Tomato RNA methylation modification recognition protein sl ect2 and application thereof

Abstract

The application discloses a tomato RNA methylation modification recognition protein SlECT2 and application thereof, and belongs to the field of plant genetic engineering and molecular breeding. The amino acid sequence of the SlECT2 protein is shown as SEQ ID No. 1. The SlECT2 gene is expressed in tomato plants, and the expression amount in reproductive organs is relatively high, and the expression amount in mature anthers is higher than that in young anthers. In the application, a tomato strain with specific knockout of the SlECT2 gene is obtained by a genetic engineering method, and the knockout mutant has abnormal pollen development at normal temperature, but the plant vegetative growth and female reproductive organs develop normally. The gene can be applied to utilization of tomato heterosis as a functional gene for regulating tomato pollen development, can reduce the hybrid seed production cost of excellent hybrid F1 generation, and has great development and utilization value.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding, specifically to the tomato RNA methylation modification recognition protein SlECT2 and its applications. Background Technology

[0002] Male sterility in crops is the genetic basis of crop hybridization breeding. The discovery and utilization of male-sterile lines has ushered in a new era of crop hybridization breeding, offering unparalleled advantages in heterosis utilization and seed production. There are two main types of plant male-sterile lines available for production: one is cytoplasmic male sterility, currently widely used in hybridization breeding of major crops such as rice and rapeseed. However, it suffers from difficulties in restorative line selection, cumbersome conversion processes, and long breeding cycles; its cytoplasm is singular and easily affected by temperature, leading to instability in fertility and conversion between fertile and sterile states. The other type is photoperiod-thermosensitive genic male sterility, which eliminates the cross-pollination process required for sterile line propagation and overcomes the side effects of sterile cytoplasm. However, its propagation and seed production processes are constrained by light and temperature conditions, so scientists have been continuously seeking and cultivating new sterile lines.

[0003] Tomato (Solanum lycopersicum L.) is a plant belonging to the genus Solanum in the family Solanaceae. It plays an important role in vegetable production and consumption in my country and is also a model plant for reproductive development research. Tomatoes were one of the earliest vegetable crops to utilize heterosis. Currently, all commercially available varieties are F1 hybrids. However, due to the lack of usable male-sterile lines, male-sterile line seed production has not been widely used in production, lagging far behind cruciferous vegetables and even behind peppers, which are also solanaceous.

[0004] The development of the anther in tomato male reproductive organs is a complex process strictly regulated by multiple genes at the transcriptional or post-transcriptional levels, involving numerous regulatory factors. However, further research is needed on the molecular mechanisms regulating tomato anther development, and the number of genes regulating male sterility identified in tomatoes is still quite limited.

[0005] N6-adenosine methylation (m 6 A) is the most conserved and widely distributed chemical modification of eukaryotic mRNA, regulating processes such as folding, splicing, nucleus exit, stability, degradation, and translation of the modified RNA, thereby affecting plant growth and development and responses to various stresses. 6 RNA modification is dynamically regulated by both the writer (methyltransferase) and the eraser (demethylase). The reader (recognition protein) can recognize and bind to RNA m. 6 A modification regulates the metabolism of the modified RNA. Therefore, studying m 6 A reader for understanding m 6A plays a crucial role in various biological processes.

[0006] SlECT2 (EVOLUTIONARILY CONSERVED C-TERMINAL REGION 2), a tomato RNA methylation modification recognition protein, is a potential m 6 A recognition protein, which is similar to m already identified in Arabidopsis. 6 The A recognition protein AtECT11 is a homologous protein possessing the conserved YTH domain, a common structural domain shared by the Arabidopsis thaliana family. Studies have shown that Arabidopsis m... 6 The reading protein ECT2 regulates fur development. Whether reading proteins are involved in tomato anther development is currently unclear. Therefore, research on tomato YTH family proteins is needed to identify tomato m proteins that can influence pollen development. 6 A recognizes the protein and analyzes m. 6 A recognition protein is involved in the molecular mechanism of regulating the reproductive growth of tomato plants, and is expected to provide genetic resources for the development of usable tomato core male sterile lines. Summary of the Invention

