An SlZFP11 gene and its application in regulating crop storage tolerance

Abstract

This invention discloses a SlZFP11 Genes and their application in regulating crop storage tolerance belong to the field of genetic engineering technology. This invention utilizes CRISPR / Cas9 gene editing technology to insert endogenous genes from tomatoes... SlZFP11 Knockout in tomatoes increases the content of sugars such as glucose, fructose, and sucrose, as well as cellulose in the resulting tomato fruits, greatly enhancing the sweetness of the fruit and thus improving fruit quality. At the same time, it reduces water loss and significantly increases storage tolerance, extending the shelf life of tomatoes and providing genetic resources and strategies for tomato germplasm innovation.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a... SlZFP11 Genes and their application in regulating crop storage tolerance. Background Technology

[0002] tomato( Solanum lycopersicum As a typical climacteric fruit, the ethylene content increases dramatically during ripening, easily causing rapid softening and post-harvest decay, resulting in significant losses. While simply suppressing ethylene or key ripening factors can delay softening, it often leads to inhibited accumulation of color and flavor compounds, resulting in the contradictory problem of "good storage life but poor taste," making it difficult to achieve a synergistic improvement in both storage life and flavor.

[0003] In recent years, genetic engineering and molecular breeding techniques have provided new avenues for improving the overall quality of tomatoes. Zinc finger proteins (ZFPs), as important transcriptional regulators, are widely involved in the regulation of growth and development, stress responses, and fruit ripening in plants. ZFPs regulate downstream gene expression through their specific DNA-binding domains, playing a crucial role, especially in abiotic stress and hormone signaling networks. Previous studies have shown that certain ZFP members influence multiple biological processes, including cell wall modification, sugar metabolism, and antioxidant synthesis, demonstrating potential in the synergistic regulation of multiple traits. However, regarding the role of ZFPs in tomatoes… SlZFP11 The specific functions of the gene are not yet clear, and its regulatory mechanisms in fruit ripening, quality formation and postharvest storage resistance still need to be elucidated.

[0004] Therefore, we should start with key transcription factors and explore... SlZFP11 Its function in synergistically improving the quality and storability of tomato fruit not only helps to deepen the understanding of the fruit ripening regulatory network, but also provides new ideas for breaking the negative correlation between "storability-flavor", providing effective genetic resources and technical approaches for upgrading fruit and vegetable quality and reducing post-harvest losses. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to provide a SlZFP11 Genes and their application in regulating crop storage tolerance to solve the technical problems of poor tomato quality and short shelf life.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a SlZFP11 Application of genes in regulating the storage tolerance of tomatoes SlZFP11 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; SlZFP11 Genes can also regulate the dry matter and sugar content of tomatoes.

[0007] Based on the above technical solution, the present invention can be further improved as follows:

[0008] further, SlZFP11 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0009] Furthermore, by inhibiting or knocking out SlZFP11 Genes that increase the dry matter and sugar content of tomatoes, while also improving their storage resistance.

[0010] The beneficial effects of this invention are as follows: This invention uses CRISPR / Cas9 gene editing technology to insert endogenous genes from tomatoes... SlZFP11 Knockout in tomatoes increases the content of sugars such as glucose, fructose, and sucrose, as well as cellulose in the resulting tomato fruits, greatly enhancing the sweetness of the fruit and thus improving fruit quality. At the same time, it reduces water loss and significantly increases storage tolerance, extending the shelf life of tomatoes. This provides genetic resources and strategies for tomato germplasm resource innovation, better meeting consumer needs. Attached Figure Description

[0011] Figure 1 Map of the constructed FastCas98 expression vector;

[0012] Figure 2 for SlZFP11 Electrophoretic detection results of CRISPR / Cas9 gene knockout plants;

[0013] Figure 3 for SlZFP11 A schematic diagram of gene editing in gene knockout strains;

[0014] Figure 4 Wild-type tomatoes and SlZFP11 Results of water loss rate detection in tomato fruits of gene knockout strains;

