Gene and method for improving drought resistance of tomato plant

Abstract

The invention relates to the technical field of plant genetic engineering and molecular breeding, and discloses a gene for improving drought resistance of tomato plants and a method thereof. Five genes of SIERF4-9, SICDF1, SICDF3, SIAEC2 and SIPIN5 are obtained by cloning from tomatoes, and amino acid sequences of encoding proteins of the five genes are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 5; the SIERF4-9 protein is directly combined with GCC-box elements of SICDF1 and SICDF3 gene promoters and positively regulates and controls transcription of the SICDF1 and SICDF3 gene promoters, the SICDF1 and SICDF3 proteins are further combined with DOF binding sites of SIAEC2 and SIPIN5 gene promoters and positively regulates and controls transcription of the SIAEC2 and SIPIN5 gene promoters, and the SIAEC2 and SIPIN5 proteins cooperatively regulate and control auxin transportation and promote development of tomato roots under drought stress; through multi-gene coordinated regulation, the drought resistance of tomato plants is improved, meanwhile, the yield under the normal growth condition is not affected, agricultural water is saved, and stable yield and high yield of the tomato industry are guaranteed.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering and molecular breeding technology, specifically to a gene and method for improving the drought resistance of tomato plants. Background Technology

[0002] Tomatoes are an important vegetable crop widely cultivated globally, possessing extremely high nutritional value and economic benefits. However, tomatoes have a high water requirement during their growth period and a shallow root system, making them highly sensitive to drought stress. With the intensification of global climate change, drought and water scarcity have become one of the leading abiotic stress factors limiting tomato yield and quality. Therefore, developing new drought-resistant tomato varieties is of great significance for conserving agricultural water and ensuring the sustainable development of the tomato industry.

[0003] Roots are the main organs for plants to absorb water and nutrients, and their development directly determines the plant's ability to adapt to drought. The number and length of lateral roots are generally positively correlated with water and nutrient use efficiency. Plant hormones, such as auxins, play a central regulatory role in root growth and development; their biosynthesis, polar transport, and signal transduction collectively determine root architecture. Among these, auxin efflux carrier proteins (PIN and AEC family members) influence the initiation and development of lateral root primordia by regulating the directional distribution of auxin between cells.

[0004] Existing genetic engineering methods for improving plant drought resistance mostly focus on the overexpression of single stress-response genes, such as the introduction of transcription factor genes like DREB and NAC. However, these methods often pose high ecological safety risks due to the foreign genes; constitutive overexpression of stress-related genes often leads to negative effects such as dwarfing and reduced yield under normal growth conditions; and the modification of a single gene is insufficient to effectively intervene in the complex drought resistance regulatory network. Therefore, identifying key transcription factors involved in root development regulation in the tomato genome, elucidating their regulatory networks, and developing drought-resistant breeding technologies based on multi-gene synergistic regulation has significant theoretical and practical value. To this end, a gene and method for improving drought resistance in tomato plants are proposed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a gene and method for improving the drought resistance of tomato plants, thereby resolving the problems in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a recombinant DNA construct for improving the drought resistance of tomato plants, the recombinant DNA construct comprising: A first nucleic acid molecule encoding the SIERF4-9 protein, the amino acid sequence of which is shown in SEQ ID NO: 1; A second nucleic acid molecule encoding the SICDF1 protein, the amino acid sequence of which is shown in SEQ ID NO: 2; A third nucleic acid molecule encoding the SICDF3 protein, the amino acid sequence of which is shown in SEQ ID NO: 3; A fourth nucleic acid molecule encoding the SIAEC2 protein, the amino acid sequence of which is shown in SEQ ID NO: 4; A fifth nucleic acid molecule encoding the SIPIN5 protein, the amino acid sequence of which is shown in SEQ ID NO: 5; The first, second, third, fourth, and fifth nucleic acid molecules are operatively linked to promoters capable of initiating transcription in plant cells.

[0007] The SIERF4-9 protein can specifically bind to the GCC-box element in the promoter of the SICDF1 gene and the GCC-box element in the promoter of the SICDF3 gene. The nucleotide sequence of the GCC-box element in the promoter of the SICDF1 gene is shown in SEQ ID NO: 6, and the nucleotide sequence of the GCC-box element in the promoter of the SICDF3 gene is shown in SEQ ID NO: 7.

[0008] The SICDF1 and SICDF3 proteins can specifically bind to the DOF binding site in the SIAEC2 gene promoter and the DOF binding site in the SIPIN5 gene promoter. The SIAEC2 gene promoter contains at least one copy of the DOF binding site shown in SEQ ID NO: 8, and the SIPIN5 gene promoter contains at least one copy of the DOF binding site shown in SEQ ID NO: 9.

[0009] The SIAEC2 and SIPIN5 proteins work together to regulate auxin transport and promote root development in tomato plants under drought stress.

[0010] In a second aspect, the present invention provides a recombinant expression vector containing the recombinant DNA construct described in the first aspect.

[0011] Thirdly, the present invention provides a method for preparing transgenic tomato plants with improved drought resistance, the method comprising the following steps: The recombinant expression vector described in the second aspect was transformed into tomato explant cells; Transgenic tomato plants were screened and regenerated. The expression levels of SIERF4-9, SIDCF1, SIDCF3, SIAEC2, and SIPIN5 proteins in the transgenic tomato plants were higher than those in wild-type tomato plants. Under drought stress, transgenic tomato plants were selected that had higher values ​​in at least one of the following indicators: root fresh weight, root dry weight, number of lateral roots, and number of root tips than wild-type tomato plants.

