Application of a reagent for inhibiting the expression level of SlABF4 in promoting fruit development

Abstract

The present invention belongs to the field of plant genetic engineering, and particularly relates to the application of a reagent for inhibiting the expression level of SlABF4 in promoting fruit development. The present invention discovers that overexpression SlABF4 will lead to a delay in the initiation of ripening and a decrease in ethylene production. On the contrary, knocking out SlABF4 will lead to premature fruit ripening and an increase in ethylene production. Therefore, it is proposed that a reagent for inhibiting the expression level of SlABF4 be applied in promoting fruit development.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and particularly relates to the application of a reagent for inhibiting the expression level of SlABF4 in promoting fruit development. Background Art

[0002] The ripening process of fleshy fruits is a series of complex physiological and biochemical processes, accompanied by changes in fruit color, texture, flavor, aroma, and other quality characteristics. Fleshy fruits can be classified into climacteric fruits and non-climacteric fruits according to whether the respiration and ethylene production increase at the beginning of ripening. Tomato, with the Latin name Solanum lycopersicum , is a typical climacteric fruit and has been selected as a model for fruit ripening due to its diploid genome, complete genome sequence annotation, 3-5 month life cycle, easy stable transformation, and availability of mutations related to natural ripening.

[0003] Tomato fruit ripening is a highly coordinated developmental process involving the expression and regulation of thousands of genes. Based on the characteristics and analysis of several well-known gene mutants, the regulatory network of tomato fruit ripening has been studied in detail, such as ripening inhibitor , non-ripening , Colorless non-ripening , and never-ripening , etc., gene mutants that inhibit tomato fruit ripening. Ethylene is the main trigger for the ripening of climacteric fruits. Inhibiting the biosynthesis of ethylene or its signal transduction can prevent the initiation of ripening. The biosynthesis of ethylene is regulated by two rate-limiting enzymes: 1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase. The abbreviation of 1-aminocyclopropane-1-carboxylic acid synthase is ACS, and the abbreviation of 1-aminocyclopropane-1-carboxylic acid oxidase is ACO. In higher plants, ACS and ACO belong to multiple gene families, among which SlACS2 , SlACS4 , and SlACO1 have been identified as key genes for ethylene biosynthesis in tomatoes. Transcription factors, with the English name transcription factors and the abbreviation TFs, also play a key role in the regulation of fruit ripening. Some fruit-related TFs have been identified as key components in the tomato ripening process, including the MADS-box proteins RIPENING INHIBITOR, TOMATO AGAMOUS-LIKE 1, FRUITFULL 1, FRUITFULL 2, and SlMADS1; the SBP-box protein COLORLESS NON-RIPENING; the NAC domain protein NON-RIPENING; and the HD-zip homeodomain protein HOMEBOX 1.

[0004] In plants, basic leucine zipper proteins, abbreviated as bZIP, and bZIP transcription factors are one of the largest transcription factor families. ABA and abiotic stresses regulate gene expression through cis - acting elements, including ABA - responsive elements, abbreviated as ABRE. The sequence of ABRE is PyACGTGG / TC. ABRE - binding factors / ABA - responsive element - binding proteins can bind to various ABRE - containing promoters. ABRE - binding factors are abbreviated as ABF, and ABA - responsive element - binding proteins are abbreviated as AREB. The ABF / AREB gene family is a branch of the bZIP TF family and is conserved in many species and is the most well - known TF in the ABA signaling pathway. It has been reported that ABF proteins can be activated by phosphorylation of SnRK2 protein kinases. In Arabidopsis thaliana, four ABF genes have been discovered and functionally characterized, and homologous genes of ABF have also been found in other species, including 35 members in wheat, 9 members in potato, 10 members in sandalwood, 8 members in jute, 14 members in poplar, and 3 members in litchi. AtABF1 is induced by low temperature, and the expression levels of AtABF2 / 3 / 4 in vegetative tissues increase in response to ABA, drought stress, and salt stress. Multiple studies have shown that the tomato gene SlAREB1 responds to drought and salt stress, and its encoded protein is regulated by ABA - dependent post - translational modification. SlAREB1 can also regulate SlDFR and SlF3'5'H transcription levels, thereby increasing the accumulation of anthocyanins under low temperature. ABA has been shown to play an important role in fruit ripening, promoting ethylene production and response by regulating various genes involved in ethylene synthesis and signal transduction. However, research on the function of ABF / AREB genes during tomato fruit ripening is still lacking, and it is still unclear whether SlABFs regulate other physiological aspects of fruit development. Summary of the Invention

[0005] To solve the above problems, the present invention provides the application of a reagent that inhibits the expression level of SlABF4 in promoting fruit development.