[0007] The purpose of this invention is to discover key genes that regulate the development of male organs in tomatoes from the tomato genome, and to create new male-sterile tomato germplasm that causes pollen abortion at room temperature without affecting vegetative growth and pistil development using gene editing technology.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides the application of the tomato RNA methylation modification recognition protein SlECT2 in regulating pollen development in tomato plants, wherein the amino acid sequence of the tomato RNA methylation modification recognition protein SlECT2 is shown in SEQ ID No. 1.

[0010] The nucleotide sequence of the gene encoding the tomato RNA methylation modification recognition protein SlECT2 is shown in SEQ ID No. 2.

[0011] This invention demonstrates that the SlECT2 protein participates in regulating the development of male reproductive organs in tomatoes. Spatiotemporal expression pattern analysis revealed that SlECT2 is expressed in all tissues except leaves (roots, stems, flowers, and fruits), with higher expression levels in reproductive organs. The expression level is significantly higher in mature flowers compared to immature flowers.

[0012] Furthermore, the application includes: using biological techniques to downregulate the expression of the tomato RNA methylation modification recognition protein SlECT2 in tomato inbred lines to obtain tomato nuclear male sterile lines.

[0013] Mutant plants obtained by mutating the nucleotide sequence of the SlECT2 encoding gene or inhibiting SlECT2 protein expression using genetic engineering methods exhibit abnormal pollen development at room temperature, but normal vegetative growth and female reproductive organ development, resulting in new tomato core male sterile lines.

[0014] Furthermore, the biological techniques include knocking out or silencing the gene encoding the tomato RNA methylation modification recognition protein SlECT2 in the tomato genome through genetic transformation.

[0015] By knocking out or silencing the SlECT2 gene in tomato, the expression level of the SlECT2 gene is reduced in wild-type tomatoes, thereby obtaining a new tomato male-sterile line that can be used to produce F1 generation hybrid seeds.

[0016] Furthermore, the knockout method includes CRISPR / Cas9 technology. The SlECT2 gene is knocked out at specific sites using the CRISPR / Cas9 system to suppress the expression of the nucleotide sequence encoding the sequence shown in SEQ ID No. 2.

[0017] Furthermore, the application includes: using biological techniques to overexpress the gene encoding the tomato RNA methylation modification recognition protein in tomato plants to obtain transgenic plants with enhanced floral organ development.

[0018] Secondly, the present invention provides a method for creating a male-sterile tomato line, comprising: using gene editing technology to knock out the gene encoding the tomato RNA methylation modification recognition protein SlECT2 with the amino acid sequence shown in SEQ ID No. 1 in the genome of a tomato inbred line, thereby obtaining a male-sterile tomato line.

[0019] Select superior tomato inbred lines and use genetic engineering methods to change the nucleotide sequence of the SlECT2 encoding gene as shown in SEQ ID No. 2, so that the amino acid sequence shown in SEQ ID No. 1 is deleted or mutated, thereby causing the protein corresponding to the amino acid sequence to lose its activity.

[0020] Furthermore, this invention provides a method for creating male-sterile tomato lines based on CRISPR / Cas9 system site-specific knockout technology, comprising the following steps:

[0021] (1) sgRNA primers were designed and synthesized based on the sgRNA target sequence of the gene encoding the tomato RNA methylation modification recognition protein SlECT2; the nucleotide sequence of the sgRNA target sequence is shown in SEQ ID No. 3;

[0022] (2) Anneal the sgRNA primers into double-stranded sgRNA, clone it into the CRISPR / Cas9 vector, and construct the tomato SlECT2 gene knockout expression vector.

[0023] (3) The target fragment was transferred into tomato varieties using Agrobacterium infection method, and mutant plants with specific knockout of the gene encoding the tomato RNA methylation modification recognition protein SlECT2 were screened.