[0015] Figure 5 Wild-type tomatoes and SlZFP11 Comparative results of storage experiments on tomato fruits from gene knockout strains;

[0016] Figure 6 Wild-type tomatoes and SlZFP11 Results of dry matter content detection in red-ripe tomato fruits of gene knockout strains;

[0017] Figure 7 Wild-type tomatoes and SlZFP11 Results of sucrose content detection in red-ripe tomato fruits of gene knockout strains;

[0018] Figure 8 Wild-type tomatoes and SlZFP11Results of fructose content detection in red-ripe tomato fruits of gene knockout strains;

[0019] Figure 9 Wild-type tomatoes and SlZFP11 Results of glucose content detection in red-ripe tomato fruits of gene knockout strains;

[0020] Figure 10 Wild-type tomatoes and SlZFP11 Gene knockout strains of tomato fruit related to sugar metabolism SlLIN5 The test results;

[0021] Figure 11 Wild-type tomatoes and SlZFP11 Gene knockout strains of tomato fruit related to sugar metabolism SlLIN7 The test results;

[0022] Figure 12 Wild-type tomatoes and SlZFP11 Gene knockout strains of tomato fruit related to sugar metabolism SlSUS3 The test results. Detailed Implementation

[0023] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0024] SlZFP11 The nucleotide sequence of the gene is as follows:

[0025] (SEQ ID NO: 1).

[0026] SlZFP11 The amino acid sequence of the protein encoded by the gene is as follows:

[0027] MENDFPNSSNSQKYQITWGPDGGKISQVKCYRCSFCKRGFSNAQALGGHMNIHRKDRAKLREISIETSDHIKKFVSPSSPDHIQALSSTDELILQHDISSDDMSNNPSKRPCVTLEEQHHNHNHHHHISKEKDENHELIIGGDVLQLPLFVDSPSKEEINKGMQLSVDDSKLDLELRLGPEP (SEQ ID NO: 2).

[0028] The primer sequences used in the following examples are shown in Table 1.

[0029] Table 1 Primer sequence listing

[0030]

[0031] Example 1 Construction SlZFP11CRISPR / Cas9 gene knockout system

[0032] 1. Design SlZFP11 gene target sequence

[0033] Target site design was performed using the website http: / / cas9.cbi.pku.edu.cn / index.jsp. The results of the target site design allowed for the evaluation of all candidate targets, including their sequence, location, GC content, and potential off-target sites. Targets with a GC content between 45% and 70%, located in the first two-thirds of the gene's CDS region (after ATG, but not on the last exon), and having at least three base mismatches with any other location in the genome were selected.

[0034] 2. Construction SlZFP11 CRISPR / Cas9 vector of genes

[0035] (1) Construction SlZFP11 CRISPR / Cas9 vector primer design for genes

[0036] ZFP11-Cas9 -F: CCAAAATACCAAATAACATGGG (gRNA1) GTTTCAGAGCTATGCTGGAAACA (SEQ ID NO: 5);

[0037] ZFP11-Cas9 -R:ATAACATGGGGTCCTGATGGAGG (gRNA2 reverse complementary sequence)CAATCACTACTTCGACTCTAGCTGT (SEQ ID NO: 6).

[0038] (2) Amplification of the target fragment: The amplification system is shown in Table 2.

[0039] Table 2 PCR amplification system

[0040]

[0041] The amplification program was as follows: 98℃ for 3 min; (98℃ for 10 s, 58℃ for 20 s, 72℃ for 30 s) for 30 cycles; 72℃ for 5 min; and stored at 4℃.

[0042] (3) FastCas9 vector digestion and fragment ligation: The ligation system is shown in Table 3.

[0043] Table 3 FastCas9 vector digestion and fragment ligation system

[0044]

[0045] The ligation procedure was: 37℃, 2 min; 16℃, 5 min. Six cycles were performed. 1 µL of the solution was used to transform competent E. coli cells.

