Method for reducing included angle of tomato leaves by gene editing

Abstract

The invention belongs to the technical field of modern agriculture, and particularly relates to a method for reducing included angles of tomato leaves by using gene editing. The invention discloses an application of an SlMYB15-like gene in regulation and control of a tomato plant type, an SlMYB15-like gene knockout mutant is constructed by utilizing a CRISPR / Cas9 gene editing technology, and the gene is found to have a remarkable influence on regulation and control of the tomato plant type. A series of observation statistics show that the stem diameter of the SlMYB15-like gene knockout mutant is obviously higher than that of wild type tomatoes, and the plant height and the leaf included angle are obviously lower than that of the wild type tomatoes, so that the tomatoes obtain a dwarf, strong and compact plant type. The research provides an important basis for tomato close planting variety breeding and tomato plant type regulation and control.

Description

Technical Field

[0001] The 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 ) is one of the important economic crops widely planted around the world. Studies have shown that increasing planting density is one of the effective strategies to increase crop yield per unit area (Lauer, S., Hall, BD, Mulaosmanovic, E., et al. 2012. Morphological changes in parental lines ofpioneer brand maize hybrids in the US central corn belt. Crop Sci. 52, 1033–1043.). However, high-density planting may cause leaves to overlap with each other, reduce interplant light transmittance, trigger plant shade avoidance response, lead to plant leggy and weak plants and increase lodging rate, and ultimately cause loss of biomass and crop yield (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, it is very important to find a germplasm resource with upward-thrusting leaves and compact plant shape.

[0003] Leaf angle is a key trait that affects plant type. Upright leaves can better capture light energy in the canopy under high-density planting, thereby improving photosynthesis and stress resistance, promoting ventilation and light transmission, and ultimately increasing crop yield. Interference with the rice OsDWARF4 gene led to a partial defect in brassinosteroid biosynthesis, which significantly reduced the leaf angle and produced 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 thus increased leaf angle (Tian, ​​J., Wang, C., Xia, J., et al. 2019. Teosinte ligule allele narrows plantarchitecture and enhances high-density maize yields. Science, 365, 658-664.). R2R3 MYB transcription factors have also recently been reported to be involved in the regulation of leaf angle. Rice OsFLP mediates brassinosteroid signaling to cause lignin deposition at leaf junctions. OsFLP mutants exhibit larger leaf angles and loose plant types (Liu, HC., Zhang, J., Wang, JX., et al. 2024. The rice R2R3 MYB transcription factorFOUR LIPS connects brassinosteroid signaling to lignin deposition and leafangle. Plant Cell, 36, 4768-4785.).

[0004] At present, the research on leaf angle is mainly focused on crops such as rice and corn, while there are few reports on tomatoes. The formation and regulation mechanism of tomato leaf angle is still unclear, so it is necessary to invent a method to reduce the 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 Use of genetically related biological material 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: In a first aspect of the present invention, there is provided an inhibition SlMYB15-like Application of genes to reduce leaf angle in tomato.

[0009] In some embodiments of the present invention, the SlMYB15-like The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0010] 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.

[0011] In some embodiments of the invention, the inhibition SlMYB15-like Gene knockout SlMYB15-like Gene, knockdown SlMYB15-like Gene, or SlMYB15-like Methods for inactivating mutations to occur.

[0012] A second aspect of the present invention provides and inhibits SlMYB15-like Use of genetically related biological material in breeding tomato varieties.

[0013] In some embodiments of the present invention, the SlMYB15-like The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0014] 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.

[0015] In some embodiments of the present invention, the biological material includes nucleic acid molecules, vectors, and cells.

[0016] 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.

[0017] In some embodiments of the present invention, the sequence of the sgRNA is shown in SEQ ID NO: 2.

[0018] In some embodiments of the present invention, sgRNA is used in conjunction with CRISPR / Cas9 vector to achieve the purpose of gene knockout. In some embodiments of the present invention, the CRISPR / Cas9 vector also 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.

[0019] In some embodiments of the present invention, the carrier includes but is not limited to pHEE401 and other common carriers in the art.

[0020] In some embodiments of the present invention, the cell includes 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.

[0021] The 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.

[0022] 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 varieties of tomatoes.

[0023] 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.

[0024] 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 described in the second aspect of the present invention SlMYB15-like The genetically related biological material is introduced into tomato tissue or tomato cells.

[0025] 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.

[0026] In some embodiments of the present invention, the step of reducing the expression level and / or activity of SlMYB15-like protein in tomatoes is specifically: (1) Design SlMYB15-like Gene target sequence sgRNA, construct tomato SlMYB15-like CRISPR / Cas9 vectors for gene editing.

