Application of HD-ZIP transcription factor in regulating tomato fruit quality

Abstract

The application discloses application of HD-ZIP transcription factors in quality regulation of tomatoes, and the protein has functional redundancy, and the functions of the protein are jointly regulated by two genes, wherein a CDS sequence of the gene Solyc03g034130.2 is shown as SEQ ID NO. 1, and a CDS sequence of the gene Solyc03g034110.2 is shown as SEQ ID NO. 2. A tomato transcription factor mutant library is constructed through CRISPR technology, and a mutant Tom6-155 which simultaneously inhibits expression of two homologous genes of the HD-ZIP transcription factor is screened. Based on observation of a phenotype of the mutant, the inventors determine that the HD-ZIP transcription factor is related to tomato fruit quality. Compared with a wild type, the brix content of the tomato fruit of the mutant is increased. It is indicated that Solyc03g034130.2 and Solyc03g034110.2 play important negative regulation roles in a tomato fruit quality formation process. The application lays a good genetic resource foundation for cultivation of a new tomato variety with high quality.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of HD-ZIP transcription factors in regulating tomato fruit quality. Background Technology

[0002] In recent years, as breeding goals have shifted towards traits such as high yield and stress resistance, modern cultivated tomatoes ( Solanum lycopersicum While tomato varieties have shown significant improvements in yield and resistance, they generally suffer from a decline in flavor and quality. Numerous studies have indicated that this phenomenon manifests primarily as a systemic deterioration in key quality indicators such as an imbalance in the sugar-acid ratio, a significant reduction in the content of characteristic aroma compounds, and changes in fruit texture. In-depth analysis reveals that this quality degradation is closely related to the disruption of the transcription factor regulatory network during fruit development and ripening. Specifically, members of multiple transcription factor families, including MYB, NAC, WRKY, and MADS-box, constitute a complex molecular network influencing fruit quality formation through precise regulation of key pathways such as sugar metabolism, organic acid synthesis, and volatile substance accumulation. However, a systematic understanding of the key transcriptional regulatory mechanisms in tomato quality formation remains lacking, particularly regarding the discovery of novel transcription factors and the analysis of their regulatory networks. To systematically improve tomato quality traits, this study identified new key transcription factors using modern molecular experimental techniques and elucidated their regulatory mechanisms on important quality traits such as sugar accumulation and aroma synthesis. This provides new theoretical basis and technical support for achieving precise improvement of tomato quality through molecular design breeding. Summary of the Invention

[0003] The purpose of this invention is to provide the application of HD-ZIP transcription factor in regulating tomato fruit quality.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows:

[0005] This application constructs a tomato transcription factor mutant library using CRISPR technology and screens for a mutant that simultaneously inhibits the expression of two homologous genes of the HD-ZIP transcription factor. Tom6-155 Based on observations of the mutant phenotype, the inventors determined that the HD-ZIP transcription factor is associated with tomato fruit quality. This protein exhibits functional redundancy, with its function jointly regulated by two genes, one of which is… Solyc03g034130.2 The CDS sequence is shown in SEQ ID NO.1, gene. Solyc03g034110.2 The CDS sequence is shown in SEQ ID NO.2. The corresponding amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0006] in, Solyc03g034130.2The gene's sequence ID in NCBI is LOC101251158, and its CDS sequence length is 675 bp, including 224 amino acids.

[0007] Solyc03g034110.2 The gene's sequence ID in NCBI is LOC101254766, and its CDS sequence length is 642 bp, containing 213 amino acids.

[0008] The DNAMAN software was used to perform sequence alignment of the two genes and their corresponding amino acids. The results showed that the CDS sequence similarity was 91.26% and the amino acid sequence similarity was 87.5%.

[0009] Through the study of tomatoes Solyc03g034130.2 and Solyc03g034110.2 Preliminary research on genes led the inventor to discover... Solyc03g034130.2 and Solyc03g034110.2 It has a certain impact on the quality of tomato fruit, the tomato Solyc03g034130.2 and Solyc03g034110.2 Genes are negatively correlated with tomato fruit quality; that is, gene knockout or knockdown increases the soluble solids content of tomato fruits, thus improving fruit quality. This lays a certain genetic resource foundation for the breeding of high-quality new tomato varieties.

[0010] The present invention also constructs a series of plant expression vectors, and the functions of expression vectors containing the above-mentioned genes, transgenic plant lines, and host cells containing the vectors in regulating tomato fruit quality also fall within the protection scope of the present invention.

