Application of reagent for inhibiting expression quantity of SlABF4 in promoting fruit development

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a reagent for inhibiting the expression quantity of SlABF4 in promoting fruit development. The invention finds that overexpression of the SlABF4 can lead to delayed maturation start and reduction of ethylene production, on the contrary, knockout of the SlABF4 can lead to premature maturation of fruits and increase of ethylene yield, so that the reagent for inhibiting the expression quantity of the SlABF4 is provided to be applied to promotion of fruit development.

Description

Technical Field

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

[0002] The ripening process of fleshy fruits is a complex series of physiological and biochemical characteristics, accompanied by changes in fruit color, texture, flavor, aroma and other quality characteristics. Fleshy fruits can be divided into climatic fruits and non-climatic fruits according to whether their respiration and ethylene production increase when they begin to ripen. Solanum lycopersicum , a typical climacteric fruit, was chosen as a model for fruit ripening due to its diploid genome, well-annotated genome sequence, 3–5 month life cycle, ease of stable transformation, and availability of mutations associated with natural ripening.

[0003] Tomato fruit ripening is a highly coordinated developmental process involving the expression and regulation of thousands of genes. Based on the characterization 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. are gene mutants that inhibit tomato fruit ripening. Ethylene is the main trigger for climacteric fruit ripening. Inhibiting ethylene biosynthesis or its signal transduction can prevent the initiation of ripening. Ethylene biosynthesis 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, including SlACS2 , SlACS4 and SlACO1 It has been identified as a key gene for ethylene biosynthesis in tomatoes. Transcription factors, also known as 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 MADS-box proteins RIPENINGINHIBITOR, TOMATO AGAMOUS-LIKE 1, FRUITFULL 1, FRUITFULL 2, and SlMADS1; SBP-box protein COLORLESS NON-RIPENING; NAC domain protein NON-RIPENING; HD-zip homology junction protein HOMEBOX 1.

[0004] In plants, basic leucine zipper proteins, abbreviated as bZIP, and bZIP transcription factors are one of the largest families of transcription factors. ABA and abiotic stresses regulate gene expression through cis-acting elements, including ABA-responsive elements, which are abbreviated as ABRE. The sequence of ABRE is PyACGTGG / TC. ABRE binding factors / ABA response element binding proteins can bind to various ABRE-containing promoters. ABRE binding factors are abbreviated as ABF, and ABA response element binding proteins are abbreviated as AREB. The ABF / AREB gene family is a branch of the bZIP TF family, which is conserved in many species and is the most famous TF in the ABA signaling pathway. It has been reported that ABF proteins can be activated by phosphorylation by SnRK2 protein kinase. In Arabidopsis, 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. Several 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 modifications. SlAREB1 can also regulate SlDFR and SlF3'5'H ABA has been shown to play an important role in fruit ripening by regulating various genes involved in ethylene biosynthesis and signal transduction to promote ethylene production and response. ABF / AREB Studies on the functions of genes in tomato fruit ripening are still lacking, and whether SlABFs regulate other physiological aspects of fruit development remains unclear. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an agent for inhibiting the expression of SlABF4 for use in promoting fruit development.

[0006] inhibition SlABF4 The reagent expressing the amount is used in promoting fruit development, and the SlABF4 The gene number is Solyc11g044560.

[0007] Preferably, by knocking out the SlABF4 Genetically modified plants were obtained to enhance fruit development.

[0008] Preferably, knocking out the SlABF4 The ways of modifying the gene include base insertion, base deletion and insertion or formation of stop codon.

[0009] Preferably, the inhibition SlABF4 The reagent for expressing the amount includes the sequences shown in SEQ ID NO.3 to SEQ ID NO.4.

[0010] Preferably, the inhibition SlABF4 The reagents for expression quantity also include pTX041 vector.

[0011] Preferably, 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.

[0012] Preferably, the transgenic plant contains the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0013] Preferably, the promoting fruit development refers to promoting fruit ripening.

[0014] Preferably, the fruit is tomato.