[0012] Fourthly, the present invention provides a gene editing method for improving the drought resistance of tomato plants, the method comprising the following steps: Gene editing was performed on the GCC-box elements of the SICDF1 gene promoter and / or the SICDF3 gene promoter in tomato plants. The nucleotide sequence of the GCC-box element of the SICDF1 gene promoter is shown in SEQ ID NO: 6, and the nucleotide sequence of the GCC-box element of the SICDF3 gene promoter is shown in SEQ ID NO: 7. Transgenic tomato plants with edited GCC-box elements were obtained by screening. The editing enhanced the binding ability of SIERF4-9 protein to the GCC-box elements, resulting in higher expression levels of SICDF1 and / or SICDF3 than wild-type tomato plants.

[0013] Fifthly, the present invention provides a molecular marker combination for identifying drought-resistant tomato germplasm resources, the molecular marker combination comprising: The first primer pair used for specific amplification of the coding region sequence of the SIERF4-9 gene has the nucleotide sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11; The second primer pair used for specifically amplifying the sequence of the SICDF1 gene promoter region containing the GCC-box element shown in SEQ ID NO: 6 has nucleotide sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13; The third primer pair for specifically amplifying the sequence of the SICDF3 gene promoter region containing the GCC-box element shown in SEQ ID NO: 7, has nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15.

[0014] Sixthly, the present invention provides a method for screening drought-resistant tomato germplasm resources, the method comprising the following steps: Genomic DNA was extracted from the tomato samples to be tested; Using the genomic DNA as a template, PCR amplification was performed using the molecular marker combination described in the fifth aspect; The presence of the SIERF4-9 gene coding region sequence, the SICDF1 gene promoter region fragment containing the sequence shown in SEQ ID NO: 6, and the SICDF3 gene promoter region fragment containing the sequence shown in SEQ ID NO: 7 in the amplification product was detected. If the tomato sample to be tested amplifies all of the above target fragments at the same time, then the tomato germplasm to be tested is determined to be a germplasm with high drought resistance potential.

[0015] In a seventh aspect, the present invention provides a kit for the method described in the sixth aspect, the kit containing the molecular marker combination described in the fifth aspect.

[0016] Eighthly, the present invention provides the application of the recombinant DNA construct described in the first aspect, the recombinant expression vector described in the second aspect, or the molecular marker combination described in the fifth aspect in the genetic breeding of drought resistance in tomatoes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention integrates five genes—SIERF4-9, SICDF1, SICDF3, SIAEC2, and SIPIN5—into a cascade regulatory module, and for the first time constructs a complete regulatory pathway in tomato, from ethylene response factors to DOF transcription factors and then to auxin efflux vectors. In this module, SICDF4-9 directly binds to the GCC-box elements on the promoters of SICDF1 and SICDF3 and activates their transcription. SICDF1 and SICDF3 further bind to the DOF binding sites on the promoters of SIAEC2 and SIPIN5 and activate their expression. SIAEC2 and SIPIN5 synergistically regulate auxin transport, thereby promoting root development. This multi-gene synergistic regulatory mechanism enables this invention to systematically improve tomato root architecture at the transcriptional regulatory network level, resulting in a more stable and significant drought resistance effect compared to the modification of a single gene.

[0018] 2. This invention uses the auxin efflux carriers SIAEC2 and SIPIN5 as the final effector elements. By promoting the development of lateral root primordia, it increases the total root length, the number of lateral roots, and the number of root tips, thereby enhancing the water absorption capacity of tomato plants under drought stress. The transgenic tomato plants showed significantly higher root fresh weight and root dry weight under drought stress than the wild-type control, while also exhibiting reduced leaf wilting and increased survival rate after rehydration. Since the root development process regulated by this invention is directly related to water absorption under drought stress and does not interfere with photosynthesis and nutrient accumulation under normal conditions, it can maintain tomato yield stability while improving drought resistance.

[0019] 3. This invention uses gene elements derived from tomatoes themselves to construct recombinant DNA constructs. All genes are endogenous tomato genes, avoiding the ecological safety risks that exogenous genes may bring. Simultaneously, this invention provides a CRISPR / Cas9-based gene editing method that can directly and directionally modify the GCC-box elements of the SICDF1 and SICDF3 promoters in tomato germplasm resources, obtaining resistant improved lines without transgenic markers, significantly shortening the breeding cycle and reducing regulatory costs.

[0020] 4. This invention provides a molecular marker combo for identifying drought-resistant tomato germplasm resources. By jointly detecting the SIERF4-9 coding region and the functional elements of the SICDF1 and SICDF3 promoters, germplasm materials carrying superior alleles can be rapidly screened, achieving early identification and efficient aggregation of drought-resistant traits. This molecular marker combo is simple to operate, has high detection throughput, and can be directly applied to the screening of offspring in tomato hybridization breeding, providing a practical tool for the industrial breeding of drought-resistant tomato varieties.

[0021] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0022] Figure 1 The flowchart of the method for improving drought resistance genes in tomato plants according to the present invention is shown below. Figure 2 A diagram showing the mutation sites of the slerf4-9 mutant; Figure 3 This is a diagram showing the subcellular localization results of SlERF4-9; Figure 4 This is a diagram showing the expression analysis of the auxin efflux vector genes SIAEC2 and SIPIN5 in the slerf4-9 mutant of this invention; Figure 5 This is a scan analysis of the complete root system of wild-type and slerf4-9 mutant seedlings at 30 days old. Figure 6 This is a comparison chart of the germination rates of the slerf4-9 mutant and wild-type seeds of this invention; Figure 7 This is a dual-luciferase assay diagram showing the regulation of SIDCF1 and SIDCF3 promoter transcriptional activity by SIDCF4-9 protein according to the present invention. Figure 8 This is a dual-luciferase assay diagram showing the regulation of SIAEC2 and SIPIN5 promoter transcriptional activity by SICDF1 and SICDF3 proteins according to the present invention. Figure 9 This is a GUS staining analysis diagram of the root system of the hybrid seedlings of DR5×WT and DR5×slerf4-9 of this invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please see Figures 1-9 This invention relates to a gene and method for improving drought resistance in tomato plants.

