Application of watermelon ClNAC55 gene in regulating sugar content of fruit

Abstract

This invention belongs to the field of biotechnology and provides watermelon CINAC 55 The application of genes in regulating fruit sugar content also provides a method for increasing the sugar content of watermelon fruit, the method comprising: CINAC 55 Manipulation of gene overexpression in plants. This invention confirms the ability of watermelons to... CINAC 55 Genes are positive regulators of sugar accumulation in watermelon fruits; overexpression CINAC 55 The gene can increase the total soluble sugar in the center of watermelon fruit by about 10%, with a significant sugar improvement effect. Using this gene can greatly shorten the breeding cycle of high-quality and stress-resistant watermelons, and has broad prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to watermelon. ClNAC55 Application of genes in regulating fruit sugar content. Background Technology

[0002] watermelon( Citrullus lanatus Watermelon is one of the top ten fruits. Sugar content is a core indicator for evaluating watermelon quality, directly affecting its commercial value and market competitiveness. Traditional watermelon quality improvement breeding mainly relies on hybridization breeding, introducing the superior traits of high-sugar germplasm into main varieties. However, this method has a long breeding cycle, low efficiency, and is prone to the cascading burden of undesirable traits. Summary of the Invention

[0003] To address the aforementioned problems in the prior art, the present invention provides a watermelon ClNAC55 Application of genes in regulating fruit sugar content.

[0004] The present invention also provides a method for increasing the sugar content of watermelon fruit, the method comprising: ClNAC55 The manipulation of gene overexpression in plants.

[0005] This invention is the first breakthrough NAC55 The traditional understanding of only participating in adversity response has been confirmed. ClNAC55 It is a positive regulator of sugar accumulation in watermelon fruit, expanding the functional boundaries of NAC transcription factors and providing new theoretical basis for elucidating the molecular mechanism of watermelon fruit quality formation. Overexpression ClNAC55 It can increase the total soluble sugar in the center of watermelon fruit by about 10%, with a significant effect on sugar improvement.

[0006] The overexpression genetic transformation system established in this invention is stable and efficient, and the genetic traits of the transgenic materials obtained are stable. It can be used directly as germplasm resources for variety breeding, or the superior haplotype of the gene can be introduced into the main cultivated varieties through molecular marker-assisted selection, which greatly shortens the breeding cycle of high-quality and stress-resistant watermelons and has broad prospects for industrial application. Attached Figure Description

[0007] Figure 1 Different time points in Example 1 ClNAC55 Line graph of gene expression levels.

[0008] Figure 2 The image shows the spectrum of the overexpression vector pMDC87d-Ub-ClNAC55 in Example 2.

[0009] Figure 3 For the eight overexpression lines in Example 2 ClNAC55 Expression quantity bar chart.

[0010] Figure 4 Comparison of fruit pulp photos of WT, OE-2 and OE-6 strains in Example 2 (left); bar chart of sugar content of WT, OE-2 and OE-6 strains (right). Detailed Implementation

[0011] To make the technical solution, objectives, and advantages of the present invention clearer, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0012] Firstly, this invention provides a watermelon ClNAC55 Application of genes in regulating fruit sugar content.

[0013] According to a first aspect of the present invention, the watermelon ClNAC55 The application of genes in regulating fruit sugar content includes:

[0014] pass ClNAC55 Gene overexpression increases the sugar content of watermelon fruit;

[0015] or:

[0016] pass ClNAC55 Gene knockout or repression ClNAC55 Gene expression, or may make ClNAC55 Gene silencing reduces the concentration of genes in plants. ClNAC55 The content and / or activity of encoded proteins reduce the sugar content of fruits.

[0017] in:

[0018] watermelon ClNAC55 The CDS sequence composition of the gene is shown in SEQ ID NO:1 in the sequence listing.

[0019] watermelon ClNAC55 The full-length DNA sequence of the gene is shown in SEQ ID NO:2 in the sequence listing.

[0020] watermelon ClNAC55 The amino acid sequence composition of the protein encoded by the gene is shown in SEQ ID NO:3 in the sequence listing.

[0021] Secondly, the present invention provides a method for increasing the sugar content of watermelon fruit, the method comprising: ClNAC55 The manipulation of gene overexpression in plants.