[0006] The application of a reagent that inhibits the SlABF4 expression level in promoting fruit development, wherein the SlABF4 gene number is Solyc11g044560.

[0007] Preferably, transgenic plants are obtained by knocking out the SlABF4 to promote fruit development.

[0008] Preferably, the method of knocking out the SlABF4 includes base insertion, base deletion, and insertion or formation of a stop codon.

[0009] Preferably, the reagent for inhibiting SlABF4 the expression level includes the sequences shown in SEQ ID NO.3 to SEQ ID NO.4.

[0010] Preferably, the reagent for inhibiting SlABF4 the expression level further includes the pTX041 vector.

[0011] Preferably, the method for obtaining the transgenic plant includes the following steps: Designing a knockout target sequence according to the SlABF4 gene sequence, cloning the knockout target sequence into the pTX041 vector, and obtaining the transgenic plant through genetic transformation;

[0012] The knockout target sequence is as shown in SEQ ID NO.3 to SEQ ID NO.4.

[0013] Preferably, the transgenic plant contains the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] Preferably, the promotion of fruit development refers to the promotion of fruit ripening.

[0015] Preferably, the fruit is a tomato.

[0016] A method for promoting fruit development, by knocking out the SlABF4 to obtain a transgenic plant to improve the promotion of fruit development;

[0017] The promotion of fruit development refers to the promotion of tomato ripening.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention discovers that overexpression of SlABF4 will lead to a delay in the initiation of ripening and a decrease in ethylene production. On the contrary, knocking out SlABF4 will lead to premature fruit ripening and an increase in ethylene production. Therefore, it is proposed to apply the reagent for inhibiting the expression level of SlABF4 in promoting fruit development.

[0020] The present invention discovers that SlABF4 inhibits the expression of SlACS2 and SlACS12 by directly binding to their promoters, thereby playing a negative regulatory role in tomato fruit ripening. In addition, SlABF4 can also affect the ripening of tomatoes by physically interacting with two key factors SlFUL1 and SlMADS1 involved in fruit ripening. The discovery of the present invention deepens the understanding of the role of the SlABF4 transcription factor and provides new insights into the regulatory network in tomato fruits. Description of the Drawings

[0021] Figure 1 Phylogenetic tree and sequence analysis of tomato SlABFs. A, Phylogenetic analysis; B, Amino acid sequence analysis of tomato SlABFs and Arabidopsis AtABF2. The positions of secondary structure amino acid sequences were determined using the crystal structure of AtABF2. C1-C4 represent conserved regions, and the bZIP region is marked with a double line.

[0022] Figure 2 Subcellular localization and expression pattern of SlABF4. A, Control GFP signal is present throughout the cell, while SlABF4-GFP is confined to the nucleus; B, Heatmap of different tissues in "Henzi" wild-type tomato SlABFs , with data based on RPKM of tomato eFP; C, Relative transcriptional levels in different tissues of "Micro-Tom" WT tomato SlABF4 , with the transcript in roots SlABF4 set to 1. Values represent the mean ± standard deviation of three biological replicates. Samples marked with different letters are significantly different, P < 0.05. D, Relative transcriptional levels of SlABF4 in response to 100 μM ABA treatment. Values represent the mean ± standard deviation of three biological replicates. Asterisks indicate significant differences determined by Student's t-test, P < 0.01.

[0023] Figure 3 Phenotypes of WT, OE- SlABF4 and CR- slabf4 lines. A, Genotype of the mutation at the SlABF4 locus generated by the CRISPR / Cas9 genome editing system. Two target sequences were specifically designed for the third exon. Mutations in transgenic plants were confirmed by sequencing the genomic regions flanking the target sites. B, Phenotypes of tomato plants of OE- SlABF4 -3, OE- SlABF4 -4, WT, CR- slabf4 -8, and CR- slabf4 -29 lines. C, qRT-PCR analysis of WT, OE, and CR fruits at the breaker stage SlABF4 , with values representing the mean ± SD of three biological replicates. Samples marked with different letters are significantly different at P < 0.05.

