Application of EMB1444-like in regulating tomato maturation and breeding
By regulating YFT1 expression through EMB1444-like, the difficult problems of tomato fruit color and ripening development in breeding were solved, precise control of tomato fruit color and ripening period was achieved, and new high-quality tomato varieties were cultivated.
Patent Information
- Application Number
- CN202310836844.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-07
- Publication Date
- 2025-09-19
- Estimated Expiration
- 2043-07-07
AI Technical Summary
In the existing technology, it is still unclear how to regulate tomato fruit color formation and fruit ripening development in breeding, especially the lack of research on regulating YFT1 expression, which makes it difficult to accurately control tomato fruit color and ripening period.
The bHLH family transcription factor EMB1444-like was used to regulate the expression of YFT1, a key gene in the ethylene signaling pathway, by specifically binding to the E-box motif (CACTTG, -1295bp to -1290bp) and its flanking sequence in the upstream promoter. An EMB1444-like down-regulation expression vector was constructed and transformed into Agrobacterium to create the EMB1444-like down-regulation expression mutant sled1, which affected ethylene synthesis, signal transduction and carotenoid synthesis, thereby regulating tomato fruit color and ripening.
Precise genetic improvement of tomato fruit color and fruit ripening has been achieved, and new varieties with excellent quality and suitable ripening period have been bred. The fruit color and ripening period are controllable, providing a theoretical basis and practical path for breeding.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the fields of molecular biology and genetics, and specifically relates to an application of EMB1444-like in regulating tomato ripening and development and breeding; in particular, it relates to an application of EMB1444-like in breeding by regulating YFT1 expression to influence tomato fruit color formation and fruit ripening and development; and particularly relates to an application of a tomato transcription factor SlEMB1444-like (EMB1444-like in Solanum lycopersicum) in controlling tomato fruit color formation and fruit ripening and development by regulating the expression of the ethylene signaling core component gene YFT1 / EIN2 (YELLOW FRUITEDTOMATO 1 / ETHYLENE INSENSITIVE 2). Background Art
[0002] Tomato (Solanum lycopersicum, 2n=24) belongs to the Lycopersicon section of the Solanaceae family and is the world's largest dual-purpose fruit and vegetable crop. Due to its small genome, short growth cycle, and ease of genetic manipulation, tomato is an important model plant for berry research. Fruit color not only affects the appearance of tomatoes but is also closely related to their nutritional quality; therefore, fruit color is a quality trait of tomatoes. Commercially available tomatoes are primarily red and yellow, and their color is related to the composition and accumulation of carotenoids. While the tomato carotenoid biosynthesis pathway has been extensively studied at the biochemical (catalytic enzyme) and molecular (gene) levels, the molecular mechanisms underlying this pathway remain unclear.
[0003] Tomatoes are typical climacteric fruits, experiencing a respiratory peak. Ethylene plays a key role in tomato fruit ripening. Downregulating the expression of the key ethylene synthesis genes ACS (ACC SYNTHASE) and ACO (ACC OXIDASE) inhibits ethylene signaling, affecting tomato fruit ripening. This also inhibits carotenoid synthesis in tomato fruits, resulting in an orange or yellow hue. Downregulating the expression of the ethylene receptor genes LeETR4 (ETHYLENE RESPONSE 4), LeETR6 (ETHYLENE RESPONSE 6), and LeCTR1 (CONSTITUTIVE TRIPLE RESPONSE 1) delays tomato fruit ripening. The ethylene-insensitive mutant Nr (Never ripe) tomato exhibits a late-ripening phenotype, with reduced lycopene accumulation. Downregulating the expression of the core transcription factor LeEIL (ETHYLENE INSENSITIVE 3-like), the ethylene signaling pathway, delays fruit ripening, resulting in a yellow phenotype.
[0004] The YFT1 (YELLOW FRUITED TOMATO 1) gene, which we cloned from tomato, encodes SlEIN2 (ETHYLENE INSENSITIVE 2) in Solanum lycopersicum, a core component of the ethylene signaling pathway. Ethylene loading into the receptor inactivates CTR1 (CONSTITUTIVE TRIPLE RESPONSE1), which loses its ability to phosphorylate SlEIN2. The carboxyl terminus (CEND) dissociates from EIN2. A portion of the CEND enters the nucleus and binds to EIN3 / EIL1, promoting its activity. A portion of the CEND forms a complex with P-bodies (processing bodies) in the cytoplasm, degrading EIN3-BINDINGF-BOX 1 / 2 (EBF1 / 2) mRNA and inhibiting EBF1 / 2 protein synthesis. This reduces nuclear import, maintains EIN3 / EIL1 homeostasis, and promotes the expression of downstream ethylene-responsive genes.
