rapeseed yield. Our research group obtained new Brassica napus germplasm with short plant height, small branching angle, and compact plant type through EMS mutagenesis. bgr Previous studies have shown that the new germplasm bgr The hybrid vigor is significant, and the F1 hybrids bred from it exhibit ideal plant type characteristics, characterized by dwarfing, compact plant type, high resistance to lodging, suitability for dense planting and mechanized harvesting, and yield increase of approximately 30%; the new germplasm bgr Characteristics BGR Gene mutations can lead to gain-of-function mutants. bgr , BGR It encodes a GSK3 protein kinase, which plays an important role in the brassinolide (BR) signal transduction pathway.

[0003] but BGR Gain-of-function mutations have also led to new germplasm of Brassica napus. bgr It exhibits undesirable agronomic traits, such as extremely dwarfed plant height, excessively small branching angles, short siliques, and small seeds. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a mutant of the bgr promoter and its application in regulating plant agronomic traits.

[0005] In a first aspect, the present invention provides a mutant of the bgr promoter, wherein the mutant is obtained by any one or more of the following mutations on the basis of the wild-type bgr promoter: (1) GG deletion of nucleotides at positions -2014 and -2015; (2) The nucleotide sequence from position -1563 to position -1538 is deleted, and a sequence identical to the nucleotide sequence from position -1659 to position -1571 is inserted at position -1571: CAAATATATCTTCATCAGCTTATGTAGACATGAAGGAAGCTGCTGCTTTAGGAGATGTTTTGTTGATCTTCATGATTCTTCTATCTTTA; (3) Insertion of nucleotide A at position -747; (4) T deletion of nucleotide -193.

[0006] Furthermore, the wild-type bgr promoter comprises any of the following nucleotide sequences: i) The nucleotide sequence shown in SEQ ID NO.1; ii) Nucleotide sequences with the same promoter function obtained by inserting, deleting, or replacing one or more nucleotides from the nucleotide sequence shown in i).

[0007] The nucleotide sequence shown in SEQ ID NO.1:

[0008] Furthermore, the mutant comprises any of the following nucleotide sequences: i) A nucleotide sequence as shown in SEQ ID NO.2, 3, 4 or 5; ii) Nucleotide sequences with the same promoter function obtained by inserting, deleting, or replacing one or more nucleotides from the nucleotide sequence shown in i).

[0009] The nucleotide sequence shown in SEQ ID NO.2:

[0010] The nucleotide sequence shown in SEQ ID NO.3:

[0011] The nucleotide sequence shown in SEQ ID NO.4:

[0012] The nucleotide sequence shown in SEQ ID NO.5:

[0013] In a second aspect, the present invention provides a biological material comprising: the aforementioned mutant; wherein the biological material is an expression cassette, vector, cell, recombinant viral particle, plant tissue or plant organ.

[0014] The expression cassette of this invention includes a promoter, a coding sequence (target gene), and a termination signal (e.g., a terminator and a polyadenylation signal) for terminating the transcription process. It also guides the cell to add a poly(A) tail to the end of the mRNA to increase mRNA stability and translation efficiency. Common examples include SV40 polyA and BGH polyA. Furthermore, various elements can be added to the expression cassette, such as enhancers, introns, Kozak sequences, Shine-Dalgarno sequences, or selectable marker genes.

[0015] The vectors described in this invention include: plasmid vectors (extrachromosomal circular DNA molecules derived from bacteria or yeast), viral vectors (modified viruses that have had their pathogenicity and self-replication capabilities removed, but retain their ability to efficiently infect cells and deliver genetic material into cells), bacteriophage vectors, or artificial chromosome vectors (e.g., bacterial artificial chromosome BAC or yeast artificial chromosome YAC).

[0016] The transgenic cells described in this invention are cells whose genetic material has undergone stable artificial alterations. These transgenic cells include animal cells, plant cells, or microbial cells.

[0017] The recombinant viral particles described in this invention are virus-like particles, consisting of a protein coat (viral capsid) encapsulating genetic material. For example, the recombinant viral particles are prepared by transfecting the aforementioned viral vector and other helper plasmids into a packaging cell line (e.g., HEK293T cells), which will then complete the expression and assembly of viral proteins and recombinant genes to obtain complete recombinant viral particles.

