Drought-resistant functional gene smhdc-like of solanum melongena and application thereof in improving drought resistance of plants

Abstract

This invention belongs to the field of plant genetic engineering and crop improvement technology, and specifically discloses a drought-resistant functional gene for eggplant. SmHDC‑ like And its application in improving plant drought resistance, the gene SmHDC‑like The nucleotide sequence of the enzyme is shown in SEQ ID NO.1; the amino acid sequence of its encoded protein, SmHDC-like, is shown in SEQ ID NO.2. This invention is the first to clone the histidine decarboxylase gene SmHDC-like from eggplant and confirm that its expression is induced by drought stress. Overexpression of the SmHDC-like gene significantly improves the plant's growth adaptability under drought stress and effectively alleviates drought-induced growth inhibition. The gene and its encoded product of this invention can be widely applied to the study of drought resistance mechanisms in eggplant, marker-assisted breeding of plants, and genetic engineering breeding, providing important gene resources and theoretical basis for drought-resistant molecular breeding of eggplant and related crops.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and crop improvement technology, and relates to the function and application of plant histidine decarboxylase (HDC) genes and their encoded proteins in regulating plant responses to drought stress. Specifically, it relates to an eggplant drought-resistance gene SmHDC-like and how regulating the expression level of this gene (overexpression) can enhance the plant's drought tolerance, water use efficiency, and post-drought recovery ability. Background Technology

[0002] Drought stress is one of the major abiotic stress factors that restrict crop growth, development, and yield formation. Against the backdrop of global climate change, the frequency and intensity of droughts are continuously increasing, posing a serious threat to global food security. Therefore, exploring plant drought-resistant gene resources, elucidating their mechanisms of action, and using biotechnology to breed new drought-resistant varieties have significant scientific value and application prospects. Plant responses to drought stress involve complex physiological, biochemical, and molecular regulatory networks, in which the synthesis and dynamic balance of small molecule signaling substances (such as hormones, polyamines, and reactive oxygen species) play a crucial role. Histamine, as a bioactive amine, is widely recognized for its function as a neurotransmitter and immunomodulator in animals, but research on it in plants remains relatively limited. In plants, histamine is mainly synthesized in one step by histidine via histidine decarboxylase (HDC). Current techniques suggest that histamine may participate in plant responses to biotic stresses (such as pathogen infection); however, its specific functions, mechanisms of action, and regulatory networks in abiotic stresses, especially drought stress, remain unclear. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide an eggplant drought-resistance functional gene, SmHDC-like. Through whole-genome analysis and molecular genetic experiments, this invention identifies for the first time an evolutionarily conserved eggplant gene, SmHDC-like, that is expressed in a drought-induced manner. Further functional validation experiments show that this gene is a positive regulator of plant drought resistance; its encoded protein significantly improves plant drought resistance by mitigating drought-induced membrane lipid peroxidation damage, maintaining cell membrane structural integrity, and regulating reactive oxygen species balance. This invention reveals the crucial role of the SmHDC-like gene in plant drought resistance, providing a new gene target and important gene resource for the genetic improvement of crop drought resistance.

[0004] This invention is achieved through the following technical solution:

[0005] A drought-resistant functional gene for eggplant, SmHDC-like, has a DNA sequence as shown in SEQ ID NO.1.

[0006] Furthermore, the protein encoded by the eggplant drought-resistant gene SmHDC-like has the amino acid sequence shown in SEQ ID NO.2.

[0007] Furthermore, the present invention provides a recombinant expression vector of the eggplant drought-resistant functional gene SmHDC-like.

[0008] Furthermore, the cloning method for the SmHDC-like gene described in this invention includes the following steps:

[0009] 1) Total RNA was extracted from the leaf tissue of *Solanum lyratum* and reverse transcribed into cDNA;

[0010] 2) Using the reference genome sequence as a template, primers cP1F and cP1R were designed;

[0011] 3) Using the cDNA reverse transcribed in step 1) as a template, and cloning with primers designed in step 2);

[0012] 4) The PCR products obtained in step 3) are recovered, sequenced, and analyzed.

[0013] The sequence of the primer cP1F is shown below:

[0014] cP1F: GGACTCTAGAGGATCCATGGGTAGCCTCTCACTTGAAATG (SEQ ID NO.3);

[0015] The sequence of the primer cP1R is shown below:

[0016] cP1R: GACCACCCGGGGATCCTAAGGACTAACGTAATCAATCTTATGATAAGAGCATG (SEQ ID NO. 4).

[0017] Furthermore, this invention protects the application of the eggplant drought-resistance gene SmHDC-like or the protein encoded by the gene in regulating plant drought resistance.

[0018] Furthermore, the present invention protects the application of the eggplant drought-resistant functional gene SmHDC-like or the protein encoded by the gene in enhancing plant drought resistance.

[0019] Furthermore, the enhancement of plant drought resistance manifests as improving poor plant growth in drought conditions and promoting good plant growth in arid environments.

[0020] Furthermore, the present invention protects the use of the eggplant drought-resistant functional gene SmHDC-like or the protein encoded by the gene in improving drought-resistant plant varieties or cultivating drought-resistant plant varieties.