[0022] According to a second aspect of the invention, the method includes constructing a structure containing ClNAC55The gene CDS sequence is overexpressed in a vector (the framework vector can be the plant expression vector pMDC87d, or other plant expression vectors), and then the vector containing the CDS sequence is... ClNAC55 The overexpression vector of the gene CDS sequence was transferred into the plant to obtain ClNAC55 Manipulation of transgenic plants with overexpressed genes.

[0023] In a preferred embodiment, the overexpression vector can be transferred into plants via Agrobacterium (e.g., Agrobacterium EHA105 strain) mediated genetic transformation of watermelon seeds.

[0024] In a preferred embodiment, ClNAC55 The CDS sequence of a gene is obtained through the following operations:

[0025] Using watermelon pulp cDNA at maturity as a template, PCR amplification was performed using the following primers to obtain... ClNAC55 CDS sequence of the gene:

[0026] Upstream primer F (SEQ ID NO:4):

[0027] 5'-ATGGACCCGCTGATGCAGCTTA-3';

[0028] Downstream primer R (SEQ ID NO:5):

[0029] 5'-TCATTGGCTGAATCCGAACATTCCTC-3'.

[0030] According to a second aspect of the invention, the method includes... ClNAC55 The CDS sequence of the gene was ligated into the pMD19-T vector to obtain the pMD19-T-ClNAC55 recombinant plasmid;

[0031] Then, using the pMD19-T- ClNAC55 Using the recombinant plasmid as a template, amplification was performed using the following primers to obtain plasmids with restriction enzyme sites. ClNAC55 Full-length CDS fragment of the gene:

[0032] F-SpeI (SEQ ID NO:6):

[0033] 5'- gagtttttctgattaacagactagt ATGGACCCGCTGATGCAGCTTA-3';

[0034] R-KpnI (SEQ ID NO:7):

[0035] 5'- ctcattttttctaccggtaccTTGGCTGAATCCGAACATTCCTC-3'.

[0036] According to a second aspect of the present invention, the method includes digesting the plant expression vector pMDC87d with SpeI and KpnI double enzymes, and then combining the double-digested plant expression vector pMDC87d with a vector containing restriction enzyme sites. ClNAC55 The full-length CDS fragment of the gene is ligated to obtain the ligation product;

[0037] The ligation product was transformed into Escherichia coli DH5α and screened to obtain the recombinant overexpression vector pMDC87d-Ub-ClNAC55;

[0038] The recombinant overexpression vector pMDC87d-Ub- ClNAC55 Recombinant Agrobacterium was obtained by introducing Agrobacterium strain EHA105.

[0039] The recombinant Agrobacterium was transferred into the cotyledonary node of watermelon seeds, and after cultivation and screening, it was obtained... ClNAC55 Watermelon plants with overexpressed genes.

[0040] ClNAC55 CDS sequence of the gene (SEQ ID NO:1):

[0041] 5'-ATGGACCCGCTGATGCAGCTTAGCTTACCGCCGGGGTTTAGATTTTTTCCGACCGACGAAGAGCTTTTAGTTCAGTATCTTTGCCGGAAAGTCGCCGGCCACCATTTCAACTTGCAGCTCATCGCTGAGATTGACTTGTATAAATTCGATCCATGGGTTTTACCTGGGAAAGCTTTATTCGGGGAAAAAGAATGGTACTTCTTCAGCCCAAGAGACCGTAAATATCCAAACGGGTCTAGACCGAACCGGGTTGCCGGCTCGGGTTACTGGAAGGCTACCGGTACGGACAAAATAATCTCGTCGGAAGGGAAGAACGTGGGGATTAAAAAGGCACTGGTTTTCTATGTGGGAAAAGCTCCGAAGGGAACAAAAACGAATTGGATTATGCACGAGTATCGCCTCATATCCTCCTCCAGAAAAACTGGAAGCTCCAAGCTGGACGATTGGGTTTTATGTCGGATTTATAAGAAGAATTCGAGTTGTCAGAAACCGACGGGGAGTATTTCAAGTAAAGAATACAGCAACGGTTCGCCGCTATCGTCGTCGTCGTCCCACATCGACGAAGTCATCGAGTCCCTACCGGAAATGGGCGACGATTTCTTCGCGTATCCAAAAACAACATTACAACAAAACGACATAATGAACAAATTCCACTTTGAAATTCCGGCGGACTCTGTAAATTCCGATTGGGCGAGTCTGGCTGGGCTCTACTCAGTGCCGGAACTCGCTCCCGTGGACCACTCGGGGACGTTCGATTACAATAACAACAACAACAACAACACCATCGCCGATCTCTACGTTCCTTCAATTACATCGCCATTTTGCCAGGTGGATTACCCACCGCCGCCGGCGTTTCGTTACTCGACGCAGCAACACGGCGGCGGAGGAATGTTCGGATTCAGCCAATGA -3'.

