Application of SlbHLH81 gene in prolonging shelf life of crop fruits

Abstract

The application discloses a kind of SlbHLH81 The application discloses application of a gene in prolonging shelf life of crop fruits, and belongs to the technical field of genetic engineering.The endogenous gene of Solanum lycopersicum is knocked out by CRISPR / cas9 gene editing technology SlbHLH81 In the tomato, the hardness of the tomato fruit is improved after knocking out, the water loss rate is reduced, the Brix, acidity and sugar-acid ratio do not change significantly, the storage resistance of the tomato fruit is greatly improved without loss of flavor quality, the postharvest loss of the fruit is reduced, thereby effectively prolonging the shelf life of the tomato fruit, providing gene resources and strategies for innovation of tomato germplasm resources, and better meeting the needs of consumers.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a... SlbHLH81 Application of genes in extending the shelf life of crop fruits. Background Technology

[0002] Tomatoes, as an important economic crop, are highly favored by consumers for their delicious flesh, rich nutrition, and unique flavor, and are widely cultivated worldwide as a fresh fruit and vegetable. The nutritional and flavor qualities of tomatoes are crucial factors determining fruit consumption. Flavor, as a subjective phenotype, is primarily composed of sugars, acids, and many volatile compounds in tomato fruit. Among these, sugar compounds are the main factors influencing the flavor and nutritional value of fruits and vegetables, and are one of the important indicators for measuring their quality and economic value.

[0003] Tomatoes, as a typical climacteric fruit, cannot be stored for long periods. During storage, the fruit's taste gradually deteriorates, wilting, shrivelting, softening, and eventually rotting. Nutrients are rapidly lost, and antioxidant capacity declines sharply, severely impacting the tomato's commercial value. Therefore, reducing post-harvest losses and improving storability without affecting fruit flavor and quality has always been a hot topic in horticultural crop research and a crucial industrial issue. Developing new, high-quality, and storable tomato varieties is a key objective of current tomato breeding. Given the shift in my country's fruit and vegetable industry's focus from yield growth to quality, utilizing genetic engineering technology to simultaneously improve fruit flavor, quality, and yield is a significant goal in Chinese horticultural crop breeding. Currently, traditional breeding methods are time-consuming and have inconsistent results, often exhibiting significant uncertainty and unpredictability. Therefore, utilizing biotechnology to simultaneously improve fruit yield and quality will be a crucial development direction for modern tomato breeding.

[0004] Basic helix-loop-helices (bHLHs) transcription factors are one of the largest families of transcription factors in plants, playing important roles in plant growth, development, and responses to abiotic stress. Previous studies have shown that certain bHLH members function in regulating drought tolerance in tomatoes, anthocyanin accumulation in fruits, and defense against leafminer moths. However, research on the role of basic helix-loop-helices (bHLHs) in tomato transcription factors remains unclear. SlbHLH81 The specific function of the gene is not yet clear, and its regulatory mechanism in the postharvest storage tolerance of fruit remains to be elucidated. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to provide a SlbHLH81 The application of genes in extending the shelf life of crop fruits can improve the storage resistance of tomatoes without reducing their flavor and quality, thus solving the technical problem of insufficient tomato fruit shelf life.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a SlbHLH81 The application of genes in extending the shelf life of tomato fruits, through inhibition or knockout. SlbHLH81 Genes that improve the firmness of tomato fruits, reduce their water loss rate, and extend the shelf life of tomato fruits; SlbHLH81 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

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

[0008] further, SlbHLH81 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0009] The beneficial effects of this invention are as follows: This invention uses CRISPR / Cas9 gene editing technology to insert endogenous genes from tomatoes... SlbHLH81 Knocking out tomatoes increases fruit firmness and reduces water loss, while maintaining no significant changes in Brix, acidity, and sugar-acid ratio. This greatly improves the storability of tomatoes without compromising flavor and quality, reduces post-harvest losses, and effectively extends the shelf life of tomatoes. It also provides genetic resources and strategies for tomato germplasm innovation, better meeting consumer needs. Attached Figure Description

[0010] Figure 1 Map of the constructed BG-plant-fast-cas9 expression vector;

[0011] Figure 2 for SlbHLH81 Positive identification results of CRISPR / Cas9 gene knockout plants;

[0012] Figure 3 for SlbHLH81 A schematic diagram of gene editing in gene knockout strains;

[0013] Figure 4 Wild-type tomatoes and SlbHLH81 Statistical chart of tomato fruit firmness in gene knockout strains;

[0014] Figure 5 Wild-type tomatoes and SlbHLH81 Comparison of tomato fruits from gene knockout strains after 60 days of storage;

[0015] Figure 6 Wild-type tomatoes and SlbHLH81 Statistical chart of water loss rate of tomato fruits from gene knockout strains;

[0016] Figure 7 Wild-type tomatoes and SlbHLH81 Statistical chart of Brix content in tomato fruits from gene knockout strains;

[0017] Figure 8 Wild-type tomatoes and SlbHLH81 A statistical chart of total acidity in tomato fruits from gene knockout strains;

[0018] Figure 9 Wild-type tomatoes and SlbHLH81 A statistical chart of the sugar-acid ratio in tomato fruits from gene knockout strains. Detailed Implementation

[0019] 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.

