A method for reducing the angle of tomato leaves using gene editing

Abstract

The present invention belongs to the field of modern agricultural technology, and specifically relates to a method for reducing the angle of tomato leaves by using gene editing. SlMYB15‑like The application of genes in regulating tomato plant type was constructed by using CRISPR / Cas9 gene editing technology SlMYB15‑like Gene knockout mutants were found to have a significant effect on regulating tomato plant type. A series of observations and statistics showed that SlMYB15‑like The knockout mutants had significantly thicker stems than wild-type tomatoes, while their plant height and leaf angle were significantly lower than those of wild-type tomatoes, resulting in a shorter, stockier, and more compact plant shape. This research provides an important foundation for the breeding of densely planted tomato varieties and the regulation of tomato plant shape.

Description

Technical Field

[0001] The present invention belongs to the field of modern agricultural technology, and specifically relates to a method for reducing the angle of tomato leaves by utilizing gene editing. Background Art

[0002] tomato( Solanum lycopersicum Maize is one of the most important economic crops cultivated worldwide. Studies have shown that increasing planting density is an effective strategy for increasing crop yield per unit area (Lauer, S., Hall, BD, Mulaosmanovic, E., et al. 2012. Morphological changes in parental lines of pioneer brand maize hybrids in the US central corn belt. Crop Sci. 52, 1033–1043.). However, high-density planting can cause overlapping leaves, reducing light transmittance between plants and triggering shade avoidance, leading to excessive growth, thinness, and increased lodging, ultimately resulting in biomass and yield losses (Cao, YY., Zhong, ZJ., Wang, HY., et al. 2022. Leaf angle: a target of genetic improvement incereal crops tailored for high-density planting. Plant Biotechnology Journal, 20, 426-436.). Therefore, identifying germplasm with upward-thrusting leaves and a compact plant shape is crucial.

[0003] Leaf angle is a key trait influencing plant architecture. Upright leaves can better capture canopy light energy under high-density planting conditions, thereby improving photosynthesis and stress tolerance, promoting ventilation and light transmission, and ultimately increasing crop yield. Interference with the rice OsDWARF4 gene resulted in a partial defect in brassinosteroid biosynthesis, significantly reducing leaf angle and resulting in higher yields than the wild type under dense planting conditions (Sakamoto, T., Morinaka, Y., Ohnishi, T., et al. 2006. Erect leaves caused by brassinosteroid deficiency increase biomassproduction and grain yield in rice. Nat. Biotechnol. 2, 105-109.). Studies have found that maize ZmRAVL1 directly activates ZmBRD1 expression, leading to increased brassinosteroid levels and, in turn, an increase in leaf angle (Tian, ​​J., Wang, C., Xia, J., et al. 2019. Teosinte ligule allele narrows plant architecture and enhances high-density maize yields. Science, 365, 658-664.). R2R3 MYB transcription factors have also recently been reported to regulate leaf angle. In rice, OsFLP mediates brassinosteroid signaling, leading to lignin deposition at leaf junctions. OsFLP mutants exhibit a larger leaf angle and a looser plant architecture (Liu, HC., Zhang, J., Wang, JX., et al. 2024. The rice R2R3 MYB transcription factor FOUR LIPS connects brassinosteroid signaling to lignin deposition and leaf angle. Plant Cell, 36, 4768-4785.).

[0004] Currently, research on leaf angle has mainly focused on crops such as rice and corn, with few reports on tomatoes. The formation and regulation mechanism of tomato leaf angle is still unclear, so it is necessary to develop a method to reduce tomato leaf angle. Summary of the Invention

[0005] The first aspect of the present invention aims to provide a method for inhibiting SlMYB15-like Application of genes to reduce leaf angle in tomato.

[0006] The second aspect of the present invention aims to provide and inhibit SlMYB15-like Application of genetically related biomaterials in breeding tomato varieties.

[0007] The third aspect of the present invention aims to provide a method.

[0008] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides an inhibitory SlMYB15-like Application of genes to reduce leaf angle in tomato.

[0010] In some embodiments of the present invention, the SlMYB15-like The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0011] In some embodiments of the present invention, the SlMYB15-like The amino acid sequence encoded by the gene is shown in SEQ ID NO: X.

[0012] In some embodiments of the present invention, the inhibition SlMYB15-like Gene knockout SlMYB15-like Gene, knockdown SlMYB15-like Genes, or SlMYB15-like Methods for generating inactivating mutations.

[0013] The second aspect of the present invention provides and inhibits SlMYB15-like Application of genetically related biomaterials in breeding tomato varieties.