[0024] Furthermore, using the mutant plant as the female parent and the tomato inbred line as the male parent, fertile F1 plants were obtained by hybridization; the tomato inbred line was used as the recurrent parent for backcrossing, and heterozygous fertile plants were selected for further backcrossing. The backcrossing was repeated until the comprehensive traits of the heterozygous fertile plants were similar to those of the recurrent parent, and then they were self-pollinated to obtain homozygous sterile lines.

[0025] Furthermore, the tomato inbred line is the tomato variety Mirco-Tom.

[0026] Thirdly, the present invention provides the application of the tomato male-sterile line obtained by the above-described creation method in tomato seed production, the application including: using the tomato male-sterile line as the female parent and the tomato inbred line as the male parent to breed F1 generation hybrid combinations.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) This invention discloses for the first time the function of the tomato RNA methylation modification recognition protein SlECT2 gene in regulating the development of tomato anther organs. By modifying the tomato SlECT2 gene sequence and the amino acid sequence of its encoded protein, the goal of controlling the male reproductive process of tomatoes can be achieved. This gene can be used as a functional gene to regulate tomato pollen development and applied to the utilization of heterosis in tomatoes, which can reduce the cost of hybrid seed production of superior hybrid F1 generation.

[0029] (2) The tomato male-sterile line prepared using the method provided in this invention exhibits no abnormal phenotype during the vegetative growth stage of tomatoes, and the plant growth and flowering / fruiting times are not significantly different from the wild type. However, during the reproductive growth stage, anther development is abnormal, and pollen abortion occurs, resulting in almost completely sterile plants. If this male-sterile line is applied to hybridization breeding, it can eliminate the need for emasculation of the female parent, improve production efficiency, reduce labor costs, and has good application prospects. Attached Figure Description

[0030] Figure 1This section presents the spatiotemporal expression characteristics of SlECT2 in tomato in Example 1. A represents the expression pattern of SlECT2 in different tissues and stages of mature tomato flowers; R: root; S: stem; L: leaf; GF: green fruit; RF: red fruit; FⅠ-Ⅵ represent the tomato pollen mother cell stage, tetrad stage, early mononuclear microspore stage, mid-to-late mononuclear microspore stage, binuclear microspore stage, and pollen maturation stage, respectively. Variance is expressed as ±SDs, based on three biological statistics. B represents the electronic expression profile of SlECT2.

[0031] Figure 2 The table shows the expression activity of the SlECT2 promoter in different organs of tomato in Example 1. A represents the root; B the stem; C the leaf; D the immature inflorescence; E the mature flower; and F the fruit. The scale bar is 5 mm. SlECT2 is expressed in most tissues of tomato, with higher expression levels in the root, stem, mature flower, and fruit. In mature flowers, the signal is strongly expressed in the petals, anthers, and sepals. However, GUS signaling is almost absent in leaves, and in immature flowers, it is only expressed in trace amounts at the budding site of the anthers at the tip.

[0032] Figure 3 This is an example of Agrobacterium-mediated genetic transformation of tomato in Example 2. A represents sowing; B represents sterile seedlings; C represents co-culture; D represents differentiation culture; E represents subculture; FG represents rooting culture; and H represents transplanting.

[0033] Figure 4 This refers to the gene editing type used in Example 2, where the homozygous knockout line was knocked out.

[0034] Figure 5 The results are shown in Example 2 and the qRT-PCR detection of the transformed tomato overexpression lines. In this diagram, A represents the PCR detection of T-DNA insertion, M is the marker, and lanes 1-10 represent the PCR positive detection products; B represents the relative expression level of SlECT2 in the stamens of different overexpression lines during the flowering period.