[0021] Furthermore, the plant is eggplant, tobacco, or Arabidopsis thaliana.

[0022] Furthermore, this study provides a method for analyzing the expression level of the eggplant drought-resistance gene SmHDC-like in eggplant response to drought, including the following steps:

[0023] S1. Obtain leaf tissues from plants after different periods of drought stress treatment and extract total RNA.

[0024] S2. Using the total RNA as a template, reverse transcription is performed to obtain cDNA;

[0025] S3. Using the first strand of cDNA as a template, fluorescence quantitative analysis was performed by amplifying the SmHDC-like gene and the internal reference gene APRT with specific primers to obtain the expression level of the SmHDC-like gene under drought stress in eggplant.

[0026] The base sequences of the specific primers for amplifying the SmHDC-like gene are shown in SEQ ID NO. 5 and SEQ ID NO. 6:

[0027] SmHDC-like-qF: 5′-TGCATGCAGTCAGCAATTTGT-3′ (SEQ ID NO. 5);

[0028] SmHDC-like-qR: 5′-TTCGCCGCGCTAAACTTCTA-3′ (SEQ ID NO. 6).

[0029] The base sequences of the specific primers for amplifying the internal reference gene APRT are shown in SEQ ID NO.7 and SEQ ID NO.8:

[0030] APRT rRNA-F: 5′-GAGATGCATGTAGGTGCTGTGCAA-3′ (SEQ ID NO.7);

[0031] APRT rRNA-R: 5′-GGCCCTTCAATTCTGGCAACTCAA-3′ (SEQ ID NO. 8).

[0032] To fully utilize the advantages of molecular breeding, this invention employs a molecular biology approach, utilizing seamless cloning, sequencing, and assembly to obtain the coding region sequence of the SmHDC-like gene from eggplant. Subsequently, bioinformatics analysis was performed to detect the expression pattern of the SmHDC-like gene in response to drought stress, further verifying its function, revealing its role in drought resistance, and identifying a drought-resistant functional gene. This invention clones the drought-resistant functional gene SmHDC-like from eggplant and performs bioinformatics analysis and its response to drought stress, aiming to provide a reference for a deeper understanding of the function and mechanism of action of SmHDC-like transcription factors in drought stress response.

[0033] Compared with the prior art, the present invention has the following advantages and effects:

[0034] (1) This invention screened a novel eggplant drought-resistant functional gene, SmHDC-like, which is the first reported sequence of an eggplant SmHDC-like gene. The homology of the gene in the amino acid sequence alignment results on NCBI was 91.71% and 92.87%, respectively. Experiments show that this gene responds to drought stress. The cloning and study of this gene have certain reference value for understanding the drought resistance mechanism of eggplant.

[0035] (2) The eggplant drought-resistant functional gene SmHDC-like provided by the present invention can improve the poor growth of yeast in drought and endow yeast with good growth characteristics in drought environment. By overexpressing this gene in plants such as Arabidopsis thaliana, tobacco or eggplant, the drought resistance of plants can be significantly improved. The acquisition of this gene can not only provide a reference gene for molecular marker-assisted breeding of eggplant drought resistance, but also enrich the gene selection for transgenic breeding of drought resistance in plants such as eggplant, and lay a preliminary foundation for molecular breeding of eggplant drought resistance. Attached Figure Description

[0036] Figure 1 This is an electrophoresis diagram of the eggplant SmHDC-like gene clone of the present invention;

[0037] Figure 2 This is a diagram showing the sequence alignment results of the SmHDC-like gene.

[0038] Figure 3 Figure showing the SmHDC-like amino acid sequence alignment results;

[0039] Figure 4 This is a diagram of eggplant SmHDC-like multiple sequence alignment analysis according to the present invention;

[0040] Figure 5 This is a phylogenetic analysis diagram of eggplant SmHDC-like phylogenetics according to the present invention;

[0041] Figure 6 This is a comparative analysis diagram of conserved motifs and domains of eggplant SmHDC-like cells and HDC cells from other species, based on the present invention.

[0042] Figure 7 This is a diagram showing the predicted secondary structure of the eggplant SmHDC-like protein of the present invention.

[0043] Figure 8 This is a diagram showing the predicted tertiary structure of the eggplant SmHDC-like protein of the present invention;

[0044] Figure 9 This is a schematic diagram of the expression pattern of the eggplant SmHDC-like gene under drought stress according to the present invention;

[0045] Figure 10 Phenotypic analysis of SmHDC-like genes in yeast under drought stress is validated.

[0046] Figure 11 Transgenic positive tobacco plants transplanted into nutrient pots;

[0047] Figure 12 Electrophoresis results of PCR detection of SmHDC-like gene in transgenic tobacco gDNA;

[0048] Figure 13 The relevant physiological indicators of wild-type and SmHDC-like plants under drought stress at different times are: (a) malondialdehyde content, (b) relative conductivity, (c) POD activity and (d) CAT activity.

