Application of the melatonin synthesis gene PtrASMT1 in improving drought resistance in citrus plants

Abstract

This invention relates to the field of plant genetic engineering, specifically to the application of the trifoliate orange melatonin synthesis gene PtrASMT1 in improving drought resistance in citrus plants. The trifoliate orange melatonin synthesis gene... PtrASMT1 The melatonin synthesis gene is used in the breeding of transgenic citrus plants to improve drought resistance. PtrASMT1 The nucleotide sequence is shown in SEQ ID NO: 1. This invention utilizes the melatonin synthesis gene... PtrASMT1 Cloning, expression analysis, and biological function verification provide new targets for molecular breeding of stress-resistant citrus plants and lay the foundation for cultivating drought-resistant citrus varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to the application of the trifoliate orange melatonin synthesis gene PtrASMT1 in improving drought resistance in citrus plants. Background Technology

[0002] Plants often face various abiotic stresses during their growth and development. Drought, as the most significant abiotic stress, severely impacts plant growth and development, leading to a significant decline in yield and quality. Citrus, as an important economic fruit tree, is highly sensitive to water shortage. Prolonged or repeated droughts inhibit new shoot growth, reduce fruit size, decrease soluble solids and juice content, and weaken the tree's resistance to pests and diseases, resulting in severe economic losses. Therefore, improving the drought resistance of citrus is an urgent problem to be solved. In view of this, this invention provides the application of the trifoliate orange melatonin synthesis gene PtrASMT1 in improving the drought resistance of citrus plants. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide the application of the trifoliate orange melatonin synthesis gene PtrASMT1 in improving drought resistance in citrus plants. The aim is to study the trifoliate orange melatonin synthesis gene through genetic engineering. PtrASMT1 The specific role of this gene in drought resistance in citrus provides a research foundation for the development of drought-resistant genetic materials for citrus.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: Firstly, the melatonin synthesis gene PtrASMT1 The application of the melatonin synthesis gene PtrASMT1 The melatonin synthesis gene is used in the breeding of transgenic citrus plants to improve drought resistance. PtrASMT1 The nucleotide sequence is shown in SEQ ID NO: 1.

[0005] Based on the above technical solution, the present invention can be further improved as follows.

[0006] Furthermore, the transgenic citrus plants enhance drought resistance by promoting the synthesis of melatonin.

[0007] Furthermore, the amino acid sequence of the melatonin is shown in SEQ ID NO: 2.

[0008] Secondly, a method for cultivating transgenic citrus plants with improved drought resistance includes the following steps: (1) The melatonin synthesis gene containing the above-mentioned melatonin PtrASMT1 The recombinant expression vector was transformed into Agrobacterium to obtain the recombinant Agrobacterium expression strain; (2) The recombinant Agrobacterium strain was transformed into the Citrus plants to be cultivated, and the melatonin synthesis gene was obtained by screening. PtrASMT1 Transgenic lines with stable genetic inheritance are known as transgenic citrus plants.

[0009] Furthermore, the vector backbone of the recombinant expression vector is pBI121.

[0010] Furthermore, the melatonin synthesis gene described in step (1) PtrASMT1 Using the cDNA of trifoliate orange as a template, the melatonin synthesis gene of trifoliate orange was amplified by PCR. PtrASMT1 .

[0011] Furthermore, the gene for melatonin synthesis was amplified by PCR. PtrASMT1 The nucleotide sequences of the primer pairs are shown in SEQ ID NO: 3-4.

[0012] Further, step (2) of transforming the recombinant Agrobacterium-expressing strain into the citrus plants to be cultivated includes the following specific steps: expanding the recombinant Agrobacterium-expressing strain into Agrobacterium infection solution; infecting the explants of the seedlings of the citrus plants to be cultivated with the Agrobacterium infection solution, and obtaining the melatonin synthesis gene by positive identification. PtrASMT1 Stable genetically inherited transgenic lines.