[0018] Those skilled in the art, having access to the sequence of the promoter mutant disclosed in this application, are fully aware of the preparation methods of the aforementioned expression cassette, vector, cell, and recombinant viral particles based on existing technology. There are no technical obstacles involved, and therefore, expression cassettes, vectors, cells, and recombinant viral particles containing the aforementioned gene mutants are also within the scope of this invention.

[0019] Thirdly, the present invention provides a plant, the plant comprising the aforementioned mutant or the aforementioned biological material; Preferably, the plant possesses any one of the following: i) The mutant was obtained directly by mutating its own wild-type bgr promoter using gene editing methods; ii) When expressing the target gene exogenously, the mutant described above is used as the promoter; iii) The offspring of i) or ii).

[0020] Fourthly, the present invention provides the application of the aforementioned mutants or the aforementioned biological materials in regulating the expression of a target gene; preferably, the target gene is... bgr Gene.

[0021] Fifthly, the present invention provides the application of the aforementioned mutants or the aforementioned biological materials in improving the agronomic traits of plants; Preferably, the agronomic traits include one or more of the following: plant height, branching angle, siliques, seed size, or yield.

[0022] In a sixth aspect, the present invention provides a method for improving the agronomic traits of plants, comprising: gene editing of the wild-type bgr promoter in the plant to transform it into the aforementioned mutant.

[0023] Furthermore, the gRNA used in the gene editing includes any one or more of the following: i)GTCTGTCATTTGTGGGGGGCT; ii) TTTTGCGATATACTACTAAT; iii) TTTTGCGATATACTACTAAT; iv) CCCTTGAGAGCTTATGAAAC.

[0024] Furthermore, the plant is a monocotyledonous plant or a dicotyledonous plant; Preferably, the plant is a plant of the genus Brassica; More preferably, the plant is Brassica napus.

[0025] The present invention has the following beneficial effects: New germplasm of Brassica napus in existing technologies bgr The plant exhibits undesirable agronomic traits, such as extremely dwarfed plant height, excessively small branching angles, short siliques, and small seeds. This invention addresses these issues. bgr Gene editing was performed on the promoter region of the gene, and after research and screening, two promoter mutants were obtained that could significantly improve the aforementioned agronomic traits and at the same time significantly increase yield.

[0026] The promoter mutants provided by this invention can be used for exogenous expression of target genes or for direct gene editing of wild-type lines. bgr starter This mutant sequence has significant application value for cultivating plant varieties that are taller, have larger branching angles, longer siliques, larger seeds, and higher yields. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 The results are the bgr promoter editing sequence analysis results provided in this embodiment of the invention; where red dashed lines represent DNA base or fragment deletions, and red squares represent DNA base or fragment insertions, with a scale bar of 200 bp per square.

[0029] Figure 2 The figure shows the T0 representative phenotype of the bgr promoter-edited strain provided in this embodiment of the invention; where A is the mature phenotype of the T0 generation transgenic plant, and the scale bar is 5cm; B is the plant height; C is the silique length; the numerical error bars in the figure represent SD (n = 3), and the significance of the experimental group and the control group was analyzed by t test (****, P<0.001; **, P<0.01; *, P<0.05).

[0030] Figure 3 This invention provides the results of greenhouse phenotype yield of the bgr mutant, which was altered by bgr promoter editing. A represents the hybrid Zhongkeyou 2 (ZKY2) and its parent 135 and parent bgr; the flowering phenotypes of four gene-edited lines (bp158, bp629, bp1430, and bp1977), with a scale bar of 5 cm; B represents the expression level of the bgr gene in the bgr mutant and gene-edited lines detected by qRT-PCR, using BnaC02.Actin7 (BnaC02g00690D) as an internal reference gene; C represents plant height; D represents branching angle; E represents yield per plant; the numerical error bars in the figure represent SD (n = 3). The significance of the experimental group and the control group was analyzed using a t-test (****, P < 0.001; ***, P < 0.005; **, P < 0.01; *, P < 0.05).