[0035] Figure 6 This study observed the plant height and floral organ morphology of the transgenic tomato lines OEECT2 23-1 and CR-slect2 in Example 3. A represents plant height; B represents sepal length; C represents petal length; D represents stamen length; E represents pistil length; F represents the morphology of the transgenic plants, from left to right: WT, OE-23-1, and CR-slect2 plants; G represents the floral organ morphology, from left to right: WT, OE-23-1, and CR-slect2, with G1 representing sepals, G2 representing petals, G3 representing pistils, G4 representing stamens, and G5 representing the morphology of the entire flower. **: P < 0.01, ***: P < 0.001 (Student t test). Scale bar: 1 cm (F), 5 mm (G).

[0036] Figure 7 The images show the fruit morphology of transgenic tomatoes OE-23-1 and CR-slect2 from Example 3. A represents fresh fruit weight; B represents fruit longitudinal diameter; C represents fruit transverse diameter; D represents the number of seeds; and E represents a cross-section of the transgenic plant fruit. *: P < 0.05, **: P < 0.01, ***: P < 0.001 (Student t test). Scale bar: 1 cm.

[0037] Figure 8 The images show the pollen morphology of the transgenic tomato plants in Example 3. A shows the pollen morphology at 4x magnification using Alexander staining; B shows the pollen morphology at 500x magnification using a scanner electron microscope; and C shows the pollen morphology at 3000x magnification using a scanner electron microscope.

[0038] Figure 9 DAPI staining observation of pollen at various levels in the tomato CR-slect2 knockout line of Example 3. MMC, pollen mother cell; Tds, tetrad; MSN, microspore nucleus; VN, vegetative nucleus; GN, reproductive nucleus.

[0039] Figure 10 The image shows a semi-thin section of the anther of the transgenic tomato CR-slect2 from Example 3. UMsp, uninucleate microspore; T, tapetum; BMsp, binucleate pollen; DBMsp, degraded binucleate pollen; PG, pollen grain; DPG, degraded pollen grain. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. Specifically, the DNA purification kit was purchased from Hangzhou Zhenbai Biotechnology Co., Ltd., and various DNA restriction endonucleases were purchased from Thermo Fisher Scientific (China) Co., Ltd. All primers used were synthesized by Youkang Company, and sequencing was performed by Hangzhou Qingke Zixi Biotechnology Co., Ltd.

[0042] Example 1: Isolation and Expression Characteristics Analysis of Tomato SlECT2 Gene

[0043] 1. The CDS sequence of the tomato SlECT2 gene described in this embodiment is 1668 bp, and the nucleotide sequence is shown in SEQ ID No. 2.

[0044] The tomato SlECT2 gene encodes 555 amino acids, and its amino acid sequence is shown in SEQ ID No. 1. Pfam analysis revealed that its conserved domain YT521-B-like domain (PF04146) is located at amino acids 327-469.

[0045] The YTH protein family shares the same conserved domain, YTH521-B-like domain (PF04146). Phylogenetic analysis was performed on the YTH protein family in humans, Arabidopsis, and tomato. The results showed that tomato SlECT2 is homologous to Arabidopsis AtECT11, both belonging to the human YTHDF family of homologous genes. Furthermore, the YTH protein families in Arabidopsis and tomato generally exhibit high homology, indicating that the YTH domain is relatively conserved during evolution.

[0046] 2. The spatiotemporal expression patterns of SlECT2 in different parts of tomato roots, stems, leaves, flowers, and fruits were investigated using qRT-PCR.

[0047] qRT-PCR amplification was performed using tomato tissue cDNA as a template. The primers are as follows:

[0048] qRT-PCR-F:5'-GCTGTCCAGTGTTCCTCTTC-3'(SEQ ID No.4);

[0049] qRT-PCR-R: 5'-CAAGCTGCCAAAAGTCCATG-3' (SEQ ID No. 5).

[0050] The results are as follows Figure 1 As shown, SlECT2 is expressed in most tissues, especially in reproductive organs, where its expression level is approximately five times higher than that in roots. Comparison of different anther developmental stages revealed higher expression levels in the later stages of anther development. Further verification confirmed the dominant expression of SlECT2 in fruits; compared to young anthers, the expression level was higher in mature anthers, indicating that SlECT2 may play an important role in the process from anther development to fruit maturity.