[0042] ClNAC55 Full-length DNA sequence of the gene (SEQ ID NO:2):

[0043]

[0044] ClNAC55 The amino acid sequence of the protein encoded by the gene (SEQ ID NO:3):

[0045] MDPLMQLSLPPGFRFFPTDEELLVQYLCRKVAGHHFNLQLIAEIDLYKFDPWVLPGKALFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKIISSEGKNVGIKKALVFYVGKAPKGTKTNWIMHEYRLISSSRKTGSSKLDDWVL CRIYKKNSSCQKPTGSISSKEYSNGSPLSSSSSHIDEVIESLPEMGDDFFAYPKTTLQQNDIMNKFHFEIPADSVNSDWASLAGLYSVPELAPVDHSGTFDYNNNNNNTIADLYVPSITSPFCQVDYPPPPAFRYSTQQHGGGGMFGFSQ.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are products that can be obtained from commercial channels; unless otherwise specified, all testing and detection methods used in the following examples are conventional testing and detection methods in the field and can be obtained from textbooks, reference books or academic journals.

[0047] The watermelon varieties used in this invention are all preserved in the Beijing Crop Germplasm Resource Bank, contact number: 010-81127107. Anyone may freely obtain these watermelon varieties to achieve the purposes of this invention.

[0048] Example 1

[0049] This embodiment is used to illustrate watermelon. ClNAC55 Analysis of gene cloning and expression patterns.

[0050] I. Using the mature pulp cDNA of the watermelon cultivar ZZJM as a template, PCR amplification was performed using the following primers to obtain... ClNAC55 CDS sequence of the gene:

[0051] Upstream primer F (SEQ ID NO:4):

[0052] 5'-ATGGACCCGCTGATGCAGCTTA-3';

[0053] Downstream primer R (SEQ ID NO:5):

[0054] 5'-TCATTGGCTGAATCCGAACATTCCTC-3'.

[0055] The PCR reaction procedure is as follows:

[0056] First, pre-denaturate at 95℃ for 5 min; then denature at 95℃ for 30 s, anneal at 57℃ for 30 s, extend at 72℃ for 30 s, for a total of 35 cycles; finally extend at 72℃ for 5 min.

[0057] The PCR reaction system (50 μL) is as follows:

[0058] 25 μL of 2×Phanta Flash Master Mix (purchased from Nanjing Novizan Biotechnology Co., Ltd., product code P510), 2 μL of upstream primer F (10 μM), 2 μL of downstream primer R (10 μM), 2 μL of template, and 19 μL of ddH2O.

[0059] After electrophoresis of the PCR product, the target fragment was recovered and sequenced to obtain the CDS sequence of the watermelon ClNAC55 gene. The full-length sequence is 909 bp (SEQ ID NO:2) and encodes 302 amino acids (SEQ ID NO:1). Protein structure analysis showed that its N-terminus contains a conserved NAM domain and its C-terminus is a transcriptional activation region, which is consistent with the typical structural characteristics of NAC transcription factors.

[0060] The watermelon obtained ClNAC55 The gene CDS sequence was ligated into the pMD19-T vector to obtain pMD19-T- ClNAC55 Recombinant plasmid; for pMD19-T- ClNAC55 The recombinant plasmid was sequenced to verify... ClNAC55 The gene CDS sequence was successfully ligated into the pMD19-T vector.

[0061] II. Analysis using qRT-PCR technology ClNAC55 Spatiotemporal expression patterns of genes:

[0062] Using cDNA reverse-transcribed from total RNA of watermelon pulp at 10, 18, 26, and 34 days post-pollination as templates, qRT-PCR amplification was performed using the following primers to detect... ClNAC55 The spatiotemporal expression of genes was analyzed.

[0063] The primers used in the qRT-PCR reaction are as follows:

[0064] Upstream primer (QPCR-F, SEQ ID NO:8):

[0065] 5'-CGAATTGGATTATGCACGAGTATCG-3';

[0066] Downstream primer (QPCR-R, SEQ ID NO:9):

[0067] 5'-CTCGATGACTTCGTCGATGTGG-3'.