[0013] Furthermore, the OD of the Agrobacterium infection solution... 600 It is 0.6-0.8.

[0014] Furthermore, the citrus plants include at least one of lemon and trifoliate orange.

[0015] This invention cloned and identified the melatonin synthesis gene from trifoliate orange (Poncirus trifoliata). PtrASMT1 Overexpression vectors and VIGS (virus-induced gene silencing) expression vectors were constructed, and corresponding transgenic plants were obtained through transgenic technology. Wild-type and transgenic plants were subjected to drought treatment, and relevant physiological indicators were measured, demonstrating that transgenic plants can promote drought resistance in citrus by promoting melatonin synthesis. The specific processes include the following: (1) Design of melatonin synthesis gene PtrASMT1 The expression patterns of the RT-qPCR primers under different treatment conditions were analyzed using real-time quantitative PCR technology. The results showed that... PtrASMT1 The expression levels were upregulated to varying degrees under dehydration, ABA and melatonin treatment, with the most significant upregulation occurring in the early stages of treatment. (2) Primers were designed based on the reference genome sequence, and gene cloning technology was used to obtain... PtrASMT1 The coding sequence of the gene (SEQ ID NO: 1) is as follows:

[0016] The amino acid sequence of melatonin (SEQ ID NO: 2): MMKKEEDDAEVEIWKYAFGFTNMAVVKCAVELGIAEAVEEKGSPITLNELASSLKCDPSLLQRIMRFLIHLKFFKEVPTSQGSMAFQQTPLSRRLMRHGENNMAAFILLESSPVMLAPWHSLGTRVLANGTSAFDKAHGKDVWSYAAEDAAHSKLINDAMACDTRLALRAIIDG CPEVFDGIETLVDIGGNDGTTLRTLTKAFPRIRGINFDLPHVVCVAEKCHGVEHVGGDMFDGVPKADAAIIKWVLHDWGDDECIKILKNCKEAITKDKGKVIIVEAIIEEDDEVDNKFKSVRLDMVMMAHTNKGKERSLKEWDYVLRQAGFSRYNITSIHAVQSLIEAFP*.

[0017] (3) The melatonin synthesis gene of trichosanthes kirilowii PtrASMT1 Overexpression vectors (pBI121) and VIGS vectors (pTRV2, virus-induced gene silencing) were constructed, and wild-type and transgenic plants were subsequently subjected to drought treatment to analyze their differences in drought resistance; the results showed... PtrASMT1 Plants with overexpression showed greater drought resistance and physiological indicators than wild-type plants. F v / F m, anti-superoxide anion activity and melatonin content were significantly increased, while relative conductivity (EL), MDA content, and H2O2 content were significantly decreased. Furthermore, after exogenous application of melatonin inhibitors, drought resistance significantly decreased, returning to wild-type levels, with some indicators showing significant changes. PtrASMT1 The phenotype and physiological indicators of the silent group were the opposite; this indicates that PtrASMT1 It mainly regulates the drought resistance of citrus by affecting the synthesis of melatonin.

[0018] The beneficial effects of this invention are: This invention targets the melatonin synthesis gene. PtrASMT1 Cloning, expression analysis, and biological function verification provide new targets for molecular breeding of stress-resistant citrus plants and lay the foundation for cultivating drought-resistant citrus varieties. Attached Figure Description

