Application of CmEXPB1 gene in melon to improve fruit resistance to cracking

Abstract

The application belongs to the field of plant genetic engineering and biotechnology, and particularly relates to application of CmEXPB1 gene of melon in improving fruit resistance to cracking. The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The gene CmEXPB1 is cloned from melon cDNA, and it is found by real-time fluorescent quantitative technology that the expression amount of the gene in the peel of melon resistant to cracking is significantly higher than that in the peel of melon susceptible to cracking. A plant expression vector is constructed, and is transformed into tomato, and it is found that the peel hardness, cortex layer toughness and tightness of the transgenic strain are significantly higher than those of the wild type, and the brittleness is significantly reduced; cell wall component determination finds that the contents of cellulose, hemicellulose and protopectin of the transgenic tomato are increased, and the contents of soluble pectin and lignin are reduced, thereby confirming that CmEXPB1 enhances the resistance to cracking by regulating the composition of cell wall. The application lays a theoretical foundation for studying the fruit resistance to cracking, and also provides a gene resource for cultivating horticultural plants with high resistance to cracking.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and biotechnology, specifically relating to the application of the CmEXPB1 gene in melon in improving fruit crack resistance. Background Technology

[0002] Fruit development in horticultural plants is synergistically regulated by internal and external factors. When the balance between internal development and external factors is disrupted, fruit cracking may occur. Fruit cracking is a common phenomenon in the fruits of horticultural crops. Expansin (EXP) is a non-enzymatic protein located on the plant cell wall that is closely related to cell swelling. It regulates the relaxation between cell wall components and increases the flexibility of the cell wall by controlling the breaking of hydrogen bonds connecting cellulose microfibrils and hemicellulose.

[0003] Studies have found that the expression level of LeEXP4 increases during seed softening in tomatoes, potentially contributing to seed coat softening and cell wall relaxation. Silencing the LeEXP1 gene reduces tomato firmness and promotes hemicellulose breakdown. In the peel of crack-resistant jujube varieties, the expression level of EXPA-like genes is significantly higher than that of crack-prone varieties, indicating that EXP plays a crucial role in regulating fruit cracking. However, the function of EXPansin in melon peel cracking remains unclear. Therefore, there is an urgent need to investigate the function of melon EXPansin genes to lay the foundation for improving horticultural plant breeding. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of the CmEXPB1 gene in melons to improve fruit crack resistance.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention first provides the application of the melon CmEXPB1 gene in improving fruit crack resistance, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] As a preferred technical solution, the amino acid sequence encoded by the melon CmEXPB1 gene is shown in SEQ ID NO.2.

[0008] As a preferred technical solution, the fruit is a tomato.

[0009] The present invention also provides primer pairs for amplifying the above-mentioned melon CmEXPB1 gene, including an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0010] The present invention also provides a recombinant expression vector containing the above-mentioned melon CmEXPB1 gene.

[0011] As a preferred technical solution, the original vector of the recombinant expression vector is pCAMBIA1300.

[0012] The present invention also provides a transformant containing the above-described recombinant expression vector.

[0013] As a preferred technical solution, the host bacterium is Agrobacterium GV3101.

[0014] The present invention also provides a method for improving fruit crack resistance, wherein the melon CmEXPB1 gene shown in SEQ ID NO.1 is ligated into an expression vector to construct a recombinant expression vector; the expression vector is transformed into microorganisms for culture and expression, and then the obtained transformants are used to infect the explants of the fruiting plants, and fruit crack-resistant plants are obtained through co-culture, screening and differentiation.

[0015] As a preferred technical solution, the plant is tomato.