[0011] The recombinant vectors include gene editing recombinant vectors and overexpression recombinant vectors.

[0012] Those skilled in the art can readily employ known methods, such as directed evolution and point mutation, to modify the present invention. Solyc03g034130.2 and Solyc03g034110.2 Genes undergo mutation. Those that have been artificially modified and possess the aforementioned genes... Solyc03g034130.2 and Solyc03g034110.2 Nucleotides with 75% or higher nucleotide sequence identity, as long as they encode the same protein and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention. Amplification of the... Solyc03g034130.2 and Solyc03g034110.2 Primer pairs for the full-length coding sequence or a fragment thereof of a gene encoding a transcription factor are also within the scope of protection of this invention.

[0013] The primary objective of this invention is to protect the aforementioned genes. Solyc03g034130.2 and Solyc03g034110.2 , Solyc03g034130.2 andSolyc03g034110.2 The application of the gene-encoded transcription factors and biological materials containing the above-mentioned coding sequences in improving the quality of tomato fruits.

[0014] The regulation of tomato quality is manifested as follows: when the... Solyc03g034130.2 Genes and Solyc03g034110.2 When genes are knocked out or knocked down simultaneously, the soluble solids content in tomatoes from the knockout or knockdown lines is significantly higher than that of wild types.

[0015] To improve fruit quality, this invention also protects a novel breeding method for improving tomato fruit quality by reducing the concentration of certain nutrients in tomato plants. Solyc03g034130.2 and Solyc03g034110.2 The expression of the gene is then disrupted to obtain tomato plants with improved fruit quality, which is manifested in an increase in soluble solids (brix) content.

[0016] Among them, reducing the content of tomatoes Solyc03g034130.2 and Solyc03g034110.2 The gene is expressed by knocking out or downexpressing the gene using gene editing technology. The target plant of this invention is the cultivated tomato AC ( Ailsa Craig ).

[0017] Regulating gene expression levels includes using DNA homologous recombination technology, CRISP-mediated gene editing technology, and Agrobacterium-mediated transformation systems. Solyc03g034130.2 and Solyc03g034110.2 Expression, obtaining transgenic plant lines, from Solyc03g034130.2 and Solyc03g034110.2 Homozygous individuals are obtained in the offspring of gene knockout or knockdown plants. Solyc03g034130.2 and Solyc03g034110.2 Mutant plants; homozygous Solyc03g034130.2 and Solyc03g034110.2 Mutant plants are those with higher fruit quality.

[0018] The above-described method for improving tomato fruit quality 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; they include not only tomatoes but also other plants with high homology, as long as they are suitable for gene transformation operations, such as various crops, flowering plants, or forestry plants. The plants mentioned can be, for example (but not limited to): dicotyledons, monocotyledons, woody plants, Rosales plants, Rosaceae plants, Prunus genus, Prunus, Brassicaceae plants, Arabidopsis genus, Arabidopsis, etc.

[0019] The term "plant" as used in this invention includes the whole plant, its parent and offspring plants, and different parts of the plant, including seeds, fruits, stems, buds, leaves, roots, flowers, tissues, and organs, all of which contain our target gene or nucleic acid. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, and pollen; similarly, each of these objects contains the target gene / nucleic acid.

[0020] This invention includes any plant cell, or any plant obtained or obtainable by the methods described herein, as well as all plant parts and their propagules. This invention also includes transfected cells, tissues, organs, or whole plants obtained by any of the foregoing methods. The only requirement is that the offspring exhibit the same genotype or phenotypic characteristics, and that offspring obtained using the methods of this invention have identical characteristics.

[0021] This invention also extends to the harvestable parts of the plants as described above, but is not limited to seeds, fruits, and peels. It further relates to other derivatives of the plant after harvest, such as organic acids, sugars, lycopene, tomatine, volatile substances, vitamins, minerals, and proteins. This invention also relates to foods or food additives obtained from the relevant plants.

[0022] Advantages of this invention:

[0023] (1) This invention uses molecular biology methods to innovatively study the effects of molecular biology on tomatoes ( Solanum lycopersicum A mutant was selected from the sample. Tom6-155 Its target Solyc03g034130.2 and Solyc03g034110.2 The gene belongs to the HD-ZIP family of transcription factors. Through phenotypic identification, the inventors have preliminarily determined... Solyc03g034130.2 and Solyc03g034110.2 It participates in the regulation of plant fruit quality. Further testing revealed that, compared to the wild type, Solyc03g034130.2 and Solyc03g034110.2 The mutant strains have higher soluble solids content.