[0019] Figure 1 The melatonin synthesis gene of this invention PtrASMT1 Flowchart of functional study; Figure 2 for PtrASMT1 Relative expression levels under ABA, drought, and melatonin treatments; where A represents ABA treatment, B represents drought treatment, and C represents melatonin treatment. Figure 3 Subcellular localization results of PtrASMT1 protein; Figure 4 for PtrASMT1 PCR identification results of lemon plants overexpressing the gene; Figure 5 for PtrASMT1 Analysis of target gene expression levels in lemon overexpression; Figure 6 for PtrASMT1 Phenotypic and physiological indicators of overexpressing lemon under drought stress were analyzed. A shows the plant control images before and after drought stress treatment; B shows the control images before and after electrical conductivity treatment; C shows the control images before and after photosynthetic efficiency treatment; D shows the control images before and after malondialdehyde (MDA) treatment; E shows the control images of leaves before and after H2O2 accumulation treatment; F shows the control images before and after H2O2 accumulation content treatment; and G shows the control images of superoxide anion (O2O2). ·－ (See the comparison chart before and after treatment of clearance ability, where H represents the comparison chart before and after treatment of melatonin (MT) content.) Figure 7 for PtrASMT1 PCR identification results of silent trifoliate orange plants; Figure 8 for PtrASMT1 Analysis of target gene expression levels in silent trifoliate orange; Figure 9 for PtrASMT1 Phenotypic and exogenous melatonin replenishment results of *Trifolium repens* under drought stress. A shows a control group of plants before and after drought stress treatment; B shows a control group of plants before and after conductivity treatment; C shows a control group of plants before and after photosynthetic efficiency treatment; D shows a control group of plants before and after malondialdehyde (MDA) treatment; E shows a control group of leaves before and after H2O2 accumulation treatment; F shows a control group of H2O2 accumulation content treatment; and G shows a control group of superoxide anion (O2) concentrations. ·－ (See the comparison chart before and after treatment of clearance ability, where H represents the comparison chart before and after treatment of melatonin (MT) content.) Detailed Implementation

[0020] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0021] Example In this embodiment, the melatonin synthesis gene of trifoliate orange (Poncirus trifoliata) was cloned and identified. PtrASMT1 Overexpression vectors and VIGS (virus-induced gene silencing) expression vectors were constructed, and corresponding transgenic plants were obtained through transgenic technology. Wild-type and transgenic plants were subjected to drought treatment, and relevant physiological indicators were measured. The results showed that the transgenic plants could promote drought resistance in citrus by promoting melatonin synthesis, such as… Figure 1 As shown.

[0022] 1. PtrASMT1 Analysis of gene expression patterns.

[0023] This invention uses *Citrus trifoliata* (a type of citrus fruit) as the material and sets up three experimental groups: the first group involves exogenously spraying 100 mM ABA (abscisic acid) onto two-month-old *Citrus trifoliata* seedlings; the second group involves subjecting two-month-old *Citrus trifoliata* seedlings to natural drought treatment; and the third group involves exogenously spraying 100 mM melatonin (N-acetyl-5-methoxytryptamine, MT) onto three-month-old *Citrus trifoliata* seedlings. Samples were taken at 0h, 1h, 3h, 6h, 9h, 12h, and 24h after treatment, with at least 15 seedlings randomly selected from each treatment group. The samples were then frozen at -80℃ for subsequent expression analysis.

[0024] Total RNA was extracted from each group of samples, and cDNA was obtained by reverse transcription. Real-time quantitative PCR analysis was performed using cDNA as a template. The quantitative primers were F: GCAGTAGTGAAATGTGCCGTAG (SEQ ID NO: 5), R: GAGGCCAGCTCGTTGAGTGTA (SEQ ID NO: 6). The enzyme used was the Hifair® Advanced One Step RT-qPCR SYBR Green Kit (Yisheng, China), and the instrument was a Bio-rad CFX96 (Bio-Ray, USA). Relative gene expression levels were calculated using a 2-1T / T ratio. －ΔΔCT Methods: Actin was used as an internal reference gene (F: CCGACCGTATGAGCAAGGAAA (SEQ ID NO: 7), R: TTCCTGTGGACAATGGATGGA (SEQ ID NO: 8)).

