Solyc05g011980 gene for increasing ventricular number and single fruit weight of tomato and application of Solyc05g011980 gene

Abstract

The invention belongs to the technical field of molecular breeding, and particularly relates to a Solyc05g011980 gene for increasing the number of tomato ventricles and the weight of a single fruit and application of the Solyc05g011980 gene. Research finds that after the Solyc05g011980 gene is knocked out, a homozygous plant can be rapidly obtained only through two generations, the number of fruit ventricles and the weight of a single fruit are both remarkably increased, exogenous gene insertion is not contained, and the homozygous plant can be directly used as a parent material for tomato breeding. A result of identifying homozygous plant fruits after gene editing proves that the number of fruit ventricles is remarkably increased, and the weight of a single fruit is synchronously and remarkably increased. According to the research, the key characters of the tomatoes are improved through precise gene editing, and a brand new effective strategy is provided for increasing the yield of the tomatoes.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to the Solyc05g011980 gene and its application for increasing the number of tomato locules and the weight of a single fruit. Background Technology

[0002] Tomatoes (Solanum lycopersicum) are an important economic vegetable. With their rich flavor and abundance of essential nutrients, they hold a vital position in vegetable supply and diet, becoming an indispensable source of nutrition in daily meals, with enormous annual consumption. In the vegetable industry, tomatoes occupy a core position, not only as a major crop cultivated in both open fields and greenhouses, but also consistently ranking among the top vegetable consumers with annual consumption exceeding tens of millions of tons. They play an irreplaceable role in ensuring vegetable market supply and meeting people's needs.

[0003] In tomato breeding research, the number of fruit locules is a key agronomical trait regulating yield and quality, and numerous studies have confirmed a significant positive correlation between it and fruit size and weight. As the core structure for tomato fruit development, an increase in the number of locules helps promote the full growth of the fruit pulp, thereby significantly increasing fruit weight and providing crucial support for yield improvement. This trait-related characteristic makes it a significant breakthrough point for high-yield tomato breeding.

[0004] It is worth noting that, in addition to their potential for increased yield, multi-chamber tomatoes also possess significant commercial advantages. Compared to fruits with fewer chambers, multi-chamber tomatoes have more densely packed flesh and a firmer texture. This structural characteristic allows them to effectively resist external pressure and impact during harvesting, packaging, and long-distance transportation, significantly reducing losses such as skin damage and flesh softening. This significantly improves the marketable fruit rate and transportation radius, providing strong support for the large-scale distribution of the tomato industry.

[0005] Based on the dual regulatory effect of the number of cavities on tomato yield and marketability, accurately identifying and analyzing the key genes that regulate the number of cavities and the weight of a single tomato not only provides a theoretical basis for the study of the molecular mechanism of tomato fruit development, but also provides core gene resources and technical support for the targeted breeding of high-yielding, transport-resistant, and high-quality tomato varieties. It has extremely high scientific research value and industrial application prospects. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, this invention has found that knocking out the Solyc05g011980 gene in tomato plants significantly increases the number of fruit locules and the weight of a single fruit, and since it does not contain any foreign gene insertion, it can be directly used as parent material for tomato breeding, providing a new and effective strategy for increasing tomato yield.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the application of the Solyc05g011980 gene in increasing the number of tomato locules and / or the weight of a single fruit, wherein the amino acid sequence of the Solyc05g011980 gene is shown in SEQ ID NO.2.

[0008] Preferably, knocking out the Solyc05g011980 gene increases the number of locules in tomato fruits and the weight of a single fruit.

[0009] The Solyc05g011980 gene is located on chromosome 5 of tomato, between 5193206 and 5191964. It consists of 1243 base pairs, including two exons, and encodes a protein containing 192 amino acids. This gene and its function or application have not been previously reported. The structure and size of tomato fruits are determined early in development, and the expression level of the Solyc05g011980 gene is highest during this stage, gradually decreasing as the fruit develops and matures. This suggests that this gene may play an important role in the formation of tomato fruit structure.

[0010] This invention utilizes CRISPR / Cas gene editing technology to knock out the Solyc05g011980 gene. After next-generation genotyping, two homozygous mutant lines were obtained. Neither of these lines can produce normal active proteins. Their fruits not only have a significantly increased number of locules, but also larger fruit size and significantly increased single fruit weight, demonstrating potential application in the field of high-yield tomato breeding.

[0011] Preferably, the nucleotide sequence of the Solyc05g011980 gene is shown in SEQ ID NO.1.

[0012] The second aspect of this invention also provides a method for increasing the number of locules and the weight of a single tomato fruit. Specifically, the Solyc05g011980 gene is knocked out in tomatoes using CRISPR technology. After screening for Solyc05g011980 gene knockout mutant lines that can be stably inherited, homozygous mutant plants are then screened to increase the number of locules and the weight of a single tomato fruit.