[0005] Plants with the loss-of-function EIN2 mutant (ein2) are insensitive to ethylene, and the "triple" response disappears during the seedling stage. EIN2 also serves as an intersection of multiple plant hormone signaling pathways; it plays an important role in salt stress, oxidative stress, and other biotic and abiotic stresses. ORE1 (oresara 1), a member of the NAC transcription factor family, promotes cell senescence and death in Arabidopsis leaves. EIN2 positively regulates ORE1 expression to promote cell senescence. EIN2 also has the function of limiting plant cell proliferation. Arabidopsis ein2 mutants exhibit significantly increased plant height, leaf area, and pod length. In particular, EIN2 can regulate the ripening and development of fruits with a respiratory burst peak. The homologous gene YFT1 of EIN2 in tomatoes regulates ethylene synthesis, ethylene signal transduction, carotenoid synthesis, and chromoplast development (Zhao, WH, et al. Yellow-fruited phenotype is caused by 573bpinsertion at 5'UTR of YFT1 allele in yft1 mutant tomato. Plant Science, 2020, 300: 110637). Although EIN2 occupies an important position in plant color formation and fruit development, especially in tomatoes, there are few reports on how to regulate YFT1 / SlEIN2. In particular, the application of improving tomato fruit color formation and fruit ripening development by regulating YFT1 expression in tomato improvement is still unclear. The patent of this invention provides a theoretical basis and practical path for revealing the application of controlling tomato fruit color formation and fruit ripening development in breeding by regulating YFT1 expression. Summary of the Invention
[0006] The purpose of the present invention is to provide an EMB1444-like for use in regulating tomato maturation and development and breeding, so as to achieve the purpose of improving tomato quality and cultivating high-quality varieties.
[0007] The technical solutions of the present invention are as follows:
[0008] The present invention provides an application of a bHLH family transcription factor EMB1444-like in regulating tomato fruit ripening and development. The amino acid sequence of the EMB1444-like is shown in SEQ ID NO.3.
[0009] The CDS sequence of EMB1444-like is shown in SEQ ID NO.4.
[0010] The transcription factor EMB1444-like regulates tomato fruit ripening and color by regulating ethylene synthesis, ethylene signal transduction, chromoplast development, carotenoid accumulation, and fruit color formation.
[0011] The EMB1444-like protein positively regulates the transcriptional expression of the gene YFT1 encoding the key component SlEIN2 of ethylene signal transduction.
[0012] The EMB1444-like specifically binds to the E-box motif (CACTTG, -1295bp to -1290bp, SEQ ID NO.1) and its flanking region of the upstream regulatory region of the upstream promoter pYFT1, a key gene in the ethylene signaling pathway, to regulate YFT1 expression; the E-box motif and its flanking sequence are shown in SEQ ID NO.2.
[0013] The present invention also provides an RNA interference sequence for down-regulating the expression of the transcription factor EMB1444-like gene. The DNA sequence of the RNA interference sequence is shown in SEQ ID NO.5.
[0014] The present invention also provides an EMB1444-like down-regulation expression vector, and the construction method of the expression vector comprises the following steps:
[0015] The 350 bp specific DNA fragment of EMB1444-like (SEQ ID NO. 5) was amplified using specific primers EMB1444-like-RNAi-F: 5′-cgcggatccgctccaggagttgatgacgag-3′ (SEQ ID NO. 44) and EMB1444-like-RNAi-R: 5′-ccggaattcggtgttttataatggaactttcac-3′ (SEQ ID NO. 45), and constructed into the RNAi vector pHELLSGATE 12 to obtain the EMB1444-like down-regulation expression vector EMB1444-like-RNAi.
[0016] Use of the expression vector as claimed in claim 7 in tomato fruit ripening.
[0017] The present invention also provides an EMB1444-like down-regulated expression mutant sled1. The EMB1444-like down-regulated expression vector is transformed into Agrobacterium, and positively transformed Agrobacterium is selected and transformed into wild-type tomato M82 (Solanum lycopersicum) to obtain the mutant sled1.
[0018] The wild-type tomato is the wild-type tomato M82.
[0019] The present invention provides an application of the transcription factor EMB1444-like in regulating tomato fruit color formation. The EMB1444-like down-regulation mutant sled1 (EMB1444-like-RNAi) alters the expression of genes related to the carotenoid synthesis pathway, restricting carotenoid synthesis in tomato fruit and changing fruit color.
[0020] The present invention provides an application of a transcription factor EMB1444-like in regulating tomato fruit ripening and development. The transcription factor EMB1444-like regulates the expression of key genes in the ethylene biosynthesis pathway and ethylene biosynthesis in tomato fruit through feedback regulation, thereby affecting the ripening and development of tomato fruit.
[0021] The present invention provides a transcription factor EMB1444-like, whose down-regulated expression mutant sled1 changes the expression of genes related to the ethylene signaling pathway, disrupts ethylene signal transduction, and affects the ripening process of tomato fruits.
[0022] The present invention provides an application of the transcription factor EMB1444-like in tomato genetic improvement and breeding. This includes controlling carotenoid synthesis, ethylene synthesis, signal transduction, and chromoplast development by altering EMB1444-like expression, thereby regulating tomato fruit color formation and fruit ripening. This application can be applied to tomato breeding to improve tomato quality and maturity.