[0025] The results showed that in the ABA treatment group, PtrASMT1 The levels were consistently increased at all time points, with the maximum induction level increasing 15-fold at 3 hours. Figure 2 (A). Dehydration treatment induced a peak expression level of approximately 6-fold at 1 hour, which then gradually decreased, returning to baseline levels at 12 hours, followed by a slight rebound. Figure 2 (B). Under exogenous MT treatment,PtrASMT1 Expression spiked approximately 10-fold at 1 and 3 hours, then gradually declined, but remained consistently higher than pre-treatment levels. Figure 2 (C). These results indicate that... PtrASMT1 It actively responds to ABA, dehydration and melatonin induction, and may participate in the melatonin-mediated drought resistance process in citrus.

[0026] 2. Cloning and sequence analysis of the PtrASMT1 gene.

[0027] This embodiment uses PCR technology to amplify the target gene. The specific process is as follows: using *Citrus aurantium* cDNA as a template, the target fragment is amplified by high-fidelity enzyme under the action of forward primer (F: ATGATGAAGAAAGAAGAAGATG (SEQ ID NO: 9)) and reverse primer (R: TCAAGGAAAAGCCTCAATAAGTG (SEQ ID NO: 10)). After gel extraction and purification, the product is cloned into the pClone007 vector (kit purchased from Beijing Qingke Biotechnology Co., Ltd.) and transformed into *E. coli* DH5α (strain purchased from Beijing Qingke Biotechnology Co., Ltd.). After antibiotic screening and positive identification, the sequence is determined using a fully automated DNA sequencer to ensure that the cloned sequence is completely consistent with the target sequence, thus obtaining the target gene. PtrASMT1 The total length.

[0028] Sequencing analysis PtrASMT1 The full-length gene is 1041 bp, as shown in SEQ ID NO: 1, encoding 346 amino acids as shown in SEQ ID NO: 2, with an isoelectric point of 5.61 and a molecular weight of 38555.53 Da.

[0029] 3. Subcellular localization of PtrASMT1 protein.

[0030] This invention obtains a product without a stop codon through primer amplification. PtrASMT1 The gene ORF sequence was obtained and fused into the pBI121-eGFP vector (plasmid purchased from Wuhan Jinkairui Biotechnology Co., Ltd.) (F: GAGAACACGGGGGACTCTAGAGATTTGGGAATCTGA (SEQ ID NO: 3), R: ATAAGGGACTGACCACCCGGGCACTTGCACTTTTGT (SEQ ID NO: 4)). To verify subcellular localization, pBI121-eGFP-PtrASMT1 and pBI121-eGFP were co-transformed into leaves of *Nicotiana benthamiana*. Laser confocal microscopy analysis showed that the green fluorescence signal completely overlapped with the chloroplast's own fluorescence, indicating that it is specifically located within the chloroplast. Figure 3 This phenotype strongly provesPtrASMT1 The site that regulates the synthesis of melatonin in citrus is the chloroplast.

[0031] 4. Construction of plant overexpression vectors, genetic transformation of lemons, and identification of positive plants.

[0032] 4.1 Construction of target gene overexpression vector: Using primer amplification to obtain homologous arms PtrASMT1 The full-length sequence (F: GAGAACACGGGGGACTCTAGAGATTTGGGAATCTGA (SEQ ID NO: 3), R: ATAAGGGACTGACCACCCGGGCACTTGCACTTTTGT (SEQ ID NO: 4)) was directionally inserted into the pBI121 vector (purchased from Thermo Fisher Scientific); the cloning plasmid was extracted and introduced into Agrobacterium. Agrobacterium tumefaciens The cells were stored in competent GV3101 cells (purchased from Beijing Qingke Biotechnology Co., Ltd.) for later use.

[0033] Select well-developed lemon fruits, remove the seeds and peel off the outer seed coat. Then, in a clean bench, disinfect the seeds first with 75% alcohol for 30 seconds, followed by surface sterilization with 2% sodium hypochlorite solution for 20 minutes, and rinse 3-5 times with sterile water. Blot off excess moisture with filter paper and inoculate onto MS solid medium (purchased from Haibo Biotechnology Co., Ltd.). After one month of dark culture and subsequent 7-10 days of light induction, seedlings for transformation are obtained.