[0016] This invention has the following advantages: A gene, CmEXPB1, was cloned from melon cDNA. Real-time quantitative PCR revealed that the expression level of this gene in crack-resistant melon peel was significantly higher than that in easily cracked peel. A plant expression vector was constructed and transformed into tomatoes. The transgenic lines showed significantly higher epidermal hardness, cortical toughness, and firmness than the wild type, while exhibiting significantly reduced brittleness. Cell wall composition analysis revealed increased cellulose, hemicellulose, and protopectin content, and decreased soluble pectin and lignin content in the transgenic tomatoes, confirming that CmEXPB1 enhances crack resistance by regulating cell wall composition. Therefore, this gene can be used to improve fruit crack resistance. The melon CmEXPB1 gene obtained in this invention lays a theoretical foundation for studying fruit crack resistance and provides a gene resource for cultivating horticultural plants with high crack resistance, demonstrating significant application value. Attached Figure Description

[0017] Figure 1 This diagram shows the expression analysis of the Expansin gene family in different pericarps of melon. A is a photograph of melon fields; B is a photograph of crack-resistant 'Xizhoumi 17' (N17) and crack-prone 'Xizhoumi 25' (N25 and C25) fruits; C is the expression analysis of the Expansin gene family in N17 and N25 using transcriptome data; D is the expression analysis of the Expansin gene family in N25 and C25 using transcriptome data; E is the differentially expressed genes in the two groups; and F is the Venn diagram analysis of the differentially expressed genes in the two groups.

[0018] Figure 2This is a diagram showing the expression analysis of the CmEXPB1 gene in different pericarps.

[0019] Figure 3 The pCAMBIA1300-CmEXPB1 recombinant plasmid was constructed. In the figure, A represents CmEXPB1 gene amplification, and B represents plasmid restriction enzyme digestion detection. In the figure, M1 is the DM2000 marker, M2 is the 1 Kb marker, 1 represents the CmEXPB1 gene amplification product, 2 represents pCAMBIA1300 vector restriction enzyme digestion, and 3 represents pCAMBIA1300-CmEXPB1 recombinant plasmid restriction enzyme digestion. The arrows indicate the target gene band.

[0020] Figure 4 The recombinant plasmid pCAMBIA1300-CmEXPB1 was transformed into Agrobacterium and identified by PCR. In the figure, M: DM2000 marker; 1-5: Agrobacterium colony PCR transformed with the recombinant plasmid; 6: negative control.

[0021] Figure 5 The CmEXPB1 gene was used to transform tomatoes.

[0022] Figure 6 To identify transgenic tomatoes of the CmEXPB1 gene, A represents PCR amplification detection of tomato leaves; M: DM2000 marker; 1-15: different transgenic lines; B represents transgenic positive tomatoes transferred and planted in the substrate.

[0023] Figure 7 The results show the detection of T3 generation transgenic tomatoes. In the figure, A represents the phenotype of transgenic tomatoes; B represents the DNA detection of transgenic tomato leaves; C represents the detection of CmEXPB1 gene-specific primer amplification products; and D represents the detection of Hyg B primer amplification products. In the figure, M represents the DM2000 marker; P1-P6 represent different transgenic tomato lines; and P0 represents non-transgenic tomatoes, which serve as the control group.

[0024] Figure 8 The mechanical properties of tomato peel were analyzed, where A represents epidermal hardness; B represents cortical hardness; C represents cortical toughness; D represents firmness; and E represents brittleness. In the figure, P1-P6 represent different transgenic tomato lines; and P0 represents a non-transgenic tomato, serving as the control group.

[0025] Figure 9 The cell wall components of tomato peel were determined, where A represents cellulose content; B represents hemicellulose content; C represents protopectin content; D represents soluble pectin content; E represents total pectin content; and F represents lignin content. In the figure, P1-P6 represent different transgenic tomato lines; and P0 represents a non-transgenic tomato, serving as the control group. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following description:

[0027] Example 1: Obtaining the CmEXPB1 gene from melon

[0028] A differentially expressed gene, CmEXPB1, was screened from the mature pericarp transcriptome data (SRA number: SRP466450) of both crack-resistant and crack-prone melon varieties. Figure 1 As shown, both transcriptomic data and quantitative real-time PCR experiments confirmed that the expression level of this gene was significantly higher in crack-resistant varieties than in crack-prone varieties. Furthermore, the expression level in the peel of non-cracking fruit of the same variety was also significantly higher than that in cracked fruit peel. Figure 2 As shown, the CmEXPB1 gene is related to the crack resistance of melon peel (Hu et al., 2024), and is therefore considered a key candidate gene. The nucleotide sequence of the CmEXPB1 gene in the melon is shown in SEQ ID NO.1:

[0029] 5’-ATGGAATATGATAATGTTGCTCAGCTTCCAAACCGTACCACAGCTTCGCATTGGCTCCCGGCCACCGCCACTTGGTACGGCAGCCCAGAAGGCGACGGCAGCGACGGTGGGGCATGTGGGTACGGTAGTTTGGTGGATGTGAAGCCATTCAAAGCAAGAGTAGGAGCGGTGAGTCCAATCCTATTCAAGGACGGGGAAGGGTGTGGTGCGTGCTACAAAGTTCGGTGCTTGGATCGAGCCATATGCGCCAGACGGGCCGTTACCATCATAGTGACCGACGAGTGTCCAGGTGGGTATTGCTCCAATGGCCGGACTCACTTCGACCTGAGTGGTGCCGCCTTCGGACGTATGGCGGTGGCTGGCGCGGGCAGCCGTCTCCGAGACCGAGGGGAGTTGACCGTCGCATACAGAAGGACTCCATGTAGGTATAGAGGAAAGAATATAGCGTTCCATGTGAATGAAGGTTCGACAGATCATTGGCTTTCGCTTTTGGTGGAGTTCGAGGATGGAGATGGAGATATTGGTTCTATGCAAATCAGACAAGCAAACTCAAAAGAGTGGATGGAAATGGCACATGTGTGGGGTGCAACATGGTGCATAAATGGAGGGCCTTTAAAGGGTCCATTCTCAGTGAAGTTAACCACATTATCCACAGCTAAAACTCTCTCAGCCAGAGATGTTATTCCAAGGAATTGGTCTCCAAAGGCAACTTACACTTCTCGCTTGAACTTCTCCTAA-3’

[0030] The amino acid sequence encoded by the melon CmEXPB1 gene is shown in SEQ ID NO.2:

[0031] MEYDNVAQLPNRTTASHWLPATATWYGSPEGDGSDGGACGYGSLVDVKPFKARVGAVSPILFKDGEGCGACYKVRCLDRAICARRAVTIIVTDECPGGYCSNGRTHFDLSGAAFGRMAVAGA GSRLRDRGELTVAYRRTPCRYRGKNIAFHVNEGSTDHWLSLLVEFEDGDGDIGSMQIRQANSKEWMEMAHVWGATWCINGGPLKGPFSVKLTTLSTAKTLSARDVIPRNWSPKATYTSRLNFS

[0032] Example 2:

[0033] 1. Materials and Methods

[0034] 1.1 Cloning of the CmEXPB1 gene and construction of plant expression vector

[0035] Based on the CmEXPB1 gene ID information (MELO3C018743.2.1) on the melon genome website, plant expression vector primers CmEXPB1-1300-F and CmEXPB1-1300-R were designed to clone the gene from melon cDNA. After purification and recovery of the target gene, it was double-digested with Sal I and BamHI, and the plant expression vector pCAMBIA1300 was also double-digested. Ligation was performed using T4 DNA ligase to obtain the recombinant plasmid pCAMBIA1300-CmEXPB1.

[0036] CmEXPB1-1300-F:

[0037] 5'-ATAGTCGACATGGAATATGATAATGTTGCTCAGC-3' SEQ ID NO.3

[0038] CmEXPB1-1300-R:

[0039] 5'-CGGGATCCTTAGGAGAAGTTCAAGCGAGAAGTG-3' SEQ ID NO.4

[0040] 1.2 Transformation of Agrobacterium with recombinant plasmids

[0041] The recombinant plasmid pCAMBIA1300-CmEXPB1 was transformed into Agrobacterium strain GV3101 using chemical transformation. Single colonies were picked on LB (Shanghai Sangon Biotech Co., Ltd., A507003) + 50 mg / L Kanamycin + 25 mg / L Rifampicin solid selection medium, and colony PCR amplification was performed using primers CmEXPB1-1300-F and CmEXPB1-1300-R. Positive colony plaques were shaken in LB + Kanamycin + Rif liquid medium, and the absorbance (OD) of the bacterial culture was measured. 600 When the bacterial cell count reaches 0.8-1.0, centrifuge at 5000 rpm for 10 minutes, collect the cells, and reserve them for explant transformation.