[0015] A method for promoting fruit development by knocking out the SlABF4 Obtain transgenic plants to enhance fruit development; The promoting fruit development refers to promoting tomato ripening.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention found that overexpression SlABF4 results in delayed onset of maturation and reduced ethylene production; conversely, knockout SlABF4 This will lead to premature fruit ripening and increased ethylene production, so it is proposed that reagents that inhibit the expression of SlABF4 be used in promoting fruit development.

[0017] The present invention found that S1ABF4 directly binds to SlACS2 and SlACS12 The discovery of the present invention deepens the understanding of the role of SlABF4 transcription factor and provides new insights into the regulatory network in tomato fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Phylogenetic tree and sequence analysis of tomato SlABFs, A is phylogenetic analysis, B is amino acid sequence analysis of tomato SlABFs and Arabidopsis AtABF2, the position of the secondary structure amino acid sequence was determined using the crystal structure of AtABF2, C1-C4 represents the conserved region, and the bZIP region is marked with a double line.

[0019] Figure 2 The subcellular localization and expression pattern of SlABF4. A shows that the control GFP signal is present in the entire cell, while SlABF4-GFP is confined to the nucleus. B shows the subcellular localization and expression pattern of SlABF4 in different tissues of the wild-type tomato "Henzi". SlABFs Heat map based on the RPKM of tomato eFPs, C for different tissues of "Micro-Tom" WT tomato SlABF4 The relative transcription level of SlABF4 The transcript of was set as 1, the values ​​represent the mean ± SD of three biological replicates, the samples marked with different letters are significantly different, P < 0.05, D is the relative transcription level of SlABF4 in response to 100 μM ABA treatment, the values ​​represent the mean ± SD of three biological replicates, and the asterisk indicates a significant difference determined by Student's t-test, P < 0.01.

[0020] Figure 3 WT, OE- SlABF4 and CR- slabf4 Phenotypes of the strains. A is the genotype of the SlABF4 locus mutation generated by the CRISPR / Cas9 genome editing system. The two target sequences were designed specifically for the third exon. The mutation of the transgenic plant was confirmed by sequencing the genomic regions flanking the target site. B is the OE- SlABF4 -3.OE- SlABF4 -4, WT, CR- slabf4 -8 and CR- slabf4 Phenotypes of tomato plants of strain -29, C is the color breaking stage of WT, OE and CR fruits SlABF4 qRT-PCR analysis, values ​​represent the mean ± SD of three biological replicates, samples marked with different letters are significantly different at P < 0.05.

[0021] Figure 4 are the tomato fruit ripening phenotypes of WT, OE-SlABF4 and CR-slabf4 lines, A is OE- SlABF4 -3.OE- SlABF4 -4, WT, CR- slabf4 -8 and CR- slabf4 Phenotypes of tomato fruits of strain -29 at different days after flowering, B is WT, OE- SlABF 4 and CR- slabf4Average number of days from flowering to fruit breaking, n>100, C is WT, OE- SlABF4 and CR- slabf4 Ethylene production in 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.

[0022] Figure 5 WT, OE- SlABF4 and CR- slabf4 Relative transcript levels of selected genes involved in ethylene production and key regulators of tomato fruit ripening in the fruit, 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 .

[0023] Figure 6is the interaction of SlABF4 with SlFUL1 and SlMADS1, A is the yeast two-hybrid interaction of SlABF4 with SlFUL1 and SlMADS1, SlABF4 was fused with pGADT7 vector, SlSnRK2s, SlRIN, SlMADS1, SlFUL1, SlCNR, SlNOR and SlAP2a were fused with pGBKT7 vector, pGADT7-T+pGBKT7-53 was the positive control; pGADT7-T+pGBKT7-Lam was the negative control; pGADT7+pGBKT7-SlABF4 was the self-activation control, DDO, SD / - Leu / -Trp is a two-deficient medium; QDO, SD / -Ade / -His / -Leu / -Trp are four-deficient medium, B is a BiFC detection of the interaction between SlABF4 and SlFUL1 and SlMADS1 in tobacco leaves, C is a firefly luciferase complementation imaging detection of the interaction of SlABF4 between SlFUL1 and SlMADS1 in tobacco leaves, D and E are pull-down assays for the interaction between SlABF4 and SlFUL1 and SlMADS1, respectively, the protein extracts were incubated with GST beads, and then the pulled fractions were analyzed by immunoblotting using anti-SlABF4 antibody.

