Application of FYVE4 gene in improving nitrogen deficiency tolerance of plants

Abstract

The application discloses application of FYVE4 gene in improving nitrogen deficiency stress tolerance of plants, and belongs to the technical field of plant genetic engineering. The nucleotide and corresponding amino acid sequence and specific application of the FYVE4 gene are provided. The application proves for the first time that the Arabidopsis gene AtFYVE4 regulates the function of plant nitrogen deficiency stress response through an autophagy pathway. It is found that the Arabidopsis nitrogen deficiency stress regulation related gene regulates the autophagy activity of Arabidopsis by interacting with the autophagy protein AtATG1C, and then regulates the tolerance of the plant to nitrogen deficiency stress. The application of the AtFYVE4 gene in the application can provide support for drought resistance research of corn, rice, wheat and other food crops and other crops.

Description

TECHNICAL FIELD

[0001] The application relates to the field of plant genetic engineering technology, and more particularly to application of FYVE4 gene in improving nitrogen deficiency stress tolerance of plants. BACKGROUND

[0002] Plants degrade damaged organelles, protein aggregates and other components through the autophagy pathway, release nitrogen sources such as amino acids (e.g., arginine, glutamine), and supply them to key physiological processes to maintain short-term survival and metabolic balance. By recycling carbon skeletons and energy, autophagy helps plants adjust metabolic flow to preferentially support root growth, nitrogen absorption-related gene expression and stress response signaling pathways. Autophagy-deficient plants (e.g., atg mutants) exhibit developmental retardation, accelerated leaf senescence and death under nitrogen deficiency, indicating that autophagy is crucial for plant stress adaptability.

[0003] As a core regulatory link of plant nitrogen deficiency response, autophagy significantly enhances the nitrogen stress tolerance and survival adaptability of plants by recycling nutrients, maintaining cell homeostasis and integrating environmental signals. Research on the mining and cloning of autophagy pathway regulatory genes helps to clarify the genetic basis of autophagy regulation, and provides guidance for the breeding and improvement of nitrogen deficiency-tolerant varieties.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide application of FYVE4 gene in improving nitrogen deficiency stress tolerance of plants. SUMMARY

[0005] Therefore, the application of FYVE4 gene in improving nitrogen deficiency stress tolerance of plants provided by the application has important value for breeding of excellent nitrogen-deficiency-resistant crop varieties.

[0006] To achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0007] The application of FYVE4 gene in improving nitrogen deficiency stress tolerance of plants, the nucleotide sequence of the FYVE4 gene is shown as SEQ ID NO. 1.

[0008] Preferably, the amino acid sequence translated and expressed by the FYVE4 gene is shown as SEQ ID NO. 2.

[0009] Preferably, the application uses an expression cassette containing the FYVE4 gene or a recombinant vector or a recombinant bacterium or a transgenic plant.

[0010] Preferably, the recombinant expression vector is a FYVE4 gene overexpression recombinant plasmid.

[0011] Preferably, the plant is Arabidopsis thaliana.

[0012] The application discloses a gene FYVE4, which is a positive regulation factor involved in regulating autophagy process and helping plants to resist nitrogen deficiency stress.

[0013] Compared with the prior art, the application discloses the application of the FYVE4 gene in improving the nitrogen deficiency stress tolerance of plants, and the following technical effects are achieved.

[0014] The application proves for the first time that the Arabidopsis gene FYVE4 has the function of regulating the response of plants to nitrogen deficiency stress through the autophagy pathway. AtFYVE4 The cloning and biological function verification of the gene have important reference significance for analyzing the genetic network of the Arabidopsis autophagy pathway regulating nitrogen stress, can provide theoretical support for cultivating new germplasm of plants with high nitrogen utilization efficiency, and have important significance for formulating scientific and effective fertilization measures and ensuring stable and high yield of crops.

[0015] The application finds that the Arabidopsis nitrogen deficiency stress regulation related gene regulates the autophagy activity of Arabidopsis by interacting with the autophagy protein AtATG1C, and then regulates the tolerance of the plant to nitrogen deficiency stress; the application is beneficial to in-depth research on the molecular mechanism of the Arabidopsis tolerance to nitrogen deficiency stress, and provides important theoretical basis and rich gene resources for cultivating new plant varieties with low nitrogen tolerance.

[0016] The application of the gene in the application can provide support for drought resistance research of grain crops such as corn, rice and wheat and other crops. AtFYVE4 The application of the gene in the application can provide support for drought resistance research of grain crops such as corn, rice and wheat and other crops. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0018] Figure 1 The drawing is a linear processing pCAMBIA1300-FYVE4-GFP vector map provided by the application.