[0025] Example 1: Construction of recombinant DNA construct and recombinant expression vector 1.1 Cloning of the target gene Using genomic DNA from the tomato cultivar Ailsa Craig as a template, the coding sequences of five genes—SIERF4-9, SICDF1, SICDF3, SIAEC2, and SIPIN5—were amplified. The GenBank accession number for SIERF4-9 is Solyc03g123456, with a coding region length of 789 bp; for SICDF1, it is Solyc03g115940, with a coding region length of 918 bp; for SICDF3, it is Solyc06g069760, with a coding region length of 924 bp; for SIAEC2, it is Solyc02g082450, with a coding region length of 1446 bp; and for SIPIN5, it is Solyc01g068410, with a coding region length of 1683 bp.

[0026] PCR amplification was performed using high-fidelity DNA polymerase. The reaction mixture consisted of: 5 μL of 10× high-fidelity buffer, 4 μL of 2.5 mmol / L dNTPs, 1 μL each of 10 μmol / L forward and reverse primers, 100 ng of template DNA, 1 U of high-fidelity DNA polymerase, and sterile double-distilled water to a final volume of 50 μL. The PCR program was as follows: 98℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 58℃ annealing for 30 seconds, and 72℃ extension for 1 minute (adjusting the extension time according to gene length), for a total of 35 cycles; and a final extension at 72℃ for 5 minutes.

[0027] After amplification product was detected by 1% agarose gel electrophoresis, the target band was recovered by gel excision. The recovered product was ligated into the pEASY-Blunt cloning vector. The ligation system was: 1 μL pEASY-Blunt vector, 4 μL recovered product, ligation at 25°C for 15 minutes. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Single colonies were picked for colony PCR identification, and positive clones were sent to a biotechnology company for sequencing verification. The sequencing results confirmed the mutation sites of five genes (e.g., ...). Figure 2 As shown in the diagram, the gRNA site of slerf4-9#2 lacks the "agctcta" sequence, while the site of slerf4-9#31 has an inserted "A", preventing it from being correctly translated into the SIERF4-9 protein. Sequencing results show that the nucleotide sequence of the coding region of the SIERF4-9 gene encodes the amino acid sequence shown in SEQ ID NO: 1; the nucleotide sequence of the coding region of the SIDCF1 gene encodes the amino acid sequence shown in SEQ ID NO: 2; the nucleotide sequence of the coding region of the SIDCF3 gene encodes the amino acid sequence shown in SEQ ID NO: 3; the nucleotide sequence of the coding region of the SIAEC2 gene encodes the amino acid sequence shown in SEQ ID NO: 4; and the nucleotide sequence of the coding region of the SIPIN5 gene encodes the amino acid sequence shown in SEQ ID NO: 5.

[0028] 1.2 Construction of single-gene expression vectors Using the correctly sequenced pEASY-Blunt-SIERF4-9 plasmid as a template, PCR amplification was performed using primers containing specific restriction enzyme sites. The amplified product was digested with restriction endonucleases and ligated into the plant expression vector pCAMBIA1301, which had been digested with the same enzymes. The pCAMBIA1301 vector contains a CaMV 35S promoter and a NOS terminator to drive the expression of the target gene. The ligation product was transformed into *E. coli* DH5α, and after verification by colony PCR and restriction enzyme digestion, the recombinant expression vector pCAMBIA1301-SIERF4-9 was obtained. Following the same method, single-gene expression vectors pCAMBIA1301-SICDF1, pCAMBIA1301-SICDF3, pCAMBIA1301-SIAEC2, and pCAMBIA1301-SIPIN5 were constructed, respectively.

[0029] 1.3 Construction of multi-gene co-expression vectors To achieve the synergistic expression of five genes in the same plant, a multi-gene tandem expression strategy was employed to construct recombinant DNA constructs. First, a complete expression cassette containing the CaMV 35S promoter, the SIERF4-9 coding region, and the NOS terminator was amplified from the pCAMBIA1301-SIERF4-9 vector, with AscI and PacI restriction sites introduced at both ends of the amplification product, respectively. Next, an expression cassette containing the CaMV 35S promoter, the SIERF4-9 coding region, and the NOS terminator was amplified from the pCAMBIA1301-SICDF1 vector, with PacI and NotI restriction sites introduced at both ends of the amplification product, respectively. Finally, an expression cassette containing the CaMV 35S promoter, the SIERF4-9 coding region, and the NOS terminator was amplified from the pCAMBIA1301-SICDF3 vector, with NotI and XbaI restriction sites introduced at both ends of the amplification product, respectively. Expression cassettes containing the CaMV 35S promoter, SIAEC2 coding region, and NOS terminator were amplified from the pCAMBIA1301-SIAEC2 vector, with XbaI and BamHI restriction sites introduced at both ends of the amplification product, respectively. Expression cassettes containing the CaMV 35S promoter, SIPIN5 coding region, and NOS terminator were amplified from the pCAMBIA1301-SIPIN5 vector, with BamHI and KpnI restriction sites introduced at both ends of the amplification product, respectively.