[0024] Figure 4 Tomato fruit ripening phenotypes of WT, OE-SlABF4, and CR-slabf4 lines. A, Phenotypes of tomato fruits of OE- SlABF4 -3, OE- SlABF4 -4, WT, CR- slabf4 -8, and CR- slabf4 -29 lines at different days after anthesis. B, WT, OE- SlABF 4, and CR-slabf4 The average number of days from flowering to fruit cracking, n > 100, C is WT, OE- SlABF4 and CR- slabf4 Ethylene production of fruits, values represent the mean ± SD of three biological replicates, asterisks indicate significant differences determined by Student's t-test, *P < 0.05, **P < 0.01, D is WT, OE- SlABF4 and CR- slabf4 Comparison of sugar content in fruits, values represent the mean ± SD of three biological replicates, samples marked with different letters are significantly different at P < 0.05.

[0025] Figure 5 For WT, OE- SlABF4 and CR- slabf4 Relative transcript levels of selected genes involved in ethylene production and key regulators of tomato fruit ripening in fruits, A is SlACO1 , B is SlACS2 , C is SlACS4 , D is SlACS12 , E is SlRIN , F is SlFUL1 , G is SlTAGL1 , H is SlNOR , I is SlCNR , J is SlAP2a , K is SlHB1 , L is SlMADS1 .

[0026] Figure 6For the interaction of SlABF4 with SlFUL1 and SlMADS1, A shows the yeast two-hybrid interaction of SlABF4 with SlFUL1 and SlMADS1. SlABF4 was fused with the pGADT7 vector, and SlSnRK2s, SlRIN, SlMADS1, SlFUL1, SlCNR, SlNOR, and SlAP2a were fused with the pGBKT7 vector. pGADT7-T + pGBKT7-53 was used as a positive control; pGADT7-T + pGBKT7-Lam was used as a negative control; pGADT7 + pGBKT7-SlABF4 was used as an auto-activation control. DDO, SD / -Leu / -Trp is a double-dropout medium; QDO, SD / -Ade / -His / -Leu / -Trp is a quadruple-dropout medium. B shows the BiFC detection of the interaction between SlABF4 and SlFUL1 and SlMADS1 in tobacco leaves. C shows the firefly luciferase complementation imaging detection of the interaction of SlABF4 between SlFUL1 and SlMADS1 in tobacco leaves. D and E are the Pull-down assays of the interaction between SlABF4 and SlFUL1 and SlMADS1, respectively. The protein extract was incubated with GST beads, and then the pulled-down fraction was analyzed by immunoblotting using an anti-SlABF4 antibody.

[0027] Figure 7 For SlABF4 negatively regulates SlACS2 and SlACS12 promoter activity. A shows the yeast one-hybrid assay of SlABF4 directly targeting SlACS2 and the promoter fragment of SlACS12. B shows the schematic diagram of the effector and reporter constructs for transient expression. C and D are the dual-luciferase assay data and imaging of SlABF4 inhibiting SlACS2 and SlACS12 promoter activity, respectively. Among them, C is the assay data, and D is the imaging. The values represent the average of three replicates. LUC: firefly luciferase activity; REN: Renilla luciferase.

[0028] Figure 8 For the electrophoretic mobility shift assay (EMSA) of the binding of SlABF4 to the promoters of target genes SlACS2 and SlACS12 “+” indicates presence; “-” indicates absence. Competitive probes used 50-fold and 100-fold unlabeled probes and mutant probes. A shows the schematic diagram of the ABRE motif in the promoters of SlACS2 and SlACS12 . B shows that the SlABF4 protein directly binds to the promoter region of SlACS2 in vitro. C shows that the SlABF4 protein directly binds to the promoter region of SlACS12 in vitro.

[0029] Figure 9 The model proposed for SlABF4 in the regulation of tomato fruit ripening is that the SlABF4 protein binds to SlACS2 and SlACS4 the AREB regions of, thereby inhibiting their transcriptional levels, further reducing ethylene production, and delaying tomato fruit ripening. SlABF4 can also directly interact with other SlFUL1 and SlMADS1, upregulating the expression of SlMADS1 while downregulating the expression of SlFUL1, thereby potentially affecting the expression of downstream target genes at the transcriptional level and resulting in delayed fruit ripening.