[0014] In some embodiments of the present invention, the SlMYB15-like The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0015] In some embodiments of the present invention, the application is to construct and inhibit SlMYB15-like Genetically related biological materials are used to obtain tomato varieties with small leaf angles.

[0016] In some embodiments of the present invention, the biological material includes nucleic acid molecules, vectors, and cells.

[0017] In some embodiments of the invention, the nucleic acid molecule comprises an inhibitory SlMYB15-like functional microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotides.

[0018] In some embodiments of the present invention, the sequence of the sgRNA is shown in SEQ ID NO: 2.

[0019] In some embodiments of the present invention, sgRNA is used in conjunction with CRISPR / Cas9 vectors to achieve the purpose of gene knockout.

[0020] In some embodiments of the present invention, the CRISPR / Cas9 vector further includes an expression vector containing the sgRNA, and of course may also include a Cas9 protein or an expression vector for expressing the Cas9 protein.

[0021] In some embodiments of the present invention, the vector includes but is not limited to pHEE401 and other common vectors in the art.

[0022] In some embodiments of the present invention, the cell comprises at least one of Escherichia coli and Agrobacterium tumefaciens, wherein Escherichia coli is a common host cell for constructing vectors and plasmids in the art, and Agrobacterium tumefaciens is a common tool for delivering DNA molecules to plants in the art.

[0023] A third aspect of the present invention provides a method comprising the step of reducing the expression level and / or activity of SlMYB15-like protein in tomatoes.

[0024] The method is at least one of a1) and a2): a1) a method for reducing the angle of tomato leaves; a2) a method for cultivating high-density planting tomato varieties.

[0025] The tomato variety comprises the following characteristics: the leaf angle becomes smaller relative to a reference level; the reference level is the level of the wild type.

[0026] The reduction in tomato SlMYB15-like The step of increasing the expression level and / or activity of the protein is to increase the expression level and / or activity of the protein by the second aspect of the present invention. SlMYB15-like Genetically related biological material is introduced into tomato tissue or tomato cells.

[0027] In some embodiments of the present invention, the introduction method comprises using at least one of Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation.

[0028] In some embodiments of the present invention, the step of reducing the expression level and / or activity of the SlMYB15-like protein in tomatoes is specifically:

[0029] (1) Design SlMYB15-like Gene target sequence sgRNA, construct tomato SlMYB15-like CRISPR / Cas9 vectors for gene editing.

[0030] (2) The CRISPR / Cas9 vector described in step (1) is transferred into Agrobacterium competent cells to obtain Agrobacterium containing the CRISPR / Cas9 vector.

[0031] (3) The Agrobacterium infection solution obtained in step (3) was used to infect the cotyledons of common wild-type tomatoes, and seedlings were obtained again through tissue culture. Tomato SlMYB15-like A stably inherited mutant strain with a gene mutation, no exogenous Cas9 protein, and a target sequence mutation.

[0032] In some embodiments of the present invention, the vector is pHEE401.

[0033] In some embodiments of the present invention, the host cell is Agrobacterium GV3101.

[0034] In some embodiments of the present invention, the tomato variety is Condine Red (CR).

[0035] The beneficial effects of the present invention are:

[0036] The present invention discovered SlMYB15-like The gene plays a significant role in regulating the leaf angle of tomatoes. CRISPR / Cas9 gene editing technology was used to knock out the tomato gene. SlMYB15-like , and obtained a knockout SlMYB15-like A series of experiments have shown that compared with the wild type, SlMYB15-like The gene knockout mutant has a smaller leaf angle and a dwarf phenotype. Therefore, the present invention provides important germplasm resources for the breeding of tomato varieties suitable for dense planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0038] Figure 1 These are the test results of tomato plants with SlMYB15-like gene knockout in Example 1; WT, wild type; myb15-like, mutant.

[0039] for Figure 2 Growth phenotype results of mutants compared with wild-type tomato plants.

[0040] for myb15-like Statistical results of stem diameter and plant height of mutant and wild-type tomato plants, where A is the statistical result of stem diameter; B is Plant height statistics of mutant and wild-type tomato plants.

[0041] Figure 3 for Phenotypic consequences of leaf angle in mutant and wild-type tomato plants.

