Drought-resistant gene acnfxl1 of actinidia chinensis and application thereof

Abstract

The present application relates to the technical field of plant molecular biology, and proposes a kiwi drought-resistant gene AcNFXL1 and application thereof.A kiwi drought-resistant gene AcNFXL1 The nucleotide sequence of the kiwi drought-resistant gene AcNFXL1 is shown as SEQ ID NO.1.Through the above technical solution, the problem of poor drought resistance of kiwi in the prior art is solved.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, specifically to a drought-resistant gene in kiwifruit. AcNFXL1 And its applications. Background Technology

[0002] Drought stress has a significant impact on kiwifruit production, serving as a crucial environmental factor limiting the industry's scale and stable output. In arid and semi-arid regions, as well as areas prone to seasonal droughts, water scarcity often leads to stunted kiwifruit plant growth and physiological metabolic disorders. Mild cases result in leaf wilting and stunted shoot growth, while severe cases cause the entire plant to wither or even die, impacting planting efficiency and industry enthusiasm. The mechanisms by which drought stress harms kiwifruit involve multiple levels, primarily including stomatal closure due to water imbalance, inhibition of photosynthesis, accumulation of reactive oxygen species, and damage to cell membrane structures. These stress responses systematically disrupt the growth and development process of kiwifruit, ultimately leading to a decline in yield and fruit quality.

[0003] Traditional breeding methods for selecting kiwifruit varieties, especially those focused on improving drought resistance, have progressed slowly, limiting their widespread cultivation in arid and semi-arid regions. With the development of molecular biology and genetic engineering, accurately identifying and utilizing drought-resistant genes, combined with genetic improvement techniques, to cultivate new drought-resistant varieties has become an important direction for expanding suitable planting areas for kiwifruit and enhancing the industry's resilience. In-depth research on the physiological regulation, gene function, and molecular mechanisms of kiwifruit's response to drought stress not only helps elucidate the biological basis of its drought adaptation but also provides crucial theoretical and technical support for identifying key drought-resistant genes, creating new drought-resistant germplasm, and developing water-saving cultivation strategies, ultimately achieving safe, high-quality, and high-yield kiwifruit production under drought adversity. Summary of the Invention

[0004] This invention proposes a drought-resistant gene for kiwifruit. AcNFXL1 Its application has solved the problem of poor drought resistance of kiwifruit in related technologies.

[0005] The technical solution of the present invention is as follows: This invention proposes a drought-resistant gene for kiwifruit. AcNFXL1 The drought-resistant gene in kiwifruit AcNFXL1 The nucleotide sequence is shown in SEQ ID NO.1.

[0006] As a further technical solution, the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0007] The present invention also proposes a recombinant vector containing the aforementioned kiwifruit drought-resistant gene. AcNFXL1 .

[0008] As a further technical solution, the recombinant vector is an overexpression vector.

[0009] This invention also proposes a recombinant strain.

[0010] As a further technical solution, the recombinant strain contains the recombinant vector.

[0011] This invention also proposes a drought-resistant gene for kiwifruit. AcNFXL1 Or a drought-resistant gene in kiwifruit AcNFXL1 The application of the encoded protein, a recombinant plasmid, or a recombinant bacterial strain in any of S1 to S2; S1, regulates plant drought resistance; S2. Cultivate transgenic plants with altered drought resistance.

[0012] As a further technical solution, the regulation of plant drought resistance is a positive regulation of plant drought resistance.

[0013] As a further technical solution, the cultivation of transgenic plants with altered drought resistance is to cultivate transgenic plants with enhanced drought resistance.

[0014] As a further technical solution, the plant includes plants of the genus Actinidis.

[0015] As a further technical solution, the plant is kiwifruit.

[0016] This invention also proposes a method for cultivating transgenic kiwifruit, comprising overexpressing the aforementioned kiwifruit drought-resistant gene in kiwifruit. AcNFXL1 .