[0024] (2) In practical applications, CRISPR / Cas9 gene editing technology can be used to obtain plants with high-quality fruits. Specifically, it can be achieved by... Solyc03g034130.2 and Solyc03g034110.2 Gene knockout or knockdown results in Solyc03g034130.2 and Solyc03g034110.2 The mutant strain has a higher soluble solids content than the target plant, providing important material for breeding new high-yield and high-quality tomato varieties, and also has important guiding significance for the genetic improvement of other crops. Attached Figure Description

[0025] Figure 1 yes Solyc03g034130.2 and Solyc03g034110.2CDS sequence alignment diagram of the gene;

[0026] Figure 2 yes Solyc03g034130.2 and Solyc03g034110.2 Image showing the alignment of the amino acid sequence encoded by the gene.

[0027] Figure 3 yes Tom6-155 Diagram of the target genes and mutation sites of the mutant.

[0028] Figure 4 yes Tom6-155 Fruit phenotype comparison with AC, scale bar = 1 cm.

[0029] Figure 5 yes Tom6-155 Analysis of soluble solids (brix) content in fruits of mutant and wild type. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0031] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0034] Unless otherwise stated, the implementation of this invention will utilize conventional botanical techniques, microbiological techniques, tissue culture techniques, molecular biology techniques, chemical techniques, biochemical techniques, DNA recombination techniques, and bioinformatics techniques that are readily apparent to those skilled in the art. These techniques have been fully explained in published literature. Furthermore, the methods employed in this invention, including DNA extraction, phylogenetic tree construction, gene editing methods, gene editing vector construction, and the acquisition of gene-edited plants, except for those used in the examples below, can all be implemented using methods already disclosed in existing literature.

[0035] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “nucleotide,” “nucleic acid molecule,” or “polynucleotide” mean, but are not limited to, isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), naturally occurring, mutant, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, and 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 of non-coding regions. These terms include a gene. “Gene” or “gene sequence” is broadly used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include coding sequences in cDNA, and / or include cDNA and its regulatory sequences. In particular embodiments, such as concerning isolated nucleic acid sequences, cDNA is preferred by default.

[0036] 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.

[0037] The inventors of this invention constructed a tomato transcriptome-scale mutant library using CRISPR-Cas9 technology. After preliminary phenotypic screening, they discovered a mutant that regulates tomato fruit quality. Tom6-155 Its SgRNA target Solyc03g034130.2 and Solyc03g034110.2 These two genes. Solyc03g034130.2 The gene sequence ID in the NCBI database is LOC101251158, the CDS sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.3. Solyc03g034110.2 The gene sequence ID in NCBI is LOC101254766, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.4.

[0038] Soluble solids content reflects the ripeness of tomato fruit and is closely related to its flavor quality and nutritional value, making it an important indicator of tomato fruit quality. It includes water-soluble compounds such as sugars (monosaccharides and disaccharides), organic acids, vitamins, and minerals. Therefore, in this study, soluble solids content was used as a primary indicator for comparing tomato quality; a higher soluble solids content indicates better quality.

[0039] Example 1: Screening for mutant strains from a tomato mutant library Tom6-155 mutant

[0040] sgRNA oligonucleotides were designed and synthesized targeting the tomato transcription factor genome. Each sgRNA targets at least two homologous genes to achieve simultaneous knockout of multiple redundant genes. Using the synthesized oligos as templates, DNA fragments containing sgRNAs encoding the target transcription factor genes were amplified by polymerase chain reaction (PCR). The amplified products were gel-cleaved and purified to remove impurities. Using the Golden-gate cloning method, the purified DNA products were cloned into the pMR284-Crimera vector. A CRISPR library of tomato transcription factors at the transcription factor genome scale was successfully constructed. The constructed CRISPR library containing tomato transcription factors was introduced into AC wild-type tomato using tissue culture technology. Positive seedlings were obtained through kanamycin resistance screening and PCR identification. These seedlings were then transplanted into greenhouses with conditions suitable for tomato growth. During the fruit ripening stage, all transgenic plants underwent comprehensive and systematic phenotypic identification, including key quality indicators such as fruit size, color, shape, and soluble solids content (brix). Among the many mutants with significant phenotypes, the mutant numbered [number missing] was selected. Tom6- 155 The mutant was used as the research subject. Compared with the AC control, its fruit brix content was significantly increased, indicating that... Tom6-155 The mutant may play an important role in the regulation of tomato fruit quality.