[0042] myb15-likefor Statistical results of leaf angles of mutant and wild-type tomato plants. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0044] Example 1 Obtaining SlMYB15-like gene knockout plants

[0045] Gene knockout mutants were constructed using CRISPR / Cas9 technology. Tomato was searched from the NCBI (http: / / www.ncbi.nlm.nih.gov / ) database. myb15-like CDS sequences of genes designed using CRISPR-P (https: / / www.genome.arizona.edu / crispr / CRISPRsearch.html) The two sgRNA sequences of the gene were amplified by PCR using a PCR instrument with the Y0014472-1 plasmid as the template for the two target sites.

[0046] Figure 4 The CDS sequence of the gene is as follows:

[0047] TTATGCTATATCAGCTCCAAGTTCCAAGTGAAAAATCAAAACAAAGTATAGAGAGAGAAAAAATAAAATGGGGAGATCTCCTTGCTGTGAGAAATTGGGGTTGAAAAGAGGTCCATGGAGCAAAGAAGAAGATTATTTACTCATCAATTACATTAAAAAGAATGGTCATCCTAATTGGCGTGCACTTCCAAAACTTGCAGGTCTATTAAGGTGCGGAAAAAGTTGTAGGCTTCGATGGACTAATTACTTGAGACCTGATATTAAGCGAGGCAATTTTACTCATCAAGAAGAAGATACAATTATCAAGTTGCATCAAGTTCTTGGAAACAGTTGGTCTGCTATTGCAGCAAGATTACCCGGAAGAACAGATAACGAAATAAAAAACATTTGGCATACTCGTCTGAAGAAAAAAAGGAATGAATCTCAACTTAAAGAAACCCAATCGGAGCCTGAAAATACTAATGTAGATGTACATTTGGAGGAGGCCAACAATTCTAATGATAAACATTCCGAAATATCGAATCTTAAAATAAACATCGAAATCCAACAACAACCAAGTCCATCATCGTCAGTATCATCATCAAGTGAAGATTCATGTTCAAATACAACTGCAACGAGTTCAGAGTCGAGAAATCAAATAATGTCCGATAATTTGTTAGAAATTGATGACGATATTTGGTCCGAGGTAGTATGGGCACAAGTCGATGACAATTATGTTGATTTGTCATTAATGGAGGATAATTACCACATTAATTCTAGCTTTGATGATAATTGGTTTTGGGATGATCTTTTTACAAGATCTAATGAGTTGATGTTAGAATTGCCTGAATTATGAGC (SEQ ID NO: 1).

[0048] The sequence of sgRNA1 is: TTGAGACCTGATATTAAGCG (SEQ ID NO: 2).

[0049] The sequence of sgRNA2 is: GCTATTGCAGCAAGATTACC (SEQ ID NO: 3).

[0050] The normal protein sequence information of MYB15-like is as follows:

[0051] MGRSPCCEKLGLKRGPWSKEEDYLLINYIKKNGHPNWRALPKLAGLLRCGKSCRLRWTNYLRPDIKRGNFTHQEEDTIIKLHQVLGNSWSAIAARLPGRTDNEIKNIWHTRLKKKRNESQLKETQSEPEN TNVDVHLEEANNSNDKHSEISNLKINIEIQQQPSPSSSVSSSSEDSCSNTTATSSESRNQIMSDNLLEIDDDIWSEVVWAQVDDNYVDLSLMEDNYHINSSFDDNWFWDDLFTRSNELMLELPEL* (SEQ ID NO: 4).

[0052] The sequence information of the MYB15-like mutant protein is as follows:

[0053] MGRSPCCEKLGLKRGPWSKEEDYLLINYIKKNGHPNWRALPKLAGLLRCGKSCRLRWTNYLRPDI myb15-like * (SEQ ID NO: 5, underline indicates different sequences).

[0054] The Y0014472-1 plasmid sequence information is as follows:

[0055]

[0056] The two purified PCR products were ligated into the pHEE401 plasmid using the Golden Gate Assembly Kit (BsaI-HFv2) (NEB, E1601).

[0057] After successful sequencing verification, the pHEE401-MYB15-like vector was electroporated into Agrobacterium tumefaciens GV3101 for tomato genetic transformation. Cotyledons of the wild-type tomato cultivar CR (Condine Red) were infected with Agrobacterium, and then cultured through plant tissue to generate complete transgenic tomato plants. Subsequently, upstream and downstream primers were designed based on the target site: cri-MYB15-like-F (CTCCTTGCTGTGAGAAATTGGG, SEQ ID NO: 7) and cri-MYB15-like-R (ACTGACGATGATGGACTTGGTT, SEQ ID NO: 8).