[0051] 3. To further confirm the spatiotemporal expression pattern of SlECT2, primers were designed by searching for the 2500bp upstream sequence of the SlECT2 gene in the plant genome database. Using tomato leaf genomic DNA as a template, specific primers were designed to amplify approximately 1500bp upstream of the gene as the promoter fragment. The primer sequences are as follows:

[0052] ProECT2-1300-F:

[0053] 5'-AACGACGGCCAGTGCCAAGCTTAGTTGTTAGGGTTGGGGGT-3'

[0054] (SEQ ID No. 6);

[0055] ProECT2-1300-R:

[0056] 5'-ATGGTACCGTGGATCCTCTAGATGAGGGAAACGAAAGAGGA-3'

[0057] (SEQ ID No.7).

[0058] The GUS signal expression vector 35S-pCAMBIA-1300-GUS was constructed by homologous recombination using a promoter fused with the restriction endonuclease HindIII and XbaI linearized to GUS signal. After successful sequencing, this vector plasmid was transformed into Agrobacterium GV3101. Genetic transformation of tomato was achieved via tissue culture. Genomic DNA was extracted from the T0 generation plants, and positive plants were screened by PCR. The PCR primer sequences used for positive plant screening are as follows:

[0059] 1300-F:5'-CTGCAAGGCGATTAAGTTGGG-3' (SEQ ID No. 8);

[0060] proECT2-JC-R:5'-AATGTCCAAAATTTGAGAAATT-3' (SEQ ID No. 9).

[0061] GUS staining was performed on various tissues of the positive plants, and the results are as follows: Figure 2 As shown, GUS signaling is highly expressed in the roots, stems, mature flowers, and fruits of tomatoes, especially in mature flowers, where the signal is strongly expressed in petals, anthers, and sepals. However, GUS signaling is almost absent in leaves, and in immature flowers, it is only expressed in trace amounts at the germination site of the apical anthers.

[0062] Example 2: Obtaining Tomato SlECT2 Gene Overexpression and Knockout Mutant Plants

[0063] 1. Constructing the SlECT2 gene overexpression vector

[0064] Using tomato leaf cDNA as a template, the CDS sequence of the SlECT2 gene was amplified using high-fidelity KOD enzyme.

[0065] Primers for CDS sequence amplification of the SlECT2 gene (including homologous arms):

[0066] OE-SlECT2-F1:5'-GCCGATATCGTCGACATGGCTGGTGAGAAGATT ATAGA-3' (SEQ ID No. 10);

[0067] OE-SlECT2-R1:5'-GTATGGGTAGGTACCGACACTACTTTTGAATGG CCTT-3' (SEQ ID No. 11);

[0068] The overexpression vector pFGC-1008 was linearized using SalⅠ and KpnⅠ. The CDS fragment and the vector were ligated using homologous recombination. The ligation product was transferred into E. coli DH5α, plated on solid LB plates containing Cmr, single colonies were selected, positive clones were identified by PCR and sent to the company for sequencing. The plasmid with correct sequencing was returned and named pFGC-1008-SlECT2.

[0069] Vector detection primers:

[0070] PFGC-1008-F2:5'-AAACCTCCTCGGATTCCATTG-3' (SEQ ID No. 12);

[0071] PFGC-1008-R2: 5'-AGGCGTCTCGCATATCTCATT-3' (SEQ ID No. 13).

[0072] 2. Constructing the SlECT2 gene knockout vector

[0073] The sgRNA target sequence sgect2 for slECT2 was designed using the online CRISPR-P 2.0 website. The sgect2 sequence begins at the 219th base of the CDS, the fourth exon, and is 20 bp in length. Based on the sgect2 sequence, a pair of complementary primers for synthesizing the sgRNA were designed.

[0074] Tomato SlECT2 gene target sequence:

[0075] sgect2:5'-TGAGCAGGCTTACTTTAATG-3' (SEQ ID No. 3).