[0049] Figure 14 Figures show the root growth phenotypes and statistical analysis results of the tested materials under different treatment conditions; where: (a) shows the root morphology of wild-type Nicotiana benthamiana (left) and the SmHDC-like gene transgenic Nicotiana line (middle and right) after 14 days of culture in MS + 3% sucrose medium; (b) shows the root morphology of the above materials in MS + 3% sucrose + 5% PEG medium. 6000 (c) shows the root morphology after 14 days of cultivation in a simulated drought medium; (d) shows the statistical analysis of the root length of each material under condition (a); and (b) shows the statistical analysis of the root length of each material under condition (b). Detailed Implementation

[0050] This invention provides an eggplant drought-resistant functional gene SmHDC-like and its application in improving plant drought resistance. In the specific embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available. The invention will be described in detail below with reference to specific embodiments.

[0051] Example 1: Cloning of the eggplant SmHDC-like gene

[0052] 1. Obtaining plant materials

[0053] The plant material used in this experiment was the superior germplasm resource of eggplant, *Solanum lyratum*. The experimental materials were provided by Huaiyin Institute of Technology. *Solanum lyratum* plants grown in greenhouse pots for approximately 60 days and of uniform size were used as the material. The second and third true leaves from the top were collected for RNA extraction.

[0054] 2. RNA extraction

[0055] RNA was extracted from treated *Solanum lyratum* leaves according to the instructions of the HiPure HP Plant RNA Kit. RNA integrity was assessed by electrophoresis, and then the purity and concentration of RNA were determined using a spectrophotometer. cDNA was obtained by reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser, which was used for subsequent gene cloning.

[0056] 3. Cloning of the SmHDC-like gene

[0057] Based on the eggplant reference genome sequence, primers for seamless cloning of the SmHDC-like gene were designed using Snapgene.

[0058] cP1F: GGACTCTAGAGGATCCATGGGTAGCCTCTCACTTGAAATG (SEQ ID NO.3)

[0059] cP1R: GACCACCCGGGGATCCTAAGGACTAACGTAATCAATCTTATGATAAGAGCATG (SEQ IDNO.4)

[0060] Using synthesized eggplant cDNA as a template, PCR was performed. After amplification to the expected length, the cDNA was recovered and ligated into the pBI121 vector, transformed into E. coli DH5α, and positive clones were screened by colony PCR. The plasmid was extracted and sent to Sangon Biotech Co., Ltd. (Nanjing) for sequencing. The sequencing results showed that the open reading frame sequence of the obtained eggplant anti-functional gene was 1392 bp. Figure 1 The gene was named SmHDC-like, and its detailed sequence is shown in SEQ ID NO.1. The amino acid sequence of SmHDC-like was deduced from the CDS open reading frame sequence; it consists of 463 amino acid residues, has a molecular weight of 51979.91 Da, and a theoretical isoelectric point (pI) of 7.53. The detailed sequence is shown in SEQ ID NO.2.

[0061] SEQ ID NO.1

[0062]

[0063] SEQ ID NO.2

[0064] MGSLSLEMDFEPSAITPRSLARRSLFLNGDNKKQKLAPAGAGPRKNLQLEVMEPTLRNDGPSLDTILVNYLDTLSQRVNFHLGYPVNLCYDHYATLAPLLQFHLNNCGDPFLQNT VDFHSKDFEVAVLDWFAKLWEIEKEQYWGYVTNGGTEGNLHGILIGRELLPNGILYASKDSHYSVLKAARMYRIDSKTINTSTNGEMDYSDLRAKLLQNKGKPAIINVTIGTTFKG AIDDLDIILETLKDCGYSQDMFYIHCDAALCGLMVPFMNNMISFKKPIGSVTISGHKFLGCPMPCGVQITRKAYINNLSRNVEYIASVDATISGSRNGLTPIFLWYSLSAKGQIGL QKDVKKCLDNAKYLKDRLQQAGISVMLNELSIIVVLERPRDHEFVRRWQLSCVKDMAHVIVMPGITREMLDNFISELVQQRKLWYQDGKAKPPCVGDDIGAQNCACSYHKIDYVSP

[0065] Example 2

[0066] This invention, after aligning the CDS sequence of the SmHDC-like gene with NCBI BLAST, found that the SmHDC-like gene showed the highest homology (up to 91.71%) with the Euphrates HDC-like gene. Figure 2 Blastp alignment revealed that the amino acid sequence encoded by the SmHDC1 gene showed the highest homology with the European white-haired HDC-like sequence (92.87% similarity). Figure 3 This indicates that the cloned eggplant HDC-like gene is a homolog of HDC-like, therefore the gene was named SmHDC-like. Further sequence phylogenetic analysis confirmed that SmHDC-like has high sequence homology with HDC-like proteins from other species. Figure 4 ), clustered together with European and American HDC-like clusters ( Figure 5 Furthermore, SmHDC-like proteins contain an AAT_I superfamily domain, and their motif arrangement is consistent with that of HDC-like proteins from other species. Figure 6 ).

[0067] Example 3: Prediction of secondary and tertiary structures of eggplant SmHDC-like structures

[0068] The secondary structure of the novel eggplant SmHDC-like protein encoded by this invention is mainly composed of three structural elements: random coils, α-helices, and extended chains, with the proportions of these three structural elements being 44.71%, 41.47%, and 13.82%, respectively. Figure 7 Using the online analysis software SWISS-MODEL, the tertiary structure of SmHDC-like transcription factors was predicted using potato aromatic amino acid decarboxylase 2 (PDB: M1CHJ3.1.A) as a template. The results showed that SmHDC-like transcription factors are mainly composed of random coils, α-helices, and extended strands, with random coils and α-helices accounting for a large proportion. Figure 8 The predictions for the tertiary structure are consistent with those for the secondary structure.