[0019] Figure 2 The drawing is a figure showing that the fyve4-1 mutant is sensitive to nitrogen deficiency stress, wherein A is phenotype analysis of different background plants grown in a nitrogen deficiency medium for 6 days, and an autophagy sensitive mutant atg5 is a positive control; B is determination of chlorophyll content in the leaves in the A figure; the analysis data are obtained from three independent repeated experiments, and letters 'a' to 'c' represent significant difference (P<0.05) of a single factor variance analysis Tukey multiple comparison test.

[0020] Figure 3The figure is the FYVE4 and ATG1C interaction diagram provided by the application, wherein A: yeast two-hybrid experiment verifies the interaction of FYVE4 and ATG1C; B: Co-IP (immunoprecipitation) experiment verifies the interaction of FYVE4 and ATG1C; C: luciferase complementation (LUC) experiment, BIK1 and XLG2 are positive controls.

[0021] Figure 4 The figure is a Western blot detection result diagram provided by the application, 0 indicates no nitrogen deficiency treatment, 12, 24, 36 represent the treatment time, and the unit is hour (h). The numerical value under the protein band represents the gray value ratio of GFP and GFP-ATG8d proteins.

[0022] Figure 5 The figure is an autophagosome observation and analysis diagram provided by the application, wherein A: confocal microscope observes the number of autophagosomes of GFP-ATG8d / WT and GFP-ATG8d / fyve4-1 plants under +N and -N treatment conditions, the scale is 20 µm; B: statistical analysis of the number of autophagosomes in the roots of the plants in A; the data are from 5 pictures taken from different plants, the numerical value represents mean ± se, *, p<0.05, t-test. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0024] The embodiments of the application disclose the application of FYVE4 gene in improving the nitrogen deficiency stress tolerance of plants.

[0025] The methods used in the embodiments are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products, for example: fyve4-1, atg5-1 mutant and Col-0 are ordered from The Arabidopsis Information Resource (ABRC). The fyve4-1 is numbered as SALK_133410c, and the atg5-1 is numbered as SAIL_129_B07, and the mutant is constructed as T-DNA insertion.

[0026] Embodiment 1

[0027] Construction of recombinant plasmid for overexpression of Arabidopsis thaliana AtFYVE4 gene

[0028] The AtFYVE4 overexpression transformation vector recombinant plasmid pCAMBIA1300-FYVE4-GFP (linearized pCAMBIA1300-FYVE4-GFP vector map is shown in FIG. 1) was constructed using the pCAMBIA1300-GFP starting vector (vector, see: Kumar S, Vishwakarma H, Ghosh G, Singh J, Padaria JC. In planta transformation in wheat: an improved protocol to develop wheat transformants. Mol Biol Rep. 2024; 51(1): 407). Figure 1

[0029] Col-0 Arabidopsis thaliana planted in Crop Institute of Guangdong Academy of Agricultural Sciences was used to extract total mRNA using a plant RNA extraction kit (Magen Company). Then, 1 μg of mRNA with high quality (OD 260 / 280 : 1.8-2.0; OD 260 / 230 ≈2.0) was used for reverse transcription to obtain cDNA using a reverse transcription kit (Novagen Company). The cDNA was used as a template for PCR amplification of the target gene using 2x SuperNova Max (GenStar Company).

[0030] Upstream primer AtFYVE4 F: 5'-GGG CCCGGG ATGTTGGAGAAGATCGGATTAC-3', as shown in SEQ ID NO. 3 (GGG is a protection base, and the underlined part is a Smal enzyme cutting site)

[0031] Downstream primer AtFYVE4 R: 5'-GGG ACTAGT ACTTGAAGCAAGAGCGTTTAGC-3', as shown in SEQ ID NO. 4 (the primer removes the stop codon, GGG is a protection base, and the underlined part is a Spel enzyme cutting site)

[0032] (1) Reaction system: 2x PCR buffer 15 μL, F primer 0.5 μL, R primer 0.5 μL, cDNA template 0.5 μL, and ddH2O was added to 30 μL. The reaction conditions were as follows: 98°C for 5 min; 98°C for 30 sec, 60°C for 30 sec, 68°C for 30 sec, 35 cycles; 72°C for 5 min. After the reaction, the PCR product was collected.

[0033] (2) Enzyme cutting reaction: the pCAMBIA1300-GFP vector was linearized by using restriction endonuclease Smal and Spel (NEB), and the enzyme cutting system was as follows: 2 μg of vector, 1 μL of Smal endonuclease, 1 μL of Spel endonuclease, 3 μL of 10x NEB Buffer, and ddH2O was added to 30 μL. The enzyme cutting conditions were as follows: 37°C for 3 hours.

[0034] (3) Homologous recombination: after the vector enzyme cutting product and the PCR product were purified and recovered, recombination was performed using a recombination kit (Novagen Company), and the recombination system was as follows: 1.5 μL of target fragment, 4.5 μL of vector fragment, 2 μL of 5x buffer, 1 μL of Exnase II, and ddH2O was added to 10 μL. The recombination conditions were as follows: 37°C for 30 min.