[0030] Figure 10 Gene characteristics of SLABF4. Among them, A is the transcriptional activation analysis of SlABF4 in the yeast system, B is the schematic diagram of pBD-SlABF4, C shows that pBD-SlABF4 significantly inhibits the expression of the LUC reporter gene compared with the pBD control group, D is the relative ratio of LUC to REN, and the values represent the average of three biological replicates. According to the Student's t-test, ** represents a significant difference at the P<0.01 level.

[0031] Figure 11 Identification of the SlABF4 overexpression line. Among them, A is the schematic diagram of the overexpression plasmid pRI101-S1ABF4, and B is the qRT-PCR analysis of the expression of SlABF4 during the shell-breaking period of wild-type and overexpressing fruits. The values represent the average ± standard deviation of three biological replicates.

[0032] Figure 12 Plant phenotypes of wild-type and transgenic tomato lines. Among them, A is plant height and plant width, and B is leaf length and leaf width. There are at least 15 plants for each transgenic line and WT. Error bars represent SD, and samples marked with different letters show significant differences at P<0.05. Detailed implementation methods

[0033] The following is a detailed description of the specific implementation methods of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation methods. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0034] In the present invention, CRISPR is a clustered regularly interspaced short palindromic repeat sequence, and Cas9 is a CRISPR-associated protein 9. The coding sequence is abbreviated as CDS.

[0035] SlACO1is named 1-aminocyclopropane-1-carboxylate oxidase 1, and its gene number is Solyc07g049530, SlACS2 is named 1-aminocyclopropane-1-carboxylate synthase 2, and its gene number is Solyc01g095080, SlACS4 is named 1-aminocyclopropane-1-carboxylate synthase4, and its gene number is Solyc05g050010, SlACS12 is named 1-aminocyclopropane-1-carboxylatesynthase 12, and its gene number is Solyc08g079750, SlRIN is named ripening inhibitor, and its gene number is Solyc05012020, SlFUL1 is named FRUITFULL 1, and its gene number is Solyc06g069430, SlTAGL1 is named TOMATO AGAMOUS-LIKE1, and its gene number is Solyc07g055920, SlNOR is named non-ripening, and its gene number is Solyc10g006880, SlCNR is named colorless non-ripening, and its gene number is Solyc02g077920, SlAP2a is named APETALA2a, and its gene number is Solyc03g044300, SlHB1 is named homebox 1, and its gene number is Solyc02g086930, SlMADS1 is named MADS-box protein 1, and its gene number is Solyc03g114840.

[0036] The overexpression vector pRI101-AN was purchased from Beijing Huayueyang Biotechnology Co., Ltd., with the model number Hyykb089; the pGADT7 vector was purchased from Beijing Huayueyang Biotechnology Co., Ltd., with the model number Hyykb105; the pBD vector was purchased from Shanghai Lianmai Bioengineering Co., Ltd., with the model number LM1658; the pGreenⅡ 62-SK vector was purchased from Wuhan Boyuan Biotechnology Co., Ltd., with the model number REC70-I; the binary vector pTX041 was the pTX041 used in the literature Cui L, Zheng F, Wang J, Zhang C, Zhang D, Gao S, Zhang C, Ye J, Zhang Y, Ouyang B et al: The tomato CONSTANS-LIKE protein SlCOL1 regulates fruit yield by repressing SFT gene expression. BMC plant biology 2022, 22(1):429.

[0037] 1. Plant materials and growth conditions

[0038] Tomato was selected as the gene transformation material, and the Latin name of the tomato Solanum lycopersicum cv., variety Micro-Tom. The wild type was denoted as WT, and WT and transgenic lines were cultured in a phytotron with a day-night temperature difference of 25°C / 20°C, a photoperiod of 16 hours of light and 8 hours of darkness. Flowers were marked during the flowering period to evaluate the fruit ripening stage. Roots, stems, leaves, seeds, flowers and pulp were collected to analyze tissue-specific gene expression. At least 10 fruits of consistent size were harvested for each biological replicate, and fruits at different stages from 15 days to 40 days after flowering were harvested in three independent replicates. After removing seeds and pectin, the peel and pulp were immediately frozen in liquid nitrogen and stored at -80°C until further use.

