Solyc08g008190 and Solyc08g008200 genes of tomatoes and application of Solyc08g008190 and Solyc08g008200 genes

Abstract

The invention discloses a tomato Solyc08g008190 gene and a tomato Solyc08g008200 gene and application of the tomato Solyc08g008190 gene and the tomato Solyc08g008200 gene. A CDS (Coding Sequence) sequence of the Solyc08g008190 gene is shown as SEQ ID NO. 1, and a CDS sequence of the Solyc08g008200 gene is shown as SEQ ID NO. 2. A tomato transporter mutant library is constructed through a CRISPR (clustered regularly interspaced short palindromic repeats) technology, and a mutant 0990-32 which simultaneously covers expression of two homologous genes of an NHE family is screened. Based on observation of the phenotype of the mutant, the inventor preliminarily determines that the two genes are related to tomato fruit quality and fruit volume increase. Compared with a wild type, the tomato fruit of the mutant has the advantages that the brix content is increased, and the fruit becomes larger. It is shown that the Solyc08g008190 gene and the Solyc08g008200 gene play an important role in regulating and controlling the growth, development and quality of the tomato fruits. The invention lays a good gene genetic resource foundation for the cultivation of new varieties of high-quality tomatoes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to two Solyc08g008190 and Solyc08g008200 genes of tomato and application thereof, and more particularly to application thereof in regulating fruit quality and fruit size of tomato. BACKGROUND

[0002] Tomato (Solanum lycopersicum) is an important economic crop in the world, and its quality has always been the core target of breeding research. However, in the modern breeding process, excessive emphasis is placed on the improvement of yield and resistance traits, which, to some extent, meets the market demand, but leads to the degradation of flavor quality in cultivated varieties. Specifically, there are prominent problems such as imbalance of sugar and acid ratio, reduction of characteristic aromatic substance content, and change of fruit texture, which not only affect the edible value of tomato, but also limit its competitiveness in the high-end market. Molecular biology research shows that this quality decline trend is closely related to the functional disorder of the material transport system during fruit development and maturation. At present, it has been found that multiple transport protein families such as SWEET, NPF, ABC and SUT directly affect the formation of fruit quality by regulating key biochemical pathways such as sugar metabolism, organic acid synthesis and volatile substance generation. At the same time, these transport proteins also participate in the regulation of fruit development-related physiological processes, and affect fruit growth and development through the regulation of hormone or nutrient distribution and transport efficiency.

[0003] In this study, CRISPR-Cas9 gene editing and multi-omics technologies are used to identify key transport proteins that regulate tomato quality and analyze their regulation mechanism on important quality traits such as sugar accumulation and aroma synthesis in tomato. By targeted editing of key transport protein genes and optimizing their expression patterns, new tomato varieties with high yield potential and excellent flavor are cultivated. SUMMARY

[0004] The purpose of the present application is to provide the application of two homologous Solyc08g008190 and Solyc08g008200 genes in regulating tomato quality and fruit size.

[0005] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0006] The present application constructs a tomato transport protein mutant library by CRISPR technology, and a mutant 0990-32 covering the expression of two homologous genes of NHE family is screened. Based on the observation of the phenotype of the mutant, the inventors preliminarily determine that the two genes are related to the fruit quality and fruit volume increase of tomato. The two tomato Solyc08g008190 and Solyc08g008200 genes found in the present application are as follows:

[0007] A1) Solyc08g008190 gene in NCBI

[0008] (https: / / www.ncbi.nlm.nih.gov / gdv / browser / genome / ?id=GCF_036512215.1) with Sequence ID of LOC101262233, CDS sequence as shown in SEQ ID NO. 1, length of 2406bp, amino acid sequence as shown in SEQ ID NO. 3, including 801 amino acids;

[0009] A2) Solyc08g008200 gene in NCBI with Sequence ID of LOC101262542, CDS sequence as shown in SEQ ID NO. 2, length of 2280bp, amino acid sequence as shown in SEQ ID NO. 4, including 759 amino acids.

[0010] Through cloning and preliminary research of Solyc08g008190 and Solyc08g008200 genes of tomato, the inventors found that Solyc08g008190 and Solyc08g008200 have certain influence on fruit quality and fruit enlargement of tomato, and the Solyc08g008190 and Solyc08g008200 genes of tomato are negatively correlated with fruit enlargement and quality of tomato; that is, after gene knockout, the tomato fruit is larger, the soluble solid content is increased, and the fruit quality is improved. Certain genetic resource foundation is laid for new breed cultivation of high-quality tomato.

[0011] The application also constructs a series of plant expression vectors, expression vectors containing the above-mentioned genes, transgenic plant lines and host cells containing the vectors also fall within the protection scope of the application in improving fruit quality and fruit enlargement of tomato.