[0023] The present invention also provides an explanation of the molecular mechanism by which a transcription factor EMB1444-like controls tomato fruit color formation and fruit ripening and development by specifically regulating the expression of the ethylene signaling core component YFT1 / EIN2 (YELLOW FRUITED TOMATO 1 / ETHYLENE INSENSITIVE 2), and its application in guiding tomato breeding practice.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] 1. For the first time, we revealed that the transcription factor EMB1444-like, independent of ethylene signaling, positively regulates the expression of the tomato YFT1 gene;
[0026] 2. Based on the regulation of ethylene synthesis, carotenoid accumulation and chromoplast development by EMB1444-like, genetic improvement of tomato fruit color formation and fruit ripening can be achieved more accurately, and new tomato varieties with suitable quality, shelf life and maturity period can be cultivated. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:
[0028] Figure 1 Bimolecular luciferase assay to screen YFT1 candidate transcription factors;
[0029] Figure 2 The EMB1444-like binding region on the YFT1 promoter was determined;
[0030] Figure 3 Identify the EMB1444-like binding site in the p7 region of the YFT1 promoter;
[0031] Figure 4 YFT1 and EMB1444-like expression in tomato materials with different genetic backgrounds;
[0032] Figure 5 Dynamic changes in fruit color of tomato M82, yft1, and sledl (EMB1444-like-RNAi) mutants (Scale bars = 1 cm);
[0033] Figure 6 PSY1, CRTISO, and CYCB are expressed in tomato M82, yft1, and sledl;
[0034] Figure 7Ultrastructural observation of chromoplasts (Scale bars = 500 nm);
[0035] Figure 8 Differences in ethylene synthesis and release among tomato M82, yft1, and sled1;
[0036] Figure 9 The genes ACO1 and ACS2 / 4 are expressed in tomato M82, yft1, and sledl fruits;
[0037] Figure 10 Expression of key genes in the tomato ethylene signaling pathway: M82, yft1, and sledl. DETAILED DESCRIPTION
[0038] The present invention is described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several changes and modifications without departing from the present invention. These all fall within the scope of protection of the present invention.
[0039] The present invention relates to the late-ripening yellow-fruited tomato mutant yft1 (n3122) and the wild-type (WT) red-fruited tomato cv. M82, both provided by Professor Dani Zamir of the Hebrew University of Jerusalem (http: / / zamir.sgn.cornell.edu / mutateds). The yft1 mutant was obtained by screening tomato cv. M82 seeds using fast neutron (60Co γ-ray) irradiation. The present invention used the TEA database (http: / / tea.solgenomics.net / ) with a similarity coefficient threshold of 0.7; a total of 2,386 genes with expression patterns similar to those of YFT1 (Solyc09g007870) were detected, including 85 transcription factors. The 3,000-bp YFT1 promoter upstream sequence (pYFT1) was submitted to PLACE (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace) to predict cis-regulatory elements within pYFT1. Twenty-three transcription factors were predicted to target pYFT1 functional elements within the 3,000-bp pYFT1. Dual luciferase assay results showed that Solyc09g066280, encoding the transcription factor EMB1444-like, increased pYFT1 activity by 4.2-fold ( Figure 1Three rounds of screening and point mutation analysis were performed using yeast one-hybrid and EMSA (electrophoretic mobility shift assay) analysis (in the first round, pYFT1 was divided into p1 to p12 fragments, each about 300 bp long, with adjacent segments overlapping by 50 bp; in the second and third rounds, the fragments were 120 bp and 50 bp long, respectively, with an overlapping region of 20 bp). It was determined that EMB1444-like specifically binds to p7-3-2 (-1319 bp to -1270 bp) of pYFT1 (defined as the first nucleotide upstream of the start codon ATG of YFT1 as -1 bp), which contains an E-box (CACTTG, -1295 bp to -1290 bp, SEQ ID NO. 1) motif ( Figure 2 and Figure 3 ).
[0040] The amino acid sequence of EMB1444-like is shown in SEQ ID NO. 3. The cDNA sequence of EMB1444-like is shown in SEQ ID NO. 4.
[0041] An EMB1444-like down-regulated expression mutant (EMB1444-like-RNAi, sled1) was constructed in the M82 background. RT-qPCR analysis of EMB1444-like and YFT1 expression revealed no significant difference in EMB1444-like expression levels between M82 and yft1 mutant fruits; however, YFT1 expression in the EMB1444-like down-regulated expression sled1 mutant fruit was significantly lower than that in M82; EMB1444-like is an upstream regulatory factor of YFT1 ( Figure 4 The fruit of the sled1 mutant tomato is delayed in maturation and development, and the fruit turns yellow 54 days after anthesis (54 days post anthesis, 54 dpa). Figure 5 ). At the same time, it was also found that the chromoplast development of the sled1 mutant was delayed compared to M82, and the number of accumulated lycopene plasmid globules was significantly reduced ( Figure 6 ).
[0042] The expression of key carotenoid biosynthesis genes PSY1 (PHYTOENE SYNTHASE 1), CRTISO (CAROTENOID ISOMERASE), and CYCB (CHROMOPLAST-SPECIFIC LYCOPENE B-CYCLASE) in the fruit of the sled1 mutant was significantly downregulated compared with that in the wild-type M82 ( Figure 7). At 54 dpa, the accumulation of lycopene and β-carotene was also significantly lower than that of M82 (Table 5). Analysis found that the expression of key ethylene synthesis genes ACS2 / 4 (ACC SYNTHESIS 2 / 4) and ACO1 (ACC OXIDASE 1) and ethylene signaling pathway genes NR (NEVER RIPE), ETR4 (ETHYLENE RECEPTOR4), AP2a (APETALA 2a) and ERF4 / 6 (ETHYLENE RESPONSIVE FACTOR 4 / 6) in sledl mutant tomatoes was significantly downregulated, inhibiting ethylene synthesis and interfering with ethylene signal transduction ( Figure 8 , Figure 9 and Figure 10 In summary, EMB1444-like inhibits ethylene and carotenoid synthesis by regulating YFT1 expression, delaying chromoplast development and thus affecting fruit color formation and ripening. This provides an important theoretical basis for genetic improvement and breeding of tomato fruit color formation and ripening.