[0039] 2. Plasmid construction and plant transformation

[0040] The SlABF4 overexpression vector was constructed using the primers shown in SEQ ID NO.5~SEQ ID NO.6 and the overexpression vector pRI101-AN, and Agrobacterium was transformed to obtain OE- SlABF4 transgenic plants.

[0041] Two independent gRNAs of SlABF4 were designed, and the gRNAs are shown in SEQ ID NO.3~SEQ ID NO.4. They were cloned into the pTX041 binary vector to construct the CRISPR / Cas9 gene editing plasmid, and Agrobacterium was transformed to obtain CR- slabf4 transgenic plants.

[0042] Among them, the gene number of SlABF4 in the tomato genome database ITAG 4.0 is Solyc11g044560, the website address of the CRISPR-P website is http: / / crispr.hzau.edu.cn / CRISPR2 / , and the address of the tomato genome database is https: / / solgenomics.net / .

[0043] 3. RNA Extraction and Real-Time Fluorescent Quantitative PCR Analysis

[0044] In the present invention, the RNAprep Pure Plant Plus Kit was used to extract total RNA from various tissues and fruits of tomato plants according to the manufacturer's instructions, reverse-transcribed into cDNA, and specific primers were designed with cDNA as a template, as shown in SEQ ID NO.7 to SEQ ID NO.34. qRT-PCR amplification was performed using Hieff Universal Blue qPCR SYBRGreen Master Mix. The relative gene expression level was calculated using the 2 SlABF4, SlACO1, SlACS2, SlACS4, SlACS12, SlRIN, SlFUL1, SlTAGL1, SlNOR, SlCNR, SlAP2a, SlHB1, SlMADS1 method with the Bio-Rad CFX Manager software, and SlSAND was used as an internal reference gene. Among them, the RNAprep Pure Plant Plus Kit was from Tiangen Biotech (Beijing) Co., Ltd., the Hieff Universal Blue qPCR SYBR Green Master Mix was from Shanghai Yesen Biotech Co., Ltd., and the gene number of SlSAND in ITAG 4.0 was Solyc03g115810. -ΔΔCt

[0045] 4. Ethylene Content Measurement

[0046] A 1 mL gas sample was drawn from the top of the container with a 1 mL syringe and then injected into the gas chromatograph to measure the ethylene production. The peak elution time was determined and calibrated using standard ethylene gas. The column temperature was 80 °C, the detector temperature was 150 °C, the carrier gas was N2, and the flow rate was 40 mL·min -1 . Among them, the gas chromatograph was from Shimadzu, GC-2014, Japan.

[0047] Statistical Analysis

[0048] The data were analyzed using IBM SPSS Statistics v.25 software. Pairwise comparisons were performed using one-way ANOVA and Student's t ​- Detection, * represents P < 0.05, ** represents P < 0.01. Multiple comparisons were performed using Duncan's test, denoted as Duncan's test. Samples marked with different letters indicate significance at P < 0.05. IBM SPSS Statistics v.25 software is from IBM Corp., Armonk, NY, USA.

[0049] Table 1 shows the primers used in the present invention.

[0050] Table 1 Primers Used in the Present Invention

[0051]

[0052] Results

[0053] 1. Characteristics of Tomato Transcription Factor SlABF4

[0054] Four tomato SlABFs genes were identified by homologous alignment, namely SlABF1, SlABF2, SlABF3, and SlABF4. Among them, the SlABF4 gene consists of 6 exons and 5 introns. This gene encodes a 400 - amino - acid protein with a conserved bZIP domain, as shown in Figure 1 A - B in it. Phylogenetic analysis showed that ABF proteins can be divided into three groups. Among them, SlABF1 and SlABF4 belong to group I, and SlABF2 and SlABF3 belong to group II respectively, as shown in Figure 1 A in it. ABF family members have four conserved domains, where the C1, C2, and C3 domains are located at the N - terminus, while the C - terminus contains a highly conserved bZIP domain and a C4 domain. The amino - acid sequences of SlABF1 - 4 were aligned with the closest Arabidopsis homolog AtABF2, and Figure 1 four highly conserved regions are shown in B in it.