[0020] SlbHLH81 The nucleotide sequence of the gene is as follows:

[0021] (SEQ IDNO:1).

[0022] SlbHLH81 The amino acid sequence of the protein encoded by the gene is as follows:

[0023] MELTQEDFLEEIVSPRIENWNTFANAWNIESPTFYQQNPEFIPSNSSLLDLIMSPSQSNYFPCPDFQESSYPFLHSFTTTTPPQLVIDSTTYNNNNIQERAIIEEGQIGHFSTDFHGHYEDSFSCYNINKVVKMEEATSRIVGEKKSKNYKVKKVEGQPSKNLMA ERRRRKRLNDRLSMLRSIVPKISKMDRTSILGDTIDYVKELLDKINRLHEENEIKDIKFLGNFKGLKTNEALVRNPPKFDVERRNEDETSIEICCGTKPGLLLSTVHTMEALGLEVQQCVVSCFSDFSMRASCSESVDHRTILSSEDVKQALFKTAGYGGRCV (SEQ ID NO: 2).

[0024] The primer sequences used in the following examples are shown in Table 1.

[0025] Table 1 Primer sequence listing

[0026]

[0027] Example 1 Construction SlbHLH81 CRISPR / Cas9 gene knockout system

[0028] 1. Design SlbHLH81 gene target sequence

[0029] CRISPR2 / CRISPR target site design was performed using the website http: / / crispr.hzau.edu.cn / cgi-bin / . The target site design results allow for the evaluation of all candidate target sites, including their sequence, location, GC content, and potential off-target sites. Target sites with a GC content between 40% and 60%, 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.

[0030] 2. Construction SlbHLH81 CRISPR / Cas9 vectors for genes

[0031] (1) The vector was constructed using homologous recombination. The gene editing vector used was BG-plant-fast-cas9, and primers were designed. SlbHLH81 -gRNA1-F (SEQ ID NO: 5) and SlbHLH81 PCR amplification was performed using -gRNA2-R (SEQ ID NO: 6).

[0032] (2) Gene amplification was performed using the gRNA expression vector osgRNA-U626 as a template, and PrimerSTAR high-fidelity enzyme and primer pairs were used. SlbHLH81 -gRNA1-F and SlbHLH81 PCR amplification was performed using 1-gRNA2-R, and the amplification system is shown in Table 2.

[0033] Table 2 PCR amplification system

[0034]

[0035] The amplification program was as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 5 min.

[0036] (3) Linearize the vector by digesting the BG-plant-fast-cas9 vector with BsmBI enzyme. The reaction system is shown in Table 3.

[0037] Table 3. Linearization reaction system of the carrier

[0038]

[0039] The reaction conditions were: 37℃ for 5 min, 65℃ for 10 min.

[0040] (4) Homologous recombination technology was used to ligate the sgRNA expression cassette into the BG-plant-fast-cas9 final vector. The reaction system is shown in Table 4.

[0041] Table 4 Homologous recombination reaction system

[0042]

[0043] The reaction conditions are: 50℃, 15-60 min.

[0044] The constructed BG-plant-fast-cas9 expression vector map is as follows: Figure 1 As shown.

[0045] Example 2 Obtained SlbHLH81 Gene knockout line plants

[0046] 1. Screening of engineered bacteria

[0047] (1) Transformation of Escherichia coli

[0048] Take the one constructed in Example 1 SlbHLH81 10 μL of the gene CRISPR / Cas9 knockout system (BG-plant-fast-Cas9 expression vector) was transformed into Escherichia coli DH5α, and the cells were screened on plates containing 50 μg / mL kanamycin. Single clones were selected, and the colonies were then identified by PCR amplification and sent to a sequencing company for sequencing screening of positive strains.

[0049] (2) Transformation of Agrobacterium

[0050] Plasmids were extracted from positive strains by shaking and transformed into Agrobacterium tumefaciens GV3101. The strains were screened on LB agar plates containing 50 μg / mL kanamycin and 100 μg / mL rifampin. Single clones were selected for colony PCR verification. The strains that were successfully verified were the engineered bacteria containing the recombinant expression vector.

[0051] 2. Transform tomato explants using Agrobacterium containing a recombinant expression vector via the leaf disc method.

[0052] (1) Seed disinfection

[0053] Wild-type tomato seeds were placed in a sterile container and soaked in a 70% (v / v) 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% (v / v) 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°C, followed by 10 hours of darkness culture at 20°C; the light intensity was 250 μmol / L. m -2 s -1 The relative humidity is 80%; the culture period is 8-10 days.

[0054] (2) Pre-culture of explants

[0055] 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.

[0056] (3) Agrobacterium infection of explants

[0057] 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.