[0013] Preferably, the gRNA used to knock out the Solyc05g011980 gene is shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0014] Preferably, the gene editing vector used to knock out the Solyc05g011980 gene is the CRISPR / Cas9 gene editing vector pAGM4723.

[0015] Preferably, the primers used for screening stable Solyc05g011980 gene knockout mutant lines are shown in SEQ ID NO. 8 and SEQ ID NO. 9.

[0016] Preferably, the primers used to screen homozygous mutant plants are shown in SEQ ID NO. 10 and SEQ ID NO. 11.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention achieves a highly efficient breakthrough in a key aspect of tomato breeding by knocking out the Solyc05g011980 gene, rapidly cultivating homozygous plants in just two generations. These homozygous plants exhibit multiple superior traits: a significantly increased number of fruit locules and a simultaneous significant increase in single fruit weight. A core advantage is the absence of exogenous gene insertion, meeting the stringent requirements for parental materials and allowing them to be directly used as high-quality parents in subsequent tomato breeding. To verify the improvement effect, the fruits produced by the gene-edited homozygous plants were systematically identified. The results clearly confirmed that the number of fruit locules was significantly increased compared to the control, and the single fruit weight also showed a significant improvement. This invention, through precise gene editing and targeted improvement of tomato yield-related traits, effectively shortens the breeding cycle and ensures material safety, providing a novel and efficient technical strategy for high-yield tomato breeding. Attached Figure Description

[0018] Figure 1 The expression level of the Solyc05g011980 gene at different stages of tomato fruit development; Figure 2 The Solyc05g011980 gene was knocked out. A. Gene knockout mutation sequence: In the cr-1 line, a single base (A) was inserted at position 266 bp of the Solyc05g011980 gene. In the cr-2 line, a 146 bp deletion occurred between positions 120 and 265 bp of the Solyc05g011980 gene. The gRNA sequence used is shown in red. The numbers above the sequence indicate the DNA sequence position of the bases, and the dashed lines indicate the position and length of the mutated base deletion. B. Protein changes caused by the mutation: Solyc05g011980 contains two exons. The wild-type protein contains 192 amino acids. The single base insertion in the cr-1 line caused premature translation termination, producing a truncated protein with 29 amino acids, of which only 25 amino acids are identical to the wild-type protein. The 146 bp deletion in the cr-2 line resulted in the loss of the start codon, preventing the production of a functional protein.

[0019] Figure 3 The mutant strain showed a significant increase in the number of fruit locules and the weight of a single fruit; A. Mutant fruit; B. Number of fruit locules in the mutant fruit; C. Weight of a single fruit (grams) in the mutant fruit. Detailed Implementation

[0020] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0022] The material used in the following examples is tomato (cv. Moneyberg), grown in the greenhouse of the Institute of Facility Agriculture, Guangdong Academy of Agricultural Sciences.

[0023] Example: The Solyc05g011980 gene for increasing the number of tomato chambers and the weight of a single fruit and its application. 1. Measurement of expression levels of the Solyc05g011980 gene at different stages of fruit development Fruit samples were collected at four stages: 15 days after flowering (15 PDA), green ripening stage (MG), color breaking stage (Br), and 7 days after color breaking (Br+7 d). Three replicates were performed at each stage, with two fruits per sample. Samples were frozen in liquid nitrogen and then ground for preservation. Total RNA was extracted using the RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biotech. RNA concentration was measured, and ODs were screened. 260 / 280 ≥2, OD 260 / 230 ≥1.8 μg of qualified RNA was used for reverse transcription. 1 μg of qualified RNA was used to synthesize cDNA using the Bio-Rad iScrip cDNA synthesis kit, followed by real-time quantitative PCR (qPCR) using Bio-Rad iQ SYBR Green ultramix buffer. The tomato Actin gene was used as an internal reference gene, and 2... -ΔΔCt The relative expression level of the Solyc05g011980 gene was calculated using the method.

[0024] The qPCR reaction system consisted of: 5 µL of iQ SYBR Green ultramixed solution, 1 µL of qPCR-F (SEQ ID NO. 3), 1 µL of qPCR-R (SEQ ID NO. 4), 0.5 µL of DNA, and 2.5 µL of sterile water.

[0025] The qPCR reaction conditions were: 95℃ for 3 minutes; 95℃ for 15 seconds, 56.9℃ for 30 seconds, for 40 cycles.

[0026] ; .

[0027] like Figure 1 As shown, the relative expression level of the Solyc05g011980 gene was highest 15 days after flowering in tomatoes, decreased significantly during the green ripening stage, and was extremely low at the color-breaking stage and 7 days after color-breaking, remaining at a relatively low level. This indicates that the expression of the Solyc05g011980 gene has a clear fruit development period specificity and may play an important role in the early stages of tomato fruit development.