[0012] Those skilled in the art can easily mutate the Solyc08g008190 and Solyc08g008200 genes described in the application by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity of the nucleotide sequence encoding the transport protein encoded by the Solyc08g008190 and Solyc08g008200 genes, as long as they encode the same protein and have the same function, are derived from the nucleotide sequence of the application and are equivalent to the sequence of the application. The primer pair for amplifying the full-length or fragment of the coding sequence of the transport protein encoded by the Solyc08g008190 and Solyc08g008200 genes also belongs to the protection scope of the application.

[0013] The most important purpose of the present application is to protect the application of the above-mentioned genes Solyc08g008190 and Solyc08g008200, the transport proteins encoded by the Solyc08g008190 and Solyc08g008200 genes and the biological materials containing the above-mentioned coding sequences in improving the quality of tomato fruits and increasing the size of fruits.

[0014] The specific performance of improving the quality of tomato fruits and increasing the size of fruits is that after the Solyc08g008190 and Solyc08g008200 genes in the tomato are mutated, the mutant strain has higher soluble solids (brix) content and larger fruits compared with the wild type.

[0015] In order to improve the quality of fruits, the present application also protects a new breeding method for improving the quality of tomato fruits, which is to obtain a plant with higher fruit quality than the target plant by reducing the expression of Solyc08g008190 and Solyc08g008200 genes in the target plant.

[0016] The way to reduce the expression of Solyc08g008190 and Solyc08g008200 genes in the target plant is to knock out or knock down Solyc08g008190 and Solyc08g008200 genes. Among them, the target plant of the present application is the cultivated species Solyc08g008190 and Solyc08g008200.

[0017] The method for regulating the expression level of the gene includes regulating the expression of Solyc08g008190 and Solyc08g008200 by using DNA homologous recombination technology, CRISP-mediated gene editing technology and Agrobacterium-mediated transformation system to obtain a transgenic plant strain, obtaining a homozygous Solyc08g008190 and Solyc08g008200 gene mutant plant from the offspring of the Solyc08g008190 and Solyc08g008200 gene knockout or knockdown plant; the homozygous Solyc08g008190 and Solyc08g008200 gene mutant plant is a plant with higher fruit quality and larger fruits.

[0018] The method for improving the quality of tomato fruits and increasing the size of fruits also applies to other receptor plants having homologous genes with tomatoes, and the receptor plants suitable for the present application are not particularly limited, and can be not only tomatoes but also other plants with high homology, as long as they are suitable for gene transformation operation, such as various crops, ornamental plants or forestry plants, etc. The plants can be, but are not limited to, dicotyledonous plants, monocotyledonous plants, woody plants, rosaceous plants, rosaceous plants, peach, cruciferous plants, Arabidopsis plants, Arabidopsis, etc.

[0019] The "plant" in the present application includes whole plants, parent and progeny plants and different parts of plants, including seeds, fruits, stems, buds, leaves, roots, flowers, tissues and organs, and the gene or nucleic acid of interest is present in each of these different parts. The "plant" mentioned herein also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions and pollen, and each of the foregoing objects also contains the gene / nucleic acid of interest.

[0020] The present application includes any plant cell, or any plant obtained or obtainable by the methods therein, and all plant parts and propagules thereof. The present application also comprises a transfected cell, tissue, organ or whole plant obtained by any of the foregoing methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics as obtained using the methods of the present application.

[0021] The present application also extends to harvestable parts of the plants as described above, but not limited to seeds, fruits, fruit peels. It also further relates to other derivatives of the plant after harvesting, such as organic acids, sugars, lycopene, tomatine, volatile substances, vitamins, minerals and proteins. The present application also relates to food or food additives obtained from the related plants.

[0022] Advantages of the present application:

[0023] (1) The present application uses molecular biology methods to innovatively screen a mutant 0990-32 in tomatoes (Solanum lycopersicum), and the target transport protein is NHE family. Through phenotype identification, the inventors preliminarily determine that Solyc08g008190 and Solyc08g008200 are involved in the regulation of fruit quality and fruit development of plants. Compared with the wild type, the soluble solid content of the Solyc08g008190 and Solyc08g008200 gene mutant strain is higher, and the volume is larger.

[0024] (2) In the actual breeding process, high fruit quality and large fruit plants can be obtained by CRISPR / Cas9 gene editing technology, specifically, Solyc08g008190 and Solyc08g008200 genes can be knocked out or knocked down to obtain Solyc08g008190 and Solyc08g008200 mutant lines, which have higher soluble solid content than the target plants, providing important materials for breeding new high-yield and high-quality tomato varieties, and having important guiding significance for genetic improvement of crops. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a coding amino acid sequence alignment map of Solyc08g008190 and Solyc08g008200 genes.