[0042] 1.3 Tomato genetic transformation

[0043] (1) Seed disinfection: MicroTOM tomato seeds were first pre-washed with sterile ddH2O (2 min), then disinfected with 75% ethanol (40 s) and 84 disinfectant (7 min), then rinsed three times with sterile ddH2O (1 min each time), and finally soaked in sterile double-distilled water for 1 h to complete the sterilization process.

[0044] (2) Sowing: Sterilized tomato seeds were placed in 1 / 2 MS medium for dark culture for 3-4 days until the seeds germinated and showed white. Then they were transferred to a light incubator to continue growing for 4-5 days.

[0045] (3) Explant treatment and pre-culture: When the cotyledons of tomato seedlings are fully expanded, use a sterilized blade to cut off the petiole and tip of the cotyledons, retain the middle part and divide it into 2-3 segments, then inoculate it into the pre-culture medium and pre-culture it at 23±2℃ for 2-3 days.

[0046] (4) Agrobacterium infection and co-culture: Agrobacterium cells obtained in step 6.1.2 were placed in an infection solution (4.43 g / L MS salt + 30 g / L sucrose + 100 μM acetylsalicylic acid + 1.25 mM MES, pH 5.8) to prepare OD. 600 The bacterial suspension was 0.1%. After 10-15 min of infection, the explants were transferred to co-culture solid medium (4.43 g / L MS salt + 100 μM acetylsalicylic acid + 1.25 mM MES + 2 mg / L AgNO3 + 8 g / L agar, pH 5.8) and cultured in the dark for 2 days at 23 ± 2℃.

[0047] (5) Callus screening and differentiation: The recovered callus was inoculated into screening medium (MS + 1.0 mg / L). 6-BA + 0.3 mg / L IAA + 80 mg / L Callus was cultured at 23°C with 100 mg / L timentin for 15-30 days under cyclic conditions of 16 hours light / 8 hours dark. Selected callus tissue was transferred to differentiation medium and cultured for another 30-40 days under the same temperature and light conditions. When the differentiated seedlings reached 2-3 cm in height, they were separated from the callus tissue and inoculated into rooting medium (1 / 2 MS + 0.6 mg / L timentin). IAA + 80 mg / L The seedlings were cultured in a light incubator at 23°C with 16 hours of light and 8 hours of darkness for approximately 30 days. Subsequently, the seedlings were transferred to a cultivation substrate of humus:vermiculite = 1:1, covered with a breathable bag to control the humidity at 70±5%, and the breathable bag was removed after the tissue culture seedlings had fully adapted to the external environment.

[0048] 1.4 Detection of genetically modified tomatoes

[0049] DNA was extracted from the selected seedlings for testing. Leaf DNA was extracted from CmEXPB1 transgenic tomatoes and non-transgenic tomatoes (wild type) using the centrifuged column-type plant genomic DNA extraction kit (DP350) from Tiangen Biotech (Beijing) Co., Ltd. Amplification was performed using 2 × Taq Plus Master Mix II (Dye Plus) (catalog number: P213-03) from Nanjing Novizan Biotechnology Co., Ltd. The primers used were gene detection primers CmEXPB1-test-F and CmEXPB1-test-R, and hygromycin primers Hyg-F and Hyg-R.