[0028] The nucleotide sequence (SEQ ID NO.1) of the Solyc05g011980 gene is shown below:

[0029] The protein sequence of Solyc05g011980 is shown in SEQ ID NO.2: MFFIFGSQTFAYFSISRIKFRVFPCIFPLKKDTIYQLIIYRDEKIGAEIINGEHRECSKSCSGEIEGDEYGGKRKGDRLKNDAVVCIGICTDLIQLVIGKFEADCRENDAGRANFIRLATEKYEGDYRENGAVCTNLISLVTGKSSSTEFDPRNSTVYLHSNRISADFVPPVLQRRRRRRKWEDDESSGKE.

[0030] 2. gRNA design of the Solyc05g011980 gene Using the DNA sequence of Solyc05g011980 (SEQ ID NO. 1), the DNA was designed using the online design tool CRISPOR. http: / / crispor.gi.ucsc.edu / Guide RNAs (gRNAs) were designed. Based on predicted efficiency and off-target probability, two gRNAs located in the first exon of Solyc05g011980 were selected.

[0031] The gRNA sequence used is as follows: ; .

[0032] 3. Constructing gene editing vectors gRNA1 and gRNA2 were ligated to vectors pICH47751 and pICH47761, respectively, with the U6 promoter. These vectors were then electroporated into *E. coli* DH5α, cultured overnight in liquid LB medium containing carbenicillin (50 mg / L), and plasmids were extracted. Sequencing was performed using primer sgRNA-R (SEQ ID NO. 7) to ensure sequence accuracy. The gRNA-containing vector was then digested with enzymes and ligated into the CRISPR / Cas9 gene editing vector pAGM4723. This was followed by electroporation into *E. coli* DH5α, and plasmids were extracted after overnight culture in liquid LB medium containing kanamycin (50 mg / L). The plasmid was then electroporated into the Agrobacterium GV3101 strain and plated on LB agar plates containing rifampicin (20 mg / L) and kanamycin (50 mg / L). Single clones were screened and cultured in liquid LB agar containing rifampicin (20 mg / L) and kanamycin (50 mg / L). This method constructs two gRNAs into the same vector, requiring only one genetic transformation to obtain multiple different mutations.

[0033] Sequencing primer sequences: .

[0034] like Figure 2 As shown in Figure A, the wild-type (WT) genotype has two gRNA (red sequence) action regions. The cr-1 line undergoes a single-base insertion mutation, inserting a base (A) at a specific position in the gRNA2 action region. The cr-2 line undergoes a 146 bp deletion mutation, resulting in the loss of a large fragment of bases in this region. The dashed line shows the location and length of the deletion. Figure 2 As shown in B, the wild-type (WT) Solyc05g011980 gene contains two exons, encoding a protein with 192 amino acids. The cr-1 line terminates translation prematurely due to a single base insertion, producing only a truncated protein with 29 amino acids, of which only the first 25 amino acids are the same as the wild-type protein. The protein is likely to lose its function. The cr-2 line loses the start codon due to a 146 bp base deletion, which prevents translation from starting and thus prevents the production of a functional protein.

[0035] 4. Genetic transformation of tomatoes Tomato seeds were first disinfected with a 1% sodium hypochlorite solution, then washed with sterile water and inoculated onto 1 / 2 MS solid medium (MS + 1 wt% sucrose + 0.8 wt% agar), and cultured for 5 days in a 16 h light / 8 h dark environment at 25°C. Afterwards, cotyledons were cut off and cultured in the dark on MS medium (MS + 3 wt% sucrose + 0.8 wt% agar) for 1 day. Additionally, Agrobacterium GV3101 containing two gRNA vectors was added to LB liquid medium containing 10 μM / mL acetylsyleugenol and cultured until OD... 600 The cotyledons were then transferred to Agrobacterium-mediated bacterial suspension for 10 min, then blotted dry on sterile filter paper. The cotyledons were then transferred to co-culture medium (MS + 3wt% sucrose + 0.05wt% MES (2-N-morpholinoethanesulfonic acid) + 0.8wt% agar) for 2 days. Afterward, the cotyledons were transferred to meristem medium (MS + 1wt% glucose + 0.8wt% agar + 2 mg / L zeatin + 500 mg / L carbenicillin + 50 mg / L kanamycin) for approximately 7 weeks. When the regenerated plantlets reached about 1 cm in length, they were cut and rooted in rooting medium (MS + 15wt% sucrose + 0.8wt% agar + 0.25 mg / L indole-3-butyric acid (IBA) + 320 mg / L termethin). After approximately 2 weeks, the well-developed seedlings were transplanted into soil for further cultivation.