[0068] The PCR reaction procedure is as follows:

[0069] First, pre-denaturate at 95℃ for 5 min; then denature at 95℃ for 10 s, anneal at 60℃ for 10 s, extend at 72℃ for 30 s, for a total of 40 cycles; finally extend at 72℃ for 10 min.

[0070] The PCR reaction system (10 μL) is as follows:

[0071] SupRealQ Ultra Hunter SYBR qPCR Master Mix (purchased from Nanjing Novizan Biotechnology Co., Ltd., product code Q713) 5μL, upstream primer (10μM) 0.5μL, downstream primer (10μM) 0.5μL, template 1μL, ddH2O 3μL.

[0072] The results are as follows Figure 1 As shown ( Figure 1 In this context, DAP refers to the number of days after watermelon pollination; 10, 18, 26, and 34 represent the four stages of watermelon maturity; 10 DAP is the immature stage, 18 DAP is the flesh color change stage, 26 DAP is the ripe stage, and 34 DAP is the late ripe stage. Figure 1 In the central flesh tissue of watermelons at four different time points, ClNAC55 The relative expression levels of genes are shown in Table 1, with three replicates at each time point.

[0073] in, ClNAC55 The relative expression level of a gene is calculated as follows:

[0074] ΔCt = Ct (Target gene) − Ct (Reference gene)

[0075] ΔCt = Treatment group ΔCt − Control group ΔCt

[0076] Relative expression level = 2 -ΔΔCt

[0077] Table 1: Four time points ClNAC55 Relative expression level of genes

[0078]

[0079] Depend onFigure 1 As shown in Table 1, starting 10 days after pollination, ClNAC55 Expression levels continue to increase, peaking at 26 days, which coincides with the fruit ripening period and the highest pulp quality. The fruit ripens further at 34 days, during which pulp quality slightly declines. As the fruit develops and matures... ClNAC55 Gene expression showed a continuous upward trend, peaking 26 days after pollination (maturity stage), and then declining in the later stages of maturity. Furthermore, simultaneous testing of fruit sugar content revealed... ClNAC55 The changes in gene expression levels were highly consistent with the trend of sugar accumulation in fruits, suggesting that it may be involved in the regulation of fruit ripening and sugar accumulation.

[0080] Example 2

[0081] This embodiment is used to illustrate... ClNAC55 Application of the gene: Overexpression in watermelon ClNAC55 Gene.

[0082] one, ClNAC55 Construction of gene overexpression vectors

[0083] 1. Design amplification primers with restriction enzyme sites (introducing a SpeI site upstream and a KpnI site downstream):

[0084] F-SpeI (SEQ ID NO:6):

[0085] 5'- gagtttttctgattaacagactagt ATGGACCCGCTGATGCAGCTTA -3';

[0086] R-KpnI (SEQ ID NO:7):

[0087] 5'- ctcattttttctaccggtacc TTGGCTGAATCCGAACATTCCTC-3'.

[0088] 2. pMD19-T- obtained in Example 1 ClNAC55 Using the recombinant plasmid as a template, PCR amplification was performed using the primers with restriction enzyme sites from step 1 above to obtain plasmids with restriction enzyme sites. ClNAC55 The full-length CDS fragment of the gene was extracted and recovered.

[0089] 3. The plant expression vector pMDC87d with the above-mentioned restriction sites was double-digested with SpeI and KpnI, and then T4 DNA ligase was used to ligate the above-mentioned expression vector with the restriction sites. ClNAC55 The full-length CDS fragment of the gene was ligated overnight at 16°C to obtain the ligation product.

[0090] 4. The ligation product obtained in step 3 above was transformed into *E. coli* DH5α. After antibiotic selection, PCR reaction, and double enzyme digestion verification, the recombinant overexpression vector pMDC87d-Ub- was obtained. ClNAC55 (like Figure 2 ).

[0091] II. The above-mentioned recombinant overexpression vector pMDC87d-Ub- ClNAC55 The Agrobacterium strain EHA105 was introduced to obtain recombinant Agrobacterium, which was then put into use.

[0092] III. Agrobacterium-mediated genetic transformation of watermelon

[0093] The genetic transformation of cotyledonary nodes (the two cotyledons of a watermelon seed) was carried out using Agrobacterium-mediated transformation.