[0069] Example 4: Analysis of the expression pattern of eggplant SmHDC-like gene in response to drought stress

[0070] 1. Treatment of plant materials

[0071] Using potted *Solanum lyratum* plants of uniform size and approximately 60 days of growth in a greenhouse (16 h light, 27℃ / 8 h darkness, 19℃), drought was simulated in a temperature-controlled plant light incubator (12 h light, 25℃ / 12 h darkness, 18℃) with 20% PEG-6000. The soil was irrigated with 20% PEG-6000 every other day. At 0 h, 3 h, 6 h, and 12 h, samples were randomly selected from 5-8 plants of different strains. The second and third true leaves from the top of the *Solanum lyratum* were collected, wrapped in aluminum foil, and immediately immersed in liquid nitrogen. These samples were then transferred to a -80℃ cryogenic freezer for storage until later use. Leaf tissue from plants without any stress treatment served as a control group. Each treatment was repeated three times.

[0072] 2. RNA extraction

[0073] RNA was extracted from treated *Solanum lyratum* leaves according to the instructions of the HiPure HP Plant RNA Kit. Integrity was assessed by conventional agarose gel electrophoresis (1.2% gel concentration; 1×TAE electrophoresis buffer; 150V, 15min). The maximum brightness of the rRNA band in the electrophoresis strip should be 1.5-2.0 times that of the second rRNA band; otherwise, it indicates rRNA sample degradation. For high-purity RNA, the A260 / A280 and A260 / A230 ratios are approximately 2.0. The OD value was measured using a spectrophotometer, and the RNA content was calculated.

[0074] 3. Obtaining cDNA

[0075] Using 1 μg of total RNA as a template, cDNA was obtained by reverse transcription using Takara Bio's PrimeScript™ RT reagent Kit with gDNA Eraser.

[0076] 4. Design specific primers for real-time quantitative PCR analysis of gene expression levels under different stresses. Based on the obtained eggplant SmHDC-like gene sequence, specific primers for real-time PCR quantification of SmHDC-like genes were designed using the primer design software Snapgene. Specifically:

[0077] SmHDC-like-qF: 5′-TGCATGCAGTCAGCAATTTGT-3′ (SEQ ID NO.5)

[0078] SmHDC-like-qR: 5′-TTCGCCGCGCTAAACTTCTA-3′ (SEQ ID NO.6)

[0079] The internal reference gene is APRT, and its primers are...

[0080] APRT rRNA-F: 5′-GAGATGCATGTAGGTGCTGTGCAA-3′ (SEQ ID NO.7)

[0081] APRT rRNA-R: 5′-GGCCCTTCAATTCTGGCAACTCAA-3′ (SEQ ID NO.8)

[0082] 5. Real-time quantitative analysis of the target gene in the sample. Using the first strand of the synthesized cDNA as a template, quantitative analysis was performed by amplification with specific primers for the target gene and the internal reference gene, respectively. The qRT-PCR experiment was conducted on a CFX96 real-time quantitative PCR instrument, with a reaction volume of 20 μL.

[0083] reagents Usage Forward primer 1.0 μL Reverse primer 1.0 μL MonAmp™ ChemoHS qPCR Mix (None Rox) 10.0 μL cDNA 1.0 μL <![CDATA[ddH2O]]> Up to 20 μL

[0084] The reaction was carried out in three steps: denaturation at 95°C for 10 min, followed by 40 cycles: 95°C for 10 s; 58°C for 10 s; 72°C for 45 s.

[0085] 6. Use 2 -ΔΔCt Relative quantitative analysis was performed. The results showed that the expression of SmHDC-like genes was induced by drought, and the overall trend was first increased and then decreased. Figure 9The relative expression level of the SmHDC-like gene was approximately 8.14 times that of the control at 0 h after drought treatment, reaching the most significant difference level. The relative expression levels at 6 h and 12 h were approximately 0.42 times that of the control at 0 h, but did not reach a significant difference compared to the control. This indicates that the SmHDC-like gene plays an important role in the drought stress response of eggplant.

[0086] Example 5: Verification of drought resistance function of eggplant SmHDC-like gene

[0087] 1. Construction of yeast expression vector for SmHDC-like gene

[0088] (1) In this experiment, TaKaRa conventional restriction enzyme (TaKaRa, Japan) was used to perform enzymatic digestion on the transformation vector pYES2-NTB (purchased from Nanjing Ruiyuan Biotechnology Co., Ltd., catalog number RY8013). The specific reaction system is as follows:

[0089] 10× QuickCut Buffer 5 μL pYES2-NTB 32 μL BamH Ⅰ 1 μL EcoR I 1 μL <![CDATA[ddH2O]]> 12 μL Total volume 50 μL

[0090] After mixing all solutions in the system, the mixture was briefly centrifuged and incubated in a 37°C water bath for 3 hours to stop the enzyme digestion reaction. The enzyme digestion bands were observed by agarose gel electrophoresis. The vector fragments were then gel-cleaved and recovered for subsequent vector ligation reactions.

