Application of Solyc02g060560 gene in regulation and control of sugar content and size of tomato fruits

Abstract

The invention discloses an application of a Solyc02g060560 gene in regulation and control of sugar content and size of tomato fruits, wherein a CDS (Coding Sequence) sequence of the Solyc02g060560 gene is as shown in SEQ ID NO. 1. A tomato transcription factor mutant library is constructed through a CRISPR (clustered regularly interspaced short palindromic repeats) technology, and a mutant TOM6-126 with unknown functions at present in a C2H2 family is screened. Based on observation of the phenotype of the mutant, the inventor determines that the gene is related to the sugar content and the fruit size of tomato fruits. Compared with a wild type, the soluble solid content (brix) of tomato fruits of the mutant is increased, and the fruits become larger. It is indicated that the Solyc02g060560 gene plays an important role in negative regulation of tomato fruit growth and development and sugar content. According to the invention, a good gene genetic resource foundation is laid for the cultivation of a new variety of tomatoes with high sugar content.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of the Solyc02g060560 gene in regulating the sugar content and fruit size of tomato fruits. Background Art

[0002] Tomato (Solanum lycopersicum) is one of the most important vegetable crops globally, and optimizing its quality is a core challenge in modern agricultural breeding. In recent years, although modern breeding techniques have significantly improved tomato yield and stress tolerance, commercial varieties generally suffer from a decline in flavor quality, manifested by an imbalance in the sugar-acid ratio, a reduction in aromatic compounds, and deteriorated texture. This phenomenon is primarily due to the directional selection pressure for high-yield and resistance traits during breeding, which has led to the loss of some flavor-related gene loci. Furthermore, postharvest processing (such as early harvesting and low-temperature storage and transportation), while extending shelf life, further inhibits the accumulation of flavor compounds, exacerbating quality degradation. Notably, genetic trade-offs often exist between yield and quality traits. For example, alleles that increase fruit size may reduce sugar content, while high sugar accumulation may inhibit fruit set. Therefore, identifying the common molecular hubs regulating yield and quality is of great significance for tomato genetic improvement.

[0003] Transcription factors (TFs), as core components of gene expression regulation, play a pivotal role in the complex regulatory network that coordinates tomato yield and flavor quality. Research has shown that multiple TF families, including MYB, NAC, WRKY, and MADS-box, play a key role in the coordinated regulation of yield-related traits (such as fruit size) and quality characteristics (such as sugar-acid balance, accumulation of volatile aromatic compounds, and fruit texture) by integrating and regulating sugar metabolism and carbon allocation pathways, hormone signaling networks, and cell expansion and differentiation. These TFs establish sophisticated spatiotemporal expression regulatory networks that dynamically balance physiological processes at different developmental stages, providing important molecular regulatory targets for overcoming the yield-quality trade-off in traditional breeding. To address this yield-quality trade-off, this study integrated multi-omics analyses (transcriptome and metabolomics) with CRISPR-Cas9 gene editing technology to systematically screen for key transcription factors that maintain high yield and enhance flavor. Furthermore, bioengineering was used to create new high-yield, high-quality tomato germplasm. Summary of the Invention

[0004] One of the objectives of the present invention is to provide the use of the Solyc02g060560 gene in regulating the sugar content and size of tomatoes.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention has found a tomato Solyc02g060560 gene, which is as follows:

[0007] The gene sequence number (Sequence ID) of the Solyc02g060560 gene in Phytozome13 (https: / / phytozome-nextjgi.doe.gov / ) is Solyc02g060560.1, the CDS sequence is shown in SEQ ID NO.1, and the length is 594 bp. The amino acid sequence is shown in SEQ ID NO.2, and includes 197 amino acids.

[0008] Through preliminary research on the tomato gene Solyc02g060560, the inventors discovered that the gene has a certain impact on tomato fruit sugar content and fruit size. The gene is negatively correlated with sugar content and fruit size. Specifically, knocking out the gene resulted in larger tomato fruits and increased soluble solids content, which in turn improved fruit sugar content and quality. This provides a genetic resource foundation for the development of new, high-sugar, high-quality tomato varieties.

[0009] The present invention also constructs a series of plant expression vectors, and the functions of the expression vectors containing the above-mentioned genes, transgenic plant lines and host cells containing the vectors in regulating the sugar content and fruit size of tomato fruits also fall within the protection scope of the present invention.

[0010] Those skilled in the art can readily mutate the Solyc02g060560 gene described herein using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence encoding the transcription factor encoded by the Solyc02g060560 gene, as long as they encode the same protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention. Primer pairs for amplifying the full-length coding sequence or fragments thereof encoding the transcription factor encoded by the Solyc02g060560 gene also fall within the scope of protection of the present invention.

[0011] The main purpose of the present invention is to protect the use of the above-mentioned Solyc02g060560 gene, the transcription factor protein encoded by the Solyc02g060560 gene, and the biological material containing the above-mentioned coding sequence in regulating the sugar content and fruit size of tomato fruits.