[0035] (4) Transformation: Take all the ligation products, add to 50 μL E. coli DH5α competent cells, transform the product coated on LB solid medium (containing kanamycin resistance, the concentration of kanamycin is 50 mg / L). Incubate at 37°C overnight, pick 4 single colonies for colony PCR identification using 2x Taq PCR StarMix (GenStar Company), then select one positive clone for sequencing, obtain a positive clone containing the AtFYVE4 gene sequence, and finally obtain the overexpression vector pCAMBIA1300-FYVE4-GFP containing the AtFYVE4 target gene.

[0036] Colony PCR identification:

[0037] PCR forward primer F: 5'-ATGTTGGAGAAGATCGGA-3'

[0038] PCR reverse primer R: 5'-TCAACTTGAAGCAAGAGC-3'

[0039] The PCR system is: 2x PCR StarMix 5 μL, F primer 0.5 μL, R primer 0.5 μL, add ddH2O to 10 μL. The reaction conditions are: 95°C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, 25 cycles; 72°C for 5 min.

[0040] Example 2

[0041] Obtaining of AtFYVE4 back complementation transgenic seedlings and analysis of nitrogen stress phenotype of AtFYVE4 back complementation and knockout plants

[0042] Agrobacterium-mediated transformation of Arabidopsis and screening of back complementation homozygous plants

[0043] The overexpression vector pCAMBIA1300-FYVE4-GFP constructed in Example 1 was transformed into the Arabidopsis fyve4-1 mutant by Agrobacterium GV3101-mediated genetic transformation method. The mature transgenic seeds were collected to obtain T0 generation transgenic seedlings. The T0 generation seeds were sown on hygromycin-resistant medium, and the resistant seedlings were screened and cultured to obtain T1 generation transgenic seeds. The T1 generation positive seeds were sown on hygromycin-resistant medium, and the resistant: sensitive ratio of the seedlings was screened to be close to 3: 1, and the resistant seedlings were cultured to obtain T2 generation positive seeds. The harvested T2 generation seeds were screened again to verify the resistance. If the resistance does not separate (100% green seedlings), it is proved to be a stable hereditary homozygous transgenic strain FYVE4-GFP / fyve4-1. The experimental method is described in: Harrison, Samuel J et al. "A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation. Plant methods. 2006; 2: 19

[0044] Verification of the response of Arabidopsis plants to nitrogen deficiency stress

[0045] 50 seeds of each material of Arabidopsis were sterilized and then placed in a 4°C refrigerator for cold treatment for 48 hours. Then the seeds were placed in a 6-well cell culture plate with 2 mL of MS liquid medium, and placed on a horizontal shaker (100 rpm) under continuous light for 5 days. Then the MS-N liquid medium (Coolaber company) was replaced, and the plants were grown under the same light intensity for 4-5 days. The degree of leaf yellowing was observed, photographed and the chlorophyll content was measured. The more obvious the yellowing of the plant leaves and the lower the chlorophyll content, the more sensitive the plant is to nitrogen deficiency stress.

[0046] The homozygous knockout plants and the complemented homozygous plants obtained above were sterilized and sown on MS+N medium and grown under long-day conditions for 7 days, then transferred to MS-N medium and grown under full-day conditions. The results are shown in Figure 2 fyve4- 1 The mutant is hypersensitive to nitrogen deficiency stress and its growth condition is significantly worse than that of the wild type, and the phenotype is similar to autophagy atg5-1 The mutant ， The chlorophyll content is low, and the FYVE4 complemented transgenic plants can completely restore their sensitive phenotype (Figure 2). It is preliminarily indicated that FYVE4 plays an important function in the response of plants to nitrogen deficiency stress.

[0047] ​Example 3

[0048] Analysis of the interaction between Arabidopsis thaliana FYVE4 gene and Arabidopsis thaliana ATG1C

[0049] Yeast two-hybrid experiment: Cloning of FYVE4 and the interaction protein coding gene ATG1C (NCBI accession number: XP_002512593.1) into pGADT7 and pGBKT7 vectors, transforming into AH109 yeast bacteria and culturing at 30°C, then picking single colonies and spotting them on different nutrient-deficient media (SD-Trp-Leu, SD-Ade-Trp-Leu-His) for growth, and analyzing and determining protein interaction. The specific experimental method is referred to the Roche yeast two-hybrid experiment manual. At2g37840 Co-immunoprecipitation experiment: FYVE4 and the interaction protein coding gene ATG1C were routinely constructed into vectors with GFP and mCherry tags, respectively transformed into Agrobacterium GV3101 strain, and GFP and mCherry-FYVE4; GFP-ATG1C and mCherry-FYVE4 were combined and transiently transformed into tobacco leaves, and after dark culture for 36-48 hours, the proteins were extracted and the protein complex was enriched with GFP-trap immunomagnetic beads for Western blotting detection.