[0024] Figure 7 Negative regulation of SlABF4 SlACS2 and SlACS12 Promoter activity, A is SlABF4 directly targeting SlACS2 Yeast one-hybrid assay of SlACS12 promoter fragments, B is a schematic diagram of effector and reporter constructs for transient expression, C and D are schematic diagrams of SlABF4 repression, SlACS2 and SlACS12 Dual luciferase detection data and imaging of promoter activity, where C is the detection data, D is the imaging, and the values ​​represent the average of three repetitions, LUC: firefly luciferase activity; REN: Renilla luciferase.

[0025] Figure 8 SlABF4 and target genes SlACS2 and SlACS12 Promoter binding electrophoretic mobility shift assay EMAS, "+" indicates presence; "-" indicates absence, competitive probes used 50-fold and 100-fold non-labeled probes and mutation probes, A is SlACS2 and SlACS12 Schematic diagram of the ABRE motif in the promoter of SlACS2 The promoter region of SlABF4 protein is directly bound to SlACS12 binds to the promoter region.

[0026] Figure 9 The proposed model for SlABF4 in the regulation of tomato fruit ripening. SlACS2 and SlACS4 SlABF4 can also directly interact with other proteins, SlFUL1 and SlMADS1, downregulating the expression of SlFUL1 while upregulating the expression of SlMADS1, which may affect the expression of downstream target genes at the transcriptional level and delay the ripening of fruit.

[0027] Figure 10 Figure 2 is the gene feature of SLABF4, where A is the transcriptional activation analysis of SlABF4 in the yeast system, B is the schematic diagram of pBD-SlABF4, C shows that compared with the pBD control group, pBD-SlABF4 significantly inhibited the expression of the LUC reporter gene, and D is the relative ratio of LUC to REN. 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.

[0028] Figure 11 Identification of SlABF4 overexpression lines, where A is a schematic diagram of the overexpression plasmid pRI101-S1ABF4, and B is qRT-PCR analysis of SlABF4 expression in wild-type and overexpressed fruits at the shell-breaking stage. Values ​​represent the mean ± SD of three biological replicates.

[0029] Figure 12 Plant phenotypes of wild-type and transgenic tomato lines, where 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. Samples marked with different letters show significant differences at P < 0.05. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0031] In the present invention, CRISPR is clustered regularly interspaced short palindromic repeats, and Cas9 is CRISPR associated protein 9. The coding sequence is abbreviated as CDS.

[0032] SlACO1The name is 1-aminocyclopropane-1-carboxylate oxidase 1, and the gene number is Solyc07g049530. SlACS2 The name is 1-aminocyclopropane-1-carboxylate synthase 2, and the gene number is Solyc01g095080. SlACS4 The name of the gene is 1-aminocyclopropane-1-carboxylate synthase4, and the gene number is Solyc05g050010. SlACS12 The name of the enzyme is 1-aminocyclopropane-1-carboxylatesynthase 12, and the gene number is Solyc08g079750. SlRIN The name is ripening inhibitor, and the gene number is Solyc05012020. SlFUL1 The name of the gene is FRUITFULL 1, and the gene number is Solyc06g069430. SlTAGL1 The name of the gene is TOMATO AGAMOUS-LIKE1, and the gene number is Solyc07g055920. SlNOR The name is non-ripening, and the gene number is Solyc10g006880. SlCNR The name is colorless non-ripening, and the gene number is Solyc02g077920. SlAP2a The name of the gene is APETALA2a and the gene number is Solyc03g044300. SlHB1 The name is homebox 1, the gene number is Solyc02g086930, SlMADS1 The name is MADS-box protein 1 and the gene number is Solyc03g114840.