[0091] (2) Refer to Trelief TM The SoSoo Cloning Kit Ver.2 (Qingke Biotechnology, China) instruction manual describes how to ligate the expression vector pYES2-NTB recovered after enzyme digestion with the SmHDC-like gene fragment, as follows:

[0092] Components Dosage SmHDC-like gene 1.5 μL pYES2-NTB (BamH I + EcoR I) 1.0 μL 2×SoSoo Mix Ver.2 5.0 μL <![CDATA[ddH2O]]> 2.5 μL Total volume 10.0 μL

[0093] The positive control system is as follows:

[0094] Components Dosage Control Vector (5 ng / μL) 2.0 μL Control Template (10 ng / μL) 3.0 μL 2×SoSoo Mix Ver.2 5.0 μL Total volume 10.0 μL

[0095] The solutions in the system were mixed in microtubes and reacted at 50°C for 15 min. The mixture was then transformed into E. coli DH5α. Positive clones were identified by PCR and sequencing, confirming the successful construction of the expression vector containing the SmHDC-like gene.

[0096] 2. Transformation of yeast (refer to the instructions for the Nanjing Ruiyuan Yeast Stress Screening Kit)

[0097] (1) Pick a single colony of BY4741 (from Nanjing Ruiyuan Yeast Stress Screening Kit, RY8013, which contains BY4741 colony and transformation vector (pYES2-NTB)) from a YPDA plate and inoculate it into 4 mL of YPDA liquid medium. Incubate at 30℃ and 225 rpm for 18-20 h (overnight) with shaking until OD. 600 >1.5.

[0098] (2) Transfer to YPDA liquid culture medium, with a culture volume of 50 mL, to allow the initial OD to rise. 600 =0.2, 30℃, 225 rpm, shake and incubate for 4-5 h until OD 600 =0.6. Centrifuge to collect bacteria at room temperature, 4000 rpm, for 5 min.

[0099] (3) Resuspend the bacterial cells in 20 mL of sterile water, mix well, centrifuge to collect the bacteria at room temperature, 4000 rpm for 5 min, and discard the supernatant.

[0100] (4) Resuspend the bacterial cells in 5 mL of 0.1 M LiAc, mix well, centrifuge to collect the bacteria at room temperature, 4000 rpm for 5 min, and discard the supernatant.

[0101] (5) Resuspend the bacterial cells in 500 μL of 0.1 M LiAc, mix well, and dispense into 1.5 mL centrifuge tubes, 50 μL each (for each transformation), for later use.

[0102] (6) Add the following reagents to each 1.5 mL centrifuge tube in sequence, mix by pipetting or vigorous shaking for about 1 min until completely mixed.

[0103] <![CDATA[50% PEG 4000 ]]> 1M LiAC salmon sperm DNA pYES2-NTB / pYES2-NTB-SmHDC-like 240 μL 36 μL 5 μL 5 μL

[0104] (7) Incubate in a water bath at 30℃ for 30 min.

[0105] (8) Heat shock in a 42℃ water bath for 25 min.

[0106] (9) 30℃ water bath for 30 min.

[0107] (10) Centrifuge to collect bacteria at room temperature, 4000 rpm for 5 min, and discard the supernatant.

[0108] (11) For each transformation, suspend the bacterial cells in 200 μL of sterile water, mix gently as much as possible, and spread on SD-Ura screening plates. Specific SD-Ura solid culture medium: 6.7 g YNB (amino acid-free), 20 g glucose, 100 mL 10× amino acid stock solution (-Ura), 20 g agar powder, add water to a final volume of 1 L, pH = 5.8, sterilize at 121℃ for 15 min, cool, and set aside.

[0109] (12) Incubate at 30℃ for 4 days.

[0110] 3. PCR verification of positive clones

[0111] Eight spots were randomly selected from the transformed plate, and PCR verification was performed using primer pairs SmHDC-like-NF / SmHDC-like-NR.

[0112] SmHDC-like-NF: CGGGATCCATGGGTAGCCTCTCACTTGAAATG (SEQ ID NO.9)

[0113] SmHDC-like-NR: GGAATTCCTAAGGACTAACGTAATCAATCTTATG (SEQ ID NO.10)

[0114] 4. Functional verification under drought stress

[0115] (1) Select clones of the above bacteria that have been verified by PCR and place them in SD-Ura liquid medium containing 2% glucose, and shake until OD. 600 Between 1.2 and 1.4.

[0116] (2) Centrifuge at 4000 rpm for 5 min to remove the supernatant.

[0117] (3) Wash the collected yeast cells with SD-Ura liquid culture medium without carbon source and shake culture for 3h.

[0118] (4) Centrifuge at 4000 rpm for 5 min to remove the supernatant.