[0058] (4) Differentiation and rooting of explants

[0059] After co-culturing explants with Agrobacterium for 2 days, the explants were transferred from KCMS medium to primary screening medium (2Z) supplemented with 20 μM trans-zeatin nucleotides, with the primary screening medium being changed every 15 days. Once differentiated buds emerged from the explants, they were transferred to subculture medium (1Z) supplemented with 10 μM trans-zeatin nucleotides, with the medium being changed every 15 days. After two subcultures, the differentiated callus tissue produced buds with independent main stems, which were cut off and inserted into rooting medium (ENR medium). After rooting, the buds were transplanted.

[0060] (5) Identification of transgenic positive plants

[0061] 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.

[0062] like Figure 2 As shown, PCR amplification was performed using the genomic DNA of T0 generation seedlings as a template, and electrophoresis detected a 400bp fragment, confirming it as a transgenic positive plant.

[0063] Subsequently, using the genomic DNA of T0 generation seedlings as a template, detection primers containing the target site were designed. SlbHLH81 -Cas9-check-F (SEQ ID NO: 7) / SlbHLH81PCR amplification was performed using Cas9-check-R (SEQ ID NO: 8). The target band was recovered after agarose gel electrophoresis. The purified PCR product was constructed into the pEASY-Blunt-Zero cloning vector for single-clone sequencing. Twenty single clones were sequenced for 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.

[0064] like Figure 3 As shown, the selected SlbHLH81 Gene knockout lines ( SlbHLH81 -KO-L1 and SlbHLH81 The mutation modes of -KO-L2 are deletion of 271 bases and insertion of 1 base.

[0065] 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.

[0066] Example 3 Knockout SlbHLH81 The effect of gene modification on tomato fruit firmness

[0067] After obtaining homozygous mutants, wild-type (WT) and gene knockout plants were collected. SlbHLH81 -KO-L1 and SlbHLH81 -KO-L2) Firmness determination of red-ripe fruit samples. Fruits were collected 7 days after color breakage, and firmness was determined using a texture analyzer. Six fruits were measured in each group, with three biological replicates. The specific procedure was as follows: a texture analyzer (General TA, Shanghai Tengba Instrument Technology Co., Ltd., Shanghai, China) with a 5mm diameter cylindrical probe was used. Firmness measurement was performed at one point on the equatorial axis of each fruit, with the puncture test parameters set as speed 0.5mm / s and depth 6mm. The maximum force applied (in Newtons) was used to determine the firmness of the fruit. The results are as follows: Figure 4 As shown.

[0068] according to Figure 4 The test results show that, in the knockout SlbHLH81 After gene therapy, the firmness of tomato fruits increased significantly.

[0069] Example 4 Knockout SlbHLH81 Effects of gene modification on water loss rate of tomato fruit

[0070] Picking wild type SlbHLH81 -KO-L1 and SlbHLH81-KO-L2 strain red-ripe fruits were harvested, labeled, and weighed initially. The fruits were then stored in the middle section of a light-incubator (60% humidity, 16 hours of light (25℃) + 8 hours of darkness (18℃)). Fruit shrinkage and water loss were observed during storage. The actual weight of each fruit was recorded every 10 days until the end of 60 days. The water loss rate was calculated based on the statistical results, as shown below. Figure 5 and Figure 6 As shown.

[0071] After 60 days of storage, observe the wild type. SlbHLH81 -KO-L1 and SlbHLH81 -KO-L2 Tomato fruit skin wrinkling condition, such as Figure 5 As shown, it was found SlbHLH81 The gene knockout tomato fruit showed less skin collapse compared to the WT group, and significantly improved storage resistance. Figure 6 Statistics were compiled on wild-type cells after 30 and 60 days of storage. SlbHLH81 -KO-L1 and SlbHLH81 The water loss rate of KO-L2 tomato fruits was found to be higher after 30 and 60 days of storage. SlbHLH81 The water loss rate of tomato fruits in the gene knockout line was significantly lower than that in the WT line, indicating that the knockout line... SlbHLH81 After genetic modification, the shelf life of tomato fruits is extended.

[0072] Example 5 Knockout SlbHLH81 Effects of gene modification on Brix content, acidity, and sugar-acid ratio of tomato fruit

[0073] Harvest wild type and SlbHLH81 Remove the ripe, red-colored fruits from the plant, 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, obtaining a crude extract. The crude extract was then analyzed using a handheld refractometer. The Brix content, acidity, and sugar / acid ratio were measured as follows: Figures 7-9 As shown. Six fruits were measured in each group, with three biological replicates.

[0074] like Figures 7-9 As shown, SlbHLH81 The Brix content, acidity, and sugar-acid ratio of the gene knockout tomato fruit did not change significantly.

[0075] In summary, this demonstrates that gene editing technology can be used to knock out the gene in tomatoes. SlbHLH81 The gene significantly improves tomato fruit firmness and reduces water loss without affecting the Brix content, acidity, and sugar-acid ratio, thus greatly enhancing the fruit's storage resistance. This provides a valuable reference for tomato germplasm resource innovation, aiming to better meet consumer needs.

Claims

1. A kind SlbHLH81 The application of genes in extending the shelf life of tomato fruits is characterized by, By inhibiting or knocking SlbHLH81 Genes that improve the firmness of tomato fruits, reduce their water loss rate, and extend the shelf life of tomato fruits; SlbHLH81 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

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