[0033] The overexpression vector pRI101 AN was purchased from Beijing Huayueyang Biotechnology Co., Ltd., model number Hyykb089; the pGADT7 vector was purchased from Beijing Huayueyang Biotechnology Co., Ltd., model number Hyykb105; the pBD vector was purchased from Shanghai Lianmai Bioengineering Co., Ltd., model number LM1658; the pGreenⅡ 62-SK vector was purchased from Wuhan Boyuan Biotechnology Co., Ltd., model number REC70-I; the pTX041 binary vector was the pTX041 used in the literature Cui L, Zheng F, Wang J, Zhang C, Zhang D, GaoS, Zhang C, Ye J, Zhang Y, Ouyang B et al: The tomato CONSTANS-LIKE proteinSlCOL1 regulates fruit yield by repressing SFT gene expression. BMC plantbiology 2022, 22(1):429.

[0034] 1. Plant materials and growth conditions Tomato was selected as the genetic transformation material. Solanum lycopersicum cv., variety Micro-Tom. The wild type was denoted as WT, and the WT and transgenic lines were cultured in an artificial climate chamber with a day-night temperature difference of 25°C / 20°C and a photoperiod of 16 h light and 8 h dark. Flowers were marked during the flowering period to assess the fruit maturity stage. Roots, stems, leaves, seeds, flowers, and pulp were collected to analyze tissue-specific gene expression. At least 10 fruits of uniform size were harvested for each biological replicate, and fruits at different stages from 15 to 40 days after flowering were harvested in three independent replicates. After removing the seeds and pectin, the peel and pulp were immediately frozen in liquid nitrogen and stored at -80°C until further use.

[0035] 2. Plasmid construction and plant transformation The S1ABF4 overexpression vector was constructed using the primers shown in SEQ ID NO.5 to SEQ ID NO.6 and the overexpression vector pRI101 AN, and Agrobacterium was transformed to obtain OE- SlABF4 Transgenic plants.

[0036] Two independent gRNAs for SlABF4 were designed, as shown in SEQ ID NO.3 to SEQ ID NO.4, and cloned into the pTX041 binary vector to construct CRISPR / Cas9 gene editing plasmids, and transformed into Agrobacterium to obtain CR- slabf4 Transgenic plants.

[0037] Among them, the gene number of SlABF4 in the tomato genome database ITAG 4.0 is Solyc11g044560, the URL of the CRISPR-P website is http: / / crispr.hzau.edu.cn / CRISPR2 / , and the address of the tomato genome database is https: / / solgenomics.net / .

[0038] 3. RNA extraction and real-time fluorescence quantitative PCR analysis The present invention uses RNAprep Pure Plant Plus Kit to extract total RNA from various tissues and fruits of tomato plants according to the manufacturer's instructions, reverse transcribes into cDNA, and designs a SlABF4, SlACO1, SlACS2, SlACS4, SlACS12, SlRIN, SlFUL1, SlTAGL1, SlNOR, SlCNR, SlAP2a, SlHB1, SlMADS1 qRT-PCR amplification was performed using Hieff Universal Blue qPCR SYBRGreen Master Mix using specific primers as shown in SEQ ID NO.7 to SEQ ID NO.34. Bio-Rad CFX Manager software was used to amplify the qRT-PCR results. -ΔΔCt The relative gene expression level was calculated by the method, and SlSAND was used as the internal reference gene. The RNAprep Pure Plant Plus Kit was from Beijing Tiangen Biotechnology Co., Ltd., and the Hieff Universal Blue qPCR SYBR Green Master Mix was from Shanghai Yesen Biotechnology Co., Ltd. The gene number of SlSAND in ITAG 4.0 is Solyc03g115810.

[0039] 4. Ethylene content measurement A 1 mL gas sample was drawn from the top of the container using a 1 mL syringe and then injected into the gas chromatograph to determine the ethylene production. The peak 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 is from Shimadzu, GC-2014, Japan.