[0058] After obtaining transgenic plants, DNA from T0 leaves was extracted and a genomic DNA fragment of approximately 400 bp, including sgRNA, was amplified using PCR technology and sent to the company for sequencing. The sequencing results were compared with the original genome sequence using Snapgene software to screen out mutant plants, which were then self-fertilized to obtain homozygous plants. The mutants were used in subsequent experiments.

[0059] The results are as follows Figure 5 As shown in Figure 2, compared to a normal, unedited tomato, the gene-edited mutant had a base insertion at the sgRNA position. Hereinafter, the normal, unedited tomato is referred to as the control. The mutant has a T insertion compared to the control.

[0060] Example 2 myb15-like Study on Gene Regulation of Tomato Plant Type

[0061] 1. Experimental methods

[0062] 1) Tomato material cultivation method

[0063] Tomato seeds (Condine Red, including mutants and wild-type) were soaked in 50°C warm water for 15 minutes and then germinated in a 28°C constant-speed shaker (200 rpm / min) for 3 days, with water changed every 12 hours. When the radicles were approximately 1 cm thick, they were sown in 72-well trays using a 3:1 mixture of peat and vermiculite. The trays were cultured in a plant factory with a temperature setting of 21 / 19°C (day / night), a photoperiod of 12 h light / 12 h dark, and a light intensity of 200 μmol·m -2 ·s-1 When the tomato seedlings grow one true leaf, transplant them into planting pots and water them with an appropriate amount of Hoagland nutrient solution every 3 days.

[0064] 2) Method for measuring tomato plant height and stem diameter

[0065] When the tomatoes have five leaves and one heart, select healthy plants with similar growth status and measure the diameter of the main stem between the second and third leaves as the stem thickness, and the length from the cotyledon to the growing point as the plant height.

[0066] 3) Tomato leaf angle measurement method

[0067] When the tomato has five leaves and one heart, select healthy plants with similar growth status and measure the angle between the third fully expanded leaf from the top and the main stem as the leaf angle.

[0068] 2. Experimental results

[0069] Plant height, stem thickness, and fruit size 、 MYB15-like As shown in A and B, the myb15-like mutant has leaves that are more upward, shorter, and more compact than the wild type. The mutant plant is significantly shorter and stockier. Its plant height is 21% lower than that of the wild type, while its stem diameter is 14% higher. Lowercase letters a and b indicate significant differences in plant height and stem diameter at the 5% level.

[0070] The blade angle results are as follows MYB15-like 、 5 Compared with the wild type, MYB15-like NAR QFYSSRRRYNYQVASSSWKQLVCYCSKITRKNR myb15-like Figure 1 myb15-like SlMYB15-like Figure 2 Figure 3 myb15-like Figure 4 myb15-like The mutant plant is significantly more compact. The leaf angle is 36% lower than that of the wild type. Lowercase letters a and b indicate significant differences in leaf angle at the 5% level (data from 8 wild-type and 8 mutant plants were included).

Claims

1. Application of SlMYB15-like gene inhibition in reducing tomato leaf angle; The nucleotide sequence of the SlMYB15-like gene is shown in SEQ ID NO: 1; The method for inhibiting the SlMYB15-like gene is to use the CRISPR / Cas9 system to cause an inactivation mutation in the SlMYB15-like gene; The CRISPR / Cas9 system includes a vector and sgRNA; The sequence of the sgRNA is shown in SEQ ID NO:

2.

2. Application of biomaterials related to the inhibition of SlMYB15-like genes in the development of tomato varieties with reduced leaf angles; The nucleotide sequence of the SlMYB15-like gene is shown in SEQ ID NO: 1; The biological materials include nucleic acid molecules, vectors, and cells; The nucleic acid molecule includes an sgRNA that inhibits SlMYB15-like function; The sequence of the sgRNA is shown in SEQ ID NO: 2; The vector comprises an sgRNA whose sequence is shown in SEQ ID NO: 2; The cell comprises the vector.

3. The use according to claim 2, characterized in that: The vector includes a CRISPR / Cas9 vector.

4. The use according to claim 2, characterized in that: The cells include at least one of Escherichia coli and Agrobacterium.

5. A method for cultivating a high-density tomato variety, comprising the step of reducing the expression level and / or activity of a SlMYB15-like protein in tomatoes; The tomato variety has the following characteristics: the leaf angle is smaller than the reference level; the reference level is the level of the wild type; The step of reducing the expression level and / or activity of the SlMYB15-like protein in tomatoes is to introduce the biological material related to the inhibition of the SlMYB15-like gene according to any one of claims 2 to 4 into tomato tissues or tomato cells.

6. The method according to claim 5, characterized in that: The introduction method includes at least one of using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation.

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