MdNAC29 protein and application of a coding gene in regulating flowering time of a plant

Abstract

The application discloses MdNAC29 protein and an application of a coding gene in regulating flowering time of plants and belongs to the technical field of biotechnology. MdNAC29 The application finds a new gene for regulating flowering time of plants, i.e. MdNAC29 By overexpressing the gene in a plant body, the gene can efficiently regulate expression of a flowering-related pathway gene of the plant, break physiological retardation of conversion of vegetative growth of the plant to reproductive growth, greatly shorten a flowering induction time length, effectively promote the plant to complete a whole process of flower bud differentiation and flower organ formation, realize early flowering of the plant and greatly shorten an overall growth cycle.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the MdNAC29 protein and its encoding gene in regulating plant flowering time. Background Technology

[0002] Apple is an important perennial woody fruit tree, and its flowering time and flowering capacity directly affect the length of the tree's juvenile period, flower bud formation, fruit set rate, alternate bearing, and the breeding cycle. Compared with annual model plants, apples have a long juvenile period, a complex flower bud differentiation cycle, and are significantly affected by fruit load and environmental conditions. Therefore, elucidating the regulatory mechanisms of genes related to apple flowering time is of great significance for shortening the breeding cycle, regulating flower bud formation, and improving orchard yield stability.

[0003] In model plants, FLOWERING LOCUS T (FT) SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) LEAFY (LFY) APETALA1 (AP1) TERMINAL FLOWER1 Genes such as (TFL1) are key factors in the flowering time regulation network. Several flowering regulatory genes homologous to or functionally related to the above-mentioned genes have also been identified in apples, for example... MdFT1 , MdFT2 , MdSOC1 , MdTFL1-1 , MdTFL1-2 , AFL1 , AFL2 , MdAP1 / MdMADS5 , MdMADS2 , MdCOL1 , MdCOL2 wait.

[0004] While existing technologies have revealed some genes regulating apple flowering time and their mechanisms of action, the apple flowering time trait is controlled by multiple genes, pathways, and environmental factors, and a large number of potential regulatory genes remain undiscovered or unverified. Therefore, there is an urgent need to further explore more regulatory genes related to apple flowering time, clarify their expression characteristics and biological functions, and provide new genetic resources and technical means for regulating apple flowering time, early flowering breeding, shortening the breeding cycle, and improving orchard production stability. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of MdNAC29 protein and its encoding gene in regulating plant flowering time.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of the MdNAC29 protein in regulating the flowering time of plants; the amino acid sequence of the MdNAC29 protein is shown in SEQ ID NO.1.

[0007] In the above applications, overexpression of the MdNAC29 protein was used to promote earlier flowering in plants.

[0008] Preferably, the plant is apple or Arabidopsis thaliana.

[0009] In a second aspect, the invention provides the application of the gene encoding the MdNAC29 protein in regulating the flowering time of plants; The gene encoding the MdNAC29 protein is MdNAC29 A gene is a nucleic acid molecule as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1.

[0010] In the above applications, overexpression in plants MdNAC29 Genes can be used to promote earlier flowering in plants.

[0011] In some preferred embodiments of the present invention, overexpression in plants MdNAC29 Genes are generated using any of the following substances: C1) contains MdNAC29 Gene expression cassettes; C2) contains MdNAC29 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains MdNAC29 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains MdNAC29 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains MdNAC29 Transgenic plant tissue containing genes, or transgenic plant tissue containing the expression cassette described in C1).

[0012] In this invention, conventional plant expression vectors in the art can be used to construct expression vectors containing... MdNAC29 Gene recombinant vectors. The plant expression vectors are not particularly limited, as long as they can achieve [the desired result]. MdNAC29 The transcription and / or expression of genes in plant cells or plant tissues are acceptable, including but not limited to binary Agrobacterium expression vectors, plasmid vectors suitable for plant genetic transformation, and expression vectors that can be used for plant microbombardment transformation.

[0013] Furthermore, the recombinant vector may include [missing information - likely related to...]. MdNAC29 The gene includes an operable promoter, terminator, and / or selectable marker gene. The promoter may be a constitutive promoter, tissue-specific promoter, inducible promoter, or developmental stage-specific promoter, as long as it can drive... MdNAC29 Gene expression in plants is acceptable. The promoters include, but are not limited to, the CaMV 35S promoter, ubiquitin promoters, floral organ or flower-related tissue-specific promoters, heat-inducible promoters, and chemically induced promoters. The terminators include, but are not limited to, the NOS terminator, the OCS terminator, the 35S terminator, or other transcription termination sequences commonly used in plant expression vectors.