[0055] To study the subcellular localization of SlABF4, the present invention generated a 35S::SlABF4 - GFP fusion vector and transiently transfected it into tobacco leaves. The results showed that the control GFP signal was distributed throughout the cell, while SlABF4 was specifically localized in the nucleus, indicating that SlABF4 has potential nuclear functions, as shown in Figure 2 A in it. In addition, the present invention also analyzed the transcriptional activation activity of SlABF4. The results showed that it did not have transcriptional activation activity in the yeast system, as shown in Figure 10 A in it. In addition, in the dual - luciferase reporter system, compared with the empty vector, SlABF4 significantly inhibited the LUC reporter activity, as shown in Figure 10 B in it, indicating that SlABF4 may have the function of a transcriptional repressor.

[0056] 2. Expression analysis of SlABF4

[0057] To reveal the expression pattern of SlABF4, the present invention analyzed the gene expression profiles at different stages of tomato fruit development using the data in Tomato eFP Browser. SlABFs The analysis results showed that among the SlABFs genes, SlABF4 had the highest expression level, such as B in Figure 2 . In addition, the present invention also conducted qRT-PCR experiments to detect the transcript accumulation of SlABF4 in different tomato tissues. SlABF4 was expressed in all tested tissues. Among the non-fruit tissues, the expression level was the highest in stems. It is worth noting that the mature green stage is abbreviated as MG. During the fruit ripening process, SlABF4 transcript levels increased rapidly and reached a peak at the MG stage, and then gradually decreased until the Br+9 stage, such as C in Figure 2 . These results indicate that SlABF4 may play a role in regulating the fruit ripening process. Among them, the website of Tomato eFP Browser is https: / / bar.utoronto.ca / efp_tomato / cgi-bin / efpWeb.cgi.

[0058] In addition, the present invention also studied whether the expression of SlABF4 was regulated by the plant hormone ABA. As shown in D in Figure 2 , the effect of spraying 100 μM exogenous ABA treatment on the expression of SlABF4 gene was shown. Compared with the control group, the expression levels of SlABF4 in tomato MG fruits treated with exogenous ABA were higher at 12 h and 24 h after treatment. These results indicate that ABA-induced SlABF4 may be involved in the regulation of tomato fruit ripening.

[0059] 3. Effects of SlABF4 on fruit ripening

[0060] To explore the physiological phenotypes of SlABF4 in the processes related to fruit ripening, the present invention generated SlABF4 gene knockout lines using the CRISPR / Cas9-mediated gene editing system, namely two homozygous CR- slabf4 lines CR- slabf4 -8 and CR- slabf4 -29. Both of these lines had a 1-bp transition in the first guide RNA. CR- slabf4 -8 had a 61-bp deletion in the second guide RNA. CR- slabf4-29 has a 5 bp deletion in the second guide RNA. The first guide RNA is abbreviated as gRNA1, and the second guide RNA is abbreviated as gRNA2. CR- slabf4 -8 has the amino acid sequence shown as SEQ ID NO.1 in Table 2. CR- slabf4 -29 has the amino acid sequence shown as SEQ ID NO.2 in Table 2. These mutations led to premature termination of translation and SlABF4 different truncations of the protein, as shown in Figure 3 A. Meanwhile, the present invention also generated two independent homologous T2 overexpression lines, namely OE- SlABF4 -3 and OE- SlABF4 -4, for further analysis, as shown in Figure 11 . The present invention observed that the expression level of SlABF4 in the OE lines was significantly increased, while the expression level of SlABF4 in the two CR gene-edited lines was significantly decreased, as shown in Figure 3 C. The plant phenotypes of the WT line and the transgenic lines are shown in Figure 3 B. Compared with the WT, the plant height of the OE-SlABF4 line was significantly increased, while the CR-slabf4 line showed a dwarf growth phenotype. However, most transgenic plants were similar to the WT in terms of plant width, leaf length, and leaf width, as shown in Figure 12 .

[0061] Table 2 Protein sequences of SlABF4 locus mutations

[0062]

[0063] Protein sequences of SlABF4 locus mutations generated by CRISPR / Cas9 genome editing compared with wild-type plants. Underlined letters indicate changes in the amino acid sequence, and * represents the terminator.