[0012] The improvement of the sugar content of tomato fruit is specifically manifested as follows: after the Solyc02g060560 gene in the tomato is mutated, the mutant strain has a higher soluble solid (brix) content and larger fruits than the wild type.

[0013] In order to increase the sugar content and fruit size of fruits, the present invention also protects a new breeding method for increasing the sugar content and enlarging the fruit of tomatoes, wherein the method is to obtain plants with higher fruit sugar content and size than the target plants by regulating the expression of the Solyc02g060560 gene in the target plants.

[0014] The method for regulating the expression of the Solyc02g060560 gene in the target plant is to knock out or knock down the Solyc02g060560 gene.

[0015] The target plant of the present invention is the cultivated tomato AC (Ailsa Craig).

[0016] Regulating the gene expression level includes using DNA homologous recombination technology, CRISP-mediated gene editing technology and Agrobacterium-mediated transformation system to regulate the expression of Solyc02g060560, obtain transgenic plant strains, and obtain homozygous Solyc02g060560 gene mutant plants from the offspring of Solyc02g060560 gene knockout or knockdown plants; the homozygous Solyc02g060560 gene mutant plants are plants with higher fruit sugar content.

[0017] The above-mentioned method for increasing the sugar content and size of tomato fruits is also applicable to other recipient plants that share homologous genes with tomatoes. There are no particular limitations on the recipient plants suitable for this invention, including not only tomatoes but also other plants with high homology, as long as they are suitable for gene transformation, such as various crops, flowers, or forestry plants. Examples of such plants include (but are not limited to): dicots, monocots, woody plants, plants of the Rosales order, plants of the Rosaceae family, Prunus, peaches, cruciferous plants, Arabidopsis, and Arabidopsis thaliana.

[0018] As used herein, "plant" includes the entire plant, its parent and progeny plants, and various parts of the plant, including seeds, fruits, stems, buds, leaves, roots, flowers, tissues, and organs, all of which contain the gene or nucleic acid of interest. "Plant" as used herein also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, and pollen, each of which may contain the gene / nucleic acid of interest.

[0019] The present invention encompasses any plant cell, or any plant obtained or obtainable by any of the methods herein, as well as all plant parts and propagules thereof. Transfected cells, tissues, organs, or whole plants obtained by any of the aforementioned methods are also encompassed by this patent. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods described herein have the same characteristics.

[0020] The present invention also extends to harvestable parts of the plants described above, including, but not limited to, seeds, fruit, and pericarp. It further relates to other post-harvest derivatives of the plants, such as organic acids, sugars, lycopene, tomatine, volatile substances, vitamins, minerals, and proteins. The present invention also relates to foods or food additives obtained from the plants.

[0021] Advantages of the present invention:

[0022] (1) The present invention uses molecular biology methods to innovatively screen a mutant TOM6-126 in tomato (Solanum lycopersicum). Its target transcription factor is the C2H2 family, whose function is currently unknown. Through phenotypic identification, the inventors preliminarily determined that Solyc02g060560 is involved in the regulation of plant fruit sugar content and fruit enlargement. Compared with the wild type, the Solyc02g060560 gene mutant strain has a higher soluble solids content.

[0023] (2) CRISPR / Cas9 gene editing technology can be used to obtain tomato plants with high fruit sugar content and increased fruit size. Specifically, the Solyc02g060560 gene can be knocked out or knocked down to obtain a Solyc02g060560 mutant strain. This strain has a higher soluble solids content than the target plant, providing important materials for breeding new high-yield and high-quality tomato varieties, and has important guiding significance for the genetic improvement of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a map of the target genes and mutation sites of the TOM6-126 mutant.

[0025] Figure 2 This is a comparison chart of the fruit phenotypes of TOM6-126 and AC. Scale bar = 1 cm.

[0026] Figure 3 The fruit length data of TOM6-126 mutant lines were compared with those of wild type.

[0027] Figure 4 The figure shows the fruit width of the TOM6-126 mutant compared with the wild type.

[0028] Figure 5This is the Brix content analysis of the fruits of the TOM6-126 mutant line compared with the wild type. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0030] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0032] Unless otherwise indicated, the practice of the present invention will utilize conventional botanical techniques, microbiology, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics techniques readily apparent to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods employed in the present invention for DNA extraction, phylogenetic tree construction, gene editing methods, gene editing vector construction, and gene-edited plant production, in addition to the methods employed in the following examples, can all be accomplished using methods disclosed in the existing literature.

[0033] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences in non-coding regions. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in a genomic sequence, and / or include coding sequences in a cDNA, and / or include cDNA and its regulatory sequences. In specific embodiments, such as with respect to isolated nucleic acid sequences, it is preferably assumed to be cDNA.

[0034] Unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0035] The inventors used CRISPR-Cas9 technology to construct a panel-scale mutant library of tomato transcription factors. After preliminary phenotypic screening, they discovered a mutant, TOM6-126, that increases sugar content and fruit size in tomato fruit. Its sgRNA targets the Solyc02g060560 gene. The Solyc02g060560 gene is listed in the Phytozome13 database as Solyc02g060560.1, with a CDS sequence shown in SEQ ID NO. 1 and an amino acid sequence in SEQ ID NO. 2.