[0034] 4.2 Preparation of Agrobacterium infection solution: Agrobacterium containing the target vector was streaked onto LB agar plates containing kanamycin (Kan) and incubated in the dark at 28°C for 2–3 days to activate the bacterial strain. Single colonies were picked and suspended in LB liquid medium (purchased from Haibo Biotechnology Co., Ltd.) and cultured with shaking at 28°C and 200 rpm. 1–2 ml of the activated bacterial solution was added to 40 ml of LB liquid medium, and the culture was continued until OD (occurrence limit) was reached. 600 The concentration was 0.6–0.8. After centrifugation, the bacterial cells were resuspended in liquid MS medium, and 20 mg / mL AS (acetylsuccinone) was added to obtain the infection solution.

[0035] 4.3 Explant cutting and infection: Remove lemon seedlings from the soil and cut them into 1-1.5 cm long stem segments at a 45° angle on sterile filter paper. Then, completely immerse the cut stem segments in the prepared Agrobacterium infection solution and shake for 15 minutes. After blotting off excess bacterial solution with sterile filter paper, spread the stem segments evenly on a co-culture medium (MS basal medium + 20 mg / mL AS + 3 mg / L 6-BA + 1 mg / L IAA) and incubate in the dark at 25°C for 3 days.

[0036] 4.4 Selection, Regeneration and Plant Survival: After co-culture, the stem segments were rinsed 3-5 times with sterile water and then transferred to selection medium (MS basal medium + 3 mg / L 6-BA + 500 mg / L Cef + 60 mg / L kanamycin (Kan)) for light culture. When the regenerated shoots reached about 1 cm in length, the shoots were cut off for elongation culture and root induction.

[0037] 4.5 Positive identification of overexpressing plants: Positive identification of transgenic plants was performed using PCR. Figure 4 Subsequently, real-time fluorescence quantitative PCR was used to analyze the genetically modified lemons. PtrASMT1 The relative expression levels showed that, compared to wild-type WT, PtrASMT1 In genetically modified lemons (#2, #5, #8, #9) PtrASMT1 The relative expression level of the gene was significantly increased. Figure 5 The resulting positive transgenic lemon (OE-) PtrASMT1 The plants were used for subsequent drought resistance analysis.

[0038] 4.6 Analysis of drought resistance in transgenic lemons: Phenotypic observation showed that OE- PtrASMT1 The degree of leaf wilting in the plants after drought stress was significantly lower than that in the wild type, while CPA (melatonin synthesis inhibitor, 100 mM) treatment partially reversed these protective effects, resulting in a degree of wilting similar to that in the wild type. Figure 6 (A) OE under drought stress PtrASMT1 The relative electrical conductivity of the plants was significantly reduced, while that of the CPA-treated plants was increased. Figure 6 (B) This is consistent with OE- PtrASMT1 The photosynthetic efficiency of the plant ( F v / F m) was better maintained, while CPA application led to F v / F m decreased significantly ( Figure 6 (C). Biochemical analysis showed that OE- PtrASMT1 The plant has a lower content of malondialdehyde (MDA). ​(D), H2O2 accumulation decreases ( ​ (E, F), and superoxide anion (O2) ·－ The ability to clear obstacles is enhanced compared to the wild type under stress. ​ (E, G). CPA treatment, however, eliminates these advantages, leading to increased oxidative damage. It is worth noting that OE- ​ The plants had significantly higher levels of melatonin (MT), which was positively correlated with their enhanced stress resistance. ​ (H). The above results indicate that ​ It promotes the biosynthesis of melatonin, and the resulting increase in melatonin levels subsequently triggers the antioxidant defense system to mitigate drought-induced oxidative stress. Inhibiting melatonin synthesis weakens this protective effect, which further highlights the key role of melatonin in responding to drought stress.

