Use of slidd7 gene in improving tomato yield and sugar content under high temperature environment

Abstract

The application discloses a kind of SlIDD7 The application discloses application of a gene in improving tomato yield and sugar content in high-temperature environment, and belongs to the technical field of genetic engineering. SlIDD7 The application discloses application of a gene in improving tomato yield and sugar content in high-temperature environment, and belongs to the technical field of genetic engineering. SlIDD7 The application discloses application of a gene in improving tomato yield and sugar content in high-temperature environment, and belongs to the technical field of genetic engineering. The application discloses application of a gene in improving tomato yield and sugar content in high-temperature environment, and belongs to the technical field of genetic engineering. The application discloses application of a gene in improving tomato yield and sugar content in high-temperature environment, and belongs to the technical field of genetic engineering.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a... SlIDD7 Application of genes in increasing tomato yield and sugar content under high temperature conditions. Background Technology

[0002] Tomatoes are an important vegetable crop worldwide, and high-temperature stress has become a key bottleneck restricting their yield and quality. Therefore, cultivating environmentally intelligent tomato strains that are "high-yielding in favorable conditions and stable-yielding in adverse conditions" and exploring their heat-resistant major functional genes have become core issues and cutting-edge hot topics in current vegetable genetics and breeding.

[0003] IDD The (INDETERMINATE DOMAIN) family of genes belongs to a subfamily of the C2H2 type zinc finger family. These are highly conserved transcription factors in plants, widely involved in growth, development, and stress responses, and are potential target genes for crop variety improvement. Currently, regarding... IDD Research on family genes has mainly focused on Arabidopsis thaliana and rice, while other species... IDD Gene functions remain largely unexplored, and tomatoes, as a model plant for studying fleshy fruits, offer a rich source of information about tomatoes. IDD Studies on family genes are rarely reported, and in tomatoes SlIDD7 The biological functions of the genes remain unexplained. Therefore, exploring tomatoes... IDD The biological functions of genes can not only elucidate IDD The regulatory mechanisms of family genes in fruit development and environmental response also provide an important theoretical basis for the quality improvement and molecular breeding of tomatoes and other fleshy fruits. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: to provide a SlIDD7 The application of genes in improving tomato yield and sugar content under high temperature conditions aims to solve the technical problem that tomato yield and quality cannot be improved in a coordinated manner under high temperature conditions.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a SlIDD7 The application of genes in increasing tomato yield and sugar content under high temperature conditions, by inhibiting SlIDD7 Gene expression or knockout SlIDD7 Genes that enhance tomato plants' tolerance to high temperatures, while also increasing fruit set rate and yield per plant, and improving the sucrose, fructose, and glucose content of tomato fruits. SlIDD7 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0006] Based on the above technical solution, the present invention can be further improved as follows:

[0007] Furthermore, the high temperature is 30-35℃.

[0008] further, SlIDD7 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... SlIDD7 In tomatoes, knockout was detected through phenotypic analysis, revealing that knockout... SlIDD7 The gene-injected lines showed significantly higher fruit set rate and yield per plant under high temperature conditions. Furthermore, the knockout lines exhibited a significant increase in the content of sugars such as sucrose, fructose, and glucose in tomato fruits, thereby improving fruit quality. This provides important genetic resources and strategies for synergistically enhancing tomato fruit yield and quality under high temperature stress conditions. Attached Figure Description

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

[0011] Figure 2 for SlIDD7 Electrophoretic detection results of CRISPR / Cas9 gene knockout plants;

[0012] Figure 3 for SlIDD7 A schematic diagram of gene editing in gene knockout strains;

[0013] Figure 4 Wild type and SlIDD7 Plant status diagram of gene knockout lines under high temperature conditions;

[0014] Figure 5 Wild type and SlIDD7 A comparison of the number of individual plants from gene knockout lines under high temperature conditions;

[0015] Figure 6 Wild type and SlIDD7 Statistical chart of fruit set rate of single plant in gene knockout lines under high temperature conditions;

[0016] Figure 7 Wild type and SlIDD7 A statistical chart of fruit yield per plant in gene knockout lines under high temperature conditions;

[0017] Figure 8 Wild type and SlIDD7 A statistical chart of sucrose content in tomato fruits of gene knockout lines under high temperature conditions;

[0018] Figure 9 Wild type and SlIDD7 A statistical chart of fructose content in tomato fruits of gene knockout lines under high temperature conditions;

[0019] Figure 10 Wild type and SlIDD7 A statistical chart showing the glucose content of tomato fruits from gene knockout strains under high-temperature conditions. Detailed Implementation

[0020] 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.

[0021] SlIDD7 The nucleotide sequence of the gene is as follows:

[0022]

[0023] SlIDD7 The amino acid sequence of the protein encoded by the gene is as follows:

[0024] (SEQ ID NO: 2).

[0025] The primer sequences used in the following examples are shown in Table 1.