[0050] Luciferase complementation experiment: FYVE4 and ATG1C genes were respectively constructed into vectors with nLUC and cLUC tags, and the constructed plasmids were transformed into Agrobacterium GV3101 strain, and then injected into 5-week-old tobacco leaves, and after dark culture for 36-48 hours, a chemiluminescence imaging system was used for shooting.

[0051] Experimental results:

[0052] Autophagy is a material turnover pathway that wraps functional abnormal proteins or damaged organelles in the cell into double-membrane autophagosomes and transports them to lysosomes for degradation. Damage to the autophagy pathway seriously affects intracellular metabolic pathways, and loss-of-function mutants exhibit a phenotype sensitive to nitrogen deficiency stress. In order to verify whether FYVE4 affects the response of plants to nitrogen deficiency stress through the regulation of the autophagy pathway, the example identified that FYVE4 and the autophagy protein ATG1C kinase have an interaction

[0053] Figure 3 ​In the A-type medium, when the two fusion proteins BD-FYVE4+AD-ATG1C are co-expressed in yeast cells, they can promote the growth of yeast cells on SD-LWHA-deficient medium. However, when the BD-FYVE4 fusion protein and AD are co-expressed (BD-FYVE4+AD), yeast cells cannot grow on SD-LWHA-deficient medium, indicating that FYVE4 and ATG1C interact. SD-LW refers to SD-Trp-Leu medium; SD-LWHA refers to SD-Trp-Leu-His-Ade medium.

[0054] The interaction between FYVE4 and ATG1C in plants was further confirmed by Co-IP (co-immunoprecipitation) and LUC (luciferase complementation assay). Figure 3 (B and C). This suggests that FYVE4 may regulate autophagy through its interaction with ATG1C, thereby helping plants resist nitrogen deficiency stress.

[0055] Example 4

[0056] Observation of autophagosomes

[0057] Grown on MS medium for 5 days GFP-ATG8d / WT and GFP-ATG8d / fyve4-1 The materials were placed in nitrogen-deficient culture medium and treated for different durations under continuous light intensity. The GFP-ATG8d-labeled autophagosomes in the root elongation region of Arabidopsis thaliana were observed using a Zeiss LSM 800 confocal microscope. GFP was excited with 488nm excitation light.

[0058] To verify whether FYVE4 affects the autophagy pathway, GFP-ATG8d transgenic plants (existing plants, see: Zhou J, Ma J, Yang C, et al. A non-canonical role of ATG8 in Golgi recovery from heat stress in plants. Nat Plants. 2023;9(5):749-765.) were used to investigate whether the loss of FYVE4 function affected the intensity of autophagic flux. GFP-ATG8d was expressed through hybridization to... fyve4-1 The mutant, grown in MS+N medium for 6 days GFP-ATG8d / WT and GFP-ATG8d / fyve4-1 Transgenic plants were transferred to MS-N medium and treated for different durations. Western blot results showed that... fyve4-1 The autophagic flux intensity of the mutant was weaker than that of the wild type (Figure 4), indicating that the absence of FYVE4 led to the obstruction of autophagic flux.

[0059] because fyve4-1Further confirmed that autophagic flux was blocked in the mutant fyve4-1 Whether the number of autophagosomes changed in vivo. The number of GFP-ATG8d labeled autophagosomes in the root elongation zone of seedlings was observed using confocal fluorescence microscopy. fyve4-1 GFP-ATG8d labeled autophagosomes in the root elongation zone of seedlings.

[0060] The results show that under nitrogen deficiency stress conditions, compared with the wild type, fyve4-1 The number of autophagosomes in the mutant was significantly increased (A and B in Figure 5). It is speculated that the loss of FYVE4 function may affect the closure of autophagosomes, thereby affecting the degradation of autophagosomes to vacuoles, resulting in accumulation of autophagosomes, and ultimately leading to reduced autophagic flux activity.

[0061] The above experimental results preliminarily prove that FYVE4 participates in the regulation of autophagy as a positive regulator, helping plants to resist nitrogen deficiency stress.

[0062] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0063] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Accordingly, the present application is not to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of overexpression of the FYVE4 gene in improving the tolerance of Arabidopsis thaliana to nitrogen deficiency stress, characterized in that, The nucleotide sequence of the FYVE4 gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The amino acid sequence of the FYVE4 gene translation is shown in SEQ ID NO.

2.

3. The application as described in claim 2, characterized in that, The application uses an expression cassette containing the FYVE4 gene, a recombinant vector, or a recombinant bacterium.

4. The application as described in claim 3, characterized in that, The recombinant expression vector is an FYVE4 gene overexpression recombinant plasmid.

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