[0039] 5. Construction of plant gene silencing expression vectors, genetic transformation of trifoliate orange and identification of positive plants.

[0040] 5.1 Construction of target gene silencing expression vector: Using primer amplification to obtain homologous arms ​ The VIGS sequence (F:gtgagtaaggttaccgaattcAGGCTGATGCTGCTA (SEQ ID NO: 11), R:cgtgagctcggtaccggatccGAACTGCATGAATAG (SEQ ID NO: 12)) was directionally inserted into the pTRV2 vector (purchased from Thermo Fisher Scientific). Positive clones were obtained through screening and identification. The cloning plasmid was extracted and introduced into Agrobacterium (…). ​ It was stored in competent GV3101 cells for later use.

[0041] 5.2 Preparation of Agrobacterium infection solution: Agrobacterium strains containing pTRV1 (responsible for viral replication and systemic spread throughout the plant), pTRV2, and the target gene recombinant vector were inoculated onto LB solid medium containing 20 mg / L rifampin and 50 mg / L kanamycin, respectively, and cultured upside down in a 28°C incubator for 2–3 days to obtain single colonies. Subsequently, single colonies were selected from each plate and inoculated into 5 mL of LB liquid medium with the same antibiotic resistance, and cultured with shaking at 28°C and 200 rpm for 24–48 hours to fully activate the cells. 2–3 mL of the activated Agrobacterium was then transferred to 80 mL of LB liquid medium and cultured further at 28°C and 200 rpm until OD was reached. 600The concentration was 1.2–1.6. The bacterial cells were resuspended in an equal volume of resuspension buffer (containing 10 mmol / L MES, 10 mmol / L MgCl2, 20 mg / L AS, pH 5.6–5.7). The pTRV1 resuspension was thoroughly mixed with the pTRV2 and the target recombinant bacterial resuspension at a 1:1 volume ratio and incubated in the dark at 28°C for 2–3 hours to complete the preparation of the infection solution.

[0042] 5.3 Seed Infection and Vacuum Infiltration: Using a sterile syringe needle, puncture the surface of the newly germinated 1-2 cm long shoots of the trifoliate orange seed, then completely immerse them in the above-mentioned infection solution. Turn on the vacuum pump to evacuate for 2-3 minutes, then release the gas for 10 minutes, repeating this process three times. Remove the seeds and place them on dry filter paper to absorb excess liquid. Then, spread them evenly in a petri dish lined with sterile water-soaked filter paper and incubate in the dark for 2-3 days at room temperature under shade.

[0043] 5.4 Sowing and Late-Stage Identification: After dark culture, the seeds were thoroughly rinsed with water to remove any residual Agrobacterium solution. The washed seeds were then sown in the substrate and grown in a room-temperature, light-controlled incubator. Approximately one month later, leaves of the trifoliate orange were collected for positive identification. Using extracted trifoliate orange DNA as a template, specific primers were used for PCR identification of positive plants (TRV2-F: AGACATGGAGTTGAAGAGT (SEQ ID NO: 13), TRV2-R: AAGAACCGCCTGAACATAA (SEQ ID NO: 14)). Real-time quantitative PCR was also used to detect the presence of [DNA] in the positive plants. ​ Gene expression levels were detected, among which... #2 , #4 , #6 , #8 , #12 Plant (TRV-) ​ The expression levels of these compounds were all less than 0.5 times that of wild-type plants, making them suitable for subsequent functional identification. ​ , ​ ).

[0044] 5.5 ​ Identification of drought resistance function in gene-silenced trifoliate orange: For TRV- ​ After natural drought treatment with TRV2 plants, TRV- ​ The plants exhibited greater drought sensitivity, manifested as severe wilting and higher electrolyte loss, which was more pronounced compared to the TRV control group. ​ (A, B). Additionally, TRV- ​ plant F v / F The m value decreased (​ (C), MDA levels increased ( ​ D). The application of exogenous MT partially restored TRV- ​ drought sensitivity phenotype of plants ( ​ (A) Specifically, it reduces the accumulation of H2O2. ​ E, F), enhanced the scavenging of superoxide anions ( ​ E, G) and increased endogenous MT content ( ​ (H). The above results indicate that ​ Gene silencing reduces melatonin biosynthesis, leading to impaired antioxidant defense systems; while the application of exogenous melatonin alleviates this symptom, further highlighting the importance of... ​ The key role of regulating melatonin in responding to drought stress.