[0036] Example 1 Screening the TOM6-126 mutant from the tomato mutant library

[0037] sgRNA oligonucleotides were designed and synthesized targeting the tomato transcription factor panel. Using these synthesized oligos as templates, polymerase chain reaction (PCR) amplification was performed to obtain DNA fragments containing sgRNAs encoding the target transcription factor genes. Amplified products were recovered by gel excision and purified to remove impurities. Purified DNA products were cloned into the pMR284-Crimera vector using the Golden-Gate cloning method. Ultimately, a CRISPR library encompassing the entire tomato transcription factor panel was successfully constructed. Using tissue culture techniques, the constructed CRISPR library containing tomato transcription factors was introduced into AC wild-type tomatoes. Positive seedlings were obtained through kanamycin resistance screening and PCR identification. These seedlings were then planted in a greenhouse suitable for tomato growth. At fruit maturity, all transgenic plants underwent comprehensive and systematic phenotypic characterization, including key quality indicators such as fruit size, color, shape, and soluble solids content (brix). Among the numerous mutants with significant phenotypes, the mutant designated TOM6-126 was selected for study. Compared with the AC wild-type control, the fruit volume was larger and the brix content was significantly increased, indicating that the TOM6-126 mutant may play an important role in the regulation of tomato fruit shape and sugar content.

[0038] Example 2 Determination of target genes of TOM6-126 mutants

[0039] To efficiently identify sgRNA and its target gene in TOM6-126 mutant plants, TOM6-126 mutant plants were sampled, genomic DNA was extracted using the CTAB method, and barcoding analysis was performed using this as a template. sgRNA and target gene were quickly identified by sequence alignment. The results showed that its target gene was Solyc02g060560 ( Figure 1 ).

[0040] Example 3 Screening of Solyc02g060560 gene mutant plants

[0041] The leaves of mutant tomato plants were taken, and genomic DNA was extracted by CTAB method. The genomic DNA was amplified by PCR using primers consisting of primers 0560-F and 0560-R. The PCR amplification products were then recovered and sequenced. The mutant plants with edited Solyc02g060560 gene were screened according to the sequencing results ( Figure 1 ).

[0042] 0560-F: 5'-CACCAGTATCAGCCCCAG-3';

[0043] 0560-R: 5'-TCCACATCATCCTCGTCATC-3'.

[0044] Example 4 Identification of Fruit Phenotypes of Tomato Solyc02g060560 Mutant Strains

[0045] The phenotypic characteristics of Solyc02g060560 mutant strains and wild-type plants were recorded by photographing ( Figure 2 ) and identification, the present invention mainly reflects the sugar content of tomatoes by the soluble solids (brix) content of tomato fruits. The higher the soluble solids (brix) content, the higher the sugar content and the better the quality. After being cultured under suitable conditions for the same number of days, the brix content of the Solyc02g060560 gene mutant strain was significantly higher than that of the wild type fruit ( Figure 5 ), and the fruit length and width increased ( Figure 3 and Figure 4 ), manifested as an increase in volume.

[0046] These results show that under suitable conditions, the fruits of the Solyc02g060560 mutant strain have higher sugar content and larger fruit size than those of wild-type plants in the same period of time, indicating that the Solyc02g060560 gene can regulate the sugar content and fruit size of tomatoes.

[0047] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

[0048] Wherein, the sequence of SEQ ID NO.1 is as follows:

[0049]

[0050]

[0051] Among them, the sequence of SEQ ID NO.2 is as follows:

[0052]

Claims

1. Application of the Solyc02g060560 gene in regulating the sugar content and size of tomato fruits, characterized in that: The CDS sequence of the Solyc02g060560 gene is shown in SEQ ID NO.

1. The regulation is manifested as follows: tomato fruits after the Solyc02g060560 gene is knocked out or knocked down are larger in volume and have a higher soluble solid content than wild-type fruits.

2. Use of a recombinant gene vector containing the Solyc02g060560 gene according to claim 1 in regulating the sugar content of tomato fruit.

3. The use according to claim 2, characterized in that The recombinant vectors include gene editing recombinant vectors and overexpression recombinant vectors.

4. A breeding method for increasing sugar content and enlarging tomato fruit, characterized in that: Tomato plants with increased fruit sugar content and volume are obtained by reducing the expression of the Solyc02g060560 gene in tomatoes to disrupt the function of the gene. The CDS sequence of the Solyc02g060560 gene is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that The method of reducing the expression of the Solyc02g060560 gene in tomatoes is to knock out the gene or knock down its expression through gene editing technology.

6. The method for increasing sugar content and enlarging tomato fruit according to claim 5, characterized in that: The Solyc02g060560 gene in the plant genome is knocked out or knocked down to obtain Solyc02g060560 gene knockout or knockdown plants, and homozygous Solyc02g060560 knockout or knockdown plants are obtained from the self-pollinated progeny of the Solyc02g060560 knockout or knockdown plants; the homozygous Solyc02g060560 knockout or knockdown plants are plants with larger fruits and higher sugar content.

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