[0076] Primers for synthesizing sgRNA:

[0077] sgect2-F:5'-GATTGTGAGCAGGCTTACTTTAATG-3' (SEQ ID No. 14);

[0078] sgect2-R:5'-AAACATTAAAGTAAGCCTGCTCAC-3' (SEQ ID No. 15).

[0079] The synthesized sgect2-F / R primers were diluted to 100 μM and annealed to form double-stranded sgRNA. PMD-18T (the vector used in CRISPR / Cas9, courtesy of Professor Zhu Jiankang from the Shanghai Institutes for Biological Sciences) was linearized using BpiⅠ, and the vector fragment was recovered via gel electrophoresis. This fragment was then ligated to the annealed and diluted sgRNA. The ligation product was transformed into *E. coli* DH5α, Amp-resistant strains. After single-cell growth, plaques were picked and sent to the company for sequencing. The correctly sequenced plasmid was named PMD-18T-SlECT2. PMD-18T-SlECT2 and pCAMBIA-1301 were double-digested using KpnⅠ and HindⅢ, and the target fragment was recovered via gel electrophoresis (the vector was larger than 10000 bp; the recovered product of PMD-18T-SlECT2 was approximately 5600 bp). The vector and the target fragment were ligated using T4 DNA ligase. The ligation product was transformed into E. coli DH5α, Kan resistant, and single colonies were selected. Positive clones were identified by PCR and sent to the company for sequencing. The sequencing results were correct and named pCAMBIA1301-SlECT2-CRISPR / Cas9.

[0080] 3. Obtaining and identifying overexpression and knockout plants

[0081] The two vectors pFGC-1008-SlECT2 and pCAMBIA1301-SlECT2-CRISPR / Cas9 were transformed into tomato "Mirco-Tom" using Agrobacterium infection. The Agrobacterium-mediated genetic transformation process of tomato is as follows: Figure 3 As shown.

[0082] Genomic DNA was extracted from T0 generation transgenic plants. The T-DNA insertion status of pCAMBIA1301-SlECT2-CRISPR / Cas9 transgenic plants was detected using universal primers for the pCAMBIA-1301 vector. Transgenic plants with T-DNA insertion were selected.

[0083] universal primers for pCAMBIA-1301 vector:

[0084] Cas9-F:5'-TGACCAGAAGCGACAAGAA-3' (SEQ ID No. 16);

[0085] Cas9-R: 5'-CTTATCGCTGTTCCTCTTGG-3' (SEQ ID No. 17).

[0086] Using specific knockout detection primers designed with approximately 300 bp upstream and downstream of the sgRNA, the genomic fragment was amplified and sent to the company for sequencing to screen for mutant plants.

[0087] Knockout type detection primers:

[0088] detect-sgect2-F:5'-GAGGGTTCGGCAGAAGTAT-3' (SEQ ID No. 18);

[0089] detect-sgect2-R:5'-TTGGAGCAGCACTATTGGT-3' (SEQ ID No. 19).

[0090] Primers for positive detection of T-DNA insertion in pFGC-1008-SlECT2 transgenic plants:

[0091] 1008-F:5'-AAACCTCCTCGGATTCATTG-3' (SEQ ID No. 12);

[0092] OE-SlECT2-R:

[0093] 5'-GTATGGGTAGGTACCGACACTACTTTTGAATGGCCTT-3' (SEQ ID No. 11);

[0094] After screening by PCR and GUS staining, a total of 14 SlECT2 overexpressing lines and 23 SlECT2 knockout positive transgenic lines were obtained.

[0095] Homozygous lines of SlECT2 knockout positive transgenic lines were screened using PCR amplification and sequencing analysis. Sequencing verification yielded three homozygous deletion expression lines, designated sg16-7, sg28-3, and sg31-1. Specifically, sg16-7 and sg31-1 each had a 5bp deletion and a 1bp addition in the SlECT2 gene coding region, while sg28-3 exhibited a large deletion mutation, deleting 98bp of the SlECT2 gene coding region. Figure 4 ).