[0046] (4) Colony PCR verification

[0047] Four single colonies were selected for PCR verification of each gene. Positive colonies were amplified and sent to a sequencing company for sequencing. Plasmids were extracted from the correctly sequenced bacterial cultures. The correct recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 and screened on plates containing 50 μg / mL kanamycin and 100 μg / mL rifampin. Single clones were selected for colony PCR verification. The successfully verified strains are the engineered bacteria for the recombinant expression vector.

[0048] The constructed FastCas98 expression vector map is as follows: Figure 1 As shown.

[0049] Example 2 Obtained SlZFP11 Gene knockout line plants

[0050] 1. Transform tomato explants using Agrobacterium containing recombinant plasmids via leaf disc method.

[0051] (1) Seed disinfection

[0052] Wild-type tomato seeds were placed in a sterile container and soaked in a 70% ethanol aqueous solution for 30 seconds to disinfect the seed surface. The ethanol aqueous solution was then drained, and the seeds were rinsed twice with sterile water. Next, they were soaked in a 5% sodium hypochlorite solution for 15 minutes, shaking constantly to ensure thorough disinfection of each seed. After rinsing 3-4 times with sterile water, the water was drained from the seeds, which were then transferred to a seed culture medium and placed in a light incubator. The culture conditions were: 14 hours of light culture at 25℃ and 10 hours of darkness culture at 20℃; the light intensity was 250 μmol•m. -2 •s -1 The relative humidity is 80%; the culture period is 8-10 days.

[0053] (2) Pre-culture of explants

[0054] The optimal time to obtain explants is about 9-10 days after the seeds germinate, when the true leaves of the tomato seedlings are about to emerge. Place the seedlings on sterile filter paper, cut the cotyledons and hypocotyls into segments with a sterile scalpel blade, and then transfer them to KCMS pre-medium for dark culture for one day.

[0055] (3) Agrobacterium infection of explants

[0056] Add 100 μL of preserved Agrobacterium tumefaciens GV3101 to 20 mL of LB medium (containing 20 μL, 50 μg / mL kanamycin and 40 μL, 100 μg / mL rifampin), and incubate overnight at 28 °C and 230 rpm until the absorbance value OD is reached. 600 Approximately 0.8-1.0. Take 1 mL of bacterial culture, centrifuge at 5000 rpm for 5 min, discard the supernatant, wash the precipitate once with KCMS liquid medium, and then dilute the bacterial culture to OD using KCMS liquid medium. 600 =0.1. Use a pipette to add one drop of Agrobacterium dilution to the wound of the explant. The bacterial solution does not need to be aspirated. Seal the petri dish and place it on KCMS medium for dark incubation for 2 days.

[0057] (4) Differentiation and rooting of explants

[0058] After co-culturing explants with Agrobacterium for 2 days, the explants were transferred from KCMS medium to a primary screening medium (2Z) supplemented with 20 μM trans-zeatin nucleotides, with the medium being changed every 15 days. Once differentiated buds emerged from the explants, they were transferred to a medium (1Z) supplemented with 10 μM trans-zeatin nucleotides, with the medium being changed every 15 days. After two cultures on 1Z medium, the differentiated callus tissue produced buds with independent main stems, which were cut off and inserted into the rooting medium ENR. After rooting, the buds were transplanted.

[0059] (5) Identification of transgenic positive plants

[0060] Transgenic T0 generation seedlings were tested using CRISPR / Cas9 vector detection primers Cas9-F (SEQ ID NO: 3) / Cas-R (SEQ ID NO: 4) to confirm whether they were transgenic positive plants. The test results are as follows: Figure 2 As shown.

[0061] like Figure 2 As shown, PCR amplification was performed using the genomic DNA of T0 generation seedlings as a template, and a 500bp fragment was detected by electrophoresis, confirming it as a transgenic positive plant.