[0026] Table 1 Primer sequence listing

[0027]

[0028] Example 1 Construction SlIDD7 CRISPR / Cas9 gene knockout system

[0029] 1. Design SlIDD7 gene target sequence

[0030] First, target site design should be performed. Target sequences should be selected with a GC content in the range of 45% to 70% and located in the first 2 / 3 of the gene's CDS region. Targets with an off-target estimate equal to or greater than 0.7 (or 0.6) have a higher risk of off-target mutations and should be avoided as much as possible.

[0031] 2. Construction SlIDD7 CRISPR / Cas9 vectors for genes

[0032] (1) Construct the vector using homologous recombination and design primers. IDD7 -gRNA1-F (SEQ ID NO: 5) and IDD7 PCR amplification was performed using -gRNA2-R (SEQ ID NO: 6).

[0033] (2) Amplification of the target fragment

[0034] Using the gRNA expression vector osgRNA-U626 as a template, PrimerSTAR high-fidelity enzyme and primer pairs were used. IDD7 -gRNA1-F and IDD7 PCR amplification was performed using -gRNA2-R, and the amplification system is shown in Table 2.

[0035] Table 2 PCR amplification system

[0036]

[0037] The amplification program was as follows: 98℃ for 1 min; (98℃ for 10 s, 58℃ for 15 s, 72℃ for 1 min) 35 cycles; 72℃ for 5 min; and stored at 4℃.

[0038] (3) FastCas9 vector digestion and fragment ligation: The ligation system is shown in Table 3.

[0039] Table 3 FastCas9 vector digestion and fragment ligation system

[0040]

[0041] The connection procedure is as follows: 37℃, 5 min; 65℃, 10 min.

[0042] (4) The sgRNA expression cassette was ligated into the BG-plant-fast-cas9 vector using homologous recombination technology. The reaction system is shown in Table 4.

[0043] Table 4 Homologous recombination reaction system

[0044]

[0045] The reaction conditions are: 50℃, 30-60 min.

[0046] The constructed BG-plant-fast-cas9 expression vector map is shown below. Figure 1 As shown.

[0047] Example 2 Obtained SlIDD7 Gene knockout line plants

[0048] 1. Screening of engineered bacteria

[0049] (1) Transformation of Escherichia coli

[0050] Take the one constructed in Example 1 SlIDD7 10 μL of the ligation product from the CRISPR / Cas9 knockout system (BG-plant-fast-Cas9 expression vector) was added to *E. coli* DH5α, incubated on ice for 25 min, followed by heat shock at 42°C for 45 s, then on ice for 2 min. 500 μL of antibiotic-free LB medium was added, and the mixture was incubated at 37°C in a shaker for 1 h. The cells were then centrifuged at 5000 rpm for 1 min, the supernatant was discarded, and 50 μL of the supernatant was used to resuspend the bacterial cells. The resuspended cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12 h. Single clones were selected for PCR amplification and identification, and positive strains were screened by sequencing.

[0051] (2) Transformation of Agrobacterium

[0052] Plasmids were extracted from positive strains by shaking. 100 ng of plasmid was added to 50 μL of Agrobacterium competent cells GV3101 and gently mixed. The mixture was then incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min. Finally, 700 μL of antibiotic-free LB medium was added and the mixture was incubated in a shaker at 28°C for 2.5 h. 100 μL of the bacterial culture was evenly spread on LB plates containing 50 μg / mL kanamycin and 100 μg / mL rifampin for screening. The plates were incubated upside down at 28°C for 2 days. Single clones were selected for colony PCR verification. The successfully verified strains were the engineered bacteria containing the recombinant expression vector.

[0053] 2. Transform tomato explants using Agrobacterium containing a recombinant expression vector via the leaf disc method.

[0054] (1) Seed disinfection

[0055] Wild-type tomato Micro-Tom seeds were placed in a sterile dish and disinfected by soaking in 70% ethanol for 30 seconds. The seeds were then rinsed twice with sterile water, followed by soaking in a 7.5% sodium hypochlorite solution for 15 minutes, shaking 2-3 times during this period. After rinsing 3-4 times with sterile water, the water was drained from the seeds. The seeds were then evenly distributed on 1 / 2 MS medium and finally placed in a light incubator. The culture conditions were: 14 hours of light incubation at 25°C, followed by 10 hours of darkness incubation at 20°C; the light intensity was 250 μmol / L. m -2 s -1The relative humidity is 80%; the culture period is 8-10 days.

[0056] (2) Pre-culture of explants

[0057] About 8-9 days after sowing tomato seeds, when the cotyledons of the tomato seedlings have expanded, is the optimal time to obtain explants. Place the seedlings on sterile filter paper, cut the cotyledons and hypocotyls into segments with a sterilized scalpel blade, and then transfer them to KCMS pre-medium for dark culture for one day.