[0017] The working principle and beneficial effects of this invention are as follows: In this invention, for the first time, a discovery was made. AcNFXL1 It is a positive regulator of drought resistance in kiwifruit, and its activity can be transiently silenced in kiwifruit using VIGS technology. AcNFXL1 The gene significantly reduced the plant's tolerance to PEG-simulated drought stress. Phenotypic observation revealed that after drought stress treatment, the gene was silenced. AcNFXL1 Kiwifruit plants exhibited earlier and more severe symptoms of leaf wilting and dehydration compared to the control group. Physiological data analysis showed that under stress, silenced plants accumulated more malondialdehyde (MDA) in their leaves, while osmotic regulators (such as proline) accumulated less. Furthermore, transient overexpression technology was used to overexpress [the substance] in kiwifruit leaf discs. AcNFXL1 The gene significantly enhances the plant's tolerance to PEG-simulated drought stress; under the same drought stress conditions, AcNFXL1 The kiwifruit leaf discs with transient overexpression showed significantly slower leaf wilting and water loss rates compared to the wild-type control group, and maintained relatively good growth even in the later stages of stress. In summary, this invention is the first to demonstrate...AcNFXL1 Genes play a key positive regulatory role in the response of kiwifruit to drought stress, providing valuable genetic resources and theoretical basis for the targeted improvement of drought resistance in kiwifruit varieties through molecular breeding methods (such as marker-assisted selection or genetic engineering). Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 In Embodiment 1 of the present invention AcNFXL1 Agarose gel electrophoresis results of gene PCR amplification products; Figure 2 For the present invention AcNFXL1 Protein domains and three-dimensional conformation; Figure 3 This invention relates to the PEG-simulated drought stress treatment of Hongyang kiwifruit and Longcheng No. 2 kiwifruit. AcNFXL1 Gene expression levels; Figure 4 Kiwifruit of the present invention AcNFXL1 Silent strains and wild types AcNFXL1 The relative expression level of genes; Figure 5 Kiwifruit of the present invention AcNFXL1 Growth status of silent lines and wild types before and after PEG-simulated drought stress treatment; Figure 6 Kiwifruit of the present invention AcNFXL1 MDA measurement results of silent lines and wild type before and after PEG-simulated drought stress treatment; Figure 7 Kiwifruit of the present invention AcNFXL1 Proline determination results of silent lines and wild types before and after PEG-simulated drought stress treatment; Figure 8 For the present invention AcNFXL1 Transient overexpression in kiwifruit leaf discs and wild-type (WT) leaf discs AcNFXL1 The relative expression level of genes; Figure 9 For the present invention AcNFXL1 Transient overexpression of kiwifruit leaf discs and wild-type (WT) leaf discs before and after air-drying experiments. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] In the following examples, all experiments were repeated three times, and the average value of the results was taken.

[0022] TRV silencing vectors are described in the following literature: Choi S, Jayaraman J, Segonzac C, Park HJ, Park H, Han SW, Sohn KH. Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or SuppressInnate Immune Responses in Nicotiana benthamiana. Front Plant Sci. 2017, 20;8:2157; The method for verifying and evaluating the drought resistance of transgenic kiwifruit is described in the following literature: Parra S, Núñez-LilloG, Tapia-Reyes P, Carrasco-Lozano EC, Porcile V, Gonzalez-Calquin C, Amaza L, Quiroz LF, Meneses C, Handford M, Norambuena L, Martínez JP, Stange C. Decoding the Hayward kiwi (Actinidia deliciosa var Hayward) genome: transcriptomic responses to drought and salinity and AdhSAP4's role insalinity stress responses. Front Plant Sci. 2025, 16:1637092; The method for determining malondialdehyde (MDA) content is described in the following literature: Zou Q. Experimental Manual on Plant Physiology. Chinese Agriculture Press; Beijing, China: 2000. pp. 127-130; The method for determining proline content is described in the following literature: Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water stress studies. Plant and Soil, 1973, 39: 205-208.