[0043] Example 1: Screening of YFT1 Highly Efficient Transcription Factors
[0044] In order to screen for transcription factors that regulate YFT1 expression, the present invention determined transcription factors with similar expression patterns to YFT1 (Solyc09g007870) based on the Tomato Expression Atlas (http: / / tea.solgenomics.net / ) transcriptome data. Submit the 3000bp sequence upstream of the YFT1 start codon ATG (pYFT1, SEQ ID NO.6) to the PLACE website (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace), predict the pYFT1 cis-regulatory elements (Table 1), and select the transcription factors with pYFT1 binding sites as candidate transcription factors. The dual luciferase assay technology was used to screen for efficient transcription factors of YFT1. The specific steps are as follows:
[0045] 1. Vector Construction
[0046] Specific primers [pYFT1-F: 5′-tccatcgatatattgatacgatatc-3′ (SEQ ID NO. 7), pYFT1-R: 5′-tatttgatttaaatggttagcaagc-3′ (SEQ ID NO. 8)] were used to PCR amplify 3,000 bp of pYFT1 and cloned into the pGreen II 0800-LUC vector (product of Shanghai Zeye Biotechnology Co., Ltd., Shanghai, China) between the Sal I and Nco I sites upstream of Firefly Luciferase (LUC) to construct pYFT1::LUC.
[0047] Based on the TEA (http: / / tea.solgenomics.net / ) transcriptome database, transcription factors (TFs) with expression patterns similar to YFT1 (Solyc09g007870) in tomato fruit were selected. Cis-acting elements of pYFT1 were predicted using the PLACE website (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace) (Table 1).
[0048] Table 1 Major cis-acting elements in the YFT1 promoter sequence*
[0049]
[0050]
[0051] *The first nucleotide upstream of the ATG start codon of YFT1 is defined as -1 bp.
[0052] A total of 23 candidate transcription factors (TFs) that bind to cis-elements in the pYFT1 sequence were screened (Table 2). The genes encoding the 23 identified candidate transcription factors were PCR amplified. The EMB1444-like CDS sequence (SEQ ID NO. 4) was amplified using specific primers (E80-BamH IF: 5′-ctctctctcaagcttggatccatggcaagccaactgcaac-3′ (SEQ ID NO. 76) and E80-Sac IR: 5′-gctcaccatactagtgagctccaagttgatctttgcctgcag-3′ (SEQ ID NO. 77)). These primers were then cloned into the BamH I and Xba I sites of the pHB vector (preserved for this experiment) to construct the expression vector pHB::TFs.
[0053] Table 2. 23 candidate transcription factors with predicted binding sites in the YFT1 promoter sequence
[0054]
[0055]
[0056] 2. Transient Transformation of Tobacco Leaves
[0057] The recombinant vectors pHB::TFs and pYFT1::LUC were transformed into competent Agrobacterium GV3101 carrying pSoup-p19 (purchased from Shanghai Yuchun Biotechnology Co., Ltd., Shanghai, China). The cells were diluted to OD 600 =0.6, and a 1:1 (v:v) mixture of pHB::TFs and pYFT1::LUC Agrobacterium (GV3101). A pHB (empty) and pYFT1::LUC mixture was used as a negative control. The mixture was drawn up using a disposable syringe with a removed needle and slowly injected into tobacco leaves. After completion, the excess solution was removed and the leaves were transferred to a dark incubation area for 24 hours. After another 24 hours of incubation in the light, samples were collected using a borer.
[0058] 3. Luciferase activity assay
[0059] The sample was ground into a powder in liquid nitrogen, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Biotechnology, Inc., Wisconsin, USA). Approximately 100 mg of sample powder was placed in a 1.5 mL pre-chilled centrifuge tube. 400 μL of 1× PLB (passive lysis buffer) was added and the tube was thoroughly vortexed for extraction. The tube was centrifuged at 9600 g for 30 seconds. 8 μL of the supernatant was transferred to a new 1.5 mL centrifuge tube and quickly mixed with 40 μL of LAR II (luciferase assay reagent II). The assay was then measured. 40 μL of Stop & Glo buffer was then added, gently mixed, and the assay was performed again. The result was the ratio of the two measured values.
[0060] By analyzing the Luciferase enzyme activity, it was found that the transcription factor EMB1444-like (Solyc09g066280) encoded by EMB1444-like regulated the transcriptional expression of pYFT1 by 4.35 times ( Figure 1 ).