[0058] (3) Agrobacterium infection of explants

[0059] Add 100 μL of preserved engineered bacteria containing the recombinant expression vector to 20 mL of LB medium (containing 50 μg / mL kanamycin and 100 μg / mL rifampin), and incubate at 28 °C and 230 rpm for 16–18 h until the absorbance OD value is reached. 600 Approximately 0.8-1.0. Take 1 mL of bacterial suspension, centrifuge at 5000 rpm for 5 min, discard the supernatant, resuspend the cells in KCMS liquid medium, discard the supernatant again, and then dilute the bacterial suspension to OD using KCMS liquid medium. 600 =0.1, which is the Agrobacterium infection solution. Use a pipette to add one drop of Agrobacterium infection solution to the wound of the explant. There is no need to aspirate the excess bacterial solution. After sealing the plate, place it in the dark and let the explant and engineered bacteria co-culture on KCMS medium for 2 days.

[0060] (4) Differentiation and rooting of explants

[0061] After co-culturing explants with Agrobacterium for 2 days, the explants were transferred to a primary screening medium (2Z) containing 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 subculture medium (1Z) containing 10 μM trans-zeatin nucleotides, with the medium being changed every 15 days. Finally, the buds that differentiated into independent main stems were cut off and inserted into rooting medium (ENR medium), and transplanted after rooting.

[0062] (5) Identification of transgenic positive plants

[0063] 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.

[0064] 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.

[0065] Subsequently, using the genomic DNA of T0 generation seedlings as a template, detection primers containing the target site were designed. IDD7 -Cr-F (SEQ ID NO: 7) / IDD7 PCR amplification was performed using Cr-R (SEQ ID NO: 8). The purified PCR product was then sequenced using the pClone007 universal TA cloning kit (catalog number: DLV103) from Qingke Biotechnology to identify the editing status of the T0 generation plants.

[0066] like Figure 3 As shown, the selected SlIDD7 Gene knockout lines ( SlIDD7- CR1 and SlIDD7- Translation of the SlIDD7 mutant protein in CR2 was prematurely terminated.

[0067] T0 generation seeds containing the Cas9 vector and edited were selected for propagation. The editing status of T1 generation plants was analyzed (using the same editing detection method as T0 generation plants). Seeds from 2-3 homozygous lines with different editing forms were collected, screened with kanamycin, and then sown into T2 generation for subsequent research.

[0068] Example 3 Knockout SlIDD7 The effect of gene modification on the yield of tomato fruit per plant

[0069] Select wild-type tomatoes (WT) that are in good growing condition and have uniform growth. SlIDD7 Five gene knockout plants were transferred to a plant culture chamber at the budding stage and cultured for four weeks under a cycle of 16 hours of light at 35°C and 8 hours of darkness at 30°C. The fruit set rate was recorded. The plants were then transferred to room temperature for further culture, and the yield of the plants was recorded when the fruits matured.

[0070] The results are as follows Figures 4-7 As shown. After 4 weeks of high-temperature treatment, SlIDD7 The number of fruits set by gene knockout lines was higher than that of wild types. Figure 4 and Figure 5 ); Statistical analysis shows that, SlIDD7 The fruit setting rate of the gene knockout lines was significantly higher than that of the WT lines. Figure 6 ), and the yield per plant has increased significantly ( Figure 7 ), further indicating SlIDD7 Genes play a negative regulatory role in regulating plant responses to high temperature stress and fruit development.

[0071] Example 4 Knockout SlIDD7 Effects of gene modification on sugar content (sucrose, fructose, and glucose) in tomato fruit

[0072] Collect wild-type (WT) and red-ripe fish. SlIDD7Mature fruit tissues from gene knockout lines were flash-frozen in liquid nitrogen and ground into powder. The sucrose, fructose, and glucose contents in tomato fruits were determined using Solarbio's Plant Sucrose Content Detection Kit (catalog number: BC2465), Plant Tissue Fructose (FT) Content Detection Kit (catalog number: BC2455), and Glucose Content Detection Kit (catalog number: BC2505), respectively.

[0073] The results are as follows Figures 8-10 As shown, analysis of sugar content revealed that fruit setting under high temperature conditions... SlIDD7 The fruit of gene knockout plants contains sucrose ( Figure 8 ),fructose( Figure 9 ) and glucose ( Figure 10 The content of these components was significantly higher than that of the wild type.

[0074] In conclusion, this demonstrates that gene editing technology can be used to knock out the gene in tomatoes. SlIDD7 Genes can improve the tomato plant's tolerance to high temperatures, specifically by: knocking out... SlIDD7 After gene injection, the fruit setting rate and yield per plant of tomatoes increased, and the content of sucrose, fructose and glucose in the fruits of the knockout lines increased, providing important genetic resources and strategies for synergistically improving the yield and quality of tomato fruits under high temperature stress conditions.

Claims

1. A kind SlIDD7 The application of genes in increasing tomato yield and sugar content under high temperature conditions is characterized by, By inhibiting SlIDD7 Gene expression or knockout SlIDD7 Genes were introduced to improve the tomato plant's tolerance to high temperatures, while simultaneously increasing fruit set rate and yield per plant, and enhancing the sucrose, fructose, and glucose content of the tomato fruit. The optimal temperature for this process was 30-35℃. SlIDD7 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

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