[0064] To determine the role of SlABF4 in tomato fruit ripening, the present invention detected the fruit development time of transgenic plants and WT plants from the flowering stage to the breaking stage. Notably, the fruit ripening period of the OE-SlABF4 line was significantly delayed by 1 to 2 days, while the fruit ripening period of the two SlABF4 gene knockout lines was 2 to 3 days earlier than that of WT fruits from the flowering stage to the color-breaking stage. Specifically, WT fruits reached the color-breaking stage at 32.12 days after pollination (DPA), while the average time from anthesis to breaking of OE- SlABF4 -3 and OE- SlABF4 -4 fruits was postponed to 33.69 days and 34.04 days respectively, and those of CR- slabf4 -8 and CR- slabf4-29 The average time from after flowering to the breaking stage of the fruits was advanced to 29.95 days and 30.47 days respectively, as shown in Figure 4 A and B in. To further determine the changes in fruit sugar composition, the present invention measured the fructose, glucose, and sucrose contents of transgenic lines and WT plants at the B+3 stage. The present invention found that the fructose content in CR fruits was significantly higher than that in WT fruits, while the fructose content in OE fruits was lower. The glucose and sucrose contents of the OE- SlABF4 -4 lines decreased slightly, on the contrary, the glucose and sucrose contents of the CR- slabf4 -29 lines increased, as shown in Figure 4 D in. These findings indicate that the sugar content of tomato fleshy fruits may be affected by the altered fruit ripening process mediated by SlABF4. DPA represents days after anthesis. B+3 represents 3 days after breaker.

[0065] 4. SlABF4 affects ethylene content and the expression of ethylene and ripening-related genes in tomatoes

[0066] As a climacteric fruit, ethylene is crucial for the ripening of tomato fruits. The present invention measured the endogenous ethylene production of WT, OE, and CR transgenic materials during fruit ripening. In the CR- slabf4 lines, ethylene release reached a peak at the B+3 stage and was higher than that of the WT lines. In contrast, the ethylene release of OE- SlABF4 fruits reached a peak at the B+6 stage and was lower than that of the WT, as shown in Figure 4 C in. In addition, the present invention used qRT-PCR technology to study the expression levels of several genes related to fruit ripening in the peels of OE- SlABF4 、CR- slabf4 and WT at 34 DPA. These genes include SlACO1 、 SlACS2 、 SlACS4 、 SlACS12 involved in ethylene biosynthesis, as well as the key transcription factor genes SlRIN 、SlFUL1、SlTAGL1、SlNOR、SlCNR、SlAP2a、SlHB1、SlMADS1 related to ripening. The key genes SlACO1 and SlACS2 / 4 / 1 related to ethylene biosynthesis were upregulated in CR- slabf4 fruits and downregulated in OE-SlABF4 fruits, as shown in Figure 5 A-D in. In addition, important regulators of fruit ripening, such as SlRIN, SlFUL1, SlTAGL1, SlNOR, SlCNR, SlAP2a, and SlHB1, showed a significant increase in transcriptional levels in CR fruits at 32 DPA, as shown in Figure 5E-K in it. On the contrary, except for SlMADS1, other transcriptional levels were down-regulated in the CR-SlABF4 lines, and SlMADS1 is a negative regulator of fruit ripening, such as Figure 5 L in it. These results suggest that SlABF4 may be a negative regulator of ethylene biosynthesis and fruit ripening in tomatoes.

[0067] 5. SlABF4 can inhibit the promoter activities of SlACS2 and SlACS12

[0068] Given that artificially enhancing or inhibiting SlABF4 in tomatoes can alter fruit ripening and affect the transcription of ethylene biosynthesis-related genes and ripening-related regulators, the present invention investigated whether SlABF4 directly binds to the promoters of these genes. Yeast cells co-transformed with pGADT7-SlABF4 and SlACS2 or SlACS12 promoters grew on synthetic defined (SD) / -His-Leu-Trp medium containing 40 mM 3-AT, while those co-transformed with pGADT7-SlABF4 and the promoters of SlACS4, SlACO1, SlRIN, SlFUL1 or SlMADS1 did not grow, which confirmed the interaction of SlABF4 with the promoters of SlACS2 and SlACS12 in yeast, such as Figure 6 A in it.

[0069] To determine whether SlABF4 regulates the transcriptional activity of SlACS2 / 12, the present invention conducted a dual-luciferase reporter assay. The present invention cloned the SlACS2 or SlACS12 promoter sequence upstream of the LUC reporter gene and co-infiltrated this construct into tobacco leaf epidermal cells with the pGreenⅡ 62-SK or pGreenⅡ 62-SK-SlABF4 effector construct, such as Figure 6 B-D in it. The results showed that SlABF4 could significantly inhibit the activities of the promoters of SlACS2 and SlACS12 compared with the empty control vector, such as Figure 6 C in it. In addition, the EMSA assay also demonstrated that SlABF4 could directly bind to SlACS2 and SlACS12, generating shifted bands in vitro, such as Figure 7 . In summary, the present invention believes that SlABF4 directly interacts with the SlACS2 / 12 promoter, negatively regulating the SlACS2 / 12 expression, thereby delaying the ripening of tomato fruits.