[0096] qRT-PCR was performed on stamens from different tomato overexpression lines during flowering. The results showed that the bud lines OE-SlECT2-1-1, OE-SlECT2-4, and OE-SlECT2-16-2 showed the highest overexpression compared to WT, exceeding 15-fold. The expression levels of bud lines OE-SlECT2-23-1 and OE-SlECT2-33-2 were approximately 5-fold higher than WT. Because a large number of seed samples were retained from the bud line OE-SlECT2-23-1 T0 generation (abbreviated as OE-23-1), this bud line was selected for further research. Figure 5 ).

[0097] Example 3: SlECT2 knockout mutants affect pollen development and fruit traits

[0098] 1. Observation of the dynamic growth of the obtained SlECT2 overexpression and knockout tomato lines revealed that, compared with WT, the transgenic plants showed no significant differences in plant type, leaf growth, and growth rate. However, the overexpression bud line OE-23-1 was taller overall, and its plant height (21.7 cm) was significantly higher than that of WT (14.7 cm) when it first started flowering.

[0099] Further observation was conducted on the effects of SlECT2 on the development of tomato flower organs, and the results were as follows: Figure 6 As shown, the flower organs of the OE-23-1 tomato plant were generally larger, with the lengths of its sepals, petals, stamens and pistils all significantly greater than those of WT; CR-slect2 (i.e., the homozygous deletion expression line sg16-7) showed significant differences from WT only in the sepals.

[0100] The results above indicate that SlECT2 overexpression significantly affects tomato plant height and floral organ development, with the plants showing an overall trend of increasing size; while the SlECT2 mutant showed no significant difference in floral organ appearance compared to the wild type.

[0101] 2. Statistical analysis was performed on the fruit characteristics of overexpression and knockout mutants and WT control plants as they grew to fruit maturity, including weight, transverse diameter, longitudinal diameter, and number of seeds.

[0102] The results are as follows Figure 7 As shown, compared with WT, the overexpressing plant OE-23-1 had larger fruits, but the difference was not significant. Only the fruit weight was significantly greater than WT, while the transverse diameter, longitudinal diameter, and number of seeds were not significantly different from WT. The mutant plant CR-slect2 had significantly smaller fruits, with its fruit weight, transverse diameter, longitudinal diameter, and number of seeds being significantly smaller than WT. This may be related to the decreased pollen viability of CR-slect2, resulting in incomplete pollination.

[0103] 3. Alexandrite staining was performed on mature pollen from SlECT2 transgenic plants, and the results are as follows: Figure 8As shown in Figure A, compared with the control plant WT pollen viability (98.6%), the overexpression line OE-23-1 showed normal pollen viability (99.2%). Although the SlECT2 mutant had normal vegetative growth and normal floral organ morphology, Alexandrine staining showed highly sterile pollen, with pollen viability reduced to 25.7%. Figure 8 As shown in B and C, scanning electron microscopy revealed that CR-slect2 pollen morphology was abnormal, with surface collapse and abnormal germination furrows. The percentage of normal morphology was only 17.0%, far lower than WT (88.0%).

[0104] The above results indicate that SlECT2 gene knockout has a significant impact on pollen viability.

[0105] 4. DAPI staining results ( Figure 9 The results showed that pollen from both CR-slect2 and WT plants could produce tetrads and release uninucleate microspores through normal meiosis. However, during the transition from the late uninucleate stage (microspore nucleus marginal stage) to the binucleate stage and the first mitosis (PMI), only some pollen grains from CR-slect2 could divide normally to produce vegetative nuclei (VN) and reproductive nuclei (GN), while some pollen grains had no detectable nucleus at all. Furthermore, during the maturation stage, some pollen grains in CR-slect2 exhibited abnormalities, preliminarily indicating that pollen abortion in CR-slect2 transgenic plants begins from the binucleate stage.