[0023] Example 1 AcNFXL1 Gene cloning: 1. Total RNA was extracted from the leaf tissue of *Actinidia chinensis* using a plant RNA extraction kit (Universal Plant RNA Extraction Kit, Beijing Huayueyang Biotechnology Co., Ltd.), and ds-cDNA of *Actinidia chinensis* was obtained using a reverse transcription kit (HiScript III 1st StrandcDNA Synthesis Kit, Vazyme). 2. Primers were designed using the obtained ds-cDNA of Hongyang kiwifruit as a template, as shown in Table 1 below; Table 1 Primer Sequences

[0024] PCR amplification was performed using kiwifruit ds-cDNA as a template to obtain AcNFXL1 The PCR amplification products of the gene were detected by electrophoresis on a 1% agarose gel. The results are shown in the figure. Figure 1 Sequencing yielded AcNFXL1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; and the protein domains and three-dimensional conformation are shown in... Figure 2 As shown.

[0025] Example 2 AcNFXL1 Analysis of gene expression patterns under drought stress: 1. Tissue culture seedlings of Hongyang kiwifruit and Longcheng No. 2 kiwifruit were cultured under the conditions of 16 h light / 8 h dark and 25 ℃; 2. Seedlings grown to 3 months of age were subjected to drought stress treatment with 10% PEG6000. Seedling samples were collected after 0, 1, 3, 5 and 7 days of treatment. Three biological replicates were set up at each time point. The samples were flash-frozen in liquid nitrogen immediately after collection and stored in an ultra-low temperature freezer at -80 ℃ for subsequent RNA extraction. 3. Total RNA was extracted using TRIzol reagent, and RNA purity and quantification were determined using a NanoDrop 2000 spectrophotometer (ThermoScientific, USA). RNA was then analyzed at different stages of drought stress treatment. AcNFXL1 Gene expression levels, and thus determine their expression patterns; The results are as follows Figure 3 As shown, after PEG6000 simulation of drought stress, AcNFXL1 The expression level of the gene showed an increasing trend in both varieties, indicating that the gene is involved in the kiwifruit's response to drought stress.

[0026] Example 3 kiwi AcNFXL1 Creation of silent strains and identification of drought resistance: 1. Recombinant plasmid pTRV2- AcNFXL1 Construction Design using SGN VIGS (https: / / vigs.solgenomics.net / ) online software. AcNFXL1 VIGS fragment, i.e. AcNFXL1 Specific interference fragments, AcNFXL1 The specific interference fragment is positions 1006-1305 shown in SEQ ID NO.1. Primers were designed based on the fragment, as shown in Table 2 below. Table 2 Primer Sequences

[0027] PCR amplification was performed using the primers in Table 2 with cDNA as a template. AcNFXL1 For some fragments, after amplification, the target fragment was purified and its concentration was measured. The amplification system is shown in Table 3 below. Table 3 Amplification System

[0028] Meanwhile, after activating pTRV2 empty vector E. coli, plasmid was extracted by shaking culture, and the concentration was detected. Double enzyme digestion was performed on the plasmid. The double enzyme digestion system is shown in Table 4 below. Table 4. Double enzyme digestion system

[0029] Enzyme digestion was performed in a water bath at 37 ℃ for 1 h. After the digestion reaction was completed, the reaction tube containing the digestion system was placed in an environment of 85 ℃ for 10 min to inactivate the enzyme. The target fragment was purified and recovered by 1.0% agarose gel electrophoresis for detection. The mixture was placed in a water bath at 37 °C for 30 min for ligation. The ligation system is shown in Table 5 below. It was then transformed into competent Escherichia coli DH5α cells (DH5α Escherichia coli, purchased from Sangon Biotech (Shanghai) Co., Ltd.), and recombinant screening and sequencing verification were performed to obtain plasmids that were correctly sequenced. Table 5 Connection System