[0040] Statistical analysis Data were analyzed using IBM SPSS Statistics v.25 software. Paired comparisons were performed using one-way analysis of variance and Student's t-test, * represents P < 0.05, ** represents P < 0.01, multiple comparisons were performed using Duncan's test, Duncan's test is denoted as Duncan's test, and samples marked with different letters represent significance of P < 0.05. IBM SPSS Statistics v.25 software was provided by IBM Corp., Armonk, NY, USA.

[0041] Table 1 shows the primers used in the present invention.

[0042] Table 1 Primers used in the present invention

[0043] result 1. Characteristics of tomato transcription factor SlABF4 Four tomato SlABFs genes were identified through homology comparison, namely SlABF1, SlABF2, SlABF3, and SlABF4, of which the SlABF4 gene consists of 6 exons and 5 introns. This gene encodes a 400-amino acid protein with a conserved bZIP domain, such as Figure 1 A~B in the phylogenetic analysis showed that ABF proteins can be divided into three groups. Among them, SlABF1 and SlABF4 belong to group I, SlABF2 and SlABF3 belong to group II, respectively. Figure 1 A in the ABF family. The ABF family members have four conserved domains, of which 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 compared with the closest homolog AtABF2 in Arabidopsis. Figure 1 Four highly conserved regions are shown in B.

[0044] In order to study the subcellular distribution 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, which means that SlABF4 has potential nuclear functions, such as Figure 2 In addition, the present invention also analyzed the transcriptional activation activity of S1ABF4. It was found that it did not have transcriptional activation activity in the yeast system, such as Figure 10 In addition, in the dual luciferase reporter system, SlABF4 significantly inhibited the LUC reporter activity compared with the empty vector, as shown in Figure 10 B in the figure indicates that SlABF4 may function as a transcriptional repressor.

[0045] 2. Expression analysis of SlABF4 In order to reveal the expression pattern of SlABF4, the present invention used the data in Tomato eFP Browser to analyze the expression patterns of SlABF4 at different stages of tomato fruit development. SlABFs The analysis results show that SlABFs In genes, SlABF4 The highest expression level is Figure 2 In addition, the present invention also conducted qRT-PCR experiments to detect SlABF4 Transcript accumulation in different tomato tissues. SlABF4 It was expressed in all tested tissues, and among non-fruit tissues, the highest expression was found in the stem. It is worth noting that the green maturity stage is abbreviated as MG, during the fruit ripening process. SlABF4 The transcript level increased rapidly and reached a peak at the MG stage, then gradually decreased until the Br+9 stage, as shown in Figure 2 These results indicate that SlABF4 It may play a role in regulating the ripening process of fruit. The URL of Tomato eFP Browser is https: / / bar.utoronto.ca / efp_tomato / cgi-bin / efpWeb.cgi.

[0046] In addition, the present invention also studies SlABF4 Is the expression of regulated by the plant hormone ABA? Figure 2 D shows the effect of spraying 100 μM exogenous ABA on SlABF4 Compared with the control group, the tomato MG fruits treated with exogenous ABA showed a significant increase in the expression of SlABF4 These results indicate that ABA-induced SlABF4 May be involved in the regulation of tomato fruit ripening.