[0027] (2) The CRISPR / Cas9 vector described in step (1) is transferred into Agrobacterium competent cells to obtain Agrobacterium containing the CRISPR / Cas9 vector.

[0028] (3) Infecting the cotyledons of common wild-type tomatoes with the Agrobacterium infection solution obtained in step (3), obtaining seedlings again through tissue culture, and screening tomatoes SlMYB15-like A mutant strain with a gene mutation, no exogenous Cas9 protein, and a stable inheritance of the target sequence.

[0029] In some embodiments of the present invention, the carrier is pHEE401.

[0030] In some embodiments of the present invention, the host cell is Agrobacterium GV3101.

[0031] In some embodiments of the present invention, the tomato variety is Condine Red (CR).

[0032] The beneficial effects of the present invention are: The present invention finds SlMYB15-like Genes play a significant role in regulating leaf angle in tomatoes. CRISPR / Cas9 gene editing technology was used to knock out tomato genes SlMYB15-like , and obtained the 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

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of the detection of tomato plants with SlMYB15-like gene knockout in Example 1; WT, wild type; myb15-like, mutant.

[0034] Figure 2 for myb15-like Growth phenotype results of mutants versus wild-type tomato plants.

[0035] Figure 3 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 myb15-like Plant height statistics of mutant and wild-type tomato plants.

[0036] Figure 4 for myb15-like Phenotypic consequences of leaf angle in mutant and wild-type tomato plants.

[0037] Figure 5 for myb15-like Statistical results of leaf angles of mutant and wild-type tomato plants. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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.

[0039] Example 1 Obtaining SlMYB15-like gene knockout plants 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) MYB15-like The two sgRNA sequences of the gene were amplified by PCR instrument using Y0014472-1 plasmid as template for the two target PCR fragments.

[0040] MYB15-like The CDS sequence of the gene is as follows: TTATGCTATATCAGCTCCAAGTTCCAAGTGAAAAATCAAAACAAAGTATAGAGAGAGAAAAAATAAAATGGGGAGATCTCCTTGCTGTGAGAAATTGGGGTTGAAAAGAGGTCCATGGAGCAAAGAAGAAGATTATTTACTCATCAATTACATTAAAAAGAATGGTCATCCTAATTGGCGTGCACTTCCAAAACTTGCAGGTCTATTAAGGTGCGGAAAAAGTTGTAGGCTTCGATGGACTAATTACTTGAGACCTGATATTAAGCGAGGCAATTTTACTCATCAAGAAGAAGATACAATTATCAAGTTGCATCAAGTTCTTGGAAACAGTTGGTCTGCTATTGCAGCAAGATTACCCGGAAGAACAGATAACGAAATAAAAAACATTTGGCATACTCGTCTGAAGAAAAAAAGGAATGAATCTCAACTTAAAGAAACCCAATCGGAGCCTGAAAATACTAATGTAGATGTACATTTGGAGGAGGCCAACAATTCTAATGATAAACATTCCGAAATATCGAATCTTAAAATAAACATCGAAATCCAACAACAACCAAGTCCATCATCGTCAGTATCATCATCAAGTGAAGATTCATGTTCAAATACAACTGCAACGAGTTCAGAGTCGAGAAATCAAATAATGTCCGATAATTTGTTAGAAATTGATGACGATATTTGGTCCGAGGTAGTATGGGCACAAGTCGATGACAATTATGTTGATTTGTCATTAATGGAGGATAATTACCACATTAATTCTAGCTTTGATGATAATTGGTTTTGGGATGATCTTTTTACAAGATCTAATGAGTTGATGTTAGAATTGCCTGAATTATGAGC (SEQ ID NO: 1).

[0041] The sequence of sgRNA1 is: TTGAGACCTGATATTAAGCG (SEQ ID NO: 2).

[0042] The sequence of sgRNA2 is: GCTATTGCAGCAAGATTACC (SEQ ID NO: 3).

[0043] The normal protein sequence information of MYB15-like is as follows: MGRSPCCEKLGLKRGPWSKEEDYLLINYIKKNGHPNWRALPKLAGLLRCGKSCRLRWTNYLRPDIKRGNFTHQEEDTIIKLHQVLGNSWSAIAARLPGRTDNEIKNIWHTRLKKKRNESQLKETQSEPEN TNVDVHLEEANNSNDKHSEISNLKINIEIQQQPSPSSSVSSSSEDSCSNTTATSSESRNQIMSDNLLEIDDDIWSEVVWAQVDDNYVDLSLMEDNYHINSSFDDNWFWDDLFTRSNELMLELPEL* (SEQ ID NO: 4).