[0094] 1. Carefully peel the seed coat off the watermelon seeds, put them into a 50mL Erlenmeyer flask, first disinfect with 70% alcohol for 30 seconds, rinse twice with sterile water, then disinfect with 3% sodium hypochlorite solution for 15 minutes, and then rinse three times with sterile water.

[0095] 2. Inoculate the sterilized seeds into BM medium and incubate in the dark at 28°C for 3 days.

[0096] 3. Cut healthy, sprouting kernels and promptly inoculate them into a 9cm petri dish containing 10mL of MS liquid medium; after all are completed, add OD to the petri dish. 600 After soaking in a bacterial solution of 0.8–1.0 for 10 minutes, the bacterial solution was filtered off to obtain the transformed explants (seed kernels).

[0097] IV. Tissue Culture of Transformed Explants

[0098] 1. Transfer the transformed explants into a co-culture medium and co-culture at 28°C for 4 days.

[0099] 2. After co-culture, the explants were transferred to selective medium and subcultured once every 1-2 weeks for a total of 3-4 times. The explants were cultured at 25℃ for 14 hours to obtain green shoots.

[0100] 3. Transfer the well-grown green shoots to the shoot elongation medium. Seedlings will be obtained in about 4 weeks.

[0101] 4. Cut off the whole seedlings and transfer them to the rooting culture medium. Rooted plants can be obtained in about 7 days.

[0102] 5. Extract DNA from the rooted plants and use the extracted DNA as a template to identify positive plants using the following primers (primers designed based on the pMDC87d vector sequence):

[0103] Upstream primer F (SEQ ID NO:10):

[0104] 5'-GTTAGTTTCTAGTTTGTGCGATCG-3';

[0105] Downstream primer R (SEQ ID NO:11):

[0106] 5'-GTATAGTTCATCCATGCCATGTGT-3'.

[0107] The PCR reaction procedure is as follows:

[0108] First, pre-denaturate at 95℃ for 5 min; then denature at 95℃ for 30 s, anneal at 57℃ for 30 s, extend at 72℃ for 30 s, for a total of 35 cycles; finally extend at 72℃ for 5 min.

[0109] The PCR reaction system (10 μL) is as follows:

[0110] 2 × Rapid Taq Master Mix (purchased from Nanjing Novizan Biotechnology Co., Ltd., product code P222) 5μL, upstream primer (10μM) 0.5μL, downstream primer (10μM) 0.5μL, template 1μL, ddH2O 3μL.

[0111] If the PCR amplification product shows a band of approximately 1.1 kbp after electrophoresis, it indicates the presence of pMDC87d-Ub- ClNAC55 The Agrobacterium EHA105 strain with the recombinant vector was successfully transferred into the plant, which was a positive strain.

[0112] 6. The above-mentioned positive plants and corresponding control plants (not transformed into the recombinant overexpression vector pMDC87d-Ub-) ClNAC55 Transplant the plants into nutrient pots (peat moss: vermiculite = 3:1), place them in a culture box for about a week to acclimate, then move them to a greenhouse for about a week, and finally plant them in a greenhouse.

[0113] 7. About 3 months after transplanting into the greenhouse, seeds of positive plants are obtained. After sowing, qPCR testing is performed during the seedling stage for screening. ClNAC55 High expression lines (overexpression lines).

[0114] qPCR test results are as follows Figure 3 As shown in Table 2.

[0115] Table 2: Eight overexpression lines ClNAC55 Relative expression level of genes

[0116]

[0117] Depend on Figure 3As shown in Table 2, among the eight overexpression lines, OE-2 and OE-6 showed the highest expression levels. ClNAC55 The relative expression level of the gene is the highest.

[0118] 8. Wild-type and two overexpression lines, OE-2 and OE-6, were planted in a greenhouse and managed using standard field practices. Sugar content was measured at fruit ripening stage, and the results are as follows: Figure 4 As shown in Table 3.

[0119] The sugar content of the fruit was determined according to the method in "NY / T 2637-2014 Determination of Soluble Solids Content in Fruits and Vegetables by Refractometer Method" using a PAL-1 handheld digital display saccharimeter purchased from Guangzhou Atago Scientific Instruments Co., Ltd.