[0036] 5. Screening of stably inherited Solyc05g011980 gene knockout mutant lines DNA was extracted from transformed plants using the Phire hot-start PCR kit from Thermo Fisher Scientific. Primers F1 (SEQ ID NO. 8) and R1 (SEQ ID NO. 9) were designed approximately 150 bp upstream and downstream of the Solyc05g011980 gene. The DNA fragment containing the mutant region was amplified by PCR using Phire hot-start II DNA polymerase. Sequencing was performed and compared with the wild-type Solyc05g011980 sequence. The mutant plants were then retained for further culture, and seeds were collected for next-generation Mendelian segregation.

[0037] The PCR amplification reaction system was as follows: 4 µL of 5×Phire buffer, 1 µL of F1 (SEQ ID NO. 8), 1 µL of R1 (SEQ ID NO. 9), 0.5 µL of DNA, 0.4 µL of Phire DNA polymerase, and 14.1 µL of sterile water.

[0038] The PCR amplification reaction conditions were: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 5 seconds, 56.4℃ annealing for 5 seconds, 72℃ extension for 15 seconds, for 40 cycles.

[0039] The primer sequences used for PCR amplification are as follows: ; .

[0040] After seed germination, DNA was extracted using the Thermo Fisher Phire hot-start PCR kit. The DNA fragment containing the mutant region was then amplified by PCR using primers F1 (SEQ ID NO. 8) and R1 (SEQ ID NO. 9). Sequencing was performed to determine if the mutation was homozygous (homozygous mutations showed a single band on electrophoresis or a single peak on sequencing). Plants containing homozygous mutations were then subjected to PCR detection using primers NPT2-F (SEQ ID NO. 10) and NPT2-R (SEQ ID NO. 11), and lines lacking NPT2 and CAS9 were selected for further cultivation. This method ensures that all homozygous mutant plants do not contain exogenous gene insertions.

[0041] The PCR amplification reaction system is the same as the previous step, and the reaction conditions are: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 5 seconds, 60℃ annealing for 5 seconds, 72℃ extension for 10 seconds, for 40 cycles.

[0042] The primer sequences used for PCR amplification are as follows: ; .

[0043] 6. Fruit phenotypic detection of mutant strains Select the second and third branches from each strain, retaining 5 fruits on each branch. Weigh 10 fruits individually and count the number of locules.

[0044] like Figure 3 As shown in Figure A, the ventral structure of the wild-type fruit cross-section is relatively simple, while the number of ventral chambers in the cross-section of the Cr-1 and Cr-2 mutant lines is significantly increased. For example... Figure 3 As shown in Figure B, the wild-type (WT) fruit has approximately 3.5 locules, the CR-1 line has approximately 6 locules, and the CR-2 line has approximately 7.5 locules. The mutant lines have significantly more locules than the wild-type. Figure 3 As shown in Figure C, the single fruit weight of the wild type is approximately 70 grams, the single fruit weight of the cr-1 line is approximately 95 grams, and the single fruit weight of the cr-2 line is approximately 100 grams. The single fruit weight of the mutant line is significantly higher than that of the wild type. This indicates that the Solyc05g011980 gene plays an important role in regulating the number of locules and single fruit weight in tomato fruits; knocking out this gene significantly increases the number of locules and improves the single fruit weight.

[0045] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Use of the Solyc05g011980 gene to increase the number of locules and / or the weight of single fruits in tomato, characterized in that, The amino acid sequence of the Solyc05g011980 gene is shown as SEQ ID NO.

2.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the Solyc05g011980 gene is shown as SEQ ID NO.

1.

3. Use according to claim 1, characterized in that, After knocking out the Solyc05g011980 gene, the number of heart chambers of tomato fruit and the single fruit weight are increased.

4. A method of increasing the number of locules and the weight of single fruits of tomato plants, characterized in that, By knocking out the Solyc05g011980 gene in tomato through CRISPR technology, screening the Solyc05g011980 gene knockout mutant lines which can be stably inherited, and then screening the homozygous mutant plants, the number of heart chambers of tomato fruit and the single fruit weight can be increased.

5. A method of increasing the number of locules and the weight of single fruit of tomato according to claim 4, characterized in that, The gRNA used for knocking out the Solyc05g011980 gene is shown as SEQ ID NO. 5 and SEQ ID NO.

6.

6. A method of increasing the number of locules and the weight of single fruit of tomato according to claim 4, characterized in that, The gene editing vector used for knocking out the Solyc05g011980 gene is the CRISPR / Cas9 gene editing vector pAGM4723.

7. A method of increasing the number of locules and the weight of single fruit of tomato according to claim 4, characterized in that, The primers used for screening the Solyc05g011980 gene knockout mutant lines which can be stably inherited are shown as SEQ ID NO. 8 and SEQ ID NO.

9.

8. A method of increasing the number of locules and the weight of single fruit of tomato according to claim 4, characterized in that, The primers used for screening the homozygous mutant plants are shown as SEQ ID NO. 10 and SEQ ID NO. 11.

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