[0062] Subsequently, using the genomic DNA of T0 generation seedlings as a template, detection primers ZFP11-Cas9check-F (SEQ ID NO: 7) / ZFP11-Cas9 check-R (SEQ ID NO: 8) containing the target site were designed for PCR amplification. After agarose gel electrophoresis, the target band was recovered, and the purified PCR product was constructed into the pEASY-Blunt-Zero cloning vector for single-clone sequencing. Twenty single clones were sequenced from each transgenic positive plant. The sequencing results were compared with the wild-type (WT) sequence to determine the editing status of the T0 generation plants. Figure 3 As shown, the selected SlZFP11 Gene knockout lines ( SlZFP11 -KO-L1 and SlZFP11 The mutation modes of -KO-L2 are the deletion of five bases in gRNA2 and the insertion of one base in gRNA1.

[0063] Seeds from the T0 generation that were sequenced as heterozygous were selected and screened with kanamycin before being sown again. The editing status of the T1 generation plants was analyzed (using the same editing detection method as the T0 generation plants). Seeds from 2-3 homozygous lines with different editing forms were collected, screened with kanamycin, and sown into the T2 generation for subsequent research.

[0064] Example 3 Knockout SlZFP11 The effect of gene modification on the storage tolerance of tomato fruit

[0065] 1. SlZFP11 Determination of water loss rate in gene knockout plants

[0066] Harvesting WT and SlZFP11 Gene knockout lines ( SlZFP11 -KO-L1 and SlZFP11 -KO-L2) Tomato fruits were washed and disinfected with distilled water, and then stored for 10 days under conditions of 60% humidity, 16 hours of light (25℃) followed by 8 hours of darkness (18℃). The storage water loss rate was calculated by measuring the fresh weight of each fruit daily, using the following formula:

[0067] ;

[0068] in, a The percentage of water loss during storage is %. b 0 represents the fresh weight of the fruit on the first day, in grams; b t The fresh weight of the fruit after storage for t days is expressed in g.

[0069] Water loss rate test results are as follows Figure 4 As shown. Figure 4 As shown, SlZFP11 The water loss rate of tomato fruits in the gene knockout line was significantly reduced.

[0070] 2. SlZFP11 Gene knockout plant storage experiment

[0071] Harvesting WT and SlZFP11 Gene knockout lines ( SlZFP11 -KO-L1 and SlZFP11-KO-L2) Tomato fruits were washed and disinfected with distilled water, and then stored for 10 days under conditions of 60% humidity, 16 hours of light (25℃) followed by 8 hours of darkness (18℃). The degree of fruit collapse was recorded and assessed on the 10th day. The storage results of the tomato fruits are as follows: Figure 5 As shown.

[0072] like Figure 5 As shown, SlZFP11 The gene knockout tomato fruit showed less skin collapse compared to the WT group, and significantly improved storage resistance.

[0073] In summary, this demonstrates that gene editing technology can be used to knock out the gene in tomatoes. SlZFP11 The gene significantly increases the cellulose content of tomato fruits while reducing water loss, further enhancing the storage resistance of tomatoes and thus improving fruit quality. This provides a valuable reference for innovation in tomato germplasm resources, aiming to better meet consumer needs.

[0074] Example 4 Knockout SlZFP11 Effects of gene modification on dry matter and sugar content of tomato fruit

[0075] 1. SlZFP11 Determination of dry matter content in gene knockout fruits

[0076] Harvest wild-type (WT) and SlZFP11 Remove ripe, red-colored fruits from the plants. Set up three biological replicates. Wash the fruit surface with distilled water, dry them, and then squeeze the tomato fruits firmly in gauze to extract the juice into a clean beaker. This is the crude extract of soluble solids. Analyze the crude extract using a handheld refractometer. The dry matter results are as follows: Figure 6 As shown.

[0077] according to Figure 6 The test results show that, in the knockout SlZFP11 After gene modification, the dry matter content in tomato fruits increased significantly.

[0078] 2. S lZFP11 Determination of sugar content in the fruit of gene knockout plants

[0079] Sugar content was determined from fruit samples collected at the red-ripe stage from wild-type and gene knockout plants. 0.1 g of fruit sample was weighed, and soluble sugars were extracted from the tomato fruit using chromatographically pure (Ar) methanol as the extractant. Three biological replicates were set up.