[0061] Example 2: Determination of EMB1444-like binding sites in pYFT1
[0062] To identify the EMB1444-like binding site on the YFT1 promoter, pYFT1 (3000 bp upstream of the YFT1 start codon ATG, with the first nucleotide upstream defined as -1 bp) was subjected to three rounds of stepwise fragmentation and verified by yeast one-hybrid and electrophoretic mobility shift assay (EMSA). The specific experimental steps are as follows:
[0063] 1. Yeast One-Hybrid Vector Construction
[0064] The CDS sequence of EMB1444-like was amplified using the specific primers EMB1444-like-EcoRI-F: 5′-gattatgcctctcccgaattcatggcaagccaactgcaac-3′ (SEQ ID NO. 22) and EMB1444-like-XhoI-R: 5′-agaagtccaaagcttctcgagctacaagttgatctttgcctgc-3′ (SEQ ID NO. 23) and cloned into the pB42AD vector (purchased from Shanghai Zeye Biotechnology Co., Ltd., Shanghai, China) between the EcoRI and XhoI sites to construct the recombinant plasmid: pB42AD-EMB1444-like. pYFT1 was amplified by three rounds of PCR. In the first round, pYFT1 (SEQ ID NO. 5) was divided into 12 segments, p1 to p12 (approximately 300 bp, with 50 bp overlap between adjacent segments) (Table 3). These segments were cloned into the pLacZi vector (Beijing Crespo Biotechnology Co., Ltd., Beijing, China) between the EcoR I and Xho I sites to construct the pLacZi-ΔpYFT1 recombinant vector series for yeast one-hybrid screening. After screening for positive fragments in the first round, the second and third rounds of segmentation were performed, further fragmenting the target DNA fragments into 120 bp and 50 bp, respectively, and similarly cloned into the pLacZi vector (Table 2).
[0065] 2. Yeast One-Hybrid Analysis
[0066] The constructed pB42AD-EMB1444-like and pLacZi-ΔpYFT1 recombinant plasmids were co-transformed into the yeast competent EGY48a (purchased from Shanghai Weidi Biotechnology Co., Ltd.). The bacterial solution was spread on the SD (-Leu / -Ura) deficiency medium with a sterilized spreading stick and inverted cultured at 28°C for 48 h. The plaques with good growth status and uniform size were selected and resuspended in 60 μL sterile ddH2O. 6 μL of bacterial solution was added dropwise to the SD (-Leu / -Ura) + X-gal (20 mg / mL) qualitative medium and inverted cultured at 28°C for 72 h. The color change of the plaques was observed ( Figure 2 ).
[0067] Table 3 Three-round segmentation of YFT1 promoter sequence*
[0068]
[0069] *The first nucleotide upstream of the ATG start codon of YFT1 is defined as -1 bp.
[0070] The results showed that EMB1444-like can bind to the p7-3-2 (-1319bp to -1270bp) region of the YFT1 promoter. The p7-3-2 region contains an E-box (CACTTG, -1295bp to -1290bp) sequence, which is predicted to be the binding motif of the EMB1444-like transcription factor. To confirm this speculation, the E-box motif of p7 (-1549bp to -1250bp) was mutated to Δp7 (CACTTG→ACACCT, SEQ ID NO.42) and the results were verified by yeast one-hybridization ( Figure 3 EMSA assay revealed that the EMB1444-like protein only bound to the probe (5′-cctaatggtctacacaccCACTTGggtaaattttatcattttttttttaaattttac-3′, SEQ ID NO.43), but could not bind to the probe containing the mutant E-box (CACTTG→ACACCT). Figure 3 ). It was demonstrated that the EMB1444-like protein specifically binds to the E-box (CACTTG, -1295 bp to -1290 bp, SEQ ID NO. 1) of pYFT1.
[0071] Furthermore, EMB1444-like specifically binds to the E-box motif (CACTTG, -1295bp to -1290bp, SEQ ID NO.1) and its flanking region of the upstream regulatory region of the upstream promoter pYFT1, a key gene in the ethylene signaling pathway, to regulate YFT1 expression; the E-box motif and its flanking sequence are shown in SEQ ID NO.2.
[0072] Example 3: EMB1444-like regulates tomato fruit ripening and color formation
[0073] A 350 bp specific fragment of EMB1444-like (SEQ ID NO. 5) was amplified using specific primers EMB1444-like-RNAi-F: 5′-cgcggatccgctccaggagttgatgacgag-3′ (SEQ ID NO. 44) and EMB1444-like-RNAi-R: 5′-ccggaattcggtgttttataatggaactttcac-3′ (SEQ ID NO. 45), and cloned into the RNAi vector pHELLSGATE 12 (Invitrogen, CA, USA) to construct the EMB1444-like down-regulated expression vector EMB1444-like-RNAi. The vector was then transformed into Agrobacterium tumefaciens EHA105 competent cells and used to transform wild-type tomato M82 to create the EMB1444-like down-regulated expression mutant sled1 in the M82 background. Total RNA was extracted from tomato fruit samples 35 dpa (days post anthesis), 47 dpa, and 54 dpa after anthesis. RT-qPCR was used to analyze the expression of EMB1444-like [EMB1444-like-qPCR-F: 5′-cttcctcccatgaggttggt-3′, SEQ ID NO.46; EMB1444-like-qPCR-R: 5′-cattctgcccctcaaccacaa-3′, SEQ ID NO.47] and YFT1 [YFT1-F: 5′-actgcggagaaggttgtg-3′, SEQ ID NO.48; YFT1-R: 5′-atggctcgtcggagaatg-3′, SEQ ID NO.49], yft1, and sledl in tomatoes with different genetic backgrounds M82 and fruit color changes. The results showed that there was no significant difference in EMB1444-like transcript expression between M82 and yft1 mutant fruits; however, the expression level of YFT1 in sled1 fruits was significantly lower than that in M82 ( Figure 4 This suggests that EMB1444-like is a transcription factor that positively regulates YFT1 expression independently of the ethylene signaling pathway.