[0026] Figure 2 is a target gene and its mutation site map of 0990-32 mutant.

[0027] Figure 3 is a fruit phenotype comparison map of 0990-32 mutant and AC, Scale bar = 1 cm.

[0028] Figure 4 is fruit length data analysis of 0990-32 mutant line compared with wild type.

[0029] Figure 5 is fruit width data analysis of 0990-32 mutant line compared with wild type.

[0030] Figure 6 is soluble solid (brix) content analysis of 0990-32 mutant line compared with wild type. DETAILED DESCRIPTION

[0031] The advantages and features of the present application will become more apparent with the description of the specific embodiments. However, the specific experimental methods involved in the following examples are conventional methods or are implemented according to the suggested conditions in the manufacturer's instructions, unless otherwise specified.

[0032] Unless specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. The experimental methods in the following examples are conventional methods, unless otherwise specified. Unless otherwise specified, the reagents and materials used are commercially available.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Also, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials are presented by way of example only.

[0034] Unless otherwise indicated, the practice of the present application will employ, unless otherwise indicated, conventional techniques of plant biology, microbiology, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics, which are within the skill of the art. Such techniques are explained fully in the literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, methods of gene editing, construction of gene editing vectors, obtaining of gene edited plants, etc. employed by the present application can be achieved using methods disclosed in the prior art, except for the methods employed in the following examples.

[0035] As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to refer to isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), naturally occurring types, mutated 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 of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene can include introns and exons in genomic sequences, and / or coding sequences in cDNA, and / or cDNA and its regulatory sequences. In particular embodiments, such as with respect to isolated nucleic acid sequences, it is preferred to assume that they are cDNA.

[0036] Unless otherwise indicated, 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 application constructed a tomato transporter group scale mutant library by using CRISPR-Cas9 technology. After preliminary phenotype screening, a mutant 0990-32 regulating tomato fruit quality was found, and the sgRNA targets of the two genes Solyc08g008190 and Solyc08g008200. The gene sequence ID of Solyc08g008190 in the NCBI database is LOC101262233, the CDS sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 3. The gene sequence ID of Solyc08g008200 in NCBI is LOC101262542, the CDS sequence is shown as SEQ ID NO. 2, and the amino acid sequence is shown as SEQ ID NO. 4.

[0038] Example 1 Screening 0990-32 mutant in tomato mutant library

[0039] sgRNA oligonucleotides were designed and synthesized for tomato transporters. Each sgRNA targets at least two homologous functional genes to achieve simultaneous knockout of multiple redundant genes. The synthesized oligos were used as templates to amplify the DNA fragments containing sgRNA of the target transporter coding genes by polymerase chain reaction (PCR). The amplification products were recovered and purified to remove impurities. The purified DNA products were cloned into pMR284-Crimera vectors using Golden-gate cloning method. Finally, a tomato transporter CRISPR library of transporter group scale was successfully constructed. The constructed CRISPR library containing tomato transporters was introduced into AC wild-type tomato using tissue culture technology. Positive seedlings were obtained by kanamycin resistance screening and PCR identification. And they were planted in a greenhouse that met the growth conditions of tomatoes. At the fruit ripening stage, all transgenic plants were comprehensively and systematically phenotyped, including fruit size, color, shape, and soluble solids content (brix) and other key quality indicators. Among the many mutants with significant phenotypes, the mutant numbered 0990-32 was chosen as the research object. Compared with the AC control, the fruit volume increased, and the brix content significantly increased, indicating that the 0990-32 mutant may play an important role in tomato fruit enlargement and quality regulation.

[0040] Example 2 Determination of 0990-32 mutant target genes

[0041] To efficiently identify sgRNA and its target gene in 0990-32 mutant plants, the 0990-32 mutant plants were sampled, the genomic DNA was extracted using the CTAB method, and Barcoding analysis was performed using the genomic DNA as a template. The sgRNA and target gene were quickly identified by sequence alignment. The results showed that the target genes were Solyc08g008190 and Solyc08g008200. The CDS sequences and amino acid sequences of Solyc08g008190 and Solyc08g008200 were obtained in phytozome 13 (https: / / phytozome-next.jgi.doe.gov / info / Slycopersicum_ITAG2_4), and further sequence alignment was performed using DNAMAN software, and the results showed that the amino acid sequence similarity was 80.9% ( Figure 1 ).It is proved that there may be functional redundancy when the two genes function, and the knockout has the phenotype of better fruit volume and quality than wild type.