[0047] 3. Effect of SlABF4 on fruit ripening In order to explore the physiological phenotype of SlABF4 in the process related to fruit ripening, the present invention used the CRISPR / Cas9-mediated gene editing system to generate SlABF4 gene knockout strains, namely two homozygous CR- slabf4 Strain CR- slabf4 -8 and CR- slabf4 -29, both strains have a 1-bp transition in the first guide RNA, CR- slabf4 -8 61 bp deletion in the second guide RNA, CR- slabf4-29 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 The amino acid sequence of -8 is shown in SEQ ID NO.1 in Table 2, CR- slabf4 The amino acid sequence of -29 is shown in SEQ ID NO.2 in Table 2. These mutations lead to premature termination of translation and SlABF4 Different truncations of the protein, e.g. Figure 3 At the same time, the present invention also generated two independent homologous T2 overexpression strains, namely OE- SlABF4 -3 and OE- SlABF4 -4, for further analysis, such as Figure 11 The present invention observed that the expression level of SlABF4 in the OE strain was significantly increased, while the expression level of SlABF4 in the two CR gene-edited strains was significantly decreased, such as Figure 3 C. The plant phenotypes of WT and transgenic lines are shown in Figure 3 B. Compared with WT, the plant height of OE-SlABF4 lines was significantly increased, while the CR-slabf4 lines showed a dwarf growth phenotype. However, most of the transgenic plants were similar to WT in terms of plant width, leaf length, and leaf width, such as Figure 12 .

[0048] Table 2 Protein sequences of S1ABF4 gene locus mutations

[0049] Protein sequences of the mutant SlABF4 locus generated by CRISPR / Cas9 genome editing compared to wild-type plants. Underlined letters indicate changes in the amino acid sequence, and * represents a terminator.

[0050] To determine the role of SlABF4 in tomato fruit ripening, the present invention detected the fruit development time from flowering to color breaking in transgenic plants and WT plants. It is worth noting that the fruit ripening period of the OE-SlABF4 strain was significantly delayed by 1 to 2 days, while the fruit ripening period of the two SlABF4 knockout strains was 2 to 3 days earlier than that of the WT fruit, from flowering to color breaking. Specifically, the WT fruit reached the color breaking stage at 32.12 days DPA, while the OE- SlABF4 -3 and OE- SlABF4 -4 The average time from flowering to fruit breaking was delayed to 33.69 days and 34.04 days, respectively, and CR- slabf4 -8 and CR- slabf4 -29 The average time from flowering to fruit breaking was advanced to 29.95 days and 30.47 days, respectively. Figure 4A, B in. To further determine the changes in the sugar composition of the fruit, the present invention measured the fructose, glucose and sucrose contents of the 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. SlABF4 -4 strain had a slight decrease in glucose and sucrose content. slabf4 -29 strains showed an increase in glucose and sucrose content, e.g. Figure 4 These findings suggest that the sugar content of tomato fleshy fruit may be affected by SlABF4-mediated alterations in the fruit ripening process. DPA stands for days after anthesis. B+3 stands for 3 days after break of color.

[0051] 4. SlABF4 affects ethylene content and the expression of ethylene and ripening-related genes in tomatoes As a climacteric fruit, ethylene is essential for the ripening of tomato fruit. The present invention measured the endogenous ethylene production of WT, OE and CR transgenic materials during the fruit ripening process. slabf4 strains, ethylene release peaked at stage B+3 and was higher than that of the WT strain. SlABF4 The ethylene release of the fruit reached a peak at stage B+6, which was lower than that of WT. Figure 4 In addition, the present invention uses qRT-PCR technology to study the OE- SlABF4 ,CR- slabf4 The expression levels of several genes related to fruit ripening in WT and WT pericarp were compared. These genes included SlACO1 , SlACS2 , SlACS4 , SlACS12 and key transcription factor genes associated with maturation SlRIN 、SlFUL1、SlTAGL1、SlNOR、SlCNR、SlAP2a、SlHB1、SlMADS1。 Key genes SlACO1 and SlACS2 / 4 / 1 related to ethylene biosynthesis were expressed in CR- slabf4 fruit, but downregulated in OE-SlABF4 fruit, such as Figure 5 In addition, the transcript levels of important regulators of fruit ripening, such as SlRIN, SlFUL1, SlTAGL1, SlNOR, SlCNR, SlAP2a, and SlHB1, were significantly increased in CR fruits at 32 DPA, as shown in Figure 5 In contrast, the transcript levels of all genes except SlMADS1 were downregulated in the CR-SlABF4 line, and SlMADS1 is a negative regulator of fruit ripening, such as Figure 5These results suggest that SlABF4 may be a negative regulator of ethylene biosynthesis and fruit ripening in tomato.