[0119] (5) Continue to culture SG-Ura with 2% galactose for 8-12 h with shaking. After adjusting OD=1.0, spread the culture at ratios of 1:1, 1:10, 1:100, and 1:1000 on SG-Ura solid medium containing 0.1M mannitol. Figure 9 Drought stress (100 mM mannitol) Figure 10 The bacterial culture was diluted to four different concentrations (1:1, 1:10, 1:100, and 1:1000) and spotted onto SG-Ura medium supplemented with 100 mM mannitol. The results showed that all experimental groups grew normally. However, when the control group was diluted to 1:100 and 1:1000, only a few single yeast colonies were observed. These results indicate that the SmHDC-like gene possesses drought tolerance.

[0120] Example 6

[0121] The plant overexpression vector constructed in Example 1 was introduced into Agrobacterium LBA4404, and genetically transformed into leaf tissues of Nicotiana benthamiana via Agrobacterium-mediated transformation. After dedifferentiation and redifferentiation culture, kanamycin-resistant plants were obtained. Figure 11 DNA was extracted from the tobacco genome using the CTAB method, and transgenic plants (A1, A2) were obtained by PCR verification. Figure 12 ), then cultured to the T2 generation, using 20% ​​PEG. 6000 Transgenic tobacco lines and wild-type Nicotiana benthamiana (control) were irrigated for 0, 2, 4, and 6 days, respectively, for subsequent physiological index determination and phenotypic analysis.

[0122] 1. Determination of malondialdehyde (MDA) content

[0123] The thiobarbituric acid (TBA) reaction method was used. 0.5 g of freshly chopped tobacco leaves were weighed and ground in a mortar with a small amount of liquid nitrogen. A 10% TCA homogenate was added and the volume was brought to 5 mL. The mixture was centrifuged at 4000 rpm for 10 min. 2 mL of the supernatant was carefully collected (2 mL of distilled water was added to the control), and 3 mL of 0.5% TBA solution was added. After mixing, the mixture was reacted in a boiling water bath for 15 min, rapidly cooled, and then centrifuged at 4000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm, 600 nm, and 450 nm.

[0124] MDA concentration (μmol / L) = 6.45 × (OD) 532 -OD 600 -0.56×OD 450

[0125] Based on experimental data ( Figure 13a) In the early stages of drought stress (0-2 days), the malondialdehyde (MDA) content in the leaves of all tested lines showed an increasing trend. The MDA content in the wild-type control (CK) increased by 200.90%, significantly higher than that in the SmHDC-like transgenic lines A1 (134.45%) and A2 (96.57%), indicating that cell membrane lipid peroxidation was more severe in wild-type plants during the early stages of stress. At the initial time point (0 days), the difference in MDA content between CK and A1 / A2 was not statistically significant; by day 2 of stress, the MDA content between CK and the two transgenic lines showed a significant difference (P < 0.05). By day 4 of stress, with the response and activation of the plant's endogenous antioxidant system, the MDA content in all lines decreased, approaching the initial level. At this time, the difference between CK and A1 was not significant, but there were significant differences between A2 and CK, and between A2 and A1 (P < 0.05). Continued stress until day 6, the MDA content in the control group (CK) increased sharply again, by 175.55%, and at this point, the differences in MDA content between CK and the two transgenic lines (A1 and A2) reached a significant level (P < 0.05). This indicates that under prolonged drought stress, the SmHDC-like gene-transgenic plants can still effectively alleviate cell membrane lipid peroxidation and maintain a low level of MDA accumulation.

[0126] In conclusion, overexpression of the SmHDC-like gene can significantly alleviate drought stress-induced membrane lipid peroxidation damage and enhance cell membrane stability in tobacco plants during stress.

[0127] 2. Determination of relative conductivity

[0128] Cut the second and third fully expanded leaves of the treated seedlings, rinse them several times with tap water, then rinse them 2-3 times with deionized water, and let the surface moisture dry. Cut 0.3g of leaf mesophyll sample, place it in a test tube, add 25 mL of deionized water, shake thoroughly, and let it stand for 24 h. Then, measure its conductivity (E) using a DDS conductivity meter. l The conductivity (E0) of the deionized water used was measured, and then the water was boiled in a 100°C water bath for 30 minutes. After cooling to room temperature, its conductivity (E2) was measured. The above test was repeated 3 times.

[0129] EL(%) = (E1-E0) / (E2-E0) × 100%

[0130] Based on experimental data ( Figure 13(b) In the early stages of drought stress (0-2 days), the relative conductivity of wild-type plants (CK) increased significantly, reaching an increase of 102.40%, which was significantly higher than that of the SmHDC-like gene transgenic lines A1 (38.32%) and A2 (39.40%). This indicates that in the early stages of stress, the cell membrane system of wild-type plants was more severely damaged, and the increase in membrane permeability was more significant. At the onset of stress (0 days), the difference in conductivity between CK and transgenic lines A1 and A2 did not reach a statistically significant level; by day 2 of stress, the conductivity between CK and the two transgenic lines showed a significant difference (P < 0.05). On day 4 of stress, with the enhancement of the plant's own antioxidant enzyme activity, the adjustment of membrane lipid composition, and the accumulation of osmotic regulatory substances, cell membrane damage and electrolyte extravasation were alleviated, and the conductivity of all lines decreased to near the initial level. At this time, the conductivity of CK was still significantly higher than that of the two transgenic lines (P < 0.05). When the stress continued for 6 days, the electrical conductivity of each line increased sharply again. The increase of CK was 69.74%, which was still significantly higher than that of transgenic lines A1 (42.99%) and A2 (40.61%). Moreover, the differences between CK and the two transgenic lines were statistically significant (P < 0.05).