[0030] Note: Exnase 5×CE II Buffer was purchased from Nanjing Novizan Biotechnology Co., Ltd. Take the recombinant plasmid (pTRV2-) that was correctly sequenced and verified. AcNFXL1 0.5 μL, inoculated into a solution containing kanamycin (Kan) + The plasmids were cultured overnight at 37 °C and 200 rpm in 5 mL of LB broth with shaking. Plasmids were extracted from *E. coli* using a plasmid recovery kit (plasmid mini-extraction kit, purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.). The obtained TRV2-... AcNFXL1 The recombinant plasmid was sequenced, and the sequencing results showed that the desired recombinant plasmid was obtained by inserting the DNA sequence at positions 1006-1305 of SEQ ID NO.1 into the multiple cloning site of the vector.

[0031] 2. Obtaining recombinant Agrobacterium Using thermal shock conversion method to convert TRV1, TRV2 and TRV2- AcNFXL1 Plasmids were transformed into Agrobacterium GV3101 to obtain TRV1 transgenic Agrobacterium, TRV2 transgenic Agrobacterium, and TRV2- AcNFXL1 Genetically modified Agrobacterium.

[0032] 3. AcNFXL1 The acquisition of Silent Red Sun Kiwifruit Hongyang kiwifruit tissue culture seedlings were cultured at 25 ℃ under 16 h light / 8 h dark conditions for 3 months. When the seedlings grew to 5-8 true leaves, the caps of the tissue culture bottles were opened and placed in an environment of 25 ℃ under 16 h light / 8 h dark conditions for 3 days. Using vacuum permeation staining, TRV1, TRV2, and TRV2- were picked separately. AcNFXL1Single clones of transgenic Agrobacterium GV3101 were cultured in liquid LB medium containing kanamycin at 28 °C with shaking for 24 h, centrifuged at 4000 r / min for 20 min, and the bacterial cells were collected. The cells were resuspended twice in MES buffer supplemented with acetylsyleugenol, and the OD of TRV1 was adjusted. 600 The value is 0.4, TRV2- AcNFXL1 TRV2's OD 600 The value is 0.5, so TRV2- AcNFXL1 TRV2 and TRV1 were mixed in equal amounts and divided into experimental groups (TRV1 + TRV2 - ...). AcNFXL1 The experimental group and the control group were inoculated with the same infection solution (TRV1+TRV2) and placed in the dark at 28 ℃ for 3.5 h. The entire tissue culture seedlings of Hongyang kiwifruit were immersed in the infection solution and the whole plant was infected by vacuum permeation method. Then, the experimental group and the control group were taken out and cultured at 25 ℃ for 10 days under the conditions of 16 h light / 8 h dark.

[0033] 4. Real-time quantitative detection AcNFXL1 Relative expression level in its silent strains Through the AcNFXL1 Quantitative gene analysis further identified the red-yellow kiwifruit. AcNFXL1 To determine whether the silent strain was successfully created, RNA was first extracted from the leaves of the experimental and control groups, and cDNA was obtained through reverse transcription. Total RNA extraction and cDNA first-strand synthesis were performed as described in Example 1. Quantitative real-time PCR analysis was conducted using a premixed quantitative real-time PCR kit from Novizan, and the results were performed using a LightCycler® 96 quantitative PCR instrument. AcNFXL1 The gene-specific upstream primer was 5′-AGGAAAAGAGAGATGCGGTGAG-3′ (SEQ ID NO.7), and the downstream primer was 5′-ACCACAACGAAACGCTTTGG-3′ (SEQ ID NO.8). Results are shown in [link to results]. Figure 4 , Figure 4 The results showed that, compared with the control group, the experimental group strains... AcNFXL1 The gene expression level was significantly reduced, indicating that the Hongyang kiwifruit was successfully obtained. AcNFXL1 Silent strain.