[0045] In summary, this invention clones [the product] from [the plant] *Citrus aurantium*. ​ The gene, whose coding sequence is shown in SEQ ID NO:1, was demonstrated through expression pattern analysis, subcellular localization, overexpression, and gene silencing experiments. It was confirmed that PtrASMT1 is located in chloroplasts and positively regulates drought resistance in citrus. Its mechanism of action depends on promoting endogenous melatonin synthesis and enhancing reactive oxygen species scavenging capacity. This invention provides new gene resources and theoretical basis for the genetic improvement of drought resistance in citrus and has important application prospects.

[0046] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Melatonin synthesis gene PtrASMT1 The application, characterized in that, The melatonin synthesis gene of the citrus plant PtrASMT1 In the breeding of transgenic citrus plants for improving drought resistance, the nucleotide sequence of the melatonin synthesis gene of the citrus plant PtrASMT1 is shown as SEQ ID NO:

1.

2. The melatonin synthesis gene according to claim 1 PtrASMT1 The application, characterized in that, The genetically modified citrus plants enhance drought resistance by promoting the synthesis of melatonin.

3. The use of a melatonin synthesis gene according to claim 2, characterized in that PtrASMT1 The amino acid sequence of the melatonin is shown in SEQ ID NO:

2. ​ 4. A method for cultivating transgenic citrus plants with improved drought resistance, characterized in that, Includes the following steps: (1) The melatonin synthesis gene containing any one of claims 1 to 4 PtrASMT1 The recombinant expression vector was transformed into Agrobacterium to obtain the recombinant Agrobacterium expression strain; (2) transforming the recombinant expression Agrobacterium strain into the citrus plant to be cultivated, and screening to obtain a citri melatonin synthesis gene PtrASMT1 The genetically stable transgenic line is a transgenic citrus plant.

5. The method for breeding a transgenic citrus plant having improved drought tolerance according to claim 4, wherein The recombinant expression vector has a vector backbone of pBI121.

6. The method for cultivating transgenic citrus plants with improved drought resistance according to claim 4, characterized in that, The melatonin synthesis gene in step (1) PtrASMT1 The melatonin synthesis gene was amplified by PCR using the cDNA of Zizyphus jujuba as template PtrASMT1 .

7. The method for cultivating transgenic citrus plants with improved drought resistance according to claim 6, characterized in that, PCR amplification of the melatonin synthesis gene of pummelo PtrASMT1 The nucleotide sequences of the primer pairs for PCR amplification of the melatonin synthesis gene of pummelo are shown as SEQ ID NOs: 3-4.

8. A method for cultivating transgenic citrus plants with enhanced drought resistance according to any one of claims 4 to 7, characterized in that, Step (2) transforming the recombinant Agrobacterium-expressing strain into the citrus plants to be cultivated includes the following specific steps: expanding the recombinant Agrobacterium-expressing strain into Agrobacterium infection solution; infecting the explants of the seedlings of the citrus plants to be cultivated with the Agrobacterium infection solution, and obtaining the melatonin synthesis gene by positive identification. PtrASMT1 Stable genetically inherited transgenic lines.

9. The method for cultivating transgenic citrus plants with improved drought resistance according to claim 8, characterized in that, OD of the agrobacterium infection solution 600 was 0.6-0.

8.

10. The method for cultivating transgenic citrus plants with improved drought resistance according to claim 8, characterized in that, The Citrus species include either lemon or trifoliate orange.

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