[0131] The above results indicate that, under drought stress, the SmHDC-like gene-transformed lines can more effectively maintain cell membrane integrity, reduce membrane permeability, and alleviate electrolyte leakage, suggesting that the expression of the SmHDC-like gene has a protective effect on the cell membrane and helps improve the drought resistance of plants.

[0132] 3. Enzyme activity assay

[0133] Preparation of crude enzyme solution: Take 0.2 g (adjust as needed) of sample (fresh leaves), wash it and place it in a pre-cooled mortar. Add 1.6 mL of 50 mmol / L pre-cooled phosphate buffer (pH 7.8) and grind it into a homogenate on an ice bath. Transfer it to a centrifuge tube and centrifuge at 4℃ and 12000 g for 20 min. The supernatant is the enzyme solution.

[0134] The activity of POD protective enzymes was determined by the guaiacol oxidation method.

[0135] (1) Sample determination: Take 3 mL of reaction solution and add 30 μL of enzyme solution. Use PBS as a control to zero the sample, and then measure the OD. 470 Value (measured in 3 min). (Add sample and measure simultaneously, wait 5 seconds before measurement, and act quickly. If slow, ensure that the time from sample addition to start timing is not significantly different for each sample.)

[0136] (2) Enzyme activity calculation: 0.01 OD value change (increase) per minute is defined as 1 enzyme activity unit (U).

[0137] POD=(ΔA 470 ×Vt) / (W×Vs×0.01×t) (U·g⁻¹·min⁻¹)

[0138] ΔA 470 : represents the change in absorbance during the reaction time; W is the fresh weight of the sample (g); t is the reaction time (min); Vt is the total volume of the extracted enzyme solution (mL, (1.6 mL)); Vs is the volume of enzyme solution taken during the determination (mL, 30 L).

[0139] Based on experimental data ( Figure 13 c) In the early stages of drought stress (0-2 days), the peroxidase (POD) activity of all tested lines increased rapidly to cope with the rapid accumulation of reactive oxygen species (ROS). Among them, the POD activity of the SmHDC-like gene-transformed lines (A1 and A2) increased significantly more than that of the wild-type control (CK), indicating that the expression of this gene may activate the expression of the POD-encoding gene in advance through epigenetic regulation, thereby increasing the enzyme activity level of POD in transgenic plants. At the onset of stress (0 days), the difference in POD activity between CK and A1 and A2 did not reach a statistically significant level; by the second day of stress, there were significant differences in POD activity between CK and the two transgenic lines, as well as between A1 and A2 (P < 0.05). During the mid-stress period (2-4 days), the CAT activity of transgenic lines increased by an average of 65.78% (average of A1 and A2), effectively maintaining intracellular ROS homeostasis and significantly reducing malondialdehyde (MDA) accumulation, thus exhibiting superior drought tolerance compared to wild-type plants. By day 4, the differences in POD activity between CK and transgenic lines A1 and A2 were statistically significant (P < 0.05). During the late-stress period (4-6 days), the POD activity of wild-type CK decreased by 54.16%, while transgenic lines maintained a high level of enzyme activity, further indicating that the SmHDC-like gene has the ability to continuously regulate POD activity, delaying the functional decline of the antioxidant system and enhancing the plant's adaptability under continuous drought stress. By day 6, the differences in POD activity between CK and A1 and A2, as well as between A1 and A2, were statistically significant (P < 0.05).

[0140] The results above indicate that the expression of SmHDC-like genes can significantly enhance the antioxidant defense capacity of plants under drought stress. The mechanism may be related to the regulation of the expression and activity of key antioxidant enzymes such as POD, thereby improving the overall drought resistance of plants.

[0141] CAT activity determination: Hydrogen peroxide method was used.

[0142] (1) Sample determination: Take 3 mL of reaction solution and add 0.1 mL (adjust as needed) of enzyme solution. Use PBS as a control to zero the sample and measure the OD. 240 (Ultraviolet) (Measured for 40 s).

[0143] (2) Enzyme activity calculation: One unit of enzyme activity (u) is defined as a decrease of 0.01 OD value per minute.

[0144] CAT=[ΔA 240 ×Vt ] / (W×Vs×0.01×t) (U·g⁻¹·min⁻¹)

[0145] ΔA 240 : represents the change in absorbance during the reaction time; W is the fresh weight of the sample (g); t is the reaction time (min); Vt is the total volume of the extracted enzyme solution (mL, 1.6 mL); Vs is the volume of enzyme solution taken during the determination (mL, 0.1 mL).