[0052] 5. SlABF4 can inhibit the promoter activity of SlACS2 and SlACS12 Given that artificial enhancement or inhibition of SlABF4 in tomatoes can change fruit ripening and affect the transcription of ethylene biosynthesis-related genes and ripening-related regulatory factors, the present invention studied whether SlABF4 directly binds to the promoters of these genes. SlACS2 or SlACS12 Yeast cells cotransformed with the promoters grew on synthetic defined (SD) / -His-Leu-Trp medium containing 40 mM 3-AT, whereas cells cotransformed with pGADT7-SlABF4 and the promoters of SlACS4, SlACO1, SlRIN, SlFUL1, or SlMADS1 did not grow, confirming the interaction of SlABF4 with the promoters of SlACS2 and SlACS12 in yeast, as Figure 6 A in.

[0053] In order to determine whether S1ABF4 regulates the transcriptional activity of S1ACS2 / 12, the present invention performed a dual luciferase reporter assay. The present invention cloned the upstream of the LUC reporter gene SlACS2 or SlACS12 The promoter sequence was co-infiltrated into tobacco leaf epidermal cells with the construct and pGreenⅡ 62-SK or pGreenⅡ 62-SK-SlABF4 effector construct, such as Figure 6 The results showed that compared with the empty control vector, SlABF4 could significantly inhibit the activity of SlACS2 and SlACS12 promoters. Figure 6 In addition, EMSA experiments also proved that SlABF4 can directly bind to SlACS2 and SlACS12, thereby generating migration bands in vitro, such as Figure 7 In summary, the present invention believes that S1ABF4 directly interacts with SlACS2 / 12 The promoter interacts with and negatively regulates SlACS2 / 12 expression, thereby delaying the ripening of tomato fruit.

[0054] 6. SlABF4 interacts with SlFUL1 and SlMADS1 in vivo and in vitro SnRK2 kinase, a core component of ABA signal transduction, can directly phosphorylate multiple downstream target proteins, including transcription factors such as ABFs, thereby mediating nuclear reactions. In order to verify whether SlABF4 is involved in the tomato ABA signaling pathway, the interaction between SlABF4 and SlSnRK2s was analyzed by Y2H assay. In this study, when BD-SlABF4 and AD-SlSnRK2.1 / 2.2 / 2.6 plasmids were spotted on QDO medium lacking Trp, Leu, His and Ade and supplemented with 5-bromo-4-chloro-3-indolyl-α-D-pyranogalactopyranoside, yeast cells grew, while in the Y2H assay, SlABF4 did not interact with SlSnRK2.8, ​​as shown in Figure 2 . Figure 8 In addition, no yeast autoactivation was observed when AD-empty and BD-SlABF4 were co-transformed in yeast. These results suggest that SlABF4 may be involved in ABA signal transduction through physical interaction with SlSnRK2 in tomato. 5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside is denoted as X-α-gal, and QDO is quadruple drop-out.

[0055] Given that 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 fruit. To verify this hypothesis, the present invention conducted Y2H, BiFC, FLCI detection and pull-down experiments. The results are shown in Figure 2. Figure 8 As shown, yeast were grown on QDO medium containing the X-α-gal indicator, indicating that SlABF4 could interact with SlFUL1 and SlMADS1. In contrast, no interaction of SlABF4 with SlRIN, SlCNR, SlNOR, or SlAP2a was observed. In addition, BiFC and Split-LUC analysis in tobacco leaves showed that SlABF4 colocalized and interacted with SlFUL1 and SlMADS1 in the nucleus, as shown in Figure 2. Figure 8 B to C in Figure 4. The interaction between SlFUL1 or SlMADS1 and SlABF4 was further verified by traction experiments in vitro, such as Fig. 9 Therefore, the present invention proposes that S1ABF4 can physically interact with S1FUL1 and S1MADS1 both in vivo and in vitro.

[0056] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0057] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0058] 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 equivalents, 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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