[0106] 5. DAPI staining and paraffin sectioning pinpointed the pollen abortion period to the binucleate stage. Therefore, semi-thin sections of anthers from late uninucleate, binucleate, and mature tomato plants were observed. The results were consistent with the above: some pollen cells underwent abnormalities during the binucleate stage until complete shrunkenness at maturity, but the tapetum degraded normally during the binucleate stage. Figure 10 ).

[0107] Example 4: Creation of a male-sterile line using the SlECT2 gene and its utilization

[0108] Using the male-sterile SlECT2 knockout mutant CR-slect2 as the female parent and a superior tomato inbred line as the male parent, fertile F1 plants were obtained through hybridization. The superior inbred line was then used as the recurrent parent for backcrossing. PCR testing revealed two genotypes in the offspring, with AA:Aa = 1:1. The heterozygous fertile plants were retained for further backcrossing. This backcrossing process was repeated until the overall traits of the heterozygous fertile plants were similar to those of the recurrent parent. These plants were then self-crossed, and the fertility ratio in the self-crossed offspring segregated at a 3:1 ratio, yielding a homozygous sterile line. This sterile line can be hybridized with a large number of tomato breeding materials (restorer lines) for F1 hybrid seed production, reducing the labor required for manual emasculation, thereby lowering the cost of F1 hybrid seed production, improving seed purity, and preventing parental loss. It has significant development and utilization value in agricultural production.

Claims

1. The application of the tomato RNA methylation modification recognition protein SlECT2 in regulating pollen development in tomato plants, characterized by, The amino acid sequence of the tomato RNA methylation modification recognition protein SlECT2 is shown in SEQ ID No. 1; The regulation involves using biological techniques to downregulate the expression of the tomato RNA methylation modification recognition protein SlECT2 in tomato inbred lines, thereby obtaining tomato nuclear male sterile lines.

2. The application as described in claim 1, characterized in that, The biological techniques include knocking out or silencing the gene encoding the SlECT2 protein, a tomato RNA methylation modification recognition protein, in the tomato genome through genetic transformation.

3. The application as described in claim 2, characterized in that, The knockout methods include CRISPR / Cas9 technology.

4. A method for creating a male-sterile tomato line, characterized in that, include: By using gene editing technology to knock out the gene encoding the tomato RNA methylation modification recognition protein SlECT2, whose amino acid sequence is shown in SEQ ID No. 1, a tomato nuclear male sterile line was obtained.

5. The method for creating a male-sterile tomato line as described in claim 4, characterized in that, Includes the following steps: (1) sgRNA primers were designed and synthesized based on the sgRNA target sequence of the gene encoding the tomato RNA methylation modification recognition protein SlECT2; the nucleotide sequence of the sgRNA target sequence is shown in SEQ ID No. 3; (2) Anneal the sgRNA primers into double-stranded sgRNA, clone it into the CRISPR / Cas9 vector, and construct the tomato SlECT2 gene knockout expression vector. (3) The target fragment was transferred into tomato varieties using Agrobacterium infection method, and mutant plants with specific knockout of the gene encoding the tomato RNA methylation modification recognition protein SlECT2 were screened.

6. The method for creating a male-sterile tomato line as described in claim 5, characterized in that, Using mutant plants with a specific knockout of the gene encoding the tomato RNA methylation modification recognition protein SlECT2 as the female parent and tomato inbred lines as the male parent, fertile F1 plants were obtained by hybridization. The tomato inbred lines were used as recurrent parents, and backcrossing was performed. Heterozygous fertile plants were selected and backcrossed again. The backcrossing was repeated until the comprehensive traits of heterozygous fertile plants were similar to those of the recurrent parents. These plants were then self-crossed to obtain homozygous sterile lines.

7. The method for creating a male-sterile tomato line as described in claim 4, characterized in that, The tomato inbred line is the tomato variety Mirco-Tom.

8. The application of the tomato male-sterile line obtained by the creation method according to any one of claims 4-7 in tomato seed production, characterized in that, The application includes: using the tomato male-sterile line as the female parent and the tomato inbred line as the male parent to breed F1 generation hybrid combinations.

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