[0034] 5. Hongyang kiwifruit AcNFXL1 Identification of drought-sensitive phenotypes in silent strains The tested Hongyang kiwifruit was TRV1+TRV2- AcNFXL1 For both the experimental group lines and the control group lines transformed with TRV1+TRV2, all experiments were performed with at least 3 biological replicates and 3 technical replicates. Methods and results of phenotypic experiments 1) Take the above-mentioned Hongyang kiwifruit AcNFXL1 The experimental group of kiwifruit seedlings was treated with a 7.5% (w / w) PEG6000 aqueous solution in hydroponics. Two days after treatment, the growth status of the tissue-cultured kiwifruit seedlings was observed and photographed. The results are shown below. Figure 5 ; 2) Take a 7.5% (w / w) PEG6000 aqueous solution to simulate drought stress in the leaves of *Actinidia chinensis*, and measure the malondialdehyde (MDA) and proline content in the leaves. The results are shown in [see attached table]. Figures 6 - 7 and Table 6 below; Table 6. Results of malondialdehyde and proline content determination

[0035] according to Figures 6 - 7 As shown in Table 6, after drought stress treatment, AcNFXL1 The growth status of the experimental group was significantly weaker than that of the control group, and the symptoms such as leaf wilting and water loss were more severe. At the same time, the malondialdehyde content was significantly higher than that of the control group, while the proline content was significantly lower than that of the control group.

[0036] The above results indicate that silence AcNFXL1 This leads to a decrease in the drought resistance of kiwifruit, specifically manifested in inhibited growth, increased malondialdehyde accumulation, and decreased proline accumulation. These results further illustrate that... AcNFXL1 The expression of this is an important mechanism by which kiwifruit responds to drought stress.

[0037] Example 4 Creation of overexpression in kiwifruit and identification of its drought resistance: 1. Construction of overexpression vectors Select pCAMBIA1302 suitable for connection AcNFXL1 Design of restriction enzyme sites (NcoI and SpeI) AcNFXL1 The primers are shown in Table 7 below: Table 7 Overexpression AcNFXL1 Primer sequence

[0038] Using the primers in Table 7, with cDNA as a template, AcNFXL1 PCR amplification was performed on a fragment without a stop codon. After amplification, the target fragment was purified and ligated into the 18-T vector (Takara). After successful transformation into *E. coli* competent cells DH5α, sequencing was performed. Clones with correct sequencing were selected, plasmids were extracted, and the amplification system is shown in Table 8 below. Table 8 Amplification System

[0039] Using NcoI and SpeI endonucleases to respectively AcNFXL1 -T was double-digested with pCAMBIA1302 (37 ℃, digestion for 0.5 h), and the digestion was then used to... AcNFXL1 The fragments were recovered via gel extraction. The NcoI and SpeI restriction enzymes in the linearized pCAMBIA1302 system were heat-inactivated (treated at 80 °C for 20 min). The double digestion procedure is shown in Table 9 below. Table 9. Double enzyme digestion system

[0040] The products after double enzyme digestion were mixed according to the ligation system in Table 10. The mixture was placed in a water bath at 37°C for 30 min for ligation, and then transformed into E. coli competent cells DH5α to obtain pCAMBIA1302- AcNFXL1 The recombinant vectors and their connection systems are shown in Table 10 below: Table 10 Connection System

[0041] 2. Obtaining recombinant Agrobacterium Using thermal shock conversion method to convert pCAMBIA1302- AcNFXL1 The plasmid was transformed into Agrobacterium GV3101 to obtain pCAMBIA1302- AcNFXL1 Genetically modified Agrobacterium.

[0042] 3. AcNFXL1 Acquisition of the leaf disc of Hongyang kiwifruit through instantaneous overexpression Will be converted to pCAMBIA1302- AcNFXL1 Agrobacterium was cultured at 28 °C with shaking until OD 600 =0.5~0.8, centrifuge to obtain resuspended bacterial cells, resuspend the bacterial cells in Agrobacterium infection medium, and adjust to OD. 600 =0.5~0.8. Mature leaves were taken and disinfected with 0.6% NaClO solution for 20 min (care should be taken to avoid damaging the leaf surface), rinsed 3 times with sterile water, and excess water was absorbed with filter paper. Leaf discs were prepared using a sterile perforator (diameter = 8 mm). The leaf discs were placed in Agrobacterium infection solution (with sterile water as a control), vacuum infiltrated for 30 min, rinsed 3 times with sterile water after vacuum infiltration, and then the water was absorbed with sterile filter paper. They were placed on 0.8% water agar plates and incubated in an artificial climate chamber at 24 ℃ for 24 h to obtain leaf discs of the Agrobacterium infection solution experimental group ( AcNFXL1 Transient overexpression leaf discs and sterile water control group leaf discs (wild-type leaf discs).