[0074] The sled1 tomato showed yellow fruit, and the fruit maturity was delayed at 54 dpa. The wild-type M82 fruit reached the red ripe stage; while the sled1 mutant fruit color only reached the color change stage, and was similar to the M82 fruit color at 47 dpa ( Figure 5 ).
[0075] Example 4: Effects of EMB1444-like on carotenoid synthesis and chromoplast development in tomato fruit
[0076] To clarify the regulatory mechanism of EMB1444-like on carotenoid synthesis in tomato fruit, the contents of total carotenoids, lycopene, β-carotene, α-carotene and lutein were measured at different developmental stages (35 dpa, 47 dpa and 54 dpa) in the fruits of M82, yft1 and sledl mutants. The differences in the ultrastructure of chromoplasts and the number of plastid globules in the fruits were also observed.
[0077] The specific experimental operations are as follows:
[0078] 1. Expression of key genes in the carotenoid pathway
[0079] Total RNA was extracted from the equatorial peel samples of M82, yft1, and sled1 at different developmental stages (35 dpa, 47 dpa, and 54 dpa), and RT-qPCR analysis was performed using a Light Cycle 96 real-time fluorescence quantitative PCR instrument (Roche Ltd., Switzerland) using specific primers (Table 4). The results showed that the expression levels of PSY1 (PHYTOENE SYNTHASE 1), CRTISO (CAROTENOID ISOMERASE), and CYCB (CHROMOPLAST-SPECIFIC LYCOPENE B-CYCLASE) in M82 fruits were significantly higher than those of yft1 and sled1, except for 35 dpa ( Figure 6 ); This indicates that EMB1444-like affects carotenoid synthesis and accumulation by regulating the expression of genes related to the carotenoid biosynthesis pathway, thereby affecting tomato fruit color formation.
[0080] Table 4 Primer sequences for analyzing the expression of key genes in the carotenoid biosynthesis pathway
[0081]
[0082] 2. Carotenoid content analysis
[0083] 2.1 Carotenoid extraction
[0084] (1) The equatorial pericarp of M82, yft1, and sled1 fruits at different developmental stages (35 dpa, 47 dpa, and 54 dpa) was ground into powder in liquid nitrogen under dark conditions.
[0085] (2) Weigh approximately 500 mg of powder into a clean 15 mL centrifuge tube and record the exact powder mass (using the weight loss method); add 1.5 mL of a mixture of KOH and methanol (w / v = 6%), mix thoroughly by inversion, and incubate in a 60°C water bath for 30 min;
[0086] (3) Cool to room temperature, add 1.5 mL of Tris-HCl buffer (50 mM Tris-HCl, 1 M NaCl, pH 7.5), mix thoroughly by inversion, and place in a refrigerator at 4°C for 10 min;
[0087] (4) Add 4 mL of chloroform, mix thoroughly by inversion, let stand on ice for 10 min, and centrifuge at 4000 rpm at 4°C for 10 min;
[0088] (5) Use a clean 5 mL syringe to draw the lower organic phase into a new 15 mL centrifuge tube; add another 4 mL of chloroform to the aqueous phase and repeat the extraction. Combine the organic phases from the two extractions and make up to 9 mL with chloroform.
[0089] (6) 1.5 mL of the extract was transferred to a 2 mL centrifuge tube and concentrated to dryness using an SPD2010 centrifugal concentrator (Thermo Fisher Scientific, Inc., Massachusetts, USA).
[0090] 2.2 Carotenoid determination
[0091] (1) Dissolve the sample in 50 μL of methyl tert-butyl ether (MTBE), shake thoroughly, and centrifuge at 12,000 rpm for 10 min.
[0092] (2) Transfer the upper liquid to the sample bottle, take 1 μL of sample, and use high performance convergence chromatography (UPC) 2 Carotenoids were determined using an ACQUITY UPC2 HSS C18 SB column (100 mm × 3.0 mm, 1.8 μm) maintained at an injection chamber temperature of 10°C. The biphasic mobile phase consisted of (A) CO2 and (B) methanol:ethanol (v:v) in a 1:2 ratio. The linear elution gradient was as follows: 95% A + 5% B (0.5 min); 70% A + 30% B (2 min); 70% A + 30% B (5 min); 95% A + 5% B (5.5 min); and 95% A + 5% B (7 min). The system flow rate was set at 1.5 mL / min, the column temperature at 45°C, and the back pressure at 22.9 MPa. The diode array detector (DAD) operated at wavelengths between 210 and 500 nm, with a compensation wavelength between 210 and 280 nm.