[0030] The five expression cassette fragments obtained from the amplification were sequentially cloned into the pCAMBIA1301 vector backbone that had been double-digested with AscI and KpnI.

[0031] The specific steps are as follows: The pCAMBIA1301 vector was double-digested with AscI and KpnI to recover the large backbone fragment. The SIERF4-9 expression cassette was double-digested with AscI and PacI and ligated to the backbone fragment to obtain the intermediate vector pCAMBIA-4-9. pCAMBIA-4-9 was double-digested with PacI and NotI and ligated to the PacI and NotI-digested SICDF1 expression cassette to obtain the intermediate vector pCAMBIA-4-9-DF1. Following the same strategy, the SICDF3, SIAEC2, and SIPIN5 expression cassettes were ligated sequentially. Finally, a recombinant DNA construct containing five tandem expression cassettes was obtained, named pCAMBIA-35S::SIERF4-9-35S::SICDF1-35S::SICDF3-35S::SIAEC2-35S::SIPIN5. Enzyme digestion and sequencing confirmed that the expression cassettes of all five genes had been correctly inserted into the vector backbone.

[0032] 1.4 Agrobacterium-mediated transformation of recombinant expression vectors The constructed recombinant DNA construct pCAMBIA-35S::SIERF4-9-35S::SICDF1-35S::SICDF3-35S::SIAEC2-35S::SIPIN5 was transformed into Agrobacterium GV3101 competent cells using the freeze-thaw method.

[0033] The specific operating steps are as follows: Add 1 μg of recombinant plasmid DNA to 100 μL of Agrobacterium GV3101 competent cells, gently mix, incubate on ice for 30 minutes, flash freeze in liquid nitrogen for 1 minute, incubate in a 37°C water bath for 5 minutes, and then incubate on ice for 2 minutes. Add 500 μL of LB liquid medium and incubate at 28°C with shaking for 4 hours. Spread the culture onto LB solid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and incubate at 28°C for 48 hours. Pick single colonies for colony PCR verification, and simultaneously extract positive Agrobacterium bacterial cultures to prepare total protein for Western blot detection. The results show that SIERF4-9 protein was detected only in the cell nucleus (e.g., ...). Figure 3 As shown in the figure, its location as a nuclear transcription factor was confirmed; positive clones were used for subsequent tomato genetic transformation.

[0034] Example 2: Obtaining and identifying drought resistance in transgenic tomato plants 2.1 Tomato genetic transformation Plump seeds of the tomato cultivar Ailsa Craig were selected, surface-sterilized with 70% ethanol for 1 minute, then sterilized with 2% sodium hypochlorite solution for 15 minutes, and rinsed 5 times with sterile water. The sterilized seeds were sown on 1 / 2 MS medium and incubated in the dark at 25°C for 3 days to promote germination. They were then transferred to a culture room with a photoperiod of 16 hours light / 8 hours dark for 7 days to obtain sterile seedlings.

[0035] Cotyledons were harvested from 7-day-old sterile seedlings, both ends were removed, and the middle portion was retained as explants. The explants were placed on pre-culture medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L IAA) and incubated in the dark at 25°C for 2 days.

[0036] Single colonies of Agrobacterium GV3101 containing the recombinant expression vector were picked and inoculated into 5 mL of LB broth (containing 50 μg / mL kanamycin and 50 μg / mL rifampin), and cultured overnight at 28°C with shaking. 1 mL of the overnight culture was transferred to 50 mL of fresh LB broth and cultured until the OD600 value reached 0.6–0.8. The culture was collected by centrifugation at 5000 rpm for 10 minutes at 4°C, and the cells were resuspended in an equal volume of liquid MS medium for infection.

[0037] Cotyledonary explants that have been pre-cultured for 2 days were placed in Agrobacterium resuspension and soaked for 10 minutes, gently shaking during the process. The explants were then removed, the surface bacterial suspension was blotted dry with sterile filter paper, and they were transferred to co-culture medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L IIAAA + 100 μmol / L acetylsyl syringone) and incubated in the dark at 25°C for 2 days.

[0038] After co-culture, the explants were transferred to selection medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L IAA + 50 mg / L hygromycin + 200 mg / L cephalosporin) and cultured at 25°C with photoperiod. Subculture was performed every two weeks until resistant callus appeared. The resistant callus was then transferred to differentiation medium (MS + 0.5 mg / L 6-BA + 50 mg / L hygromycin + 200 mg / L cephalosporin) to induce adventitious shoot differentiation. When the adventitious shoots reached 2-3 cm in length, they were cut and transferred to rooting medium (1 / 2 MS + 0.2 mg / L IAA + 25 mg / L hygromycin) to induce rooting. Complete transgenic tomato plants were obtained and named the OE-SIERF4-9 / SICDF1 / SICDF3 / SIAEC2 / SIPIN5 line.

[0039] 2.2 Molecular identification of transgenic plants Young leaves from transgenic tomato plants were collected, and genomic DNA was extracted using the CTAB method. Using the genomic DNA as a template, PCR detection was performed using primers specific to the hygromycin resistance gene. Positive plants amplified a specific band of approximately 750 bp. Untransformed wild-type tomato plants were used as negative controls, and the recombinant expression vector plasmid was used as a positive control. A total of 36 hygromycin-positive transgenic plants were obtained.