[0041] Example 2 Tom6-155 Identification of target genes in mutants

[0042] For efficient identification Tom6-155 The sgRNA and its target genes in mutant plants have the effect on Tom6-155 Mutant plants were sampled, and genomic DNA was extracted using the CTAB method. This DNA was then used as a template for barcoding analysis. sgRNA and target genes were rapidly identified through sequence alignment. The results showed that the target gene was... Solyc03g034130.2 and Solyc03g034110.2It is available in Phytozome 13 (https: / / phytozome-next.jgi.doe.gov / info / Slycopersicum_ITAG2_4). Solyc03g034130.2 and Solyc03g034110.2 The CDS and amino acid sequences of the two genes were further aligned using DNAMAN software, and the results showed that the CDS sequence similarity was 91.26%. Figure 1 The amino acid sequence similarity was 87.5%. Figure 2 This demonstrates that there may be functional redundancy when these two genes are functioning, and that knocking out these genes results in a phenotype with superior fruit quality compared to the wild type.

[0043] Example 3 Screening Solyc03g034130.2 and Solyc03g034110.2 Mutant plants

[0044] Leaves of mutant tomato plants were collected, and genomic DNA was extracted using the CTAB method, employing primers 4130-F and 4130. - PCR amplification was performed using primers consisting of R and primers 4110-F and 4110-R. The PCR amplification products were then recovered and sequenced, and screening was performed based on the sequencing results. Solyc03g034130.2 and Solyc03g034110.2 Mutant plants with gene editing ( Figure 3 The primers are as follows:

[0045] 4130-F: 5'-CTTCAATTTCATTACATCAAGAGA-3';

[0046] 4130-R: 5'-ATAGTGTCACATCAAGAAGGA-3'.

[0047] 4110-F: 5'-GTATTCCTTTCTTCACTTTCTAG-3';

[0048] 4110-R: 5' - CATCAAGGGCCACATAG -3'.

[0049] Example 4 Tomato Solyc03g034130.2 and Solyc03g034110.2 Phenotypic identification of fruit from gene mutant strains

[0050] right Solyc03g034130.2 / Solyc03g034110.2 Phenotypic photographs were taken of gene mutant lines and wild-type plants. Figure 4 The present invention mainly uses the Brix content of soluble solids in tomato fruit for identification and determination. Figure 5The quality of tomatoes is reflected by their Brix content; the higher the Brix content, the better the quality. For example... Figure 5 As shown, after culturing for the same number of days under suitable conditions, Solyc03g034130.2 / Solyc03g034110.2 The soluble solids content (brix) of the mutant strain was significantly higher than that of the wild-type fruit.

[0051] These results indicate that, under suitable conditions, within the same time period, Solyc03g034130.2 / Solyc03g034110.2 The fruits of the mutant strain had a higher soluble solids (Brix) content than those of the wild-type plant. This indicates that... Solyc03g034130.2 / Solyc03g034110.2 Genes can regulate the fruit quality of tomatoes.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. The application of HD-ZIP transcription factor in regulating tomato quality, characterized in that, The amino acid sequences of the HD-ZIP transcription factors are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and the encoding genes are respectively Solyc03g034130.2 Genes and Solyc03g034110.2 Genes, the ones mentioned Solyc03g034130.2 The CDS sequence of the gene is shown in SEQ ID NO.

1. Solyc03g034110.2 The CDS sequence of the gene is shown in SEQ ID NO.2; the regulation of tomato quality is manifested as follows: when the... Solyc03g034130.2 Genes and Solyc03g034110.2 When the gene was knocked out, the soluble solids content in the knockout tomato strain increased.

2. A breeding method for improving the quality of tomato fruits, characterized in that, By simultaneously knocking out the tomato plants Solyc03g034130.2 Genes and Solyc03g034110.2 Genes were used to obtain plants with improved tomato fruit quality. Solyc03g034130.2 The CDS sequence of the gene is shown in SEQ ID NO.

1. Solyc03g034110.2 The CDS sequence of the gene is shown in SEQ ID NO.2; the improvement in tomato fruit quality is manifested by an increase in soluble solids content.

3. The breeding method for improving tomato fruit quality according to claim 2, characterized in that, In the tomato genome Solyc03g034130.2 Genes and Solyc03g034110.2 Gene knockout yields Solyc03g034130.2 Genes and Solyc03g034110.2 Gene knockout plants, from Solyc03g034130.2 Genes and Solyc03g034110.2 Homozygous individuals were obtained from the self-pollination offspring of gene knockout plants. Solyc03g034130.2 Genes and Solyc03g034110.2 Gene knockout plants.

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