[0031] Figure 4 The results of field yield trait analysis of the bgr promoter-edited lines provided in this embodiment of the invention are shown below. A represents the phenotypic characteristics of the bp158 and bp1430 promoter-edited lines and the bgr mature phenotype, with a scale bar of 5 cm; B represents the siliques, with a scale bar of 0.5 cm; C represents plant height; D represents silique length; E represents the number of seeds per silique; and F represents yield per plant. The numerical error bars in the figure represent SD (n = 3). The significance of the experimental and control groups was analyzed using a t-test (***, P < 0.005; **, P < 0.01; *, P < 0.05; no significant difference, ns). Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0034] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0035] Example 1: Brassica napus bgr Identification of gene promoter gene editing target sites and vector construction (1) bgr promoter cloning Based on bgr The genome sequencing and de novo assembly results of the mutant yielded... bgr The 2168 bp upstream of the ATG gene is the promoter reference sequence. Specific primers bP.F (5´-agcaggctttgactttGGATGAGCCAATAAAGTTTAGTTTG-3´) and bP.R (5´-tgggtctagagactttccGTGCTATTCTTCTCTCTCTCTCTCTAAC-3´) with homologous arms of the cloning vector pGWC were designed, and the CTAB method was used for extraction. bgr The mutant DNA was amplified by PCR using the high-fidelity enzyme KOD (TOYOBO, KMM). The promoter fragment was ligated into the linear vector pGWC (digested with AhdⅠ, NEB) via homologous recombination (Biomed, CL117). This pGWC was then transformed into competent *E. coli* cells DH5α. Positive single clones were selected and sequenced to obtain... bgr Gene promoter sequence.

[0036] (1) bgr Design of promoter gene editing targets This invention designs gRNAs at different sites, in bgr Twenty gRNAs (Table 1, named by position number, e.g., -2034 bp indicates 2034 bp upstream of the start codon ATG, and the target site is named BP158) were designed as gene editing sites on the promoter sequence of the gene.

[0037] Table 1 bgr List of gRNA designs for gene promoter regions

[0038] (2) Carrier construction Two 5' end bands were synthesized by Shanghai Sangon Biotech Co., Ltd. Bsa I. Base-complementary single-stranded DNA at the restriction enzyme sites was denatured at 95 °C for 5 min, followed by denaturation at 30 °C for 5 min to form double strands with adapters at both ends. Simultaneously, the pHSE401 vector (a gift from Professor Chen Qijun of China Agricultural University) was digested with a single enzyme. Bsa I. Enzyme digestion (NEB): The linear vector and double-stranded DNA were reacted at 25 °C for 1 h using T4 ligase (NEB) to construct a gene knockout vector. The plasmid with the correct sequence was transformed into Agrobacterium GV3101 and is ready for use.

[0039] Example 2 bgr Genetic transformation in the context Gene editing vectors were transformed into rapeseed using Agrobacterium tumefaciens infection. bgr The genetic transformation method for the mutant (disclosed in ZL202211177399.1) is the same as that in ZL202211177399.1.

[0040] Example 3: Identification of editing sites and phenotypic analysis of gene-edited lines After obtaining T0 generation transgenic rapeseed, plant genomic DNA was extracted, and PCR identification was performed using specific primers. A total of 10 positive edited lines with different target sites were obtained. The editing methods included single base insertion, deletion, fragment deletion, and insertion. Figure 1 Compared to bgr-D Seven of the gene-edited strains bp158, bp549, bp629, bp1163, bp1430, bp1737, bp1977 The plant shape has changed, and traits such as dwarfing, leaf curling, and small branching have partially recovered. Figure 2 (The results show that) some of the edited lines were heterozygous and require further selection through multiple generations. This finding indicates that gene editing of the promoters of key regulatory genes can induce changes in rapeseed plant morphology.

[0041] Example 4 bgr Promoter gene editing on rapeseed mutants bgr Influence of plant type To evaluate the breeding and production potential of promoter-edited lines, under greenhouse pot conditions, "Zhongkeyou 2" and its paternal tall-stemmed material 135 and maternal dwarf mutant were used. bgr As a control, the traits of the gene-edited lines were examined. Gene-edited lines bp158, bp629, bp1430 and bp1977 The plant height ranges from 56 to 130 cm, and the branching angle ranges from 30 to 50°. bgr The mutant showed significantly increased plant height and branching angle compared to the previous strain, and was consistent with that of Zhongke Oil No. 2. Figure 3 (A, C, and D). Other gene-edited lines showed poor yield traits, with drawbacks such as short siliques, small seeds, and no significant increase in plant height.

[0042] Furthermore, the results showed that the phenotypic data of the promoter-edited lines were consistent with those in vivo. bgr Gene expression levels are related ( Figure 3 (B) This further confirms that the traits of genetically modified rapeseed are affected. bgr The impact of gene expression levels.

[0043] in: bp158 : GG deletion of nucleotides at positions -2014 and -2015.