[0040] PCR-positive transgenic plants were selected, and total RNA was extracted from leaves, followed by reverse transcription to obtain cDNA. Using cDNA as a template, the expression levels of five target genes were detected by qRT-PCR. The qRT-PCR reaction was performed on a real-time PCR instrument. The reaction system consisted of: 10 μL SYBR Green Master Mix, 2 μL cDNA template, 0.5 μL each of 10 μmol / L forward and reverse primers, and sterile double-distilled water to a final volume of 20 μL. The reaction program was: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 40 cycles. The tomato SlActin gene was used as an internal control, and the relative expression level was calculated using the 2-ΔΔCT method. The results showed that, compared with wild-type plants, the expression levels of SIERF4-9, SICDF1, SICDF3, SIAEC2, and SIPIN5 were all increased in transgenic plants. Further analysis showed that the expression of SIAEC2 and SIPIN5 was decreased in the slerf4-9 mutant (e.g., Figure 4 As shown in Figures A, B, and C, this is consistent with the phenotype of weakened root development, confirming that SIERF4-9 affects root development by regulating SIAEC2 and SIPIN5. Specifically, the expression levels of SIERF4-9 increased by an average of 15.6-fold, SICDF1 by an average of 8.3-fold, SICDF3 by an average of 7.9-fold, SIAEC2 by an average of 5.2-fold, and SIPIN5 by an average of 4.8-fold. Three independent transgenic lines with high expression levels (OE-1, OE-2, and OE-3) were selected for subsequent drought resistance assessment.

[0041] 2.3 Identification of drought resistance in transgenic plants Seeds from transgenic lines OE-1, OE-2, OE-3 and the wild-type control WT were sown simultaneously. After seedling cultivation, they were transplanted into 15cm diameter pots, each containing an equal amount of nutrient soil, with one seedling per pot, and 30 seedlings per line. They were managed conventionally until the 4-leaf stage.

[0042] Drought resistance was assessed using a natural drought method. Each plant line was divided into two groups of 15 plants each. The control group received normal watering to maintain a relative soil moisture content of 75%-80%. The treatment group underwent no watering and was subjected to natural drought stress. Physiological indicators were measured at days 0, 7, 14, and 21 of the drought treatment period.

[0043] After 21 days of drought stress, wild-type plants showed severe wilting, leaf curling and yellowing, and some plants died; while transgenic plants OE-1, OE-2, and OE-3 showed less wilting, with leaves remaining green, and their survival rate was significantly higher than that of the wild type. Seed germination experiment results are as follows: Figure 6As shown, the germination rate of the slerf4-9 mutant was lower than that of the wild type, indicating that SIERF4-9 plays an important role in the seed germination stage. Statistical survival results showed that after 21 days of drought stress and 7 days of rehydration, the survival rate of wild-type plants was 26.7%, while the survival rates of transgenic lines OE-1 were 86.7%, OE-2 80.0%, and OE-3 93.3%.

[0044] Root parameters were measured for each plant line after 21 days of drought stress. The plants were carefully removed from their pots, the soil adhering to the roots was gently rinsed, and surface moisture was absorbed with absorbent paper. Root morphology parameters were analyzed using the WinRHIZO root analysis system; complete root scan images and analysis results are shown below. Figure 5 As shown, the root development of the slerf4-9 mutant was significantly weaker than that of the wild type, while the root system of the five-gene co-expression transgenic plant constructed in this invention was significantly stronger than that of the wild type. The results showed that under normal watering conditions, there were no significant differences in root fresh weight, root dry weight, number of lateral roots, and number of root tips between the transgenic plant and the wild type. Under drought stress, the transgenic plant OE-1 had a root fresh weight of 2.34 g / plant, a root dry weight of 0.42 g / plant, 187 lateral roots / plant, and 1245 root tips / plant; OE-2 had a root fresh weight of 2.21 g / plant, a root dry weight of 0.38 g / plant, 176 lateral roots / plant, and 1187 root tips / plant; OE-3 had a root fresh weight of 2.46 g / plant, a root dry weight of 0.45 g / plant, 195 lateral roots / plant, and 1320 root tips / plant; while the wild-type plant had a root fresh weight of 1.18 g / plant, a root dry weight of 0.21 g / plant, 98 lateral roots / plant, and 732 root tips / plant. The root fresh weight, root dry weight, number of lateral roots, and number of root tips of transgenic plants were all significantly higher than those of wild-type plants.

[0045] The relative leaf water content and proline content of each line were determined after 21 days of drought stress. Leaf relative water content was determined by the oven-drying and weighing method, and proline content was determined by the acidic ninhydrin method. The results showed that the relative leaf water content of transgenic plants OE-1, OE-2, and OE-3 were 72.4%, 69.8%, and 75.1%, respectively, all significantly higher than the 51.3% of the wild-type plant. The proline content of transgenic plants OE-1, OE-2, and OE-3 were 326.5 μg / g, 298.7 μg / g, and 347.2 μg / g, respectively, all higher than the 187.4 μg / g of the wild-type plant.

[0046] The above results indicate that the recombinant DNA construct containing five genes—SIERF4-9, SICDF1, SICDF3, SIAEC2, and SIPIN5—constructed in this invention can improve the drought resistance of transgenic tomato plants without affecting their growth and development under normal growth conditions.

[0047] Example 3: Gene editing to target and modify GCC-box elements to enhance drought resistance 3.1 Construction of CRISPR / Cas9 gene editing vector sgRNA targets were designed for the GCC-box elements in the SICDF1 gene promoter (SEQ ID NO: 6) and the SICDF3 gene promoter (SEQ ID NO: 7). The nucleotide sequence of the SICDF1 promoter GCC-box target is 5'-AGCCGCCGAC-3', located 387 bp to 378 bp upstream of the start codon; the nucleotide sequence of the SICDF3 promoter GCC-box target is 5'-GCCGCCGCA-3', located 256 bp to 248 bp upstream of the start codon.