[0044] The sequence information of MYB15-like mutant protein is as follows: MGRSPCCEKLGLKRGPWSKEEDYLLINYIKKNGHPNWRALPKLAGLLRCGKSCRLRWTNYLRPDI NAR QFYSSRRRYNYQVASSSWKQLVCYCSKITRKNR * (SEQ ID NO: 5, underline indicates different sequences).

[0045] The Y0014472-1 plasmid sequence information is as follows:

[0046] The two purified PCR products were ligated into pHEE401 plasmid using Golden Gate Assembly Kit (BsaI-HFv2) (NEB, E1601).

[0047] After successful sequencing verification, the pHEE401-MYB15-like vector was electroporated into Agrobacterium GV3101 for genetic transformation of tomato. The cotyledons of the wild-type tomato variety CR (Condine Red) were infected with Agrobacterium, and the cotyledons were then cultured into complete transgenic tomato plants through plant tissue culture. Subsequently, primers were designed upstream and downstream according to the target location. The primers were cri-MYB15-like-F (CTCCTTGCTGTGAGAAATTGGG, SEQ ID NO: 7) and cri-MYB15-like-R (ACTGACGATGATGGACTTGGTT, SEQ ID NO: 8).

[0048] After obtaining transgenic plants, extract T0 leaf DNA, use PCR technology to amplify the genomic DNA fragment of about 400bp including sgRNA, and send it to the company for sequencing. Use Snapgene software to compare the sequencing results with the original genome sequence, screen out mutant plants, and then obtain homozygous plants through self-pollination. myb15-like The mutants were used in subsequent experiments. The results are as follows Figure 1 As shown. Compared with the common tomato that has not been gene-edited, the gene-edited mutant has a base insertion at the position of the sgRNA. The common tomato that has not been gene-edited is referred to as the control. myb15-like The mutant has a T inserted compared to the control.

[0049] Example 2 SlMYB15-like Study on Gene Regulation of Tomato Plant Type 1. Experimental methods 1) Tomato material cultivation method Tomato seeds (Condine Red, including mutants and wild type) were soaked in 50°C warm water for 15 minutes and then placed in a 28°C constant speed shaker (200 rpm / min) for germination for 3 days, with water changed every 12 hours. When the radicle of the seeds was about 1 cm long, they were sown in a 72-hole tray with a substrate of a 3:1 mixture of peat and vermiculite. The tray was placed in a plant factory with a temperature 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 -1When the tomato seedlings grow a true leaf, transplant them into planting pots and water them with an appropriate amount of Hoagland nutrient solution every 3 days.

[0050] 2) Method for measuring tomato plant height and stem diameter When the tomatoes grow to 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.

[0051] 3) Method for measuring tomato leaf angle 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.

[0052] 2. Experimental results Plant height, stem thickness, and fruit Figure 2 , Figure 3 As shown in A and B. The myb15-like mutant has leaves that are more protruding, shorter, and more compact than the wild type. myb15-like The mutant plant type is obviously shorter and stronger. The plant height is 21% lower than that of the wild type, and the stem diameter is 14% higher than that of the wild type. Lowercase letters a and b represent significant differences in plant height and stem diameter at the 5% level.

[0053] The blade angle results are as follows Figure 4 , 5 Compared with the wild type, 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 represent significant differences in the leaf angle values ​​of different plants at the 5% level (8 plants of the wild type and mutant were selected for data statistics).

Claims

1. Inhibition SlMYB15-like The application of genes in reducing the angle of tomato leaves; Said SlMYB15-like The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The inhibition SlMYB15-like Gene knockout SlMYB15-like Gene, knockdown SlMYB15-like Gene, or SlMYB15-like Methods for inactivating mutations to occur.

3. With inhibition SlMYB15-like The use of genetically related biological materials in breeding tomato varieties; Said SlMYB15-like The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The application is to construct and inhibit SlMYB15-like Genetically related biological materials are used to obtain tomato varieties with small leaf angles.

4. The use according to claim 3, characterized in that: The biological materials include nucleic acid molecules, vectors, and cells.

5. The use according to claim 4, characterized in that: The nucleic acid molecule comprises an inhibitory SlMYB15-like Functional microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotides.

6. The use according to claim 5, characterized in that: The sequence of the sgRNA is shown in SEQ ID NO:

2.

7. The use according to claim 4, characterized in that: The vector includes a CRISPR / Cas9 vector.

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

9. A method for cultivating high density planting varieties of tomatoes, comprising reducing SlMYB15-like The step of measuring the expression level and / or activity of the protein; 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; The reduction in tomato SlMYB15-like The step of increasing the expression level and / or activity of the protein is to inhibit the expression level and / or activity of the protein according to any one of claims 3 to 8. SlMYB15-like The genetically related biological material is introduced into tomato tissue or tomato cells.

10. The method according to claim 9, 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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