Application of HD-ZIP transcription factor in regulation and control of tomato fruit quality

Abstract

The invention discloses an application of an HD-ZIP transcription factor in regulation of tomato quality, the protein has functional redundancy, the function of the protein is jointly regulated by two genes, the CDS sequence of the gene Solyc03g034130.2 is as shown in SEQ ID NO.1, and the CDS sequence of the gene Solyc03g034110.2 is as shown in SEQ ID NO.2. The invention also discloses an application of the HD-ZIP transcription factor in regulation of tomato quality. A tomato transcription factor mutant library is constructed through a CRISPR (clustered regularly interspaced short palindromic repeats) technology, and a mutant Tom6-155 capable of simultaneously inhibiting expression of two homologous genes of an HD-ZIP transcription factor is screened. Based on observation of the phenotype of the mutant, the inventor determines that the HD-ZIP transcription factor is related to the tomato fruit quality. Compared with a wild type, the tomato fruit brix content of the mutant is increased. The result shows that the Solyc03g034130.2 and the Solyc03g034110.2 play an important negative regulation and control role in the formation process of the tomato fruit quality. The invention lays a good gene genetic resource foundation for the cultivation of new varieties of high-quality tomatoes.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to the application of HD-ZIP transcription factor in regulating tomato fruit quality. Background Art

[0002] In recent years, with breeding efforts focused on traits such as high yield and stress tolerance, modern cultivated tomato (Solanum lycopersicum) varieties have experienced significant improvements in yield and resistance, but have also faced a significant decline in flavor quality. Numerous studies have shown that this phenomenon manifests itself in a systematic deterioration of key quality indicators, including an imbalance in the sugar-acid ratio, a significant decrease in the content of characteristic aroma compounds, and changes in fruit texture. In-depth analysis has revealed that this quality degradation is closely linked to disruptions in the transcription factor regulatory network during fruit development and ripening. Specifically, members of multiple transcription factor families, such as MYB, NAC, WRKY, and MADS-box, finely regulate key pathways such as sugar metabolism, organic acid synthesis, and volatile compound accumulation, forming a complex molecular network that influences fruit quality. However, a systematic understanding of the key transcriptional regulatory mechanisms underlying tomato quality remains lacking, particularly with the urgent need to identify novel transcription factors and analyze their regulatory networks. To systematically improve tomato quality traits, this study used modern molecular experimental techniques to identify new key transcription factors and elucidate their regulatory mechanisms for important tomato quality traits, such as sugar accumulation and aroma synthesis. This provides a 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 the present invention is to provide an application of HD-ZIP transcription factor in regulating tomato fruit quality.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows:

[0005] This application used CRISPR technology to construct a library of tomato transcription factor mutants, identifying a mutant, Tom6-155, that simultaneously inhibited the expression of two homologous HD-ZIP transcription factor genes. Based on observations of this mutant's phenotype, the inventors determined that the HD-ZIP transcription factor is associated with tomato fruit quality. This protein exhibits functional redundancy, with its functions co-regulated by two genes. The CDS sequence of the gene Solyc03g034130.2 is shown in SEQ ID NO. 1, and the CDS sequence of the gene Solyc03g034110.2 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] The gene sequence ID (Sequence ID) of the Solyc03g034130.2 gene in NCBI (https: / / www.ncbi.nlm.nih.gov / gdv / browser / genome / ?id=GCF_000188115.5) is LOC101251158, and the CDS sequence length is 675 bp, including 224 amino acids.

[0007] The gene sequence ID (Sequence ID) of the Solyc03g034110.2 gene in NCBI is LOC101254766, and the CDS sequence length is 642 bp, including 213 amino acids.

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

[0009] Through preliminary research on the tomato genes Solyc03g034130.2 and Solyc03g034110.2, the inventors discovered that Solyc03g034130.2 and Solyc03g034110.2 have a certain impact on tomato fruit quality. These genes are negatively correlated with tomato fruit quality; that is, knocking out or knocking down these genes increases the soluble solids content in tomato fruit, improving fruit quality. This provides a genetic resource foundation for the cultivation of high-quality new tomato varieties.

[0010] 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 tomato fruit quality also fall within the protection scope of the present invention.

[0011] Among them, the recombinant vector includes a gene editing recombinant vector and an overexpression recombinant vector.

[0012] Those skilled in the art can readily mutate the Solyc03g034130.2 and Solyc03g034110.2 genes 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 sequences of the Solyc03g034130.2 and Solyc03g034110.2 genes, as long as they encode the same proteins and have the same function, are derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention. Primer pairs for amplifying the full-length coding sequences or fragments thereof of the transcription factors encoded by the Solyc03g034130.2 and Solyc03g034110.2 genes also fall within the scope of protection of the present invention.

[0013] The main purpose of the present invention is to protect the above-mentioned genes Solyc03g034130.2 and Solyc03g034110.2, the transcription factors encoded by the Solyc03g034130.2 and Solyc03g034110.2 genes, and the use of biological materials containing the above-mentioned coding sequences in improving the quality of tomato fruits.