[0070] 6. SlABF4 interacts with SlFUL1 and SlMADS1 in vivo and in vitro

[0071] The core component of ABA signal transduction, the SnRK2 kinase, can directly phosphorylate multiple downstream target proteins, including transcription factors such as ABFs, thereby mediating nuclear responses. To verify whether SlABF4 is involved in the tomato ABA signaling pathway, the present invention analyzed the interaction between SlABF4 and SlSnRK2s through a Y2H assay. In this study, when the BD-SlABF4 and AD-SlSnRK2.1 / 2.2 / 2.6 plasmids were spotted onto QDO medium lacking Trp, Leu, His, and Ade and supplemented with 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside, yeast cells grew, while in the Y2H assay, there was no interaction between SlABF4 and SlSnRK2.8, as shown in Figure 8 A in. In addition, no yeast self-activation was observed when co-transforming AD-empty and BD-SlABF4 in yeast. These results indicate that SlABF4 may be involved in ABA signal transduction through physical interaction with SlSnRK2 in tomatoes. 5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside is denoted as X-α-gal, and QDO is quadrupledrop-out.

[0072] Given that the tomato ripening time is altered in OE-SlABF4 and CR-slabf4 transgenic plants, the present invention hypothesizes that SlABF4 may directly interact with ripening-related proteins to regulate the development and ripening of tomato fruits. To verify this hypothesis, the present invention conducted Y2H, BiFC, FLCI assays, and pull-down experiments. The results are as shown in Figure 8 Yeast grew on QDO medium containing the X-α-gal indicator, indicating that SlABF4 can interact with SlFUL1 and SlMADS1. In contrast, no interaction between SlABF4 and SlRIN, SlCNR, SlNOR, or SlAP2a was observed. In addition, BiFC and Split-LUC analyses conducted in tobacco leaves showed that SlABF4 co-localized and interacted with SlFUL1 and SlMADS1 in the nucleus, as shown in Figure 8 B - C in. The interaction between SlFUL1 or SlMADS1 and SlABF4 was further verified by pull-down experiments in vitro, as shown in Figure 9 . Therefore, the present invention believes that SlABF4 can physically interact with SlFUL1 and SlMADS1 both in vivo and in vitro.

[0073] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent repetition, the present invention describes preferred embodiments.

[0074] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made by those skilled in the art once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Inhibition SlABF4 The reagent for expressing the amount of fruit is used in promoting fruit development, characterized in that: Said SlABF4 The gene number is Solyc11g044560.

2. The use according to claim 1, characterized in that: By knocking out the SlABF4 Obtain transgenic plants to enhance fruit development.

3. The use according to claim 2, characterized in that: Knock out the SlABF4 The ways of modifying the gene include base insertion, base deletion and insertion or formation of stop codon.

4. The use according to claim 1, characterized in that: The inhibition SlABF4 The reagent for expressing the amount includes the sequences shown in SEQ ID NO.3 to SEQ ID NO.

4.

5. The use according to claim 4, characterized in that: The inhibition SlABF4 The reagents for expression quantity also include pTX041 vector.

6. The use according to claim 5, characterized in that: The method for obtaining the transgenic plant comprises the following steps: SlABF4 Design the knockout target sequence of the gene sequence, clone the knockout target sequence into the pTX041 vector, and obtain transgenic plants through genetic transformation; The knockout target sequence is shown in SEQ ID NO.3 to SEQ ID NO.

4.

7. The use according to claim 6, characterized in that: The transgenic plant contains the amino acid sequence shown by SEQ ID NO.1 or SEQ ID NO.

2.

8. The use according to claim 1, characterized in that: The promoting fruit development refers to promoting fruit ripening.

9. The use according to claim 8, characterized in that: The fruit is a tomato.

10. A method for promoting fruit development, characterized in that: By knocking out the SlABF4 Obtain transgenic plants to enhance fruit development; The promoting fruit development refers to promoting tomato ripening.

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