[0120] Table 3: Sugar content of WT, overexpression lines OE-2 and OE-6 (unit: Brix)

[0121]

[0122] Depend on Figure 4 As can be seen from Table 3, ClNAC55 The fruit of the overexpression strain had approximately 10% higher sugar content than the wild-type fruit. This indicates that overexpression in watermelon plants... ClNAC55 Genes can increase the sugar content of watermelon fruits.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Watermelon ClNAC55 Application of genes in regulating fruit sugar content.

2. The watermelon according to claim 1 ClNAC55 The application of genes in regulating fruit sugar content is characterized by: The watermelon ClNAC55 The application of genes in regulating fruit sugar content includes: pass ClNAC55 Gene overexpression increases the sugar content of watermelon fruit; or: pass ClNAC55 Gene knockout or repression ClNAC55 Gene expression, or may make ClNAC55 Gene silencing reduces the concentration of genes in plants. ClNAC55 The content and / or activity of encoded proteins reduce the sugar content of fruits.

3. The watermelon according to claim 1 or 2 ClNAC55 The application of genes in regulating fruit sugar content is characterized by: watermelon ClNAC55 The CDS sequence composition of the gene is shown in SEQ ID NO:1 in the sequence listing; watermelon ClNAC55 The full-length DNA sequence of the gene is shown in SEQ ID NO:2 in the sequence listing; watermelon ClNAC55 The amino acid sequence composition of the protein encoded by the gene is shown in SEQ ID NO:3 in the sequence listing.

4. A method for increasing the sugar content of watermelon fruit, the method comprising: ClNAC55 The manipulation of gene overexpression in plants.

5. The method according to claim 4, characterized in that: The method includes constructing a... ClNAC55 Overexpression vector of gene CDS sequence, and then the vector containing the gene CDS sequence. ClNAC55 The overexpression vector of the gene CDS sequence was transferred into the plant to obtain ClNAC55 Manipulation of transgenic plants with overexpressed genes.

6. The method according to claim 5, characterized in that: The overexpression vector was transferred into plants via Agrobacterium-mediated genetic transformation of watermelon seeds.

7. The method according to claim 6, characterized in that: ClNAC55 The CDS sequence of a gene is obtained through the following operations: Using watermelon pulp cDNA at maturity as a template, PCR amplification was performed using the following primers to obtain... ClNAC55 CDS sequence of the gene: Upstream primer F (SEQ ID NO:4): 5'-ATGGACCCGCTGATGCAGCTTA-3'; Downstream primer R (SEQ ID NO:5): 5'-TCATTGGCTGAATCCGAACATTCCTC-3'.

8. The method according to claim 7, characterized in that: The method includes... ClNAC55 The CDS sequence of the gene was ligated into the pMD19-T vector to obtain pMD19-T- ClNAC55 Recombinant plasmids; Then, using the pMD19-T- ClNAC55 Using the recombinant plasmid as a template, amplification was performed using the following primers to obtain plasmids with restriction enzyme sites. ClNAC55 Full-length CDS fragment of the gene: F-SpeI (SEQ ID NO:6): 5'-GAGTTTTCTGATTAACAGACTAGTATGGACCCGCTGATGCAGCTTA-3'; R-KpnI (SEQ ID NO:7): 5'-CTCATTTTTTTACCGGTACCTTGGCTGAATCCGAACATTCCTC-3'.

9. The method according to claim 8, characterized in that: The method includes digesting the plant expression vector pMDC87d with SpeI and KpnI, and then combining the double-digested plant expression vector pMDC87d with the enzyme-containing restriction sites. ClNAC55 The full-length CDS fragment of the gene is ligated to obtain the ligation product; The ligation product was transformed into *E. coli* DH5α, and the recombinant overexpression vector pMDC87d-Ub- was obtained through screening. ClNAC55 ; The recombinant overexpression vector pMDC87d-Ub- ClNAC55 Recombinant Agrobacterium was obtained by introducing Agrobacterium strain EHA105. The recombinant Agrobacterium was transferred into the cotyledonary node of watermelon seeds, and after cultivation and screening, it was obtained... ClNAC55 Watermelon plants with overexpressed genes.

10. The method according to any one of claims 4-9, characterized in that: watermelon ClNAC55 The CDS sequence composition of the gene is shown in SEQ ID NO:1 in the sequence listing; watermelon ClNAC55 The full-length DNA sequence of the gene is shown in SEQ ID NO:2 in the sequence listing.

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