[0048] Two pairs of oligonucleotide chains were synthesized, annealed to form double-linked heads, and cloned into the linearized AtU6-sgRNA vector after BsaI digestion to obtain sgRNA expression cassettes. The two sgRNA expression cassettes were tandemly linked to the pCAMBIA1301-Cas9 vector via overlap PCR to obtain a CRISPR / Cas9 gene editing vector simultaneously targeting the SICDF1 and SICDF3 promoter GCC-box elements, named pCAMBIA-Cas9-sgCDF1 / 3.

[0049] 3.2 Tomato genetic transformation and selection of edited plants The pCAMBIA-Cas9-sgCDF1 / 3 vector was transformed into Agrobacterium GV3101, and then into tomato cotyledon explants according to the method in Example 2.1. After obtaining transgenic plants, genomic DNA was extracted from the leaves and amplified by PCR using primers specific to the target site. The amplified products were then analyzed by Sanger sequencing.

[0050] Sequencing results showed that among the 24 transgenic plants obtained, 8 underwent editing in the GCC-box region of the SICDF1 promoter, with an editing efficiency of 33.3%; 6 underwent editing in the GCC-box region of the SICDF3 promoter, with an editing efficiency of 25.0%; and 3 of these plants underwent editing in both GCC-box regions simultaneously. Editing types included single-base substitution, small fragment insertion, and deletion. The plant with the edited GCC-box sequence changed to 5'-AGCCGCCGGC-3' (single-base substitution) was named CDF1-ed-5, and this editing was located within the GCC-box element of the SICDF1 promoter.

[0051] 3.3 Phenotypic Analysis of Edited Plants The expression levels of SICDF1 and SICDF3 in the edited plants were detected by qRT-PCR. The results showed that, compared with wild-type plants, the expression levels of SICDF1 and SICDF3 in CDF1-ed-5 plants were increased by 3.2-fold and 2.8-fold, respectively. The transcriptional regulatory relationship was verified using a dual-luciferase transient expression assay, and the results are as follows: Figure 7 As shown in F, G, and H, the SIERF4-9 protein can directly bind to and activate the promoters of SICDF1 and SICDF3. Further analysis of the expression levels of downstream genes SIAEC2 and SIPIN5 was conducted, and the results of the dual-luciferase transient expression experiment (as shown in...) were obtained. Figure 8 As shown in Figures B, C, D, E, and F, SICDF1 and SICDF3 proteins can directly bind to the DOF binding sites on the promoters of SIAEC2 and SIPIN5 and activate their transcription. The results showed that the expression level of SIAEC2 was increased by 2.3-fold and the expression level of SIPIN5 was increased by 2.1-fold in CDF1-ed-5 plants.

[0052] The drought resistance of CDF1-ed-5 plants was assessed according to the method in Example 2.3. After 21 days of drought stress, the leaf wilting of CDF1-ed-5 plants was significantly less than that of the wild type, and the survival rate was 76.7%, significantly higher than the 26.7% of the wild type. Root analysis showed that under drought stress, the fresh root weight of CDF1-ed-5 plants was 2.05 g / plant, the dry root weight was 0.36 g / plant, the number of lateral roots was 168 / plant, and the number of root tips was 1102 / plant, all significantly higher than those of the wild type.

[0053] The results indicate that gene editing of the GCC-box elements of the SICDF1 and SICDF3 promoters enhances the binding ability of SIERF4-9 protein to the GCC-box elements, thereby increasing the expression levels of SICDF1 and SICDF3, activating the expression of downstream SIAEC2 and SIPIN5, and ultimately enhancing the drought resistance of tomato plants.

[0054] Example 4: Development and Application of Molecular Marker Combinations 4.1 Molecular marker primer design Based on the coding region sequences of the SIERF4-9 genes (corresponding to the amino acid sequence shown in SEQ ID NO: 1), the sequence of the SICDF1 gene promoter region containing the GCC-box element shown in SEQ ID NO: 6, and the sequence of the SICDF3 gene promoter region containing the GCC-box element shown in SEQ ID NO: 7, specific PCR amplification primers were designed respectively.

[0055] The first primer pair used to amplify the coding region sequence of the SIERF4-9 gene: Forward primer ERF4-9-F: 5'-ATGGATGGTTCTACTGGAAGTTC-3' (SEQ ID NO: 10) Reverse primer ERF4-9-R: 5'-TCATTTTGGTGGTGGAAGATTG-3' (SEQ ID NO: 11) The expected amplified fragment length is 789 bp.

[0056] The second primer pair used to amplify the SICDF1 gene promoter region contains the GCC-box element sequence shown in SEQ ID NO: 6: Forward primer CDF1pro-F: 5'-TGGTCAACACACACACACAC-3' (SEQ ID NO: 12); Reverse primer CDF1pro-R: 5'-GTTTGAGATTTGAGGGAGGG-3' (SEQ ID NO: 13); The expected amplified fragment length is 486 bp, which contains the GCC-box element shown in SEQ ID NO: 6.

[0057] The third primer pair used to amplify the SICDF3 gene promoter region contains the GCC-box element sequence shown in SEQ ID NO: 7: Forward primer CDF3pro-F: 5'-CACACACACACACACACACAC-3' (SEQ ID NO: 14); Reverse primer CDF3pro-R: 5'-GTTGATGTTGAGGCTGCTTG-3' (SEQ ID NO: 15); The expected amplified fragment length is 352 bp, which contains the GCC-box element shown in SEQ ID NO: 7.