[0044] bp629 The sequence TATGATTATTCCATGGTGGTTTTTG is deleted from position -1563 to -1538, and a sequence identical to the sequence from position -1659 to -1571, CAAATATATCTTCATCAGCTTATGTAGACATGAAGGAAGCTGCTGCTTTAGGAGATGTTTTGTTGATCTTCATGATTCTTCTATCTTTA, is inserted from position -1571.

[0045] bp1430 : Insertion of nucleotide A at position -747.

[0046] bp1977 : T deletion at position -193.

[0047] Example 5 bgr Promoter gene editing on mutants bgr Impact on production To evaluate the productivity of the gene-edited lines, this invention was field-planted in the Olympic Science and Technology Park of the Chinese Academy of Sciences. bp158 and bp1430 Two gene-edited lines were examined for traits in mature transgenic rapeseed. The results showed that the branching of the two edited lines was looser than that of the mutant. Figure 4 (A) Plant height increased significantly ( Figure 4 (C) The increase reached over 86.4%; the length of the siliques significantly increased ( Figure 4 (B and D in the middle), the increase reached over 48.5%; and bp158 The number of seeds per silique of the strain increased significantly. Figure 4 In the middle E), the increase reached over 33.7%; due to changes in yield traits, the yield per plant from promoter editing was ultimately significantly increased ( Figure 4 (For example, the increase in the middle F) reached over 98%.

[0048] These results indicate that... bp158 and bp1430 Two promoter editing methods can alter mutants bgr The plant type traits of rapeseed can be further optimized, increasing breeding potential and providing the possibility of screening out stable and high-yielding rapeseed germplasm in the later stage.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant of the bgr promoter, characterized in that, The mutant is obtained by performing any one or more of the following mutations on the wild-type bgr promoter: (1) GG deletion of nucleotides at positions -2014 and -2015; (2) The nucleotide sequence from position -1563 to position -1538 is deleted, and a sequence identical to the nucleotide sequence from position -1659 to position -1571 is inserted at position -1571: CAAATATATCTTCATCAGCTTATGTAGACATGAAGGAAGCTGCTGCTTTAGGAGATGTTTTGTTGATCTTCATGATTCTTCTATCTTTA; (3) Insertion of nucleotide A at position -747; (4) T deletion of nucleotide -193.

2. The mutant according to claim 1, characterized in that, The wild-type bgr promoter includes any of the following nucleotide sequences: i) The nucleotide sequence shown in SEQ ID NO.1; ii) Nucleotide sequences with the same promoter function obtained by inserting, deleting, or replacing one or more nucleotides from the nucleotide sequence shown in i).

3. The mutant according to claim 1 or 2, characterized in that, The mutant includes any of the following nucleotide sequences: i) A nucleotide sequence as shown in SEQ ID NO.2 or 3; ii) Nucleotide sequences with the same promoter function obtained by inserting, deleting, or replacing one or more nucleotides from the nucleotide sequence shown in i).

4. A biomaterial, characterized in that, include: The mutant according to any one of claims 1-3; the biological material is an expression cassette, vector, cell, recombinant viral particles, plant tissue or plant organ.

5. A plant, characterized in that, The plant includes the mutant according to any one of claims 1-3, or the biological material according to claim 4; Preferably, the plant possesses any one of the following: i) The mutant was obtained directly by mutating its own wild-type bgr promoter using gene editing methods; ii) When expressing the target gene exogenously, the mutant described above is used as the promoter; iii) The offspring of i) or ii).

6. The application of the mutant according to any one of claims 1-3, or the biological material according to claim 4, in regulating the expression of a target gene; preferably, the target gene is the bgr gene.

7. The use of the mutant according to any one of claims 1-3, or the biomaterial according to claim 4, in improving the agronomic traits of plants; Preferably, the agronomic traits include: One or more of the following: plant height, branching angle, siliques, seed size, or yield.

8. A method for improving the agronomic traits of plants, characterized in that, include: Gene editing of the wild-type bgr promoter in plants to transform it into the mutant described in any one of claims 1-3.

9. The method according to claim 8, characterized in that, The gRNA used in the gene editing includes any one or more of the following: i)GTCTGTCATTTGTGGGGGGCT; ii) TTTTGCGATATACTACTAAT; iii) TTTTGCGATATACTACTAAT; iv) CCCTTGAGAGCTTATGAAAC.

10. The method according to claim 8 or 9, characterized in that, The plant is a monocotyledonous plant or a dicotyledonous plant; Preferably, the plant is a plant of the genus Brassica; More preferably, the plant is Brassica napus.

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