[0093] 2.3 Standard curve preparation
[0094] (1) Lycopene, β-carotene, α-carotene, and lutein standards (Sigma Chemical Co., CA, USA) were dissolved in MTBE (Thermo Fisher Scientific Inc., MA, USA) to prepare a concentration gradient (5 μg / mL, 10 μg / mL, 50 μg / mL, 250 μg / mL, and 500 μg / mL);
[0095] (2) Each standard sample was injected in order from low to high concentration to establish a standard curve of concentration and peak area to estimate the concentration of the carotene component in the sample.
[0096] The results of carotenoid component analysis showed that the total carotenoid content of yft1 and sledl was significantly lower than that of M82, indicating that EMB1444-like controls the accumulation of carotenoids in tomatoes by regulating YFT1 (Table 5).
[0097] Table 5 Carotenoid contents in fruits of tomatoes M82, yft1 and sled1 at different developmental stages (μg·g -1 FW)*
[0098]
[0099]
[0100] FW (fresh weight): fresh weight.
[0101] 3. Chromoplast Development and Ultrastructure
[0102] 3.1 Sectioning and ultrastructural observation
[0103] (1) Sampling and fixation: The equatorial peel of M82, yft1 and sled1 tomatoes at different developmental stages (35 dpa, 47 dpa and 54 dpa) was cut into approximately 1 mm 3 Tissues were fixed in 2.5% glutaraldehyde and vacuum treated with a vacuum pump (Millipore Co., Massachusetts, USA) for 30 min until the tissue blocks were completely immersed in the fixative, and then fixed at 4°C for 24 h.
[0104] (2) Rinsing and dehydration: Aspirate the fixative and rinse the sample with 0.1M PB (phosphate buffer), incubate at 4°C for 15 min, and repeat the operation three times. Discard the rinse solution, add an appropriate amount of osmium phosphate (Zhongjing Science and Technology Co., Ltd., Beijing, China), incubate at 4°C for 2 h, discard the osmium phosphate into a dedicated waste bottle, then rinse the sample with 0.1M PB, incubate at 4°C for 15 min, and repeat the operation three times. Dehydrate the sample with 50% ethanol, 70% ethanol, and 90% ethanol at 4°C for 15 min respectively; replace with 90% ethanol:90% acetone (v:v = 1:1) for 20 min, and then replace with 90% acetone for 20 min.
[0105] (3) Embedding and curing: Replace with 100% acetone for 20 min, 3 times; replace with a mixed solution of acetone: epoxy resin (Ted Pella Inc., California, USA) (v:v = 1:1) for 1 h, replace with acetone: epoxy resin (v:v = 1:2) overnight; replace with pure resin for 7 h; remove the embedding plate, add an appropriate amount of resin, carefully place the sample on the appropriate position on the embedding plate with a toothpick (record the sample position), and place it in a 60°C electric heated blast drying oven for polymerization for 48 h.
[0106] (4) Sample sectioning, staining, and electron microscopy observation: The solidified embedded blocks were trimmed to expose the sample surface. The samples were then sliced using a UC6-FC6 cryo-ultrathin microtome (Leica Instruments GmbH, Germany) and stained with 2% lead citrate (Zhongjing Science and Technology Co., Ltd., Beijing, China). The ultrastructure of the chromoplasts was observed using a 120 kV biological transmission electron microscope (FEI, Oregon, USA).
[0107] Observation of the chromoplast development process revealed that the development of yft1 and sled1 chromoplasts was delayed compared with the wild-type M82, indicating that EMB1444-like affects the development of chromoplasts in tomato fruit by regulating the expression of YFT1. Figure 7 ).
[0108] 3.2 Liposome sphere count
[0109] The number of lipid globules was counted in three different fields of view for each sample under transmission electron microscopy (three biological replicates, n=3). The results showed that at 54 dpa, the number of lipid globules in yft1 and sled1 was significantly lower than that in wild-type M82. At the same time, the number of lipid globules in sled1 fruit was significantly higher than that in yft1. EMB144-like can affect the number of lipid globules in tomato fruit by regulating YFT1 expression ( Figure 7 ).
[0110] Example 5: EMB1444-like regulates ethylene biosynthesis
[0111] To clarify the effects of EMB1444-like regulation of YFT1 on ethylene synthesis and signal transduction in tomato fruit, the expression differences of key genes involved in ethylene synthesis and ethylene signal transduction in the fruits of M82, mutant yft1, and sledl were analyzed. The specific steps are as follows:
[0112] 1. Determination of ethylene release from tomato fruits
[0113] Tomato fruits (n=3) from M82, yft1, and sled1 varieties at different developmental stages (35, 47, and 54 dpa) were collected, and the weight of each fruit was recorded. The fruits were then placed at a constant temperature of 25°C for 2 hours to eliminate any changes in ethylene release levels caused by physiological stress from harvesting. The fruits were carefully sealed in a 500 mL gas collection bottle and the gas was collected at a constant temperature of 25°C for 4 hours. The gas was slowly aspirated using a 1 mL disposable syringe and rapidly injected into a GC-2010 gas chromatograph (Shimadzu Instruments Co., Ltd., Japan) for determination of ethylene content. The ethylene concentration of the samples was calculated using standard ethylene gas (10 ppm). A standard curve was prepared by injecting 0.1 mL, 0.3 mL, 0.5 mL, 0.6 mL, and 0.9 mL of standard ethylene gas (10 ppm) at each time point. The standard curve was constructed with concentration as the ordinate and peak area as the abscissa. Ethylene release from tomatoes was calculated using the following formula.