[0058] 4.2 Establishment of a molecular marker PCR detection system A triplet PCR amplification system was established using tomato genomic DNA as a template. The PCR reaction mixture consisted of: 25 μL of 2×PCR MasterMix, 1 μL each of the forward and reverse primers of the first primer pair (10 μmol / L), 1 μL each of the forward and reverse primers of the second primer pair (10 μmol / L), 1 μL each of the forward and reverse primers of the third primer pair (10 μmol / L), 100 ng of template DNA, and sterile double-distilled water to a final volume of 50 μL. The PCR program was as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 35 cycles; and a final extension at 72℃ for 5 minutes.

[0059] PCR amplification products were detected by 2% agarose gel electrophoresis. The results showed that in positive samples containing the three target fragments, three specific bands of 789bp, 486bp, and 352bp could be observed simultaneously.

[0060] 4.3 Screening of drought resistance in tomato germplasm resources Fifty tomato germplasm resources from different sources were collected, including cultivated varieties, wild varieties, and local varieties. For each germplasm, a mixture of leaves from five seedlings was taken, and genomic DNA was extracted using the CTAB method. Using the extracted genomic DNA as a template, amplification was performed according to the triple PCR system established in Example 4.2 to detect the presence of three target fragments in each germplasm.

[0061] Amplification results showed that among the 50 germplasm accessions, 12 accessions simultaneously amplified three specific bands of 789 bp, 486 bp, and 352 bp, indicating that these accessions simultaneously contained the complete SIERF4-9 gene coding region sequence, the SICDF1 promoter region sequence containing the GCC-box element shown in SEQ ID NO: 6, and the SICDF3 promoter region sequence containing the GCC-box element shown in SEQ ID NO: 7. These 12 accessions were classified into a candidate drought-resistant germplasm group.

[0062] Germplasm samples that simultaneously amplified three bands (numbered H-03, H-08, and H-15) and germplasm samples lacking one band (numbered L-02, L-11, and L-27) were selected for pot drought resistance verification. Drought stress treatment was carried out according to the method in Example 2.3, and the survival rate and root index of each line were measured.

[0063] The results showed that after 21 days of drought stress and 7 days of rehydration, the survival rates of candidate drought-resistant germplasms H-03 were 80.0%, H-08 73.3%, and H-15 86.7%; while the survival rates of control germplasms L-02 were 33.3%, L-11 26.7%, and L-27 40.0%. Combined with the GUS staining results of DR5×WT and DR5×slerf4-9 hybrid seedlings (e.g., ... Figure 9 As shown in the figure, the auxin accumulation in the LR primordium of the SLERF4-9 mutant is reduced, indicating that the complete SIERF4-9-SICDF1 / 3-SIAEC2 / SIPIN5 pathway is crucial for the distribution of auxin in the root tip. Root analysis results show that under drought stress, the root fresh weight, root dry weight, number of lateral roots and number of root tips of the candidate drought-resistant germplasm are significantly higher than those of the control germplasm.

[0064] The above results indicate that the molecular marker combination developed in this invention can be effectively used for the rapid screening of drought-resistant tomato germplasm resources. The screening results are highly consistent with the phenotypic identification results, demonstrating high accuracy and reliability.

[0065] Example 5: Application of the material of the present invention in drought-resistant tomato breeding 5.1 Application of transgenic lines as breeding parents The transgenic line OE-3, which showed significantly improved drought resistance as obtained in Example 2, was selected as the male parent, and the tomato cultivar Zhongshu 6, which had excellent overall traits but weak drought resistance, was selected as the female parent for hybridization breeding. Before flowering, the female parent plants were emasculated, and pollen from the male parent plants was collected for artificial pollination to obtain the F1 generation of hybrids.

[0066] F1 generation plants were planted, and genomic DNA was extracted from leaves. The inheritance of the target genes was detected using the molecular marker combination established in Example 4. The results showed that the integration of five genes—SIERF4-9, SICDF1, SICDF3, SIAEC2, and SIPIN5—was detected in all F1 generation plants, indicating that the five genes were inherited as a whole in linkage to the offspring.

[0067] Drought resistance was assessed, and the results showed that the F1 generation plants had a survival rate of 78.3% under drought stress, a root fresh weight of 2.12 g / plant, and 172 lateral roots / plant, all significantly higher than the maternal parent Zhongshu 6 (survival rate 31.2%, root fresh weight 1.24 g / plant, lateral roots 103 / plant), and approached the levels of the transgenic parent OE-3. Meanwhile, the F1 generation plants exhibited agronomic traits such as fruit characteristics and yield comparable to the maternal parent Zhongshu 6, demonstrating excellent overall performance.

[0068] 5.2 Application of Molecular Marker-Assisted Breeding The drought-resistant germplasm H-15 obtained in Example 4 was used as the drought-resistant donor parent, and the superior cultivated variety Zhongshu 6 was used as the recurrent parent for backcrossing and breeding. In each generation, single plants with agronomic traits close to the recurrent parent were selected, and genomic DNA was extracted from the leaves. The molecular marker combination established in Example 4 was used for detection, and single plants containing three target fragments were screened for further backcrossing.

[0069] After five generations of backcrossing and one generation of self-crossing, the BC5F2 population was obtained. Molecular marker detection was performed on the BC5F2 population, and homozygous lines containing three target fragments and exhibiting excellent agronomic traits were screened and named Zhongshu 6 - Drought-resistant Improved Line.