[0014] The tomato quality regulation is manifested as follows: when the Solyc03g034130.2 gene and the Solyc03g034110.2 gene are simultaneously knocked out or knocked down, the soluble solid content in the tomato of the knocked out or knocked down strain is significantly higher than that of the wild type.

[0015] In order to improve fruit quality, the present invention also protects a new breeding method for improving tomato fruit quality, by reducing the expression of Solyc03g034130.2 and Solyc03g034110.2 genes in tomato plants and thereby destroying the function of the genes to obtain tomato plants with improved fruit quality. The improved fruit quality is manifested as an increase in the soluble solids (brix) content.

[0016] The expression of the Solyc03g034130.2 and Solyc03g034110.2 genes in tomatoes is reduced by knocking out or knocking down the expression of the genes using gene editing technology. The target plant of the present invention is the cultivated tomato cultivar AC (Ailsa Craig).

[0017] Regulating gene expression levels includes utilizing DNA homologous recombination technology, CRISP-mediated gene editing technology, and Agrobacterium-mediated transformation system to regulate the expression of Solyc03g034130.2 and Solyc03g034110.2, thereby obtaining transgenic plant lines, and obtaining homozygous Solyc03g034130.2 and Solyc03g034110.2 gene mutant plants from the offspring of Solyc03g034130.2 and Solyc03g034110.2 gene knockout or knockdown plants; homozygous Solyc03g034130.2 and Solyc03g034110.2 gene mutant plants are plants with higher fruit quality.

[0018] The above-mentioned 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 the present 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.

[0019] 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.

[0020] The present invention encompasses any plant cell, or any plant obtained or obtainable by any of the methods herein, and all plant parts and propagules thereof. The present invention also encompasses transfected cells, tissues, organs, or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods of the present invention have the same characteristics.

[0021] 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.

[0022] Advantages of the present invention:

[0023] (1) The present invention uses molecular biology methods to innovatively screen a mutant, Tom6-155, in tomato (Solanum lycopersicum). Its target genes, Solyc03g034130.2 and Solyc03g034110.2, belong to the HD-ZIP family of transcription factors. Through phenotypic identification, the inventors preliminarily determined that Solyc03g034130.2 and Solyc03g034110.2 are involved in regulating plant fruit quality. Further testing found that compared with the wild type, the mutant strains of Solyc03g034130.2 and Solyc03g034110.2 had 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, Solyc03g034130.2 and Solyc03g034110.2 genes can be knocked out or knocked down to obtain Solyc03g034130.2 and Solyc03g034110.2 mutant strains. These strains have a higher soluble solids content than the target plants, providing important materials for breeding new high-yield and high-quality tomato varieties, and also have important guiding significance for the genetic improvement of other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a CDS sequence alignment of Solyc03g034130.2 and Solyc03g034110.2 genes;

[0026] Figure 2 This is an alignment of the amino acid sequences encoded by the Solyc03g034130.2 and Solyc03g034110.2 genes.

[0027] Figure 3 This is a map of the target genes and mutation sites of the Tom6-155 mutant.

[0028] Figure 4 This is a comparison chart of the fruit phenotypes of Tom6-155 and AC. Scale bar = 1 cm.

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

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[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 the present invention used CRISPR-Cas9 technology to construct a large-scale mutant library of tomato transcription factors. After preliminary phenotypic screening, they discovered a mutant, Tom6-155, that regulates tomato fruit quality. Its sgRNA targets two genes, Solyc03g034130.2 and Solyc03g034110.2. The Solyc03g034130.2 gene has a gene sequence ID of LOC101251158 in the NCBI database, a CDS sequence as shown in SEQ ID NO.1, and an amino acid sequence as shown in SEQ ID NO.3. The Solyc03g034110.2 gene has a gene sequence ID of LOC101254766 in the NCBI database, a CDS sequence as shown in SEQ ID NO.2, and an amino acid sequence as shown in SEQ ID NO.4.

[0038] Soluble solids content reflects the maturity of tomato fruits and is closely related to the flavor quality and nutritional value of the fruit. It is an important indicator for measuring tomato fruit quality. It includes water-soluble compounds such as sugars (monosaccharides and disaccharides), organic acids, vitamins, and minerals. Therefore, in the research process of this paper, soluble solids content was used as a primary indicator for comparing tomato quality. The higher the soluble solids content, the better the quality.