[0042] Example 3: Screening of Solyc08g008190 and Solyc08g008200 gene mutant plants

[0043] The mutant tomato plant leaves were taken, the genomic DNA was extracted by the CTAB method, and the primer pair composed of primers 8190-F, 8190-R and primers 8200-F, 8200-R was used for PCR amplification, then the PCR amplification product was recovered and sequenced, and according to the sequencing results, the mutant plants in which Solyc08g008190 and Solyc08g008200 genes were edited were screened ( Figure 2 ).

[0044] 8190-F: 5'-GACTTTTGAGCATCTTAGTAAGG-3';

[0045] 8190-R: 5'-GATTGAATCATTCTTGGACATGT-3'.

[0046] 8200-F: 5'-GTTTGAAAATCTCAGCAAGGTG-3';

[0047] 8200-R: 5'-GGAGCTGTTCTTAGGAAGTC-3'.

[0048] Example 4: Phenotypic identification of tomato Solyc08g008190 and Solyc08g008200 gene mutant lines

[0049] ​The Solyc08g008190 / Solyc08g008200 gene mutant strain and wild type plant are taken photos for phenotype record Figure 3 The fruit length and width of the Solyc08g008190 / Solyc08g008200 gene mutant strain are compared with those of the wild type plant, and it can be seen that the fruit length and width are increased, and the fruit volume is increased Figure 4 and Figure 5 ).

[0050] The quality of the tomato is reflected by the soluble solid brix content of the tomato fruit, and the higher the soluble solid brix content, the better the quality. Figure 6 As shown in the table, the brix content of the Solyc08g008190 / Solyc08g008200 gene mutant strain is significantly higher than that of the wild type fruit after the same number of days of culture under suitable conditions.

[0051] These results show that under suitable conditions, the soluble solid content of the fruit of the Solyc08g008190 / Solyc08g008200 gene mutant strain is higher than that of the wild type plant fruit, and the fruit is larger, within the same time. It is shown that the Solyc08g008190 / Solyc08g008200 gene can regulate the fruit quality and the fruit size of the tomato.

[0052] The above-described embodiments are only preferred embodiments of the present application, and are used to explain the present application, but do not limit the scope of the present application. For those skilled in the art, of course, other embodiments can be easily made by substitution or change based on the technical content disclosed in the present specification, and therefore, any changes and improvements made on the principle of the present application should be included in the scope of the present application.

Claims

1. The tomato Solyc08g008190 and Solyc08g008200 genes characterized in that, The CDS sequence of the Solyc08g008190 gene is shown as SEQ ID NO. 1, and the CDS sequence of the Solyc08g008200 gene is shown as SEQ ID NO.

2.

2. Tomato Solyc08g008190 and Solyc08g008200 genes according to claim 1, characterized by, The amino acid sequence of the protein encoded by the Solyc08g008190 gene is shown as SEQ ID NO. 3, and the amino acid sequence of the protein encoded by the Solyc08g008200 gene is shown as SEQ ID NO.

4.

3. A gene expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic plant cell line containing the Solyc08g008190 and Solyc08g008200 genes of claim 1.

4. Use of Solyc08g008190 and Solyc08g008200 genes in modulating tomato fruit quality and fruit size according to claim 1, characterized in that, The regulation specifically manifests that the soluble solid content of the Solyc08g008190 and Solyc08g008200 gene knockout lines is higher, and the fruits are larger, compared with the wild type.

5. Use according to claim 4, characterized in that, The larger fruits manifest that the fruit volume is larger.

6. Application of the gene expression cassette, the recombinant vector, the recombinant microorganism, or the transgenic plant cell line containing the Solyc08g008190 and Solyc08g008200 genes of claim 1 in regulating the fruit quality and the fruit size of tomatoes.

7. A breeding method for improving the quality and increasing the size of tomato fruits, characterized by, The plants with higher fruit quality and fruit volume than the target plants are obtained by reducing the expression of the Solyc08g008190 and Solyc08g008200 genes in the target plants, wherein the target plants are tomatoes, the CDS sequence of the Solyc08g008190 gene is shown as SEQ ID NO. 1, and the CDS sequence of the Solyc08g008200 gene is shown as SEQ ID NO.

2.

8. The breeding method according to claim 7, characterized in that, The method for reducing the expression of the Solyc08g008190 and Solyc08g008200 genes in tomatoes is knockout or knockdown of the expression.

9. The breeding method according to claim 8, characterized in that, The Solyc08g008190 and Solyc08g008200 genes in the plant genome are knocked out or knocked down to obtain Solyc08g008190 and Solyc08g008200 knockout or knockdown plants, and the homozygous Solyc08g008190 and Solyc08g008200 knockout or knockdown plants are obtained from the selfed offspring of the Solyc08g008190 and Solyc08g008200 knockout or knockdown plants; the homozygous Solyc08g008190 and Solyc08g008200 knockout or knockdown plants are the plants with larger fruits and higher quality.

Images