[0050] CmEXPB1-test-F:

[0051] 5'-ATTCAAAGCAAGAGTAGGAGCGG-3' SEQ ID NO.5

[0052] CmEXPB1-test-R:

[0053] 5'-TTGAGTTTGCTTTGTCTGATTTGC-3' SEQ ID NO.6

[0054] Hyg-F:

[0055] 5'-ACGGTGTCGTCCATCACAGTTTGCC-3' SEQ ID NO.7

[0056] Hyg-R:

[0057] 5'-TTCCGGAAGTGCTTGACATTGGGGA-3' SEQ ID NO.8

[0058] 1.5 Analysis of the mechanical properties of transgenic tomato peel

[0059] During the tomato fruit ripening period, the mechanical properties of the tomato peel were determined by puncture test using the British SMS texture analyzer (TA-XT plus). Ten fruits were selected from each transgenic line and wild-type tomato for the test.

[0060] 1.6 Determination of Cell Wall Composition in Transgenic Tomato Peel

[0061] The contents of cellulose, hemicellulose, total pectin, soluble pectin, protopectin, and lignin in the peel of genetically modified and wild-type tomatoes were determined according to the kits provided by Yunzhi (Hainan) Biomedical Technology Co., Ltd. (Catalog No.: Cellulose: YX-W-B634; Hemicellulose: YX-W-B633; Total pectin: YX-W-A517; Soluble pectin: YX-W-WSP; Protopectin: YX-C-C113; Lignin: YX-W-B636).

[0062] 2 Results and Analysis

[0063] 2.1 Construction of CmEXPB1 gene plant expression vector

[0064] Using melon cDNA as a template, the CmEXPB1 gene was cloned, such as... Figure 3 As shown, the gene CDS sequence length is 738 bp ( Figure 3 A). After double digestion with Sal I and BamHI, the plasmid was ligated into the plant expression vector pCAMBIA1300 to obtain the recombinant plasmid pCAMBIA1300-CmEXPB1. The enzyme digestion results of the recombinant plasmid are as follows. Figure 3 As shown in Figure B, the results indicate that the recombinant plasmid excised both the empty vector band and the target gene band, demonstrating that the CmEXPB1 gene was successfully inserted into the vector. Subsequent sequencing further confirmed the sequence accuracy.

[0065] 2.2 Transformation of Agrobacterium with recombinant plasmids

[0066] The recombinant plasmid pCAMBIA1300-CmEXPB1 was introduced into Agrobacterium GV3101 via chemical transformation, followed by screening in LB medium containing Kana and Rif. Single colonies were selected for liquid culture and then subjected to PCR detection. Figure 4As shown, the electrophoresis patterns all exhibited clear, specific bands with molecular weights as expected, while the negative control showed no bands. These results indicate that the transformation process was successfully completed, and strain number 1 obtained from the screening was used for subsequent large-scale culture.

[0067] 2.3 Genetic transformation of tomatoes

[0068] Agrobacterium-mediated transformation was used to co-culture tomato explants with Agrobacterium containing the pCAMBIA1300-CmEXPB1 plasmid. The process involved pre-culture, co-culture, extended screening, selection, differentiation, and rooting. Figure 5 ), and transgenic plants were obtained.

[0069] DNA was extracted from the leaves of the transformed plants and amplified using CmEXPB1-test-F and CmEXPB1-test-R primers to initially screen for positive plants. The results are as follows: Figure 6 As shown. Figure 6 As shown in Figure A, among the leaves of 15 plants, except for plants 5 and 7 which did not amplify the target gene, and plant 11 which showed a weak target gene band, the remaining 12 plants all showed the target gene band, and were preliminarily identified as positive transgenic plants. The positive plants were then transferred to flowerpots for further cultivation. Figure 6 B).

[0070] 2.4 PCR identification of T3 generation CmEXPB1 gene-transformed tomatoes

[0071] After multiple generations of screening and identification, transgenic plants were obtained. Figure 7 A). Four transgenic lines (P1, P2, P5, and P6) were selected for study in the T3 generation. These four lines were further identified, and DNA was extracted from leaves of the four transgenic lines and non-transgenic tomato plants. Figure 7 B). Amplification using gene-specific primers and hygromycin primers revealed that the target gene could be amplified in all four transgenic lines. Figure 7 C) and hygromycin gene ( Figure 7 (D) indicates that the target gene transformation was successful.