[0114]
[0115] The test results showed that the peak of ethylene release in the fruit of the sled1 mutant was delayed compared with that of M82, and the release amount was significantly decreased; while the ethylene release amount in the fruit of the sled1 mutant was significantly higher than that of the yft1 tomato ( Figure 8 ).
[0116] 2. EMB1444-like regulates the expression of key genes for ethylene synthesis
[0117] Total RNA (n=3) was extracted from the equatorial pericarp of M82, yft1, and sled1 fruits at different developmental stages (35, 47, and 54 dpa). Reverse transcription was performed to prepare cDNA libraries, which were used as templates for RT-qPCR. RT-qPCR analysis of the key ethylene biosynthesis genes ACS2 / 4 (ACCSYNTHASE 2 / 4) and ACO1 (ACC OXIDASE 1) was performed using a LightCycle 96 real-time fluorescence quantitative PCR instrument (Roche GmbH, Switzerland) using gene-specific primers (Table 6). The results showed that the expression levels of ACS2 / 4 and ACO1 in the sled1 mutant were lower than those in M82, but significantly higher than those in yft1. This suggests that EMB1444-like affects the expression of key ethylene biosynthesis genes in tomato fruit by regulating YFT1 expression. Figure 9 ).
[0118] 3. Analysis of key gene expression in the ethylene signal transduction pathway
[0119] The cDNA library prepared in 2. was used as a template to analyze the expression of key genes in the ethylene signaling pathway, including NR (NEVER RIPE), ETR4 (ETHYLENE RECEPTOR 4), AP2a (APETALA 2a), and ERF4 / 6 (ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 4 / 6). The results showed that the expression levels of NR, ETR4, AP2a, and ERF4 / 6 in the sled1 mutant were significantly lower than those in M82 after yft1 and EMB1444-like-RNAi, but significantly higher than those in the yft1 mutant. This suggests that EMB1444-like affects ethylene signaling in tomato fruit by regulating YFT1 expression. Figure 10 ).
[0120] Table 6 Primer sequences for RT-qPCR analysis of key genes in ethylene biosynthesis and ethylene signal transduction
[0121]
[0122]
[0123] In summary, the present invention, based on a transcription factor EMB1444-like, specifically binds to an E-box (CACTTG, -1295bp to -1290bp) functional element in the pYFT1 regulatory region upstream of the promoter encoding the gene YFT1, a core component of the ethylene signaling pathway, SlEIN2. This positively regulates YFT1 expression, controlling ethylene synthesis, carotenoid synthesis, and chloroplast / chromoplast development in tomato fruit, thereby influencing tomato fruit color formation and fruit ripening. This invention can provide a theoretical basis for improving tomato quality and maturity, and can be used to develop tomato varieties with high quality and shelf life suitable for production applications.
[0124] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. The embodiments of this application and the features in the embodiments may be combined with each other in any manner unless there is a conflict.
Claims
1. An application of a bHLH family transcription factor EMB1444-like in regulating tomato fruit ripening and development, characterized in that: The amino acid sequence of EMB1444-like is shown in SEQ ID NO. 3; the regulation is to delay the ripening of tomato fruit by downregulating the gene expression of the transcription factor EMB1444-like.
2. The use according to claim 1, characterized in that The CDS sequence of EMB1444-like is shown in SEQ ID NO.
4.
3. The use according to claim 1, characterized in that The transcription factor EMB1444-like regulates tomato fruit ripening and color by regulating ethylene synthesis, ethylene signal transduction, chromoplast development, carotenoid accumulation, and fruit color formation.
4. The use according to claim 1, characterized in that The EMB1444-like protein positively regulates the transcriptional expression of the gene YFT1 encoding the key component SlEIN2 of ethylene signal transduction.
5. The use according to claim 4, characterized in that The EMB1444-like specifically binds to the E-box motif and its flanking sequence of the upstream promoter pYFT1 of the key gene YFT1 in the ethylene signaling pathway to regulate the expression of YFT1; the E-box motif is shown in SEQ ID NO.1; the E-box motif and its flanking sequence are shown in SEQ ID NO.
2.
6. An RNA interference sequence for downregulating the expression of the transcription factor EMB1444-like gene, characterized in that: The DNA sequence of the RNA interference sequence is shown as SEQ ID NO.
5.
7. An EMB1444-like down-regulation expression vector, characterized in that: The method for constructing the expression vector comprises the following steps: The EMB1444-like specific DNA fragment was amplified using specific primers EMB1444-like-RNAi-F and EMB1444-like-RNAi-R, as shown in SEQ ID NO. 5, and constructed into the RNAi vector pHELLSGATE 12 to obtain an EMB1444-like down-regulation expression vector, named EMB1444-like-RNAi; the sequence of the EMB1444-like-RNAi-F is shown in SEQ ID NO. 44; the sequence of the EMB1444-like-RNAi-R is shown in SEQ ID NO.
45.
8. Use of the expression vector according to claim 7 in delaying the ripening of tomato fruits.