[0014] Preferably, a plant binary expression vector is used to construct a structure containing... MdNAC29 Gene recombination vectors.

[0015] In some preferred embodiments of the present invention, the plant binary expression vector is the pPZP211 plasmid.

[0016] Preferably, the plant is apple or Arabidopsis thaliana.

[0017] The beneficial effects of this invention are: This invention has discovered a new gene that regulates the flowering time of plants. MdNAC29 Genes, through overexpression within the plant MdNAC29 Genes can efficiently regulate the expression of genes in flowering-related pathways in plants, break the physiological blockage in the transition from vegetative growth to reproductive growth, significantly shorten the flowering induction time, effectively promote the early completion of the entire process of flower bud differentiation and flower organ formation, achieve early flowering, and significantly shorten the overall growth cycle. Attached Figure Description

[0018] Figure 1 Analysis of the time course of relative expression levels of flowering genes in apple shoot tip tissue after girdling treatment; in the figure, A represents... MdAFL1 The relative expression level, B is MdAFL2 The relative expression level, C is MdFT1 The relative expression level, D is MdAFUL The relative expression level, E is MdSOC1 The relative expression level, F is MdTFL1 The relative expression level.

[0019] Figure 2 : Apple stem tip tissue after girdling MdNAC29 Time-series analysis of relative expression levels.

[0020] Figure 3 : MdNAC29Pearson correlation analysis between expression levels and relative expression levels of flowering genes; in the figure, A represents... MdAFL1 Relative expression level and MdNAC29 Pearson correlation analysis results for relative expression levels, B is... MdAFL2 Relative expression level and MdNAC29 Pearson correlation analysis results for relative expression levels, C is MdFT1 Relative expression level and MdNAC29 Pearson correlation analysis results of relative expression levels, D is MdAFUL Relative expression level and MdNAC29 Pearson correlation analysis results for relative expression levels, E is MdSOC1 Relative expression level and MdNAC29 Pearson correlation analysis results for relative expression levels, F = MdTFL1 Relative expression level and MdNAC29 Pearson correlation analysis results of relative expression levels; R is the correlation coefficient, showing a highly significant statistical correlation between the two; P < 0.05 is considered statistically significant; data are mean ± standard deviation (n = 3).

[0021] Figure 4 In transgenic Arabidopsis thaliana MdNAC29 The relative expression level.

[0022] Figure 5 In transgenic Arabidopsis thaliana MdNAC29 PCR electrophoresis verification; in the figure, lane 1 is transgenic Arabidopsis N1, lane 2 is transgenic Arabidopsis N2, lane 3 is transgenic Arabidopsis N3, and lane 4 is wild-type Arabidopsis; M is the molecular weight standard (Marker).

[0023] Figure 6 Flowering performance of transgenic Arabidopsis thaliana (OE-MdNAC29) and wild-type Arabidopsis thaliana (WT) at bolting stage.

[0024] Figure 7 Flowering phenotype statistics of transgenic Arabidopsis thaliana (MdNAC29) and wild-type Arabidopsis thaliana (WT); in the figure, A is the statistical result of early flowering rate; B is the statistical result of flowering time. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] As mentioned earlier, the flowering time trait in apples is controlled by multiple genes, pathways, and environmental factors, and a large number of potential regulatory genes have yet to be discovered or validated. Therefore, it is urgent to further explore more regulatory genes related to apple flowering time.

[0027] In view of this, the present invention performs transcriptome sequencing on the red-fleshed apple tissue after girdling and screens out a gene encoding an NAC transcription factor whose expression pattern is highly positively correlated with the key stage of flowering, and names it as MdNAC29 The gene encodes the MdNAC29 protein.

[0028] The amino acid sequence of the MdNAC29 protein is shown in SEQ ID NO.1; specifically as follows: MGNISSSTATSTTGQSVPDMVLAIRFHPTEEEMADFLWKKMKGHNSQACHSLPVIDVCKFEPWDLPERMFPDTRYQAKAWYSFSQPDYKYINSPRCNRITEKGFWKITGKPRDISSTHVTCKKRTLTFYKGRVSKSERTGWVMHEYYFTKPK QGSNTSQGRDPLRDFVLYRLKKKPANSMSDNKNLQDTLIGDDLTDPGNGSCIASSSGNDQAATAAALNHDMTPAAEELLAYQELEDDLFDSGNHDRGEPGGGISSDSTDQVLRDMIQQWKSCAPVGEYLNSLLTLPESTSATSATSAWKCS.