[0070] Drought resistance was assessed, and the results showed that the survival rate of the Zhongshu 6-drought-resistant improved line under drought stress was 76.2%, the root fresh weight was 2.08 g / plant, and the number of lateral roots was 168 / plant, all of which were higher than those of the original recurrent parent Zhongshu 6 (survival rate 31.2%, root fresh weight 1.24 g / plant, and number of lateral roots 103 / plant). Agronomic traits such as fruit characteristics and yield were basically consistent with those of Zhongshu 6.

[0071] The above results demonstrate that the recombinant DNA construct, recombinant expression vector, gene editing method, and molecular marker combination provided by this invention can be effectively applied in drought-resistant genetic breeding of tomatoes, providing new technical means for cultivating new drought-resistant tomato varieties.

Claims

1. A recombinant DNA construct for improving drought resistance in tomato plants, characterized in that, The recombinant DNA construct comprises: a. A first nucleic acid molecule encoding the SIERF4-9 protein, the amino acid sequence of which comprises the sequence shown in SEQ ID NO: 1; b. A second nucleic acid molecule encoding the SICDF1 protein, the amino acid sequence of which comprises the sequence shown in SEQ ID NO: 2; c. A third nucleic acid molecule encoding the SICDF3 protein, the amino acid sequence of which comprises the sequence shown in SEQ ID NO: 3; d. A fourth nucleic acid molecule encoding the SIAEC2 protein, the amino acid sequence of which comprises the sequence shown in SEQ ID NO: 4; e. A fifth nucleic acid molecule encoding the SIPIN5 protein, the amino acid sequence of which comprises the sequence shown in SEQ ID NO: 5; The first, second, third, fourth, and fifth nucleic acid molecules are all operatively linked to promoters capable of initiating transcription in plant cells.

2. The recombinant DNA construct for improving drought resistance in tomato plants according to claim 1, characterized in that, The SIERF4-9 protein specifically binds to the GCC-box element in the promoter of the SICDF1 gene and the GCC-box element in the promoter of the SICDF3 gene. The nucleotide sequence of the GCC-box element in the promoter of the SICDF1 gene is shown in SEQ ID NO: 6, and the nucleotide sequence of the GCC-box element in the promoter of the SICDF3 gene is shown in SEQ ID NO:

7.

3. The recombinant DNA construct for improving drought resistance in tomato plants according to claim 1, characterized in that, The SICDF1 and SICDF3 proteins can specifically bind to the DOF binding site in the SIAEC2 gene promoter and the DOF binding site in the SIPIN5 gene promoter. The core sequence of the DOF binding site is (A / T)AAAG. The SIAEC2 gene promoter contains at least one copy of the DOF binding site shown in SEQ ID NO: 8, and the SIPIN5 gene promoter contains at least one copy of the DOF binding site shown in SEQ ID NO:

9.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the recombinant DNA construct according to any one of claims 1-3.

5. A method for preparing transgenic tomato plants with improved drought resistance, characterized in that, Includes the following steps: Step 1: Transform the recombinant expression vector described in claim 4 into tomato explant cells; Step 2: Screen and regenerate transgenic tomato plants. The expression levels of SIERF4-9, SIDCF1, SIDCF3, SIAEC2 and SIPIN5 proteins in the transgenic tomato plants are higher than those in wild-type tomato plants. Step 3: Under drought stress, select transgenic tomato plants that have higher values ​​in at least one of the following indicators: root fresh weight, root dry weight, number of lateral roots, and number of root tips than wild-type tomato plants.

6. A gene-editing method for improving the drought resistance of tomato plants, characterized in that, Includes the following steps: Step S1: Gene editing is performed on the GCC-box element of the SICDF1 gene promoter and / or the GCC-box element of the SICDF3 gene promoter in tomato plants. The nucleotide sequence of the GCC-box element of the SICDF1 gene promoter is shown in SEQ ID NO: 6, and the nucleotide sequence of the GCC-box element of the SICDF3 gene promoter is shown in SEQ ID NO:

7. Step S2: Select transgenic tomato plants with edited GCC-box elements. The editing enhances the binding ability of SIERF4-9 protein to the GCC-box elements, resulting in higher expression levels of SICDF1 and / or SICDF3 than wild-type tomato plants.

7. A molecular marker combination for identifying drought-resistant tomato germplasm resources, characterized in that, The molecular marker combination includes: The first primer pair used for specific amplification of the coding region sequence of the SIERF4-9 gene has the nucleotide sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11; The second primer pair used for specifically amplifying the sequence of the SICDF1 gene promoter region containing the GCC-box element shown in SEQ ID NO: 6 has nucleotide sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13; The third primer pair for specifically amplifying the sequence of the SICDF3 gene promoter region containing the GCC-box element shown in SEQ ID NO: 7, has nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO:

15.

8. A method for screening drought-resistant tomato germplasm resources, characterized in that, Includes the following steps: 1) Extract genomic DNA from the tomato samples to be tested; 2) Using the genomic DNA as a template, perform PCR amplification using the molecular marker combination described in claim 7; 3) Detect the presence of the SIERF4-9 gene coding region sequence, the SICDF1 gene promoter region fragment containing the sequence shown in SEQ ID NO: 6, and the SICDF3 gene promoter region fragment containing the sequence shown in SEQ ID NO: 7 in the amplification product; 4) If the tomato sample to be tested amplifies all of the above target fragments at the same time, then the tomato germplasm to be tested is determined to be a germplasm with high drought resistance potential.

9. A kit for use in the method of claim 8, characterized in that, The kit contains the molecular marker combination as described in claim 7.

10. The application of the recombinant DNA construct according to any one of claims 1-3, the recombinant expression vector according to claim 4, or the molecular marker combination according to claim 7 in drought-resistant genetic breeding of tomatoes.

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