[0146] Based on experimental data ( Figure 13 Unlike the trends in malondialdehyde (MDA) content and relative conductivity, the catalase (CAT) activity of the SmHDC-like gene transgenic lines (A1 and A2) was generally higher than that of the wild-type control (CK) throughout the stress period. At the onset of stress (0 d), the CAT activity levels of each line were similar, and the difference between CK and A1 and A2 was not statistically significant. On day 2 of stress, the CAT activity of all lines increased, but the SmHDC-like gene transgenic lines responded more rapidly, with an activity increase of 5.36 times that of the control group CK. At this time, the difference in CAT activity between CK and the two transgenic lines was significant (P < 0.05). In the middle of the stress period (2-4 d), with the intensification of drought stress and the synergistic regulation of the endogenous antioxidant system, the CAT activity of transgenic lines A1 and A2 remained at a high level. By day 4, the activity difference between CK and A1 and A2 remained significant (P < 0.05). During the later stages of stress (4-6 days), the persistent drought caused the CAT activity of the wild-type CK to decrease from a peak of 397.72 U·g⁻¹·min⁻¹ FW to 249.53 U·g⁻¹·min⁻¹ FW, a decrease of 148.16 U·g⁻¹·min⁻¹ FW. Although the transgenic lines also showed a decrease, their activity was still 3.34 times that of the CK group, and the differences between CK and A1 and A2 were all significant (P < 0.05).

[0147] The above results indicate that heterologous expression of the SmHDC-like gene can significantly enhance catalase activity in plants under drought stress, thereby effectively alleviating membrane lipid peroxidation, maintaining cell membrane structural integrity, and thus improving the overall drought resistance of transgenic plants.

[0148] Example 7

[0149] The tobacco seeds (CK) and SmHDC-like gene-transgenic tobacco seeds (A1 and A2) harvested in Example 6 were first soaked in 75% alcohol for 90 seconds for preliminary surface disinfection, and rinsed 1-2 times with sterile water; then, they were surface disinfected with 20% sodium hypochlorite for 42 minutes and rinsed 5-7 times with sterile water. They were then inoculated into seed germination medium (MS + 3% sucrose, Ms + 3% sucrose + 5% PEG) using the flat-laying method. 6000 Place them under light conditions at 25℃ to cultivate sterile seedlings.

[0150] When sown on Ms+3% sucrose medium for 14 days, the transgenic tobacco (A1 and A2) showed significant differences compared to the CK group, and there was a significant difference between the A1 and A2 groups. Figure 14 a) and c); Sowing in Ms + 3% sucrose + 5% PEG 6000 At 14 days on culture medium, the transgenic genome and the control group also reached a significant difference, but there was no significant difference between groups A1 and A2. Figure 14 (b and d). The results showed that heterologous expression of the SmHDC-like gene significantly improved the drought resistance of tobacco by increasing root elongation, indicating that overexpression of the SmHDC-like gene can improve the drought resistance of eggplant.

[0151] The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A drought-resistant gene in eggplant SmHDC-like Its characteristics are, The DNA sequence of the gene is shown in SEQ ID NO.

1.

2. The eggplant drought-resistant functional gene as described in claim 1 SmHDC-like The encoded protein is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Contains the eggplant drought-resistant functional gene as described in claim 1 SmHDC-like Recombinant expression vectors.

4. The gene cloning method as described in claim 1, characterized in that, Includes the following steps: 1) Total RNA was extracted from the leaf tissue of *Solanum lyratum* and reverse transcribed into cDNA; 2) Using the reference genome sequence as a template, primers cP1F and cP1R were designed; 3) Using the cDNA reverse transcribed in step 1) as a template, and cloning with primers designed in step 2); 4) The PCR products obtained in step 3) are recovered, sequenced, and analyzed. The sequence of primer cP1F is shown in SEQ ID NO.3; the sequence of primer cP1R is shown in SEQ ID NO.

4.

5. The eggplant drought-resistant functional gene as described in claim 1 SmHDC-like Or the application of the protein described in claim 2 in regulating plant drought resistance.

6. The eggplant drought-resistant functional gene as described in claim 1 SmHDC-like Or the application of the protein described in claim 2 in enhancing plant drought resistance.

7. The application according to claim 6, characterized in that: The enhancement of plant drought resistance manifests as improving poor plant growth in drought conditions and promoting good plant growth in arid environments.

8. The eggplant drought-resistant functional gene as described in claim 1 SmHDC-like Or the protein of claim 2 may be used in improving drought-resistant plant varieties or in cultivating drought-resistant plant varieties.

9. The application according to any one of claims 5-8, characterized in that: The plant in question is eggplant, tobacco, or Arabidopsis thaliana.

10. A drought-resistant gene in eggplant SmHDC-like A method for analyzing the expression levels of eggplant in response to drought, characterized by: Includes the following steps: S1. Obtain leaf tissues from plants after different periods of drought stress treatment and extract total RNA. S2. Using the total RNA as a template, reverse transcription is performed to obtain cDNA; S3. Using the first strand of cDNA as a template, respectively... SmHDC-like Genes and internal reference genes APRT Specific primers were used for amplification, followed by fluorescence quantitative analysis to obtain the... SmHDC-like Gene expression levels in eggplant under drought stress; The amplification SmHDC-like The base sequences of the gene-specific primers are shown in SEQ ID NO.5 and SEQ ID NO.6; The amplified internal reference gene APRT The base sequences of the specific primers are shown in SEQ ID NO.7 and SEQ ID NO.8.

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