[0080] The specific procedure was as follows: The sample was derivatized using methoxyamine hydrochloride and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Determination was performed using a Shimadzu GC 2010 Pro chromatographic instrument. The chromatographic parameters were: SPL temperature 250℃, split ratio 10:1; column flow rate 1 mL / min; temperature program: initial temperature 130℃, increased to 185℃ at a rate of 5℃ / min, then increased to 190℃ at a rate of 0.5℃ / min, and finally increased to 300℃ at a rate of 8℃ / min, held for 10 min. The detector temperature was 320℃, hydrogen flow rate was 40 mL / min, dry air flow rate was 400 mL / min, and nitrogen flow rate was 40 mL / min. The results of glucose, sucrose, and fructose content determination are as follows: Figures 7-9 As shown, the horizontal axis represents the fruit ripening time point. "Br" represents the Breaker stage, which is the moment when the fruit begins to change from green to light yellow or orange, and is the starting point of tomato fruit ripening. "Br+3" represents the 3rd day after the color breaks, which is the ripening stage of the fruit from the 3rd day after the color breaks. "Br+7" represents the 7th day after the color breaks, which is the late ripening stage.

[0081] according to Figures 7-9 The test results show that, in the knockout SlZFP11 After gene therapy, the glucose, sucrose, and fructose content in tomato fruits increased significantly.

[0082] 3. SlZFP11 Detection of the expression of genes affecting sugar metabolism in tomato fruits from gene knockout strains

[0083] (1) Take the same developmental stage SlZFP11 Gene knockout strains ( SlZFP11 -KO-L1 and SlZFP11 -KO-L2) and wild-type (WT) tomato fruits, each with more than 3 biological replicates;

[0084] (2) Frozen tissues were ground in liquid nitrogen, and total RNA was extracted from the samples using the Tiangen RNAprep Pure Plant Total RNA Extraction Kit (DP432). The purity and integrity of the RNA were then assessed. The purity (OD260 / 280≈1.8-2.0) and integrity (clear electrophoretic bands) were detected using a micro-ultraviolet spectrophotometer, indicating good RNA purity. 1 μg of RNA was used to perform reverse transcription to synthesize cDNA using the Novizan HiScriptIII All-in-one RT SuperMix Perfect for qPCR Kit (R333-C1), and then diluted for later use.

[0085] (3) Perform qRT-PCR detection and calculate the relative expression level. Statistical analysis (t-test).P <0.05 is considered significant. SlZFP11 The expression detection results of genes affecting cell wall metabolism in tomato fruits from gene knockout strains are as follows: Figures 10-12 As shown.

[0086] like Figures 10-12 As shown, genes related to glucose metabolism SlLIN5 , SlLIN7 and SlSUS3 The level of expression at SlZFP11 -KO-L1 and SlZFP11 The significant increase in the fruit of -KO-L2 plants indicates SlZFP11 Genes influence the sugar content in tomato fruits by regulating the expression of genes involved in sugar metabolism.

[0087] In summary, this demonstrates that gene editing technology can be used to knock out the gene in tomatoes. SlZFP11 The gene significantly increases the sugar content of tomato fruit, greatly enhancing its sweetness and thus improving fruit quality. This provides a valuable reference for innovation in tomato germplasm resources, aiming to better meet consumer demands.

Claims

1. A kind SlZFP11 The application of genes in regulating the storage tolerance of tomatoes is characterized by, By inhibiting or knocking out SlZFP11 the gene, the dry matter and sugar content of the tomato is increased, and the storage resistance of the tomato is also increased; the nucleotide sequence of the gene is shown as SEQ ID NO:

1. SlZFP11 the gene, the dry matter and sugar content of the tomato is increased, and the storage resistance of the tomato is also increased; the nucleotide sequence of the gene is shown as SEQ ID NO: 1.

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