[0072] 2.5 Determination of mechanical properties of tomato peel transgenic with CmEXPB1 gene

[0073] To investigate the effects of CmEXPB1 on tomato peel, the peel characteristics of four transgenic tomatoes and one non-transgenic tomato were analyzed. The skin firmness of the transgenic tomatoes was significantly higher than that of the non-transgenic tomatoes. Analysis revealed that the skin firmness of the four transgenic tomatoes was 4.5, 4.0, 4.5, and 3.4 times that of the non-transgenic tomatoes, respectively. Figure 8A). Cortical hardness and cortical toughness showed a similar pattern to epidermal hardness: the skin of transgenic tomatoes was significantly harder than that of non-transgenic tomatoes, but the value of P2 in the transgenic line was lower in all four lines, although it was still significantly higher than that of the skin of non-transgenic tomatoes. Figure 8 B, Figure 8 C). The cortical toughness of the four transgenic tomatoes was 2.7, 1.7, 1.9, and 1.9 times that of the non-transgenic tomatoes, respectively. Firmness reflects the tightness of cell arrangement and the size of intercellular spaces. The results showed that the cortical skin of transgenic tomatoes was significantly firmer than that of non-transgenic tomatoes, requiring more force to break open. Figure 8 D). From Figure 8 As can be seen from E, the skin of non-GMO tomatoes is significantly more brittle than that of GMO tomatoes, suggesting that non-GMO tomatoes are more prone to cracking when subjected to adverse environmental conditions.

[0074] 2.6 Determination of cell wall components in tomato pericarp transgenic with CmEXPB1 gene

[0075] To further investigate the effects of CmEXPB1 on tomato peel structure, the contents of cell wall-related components in the peels of transgenic and non-transgenic tomatoes were measured. Figure 9 As can be seen, compared with non-GMO tomatoes, the cellulose content in the peel of GMO tomatoes is higher ( Figure 9 A) Hemicellulose content ( Figure 9 B) and protopectin content ( Figure 9 C) The levels of cellulose and hemicellulose were significantly increased in all transgenic lines, with the highest cellulose content in transgenic line P1, the highest hemicellulose content in transgenic line P5, and the highest protopectin content in transgenic line P2. Conversely, the soluble pectin content in the peel of transgenic tomatoes was significantly lower than that in non-transgenic tomatoes, while there was no significant difference among the transgenic tomato lines. Figure 9 D). No significant pattern was found in total pectin content between transgenic and non-transgenic tomatoes. Among the four transgenic lines, the total pectin content in P1 was significantly higher than that in P2, P5, and P6, while the total pectin content in these three transgenic lines did not differ significantly from that in the non-transgenic P0. Simultaneously, the lignin content in the peels of different types of tomatoes was also measured, and the results showed that the lignin content in the peels of all four transgenic tomatoes was significantly lower than that in the non-transgenic tomatoes. Figure 9 F).

[0076] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. Cucumis melo CmEXPB1 application of the gene in improving fruit resistance to cracking, the fruit being tomato, the Cucumis melo CmEXPB1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the application is overexpression of the Cucumis melo CmEXPB1 gene in tomato.

2. Use according to claim 1, characterized in that, The melon CmEXPB1 The amino acid sequence encoded by the gene is shown as SEQ ID NO.

2.

3. A method of increasing the resistance of a fruit to cracking, characterized in that, The melon gene shown in SEQ ID NO. 1 CmEXPB1 CmEXPB1 CmEXPB1 CmEXPB1 CmEXPB1 CmEXPB1 Cm The gene is connected to an expression vector to construct a recombinant expression vector; the recombinant expression vector is transformed into microorganisms for culture and expression, and then the obtained transformants are used to infect tomato explants, and through co-culture, screening and differentiation, tomato fruit cracking resistance plants are obtained.

Images