[0043] 4. Real-time quantitative detection AcNFXL1Relative expression level in transiently overexpressed kiwifruit leaf disc Extraction and preparation AcNFXL1 RNA from leaf discs and wild-type leaf discs was transiently overexpressed, and cDNA of the strain was obtained by reverse transcription. Total RNA extraction and first-strand cDNA synthesis were performed as described in Example 1. Quantitative real-time PCR analysis was performed using a premixed kit (Novizan) and detected using a LightCycler® 96 PCR instrument. AcNFXL1 The gene-specific upstream primer sequence is 5′-AGGAAAAGAGAGATGCGGTGAG-3′ (SEQ ID NO.11), and the downstream primer sequence is 5′-ACCACAACGAAACGCTTTGG-3′ (SEQ ID NO.12). Figure 8 This indicates that, using the wild type as a reference, transient overexpression of kiwifruit leaf disc... AcNFXL1 The gene expression level was significantly increased, indicating that the desired result was successfully obtained. AcNFXL1 Instantaneous overexpression of kiwi leaf disc.

[0044] 5. AcNFXL1 Identification of drought resistance phenotypes in kiwifruit leaf discs by transient overexpression Air drying experimental method: The above-prepared AcNFXL1 Transient overexpression of kiwifruit leaf discs and wild-type leaf discs was performed. The wrinkling was observed in a 30 ℃ artificial climate chamber with 40% humidity. Growth was observed and photographed at 0 h, 4 h, and 8 h. All experiments were performed with at least three biological replicates and three technical replicates. Results are shown below. Figure 9 ; Figure 9 The results showed that after in vitro treatment, AcNFXL1 Transient overexpression resulted in significantly better leaf disc condition in kiwifruit compared to wild-type leaf discs, with wild-type leaf discs showing less wilting and dehydration. AcNFXL1 Transient overexpression is more severe in kiwifruit leaf discs.

[0045] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A drought-resistant gene in kiwifruit AcNFXL1 Its characteristics are, The drought-resistant gene in kiwifruit AcNFXL1 The nucleotide sequence is shown in SEQ ID NO.

1.

2. A drought-resistant gene for kiwifruit according to claim 1. AcNFXL1 The encoded protein is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector contains the kiwifruit drought-resistant gene as described in claim 1. AcNFXL1 .

4. A recombinant vector according to claim 3, characterized in that, The recombinant vector is an overexpression vector.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the recombinant vector as described in claim 3.

6. A drought-resistant gene for kiwifruit according to claim 1 AcNFXL1 Or a drought-resistant gene for kiwifruit as described in claim 2 AcNFXL1 The application of the encoded protein or the recombinant vector of claim 4 or the recombinant strain of claim 5 in any one of S1 to S2: S1, regulates plant drought resistance; S2. Cultivate transgenic plants with altered drought resistance.

7. The application according to claim 6, characterized in that, The regulation of plant drought resistance is a positive regulation of plant drought resistance.

8. The application according to claim 6, characterized in that, The term "cultivating transgenic plants with altered drought resistance" refers to cultivating transgenic plants with enhanced drought resistance.

9. The application according to claim 6, characterized in that, The plants mentioned include those of the genus Actinidis.

10. A method for cultivating transgenic kiwifruit, characterized in that, include: Overexpression of the drought-resistant kiwifruit gene of claim 1 in kiwifruit AcNFXL1 .

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