[0039] Example 1 Screening Tom6-155 mutants from a tomato mutant library

[0040] sgRNA oligonucleotides were designed and synthesized for the tomato transcription factor panel. Each sgRNA targeted at least two genes with homologous functions to achieve simultaneous knockout of multiple redundant genes. Using the synthesized oligos as templates, polymerase chain reaction (PCR) amplification was performed to obtain DNA fragments containing sgRNAs encoding the target transcription factor genes. The amplified products were recovered by gel excision and purified to remove impurities. Using the Golden-gate cloning method, the purified DNA products were cloned into the pMR284-Crimera vector. Ultimately, a CRISPR library of tomato transcription factors was successfully constructed. Using tissue culture technology, 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. They were then planted in a greenhouse suitable for tomato growth conditions. At the fruit maturity stage, all transgenic plants were comprehensively and systematically phenotypicly characterized, 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 Tom6-155 was selected for research. Compared to the AC control, its fruit brix content was significantly increased, suggesting that the Tom6-155 mutant may play an important role in regulating tomato fruit quality.

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

[0042] In order to efficiently identify sgRNA and its target gene in Tom6-155 mutant plants, Tom6-155 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 genes were quickly identified by sequence alignment. The results showed that the target genes were Solyc03g034130.2 and Solyc03g034110.2. The CDS sequences and amino acid sequences of the two genes Solyc03g034130.2 and Solyc03g034110.2 were obtained in phytozome 13 (https: / / phytozome-next.jgi.doe.gov / info / Slycopersicum_ITAG2_4). Sequence alignment was further performed using DNAMAN software, and the results showed that the CDS sequence similarity was 91.26% ( Figure 1 ), the amino acid sequence similarity is 87.5% ( Figure 2 ). This suggests that there may be functional redundancy between the two genes, and knocking out both genes results in a phenotype with fruit quality superior to that of the wild type.

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

[0044] 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 4130-F, 4130-R and primers 4110-F, 4110-R. The PCR amplification products were then recovered and sequenced. The mutant plants with edited Solyc03g034130.2 and Solyc03g034110.2 genes were screened based on the sequencing results. Figure 3 ), and the primers are:

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

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

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

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

[0049] Example 4 Identification of Fruit Phenotypes of Tomato Solyc03g034130.2 and Solyc03g034110.2 Mutant Strains

[0050] The phenotypic characteristics of Solyc03g034130.2 / Solyc03g034110.2 mutant lines and wild-type plants were recorded by photographing ( Figure 4 ) and identification, the present invention mainly through the tomato fruit soluble solids brix content ( Figure 5 ) to reflect the quality of tomatoes. The higher the soluble solids Brix content, the better the quality. Figure 5 As shown in the figure, after being cultured for the same number of days under suitable conditions, the soluble solid brix content of the Solyc03g034130.2 / Solyc03g034110.2 gene mutant strain was significantly higher than that of the wild type fruit.

[0051] These results indicate that under suitable conditions, the fruits of the Solyc03g034130.2 / Solyc03g034110.2 mutant line had a higher soluble solids (Brix) content than the wild-type fruit within the same timeframe, suggesting that the Solyc03g034130.2 / Solyc03g034110.2 gene can regulate tomato fruit quality.

[0052] 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.

Claims

1. Application of HD-ZIP transcription factor in regulating tomato quality, characterized in that: The amino acid sequences of the HD-ZIP transcription factor are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, corresponding to the Solyc03g034130.2 gene and the Solyc03g034110.2 gene, respectively. The CDS sequence of the Solyc03g034130.2 gene is shown in SEQ ID NO.1, and the CDS sequence of the Solyc03g034110.2 gene is shown in SEQ ID NO.

2. The tomato quality regulation is manifested as follows: when the Solyc03g034130.2 gene and the Solyc03g034110.2 gene are simultaneously knocked out or knocked down, the soluble solids content in the tomatoes of the knockout or knockdown strains is significantly higher than that of the wild type.

2. Use of a recombinant vector containing the Solyc03g034130.2 gene and the Solyc03g034110.2 gene according to claim 1 in regulating tomato fruit quality.

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 improving tomato fruit quality, characterized in that: Tomato plants with improved fruit quality are obtained by reducing the expression of Solyc03g034130.2 and Solyc03g034110.2 genes in tomato plants, thereby disrupting the functions of the genes. The CDS sequence of the Solyc03g034130.2 gene is shown in SEQ ID NO. 1, and the CDS sequence of the Solyc03g034110.2 gene is shown in SEQ ID NO.

2. The improved fruit quality is reflected in the increased soluble solids content.

5. The method for improving tomato fruit quality according to claim 4, characterized in that: The method of reducing the expression of Solyc03g034130.2 and Solyc03g034110.2 genes in tomatoes is to knock out the genes or knock down their expression through gene editing technology.

6. The method for improving tomato fruit quality according to claim 5, characterized in that: The Solyc03g034130.2 and Solyc03g034110.2 genes in the tomato genome were knocked out or knocked down to obtain plants with Solyc03g034130.2 and Solyc03g034110.2 gene knockout or knockdown, and homozygous Solyc03g034130.2 and Solyc03g034110.2 knockout or knockdown plants were obtained from the self-pollinated progeny of the Solyc03g034130.2 and Solyc03g034110.2 knockout or knockdown plants.

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