[0029] MdNAC29 The nucleotide sequence of the gene is shown in SEQ ID NO.2; specifically as follows: ATGGGAAACATCAGCAGCAGCACCGCCACATCAACAACCGGACAATCGGTGCCGGACATGGTGCTTGCGATCAGATTCCATCCCACTGAAGAGGAAATGGCAGACTTCTTGTGGAAGAAAATGAAAGGTCACAACTCCCAAGCCTGCCATTCCCTCCCTGTCATCGATGTCTGCAAGTTCGAGCCTTGGGATTTGCCTGAGCGCATGTTCCCTGACACTCGATATCAAGCTAAGGCATGGTACTCATTCAGCCAGCCTGATTACAAATACATCAATAGTCCTCGCTGCAACAGGATCACTGAGAAAGGCTTCTGGAAAATCACAGGCAAGCCACGAGATATCAGCTCCACACATGTCACTTGCAAAAAGAGAACCTTGACCTTCTACAAAGGTCGTGTGTCTAAATCCGAGAGGACTGGCTGGGTGATGCACGAGTACTATTTCACTAAACCTAAACAGGGTTCTAATACCAGTCAGGGGAGGGATCCGCTGAGGGACTTTGTTCTTTACCGCTTAAAGAAGAAGCCAGCTAATTCTATGTCTGATAACAAGAATCTTCAGGATACTTTGATCGGTGATGATTTAACTGATCCTGGTAATGGTAGTTGCATTGCATCTAGTTCCGGAAATGATCAAGCAGCTACTGCTGCTGCATTGAATCATGATATGACTCCAGCTGCAGAAGAACTTCTTGCATACCAAGAGCTAGAAGATGATCTGTTTGACAGTGGAAATCATGATCGTGGCGAACCTGGTGGCGGCATCTCATCTGATTCTACTGATCAAGTTCTACGTGACATGATTCAACAGTGGAAGTCGTGTGCTCCGGTAGGTGAATATCTGAATTCACTCTTGACTCTTCCTGAGTCCACCTCAGCCACCTCAGCCACCTCAGCTTGGAAATGCTCCTGA。

[0030] To systematically reveal MdNAC29The biological function of the gene in apple flowering was investigated through heterologous genetic transformation in the model plant Arabidopsis thaliana, revealing that overexpression of the gene in Arabidopsis thaliana was crucial. MdNAC29 Genes can significantly promote earlier flowering.

[0031] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0032] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: The plant binary expression vector (pPZP211-3Flag) is described in the journal article (Arabidopsis FHY3 and FAR1 Regulate Light-Induced myo-Inositol Biosynthesis and Oxidative Stress Responses by Transcriptional Activation of MIPS1. Molecular Plant, 2016, Vol. 9, No. 4, 541–557); it is available to the public from the applicant for use in replicating this experiment.

[0033] Example 1: Gene screening related to plant flowering time 1. Test method: The experiment was conducted in 2022 in Wenyang County, Tai'an City, Shandong Province, and the State Key Laboratory of Shandong Agricultural University. The tested apple variety was Manhong, with a row spacing of 3.0m × 1.5m. The experimental sites were all sandy soil, flat in terrain, and under consistent management conditions. Apple trees with similar trunk thickness, tree height, and crown width were selected as test materials.

[0034] The experiment included three treatments: A. Severe girdling (GHB); B. Mild girdling (HB); C. Control (CK).

[0035] The experimental specimens underwent girdling treatment on June 5, 2022. Girdling was performed on main trunks with similar growth patterns, with each trunk representing one replicate, for a total of three replicates. Girdling was performed at a point 15-20 cm above the ground. Light girdling (HB) had a width of approximately 0.5 cm, while heavy girdling (GHB) had a width of approximately 1 cm, with the cut reaching the xylem. The control (CK) specimens did not undergo girdling treatment.

[0036] A total of 7 samples were taken from the fruit tree tissue. The first sample group was taken before the girdling treatment, and then the stem samples were taken every 15 days or so. The sampling times were June, July, August and September 2022.

[0037] Transcriptome sequencing was performed on red-fleshed apple tissue after girdling; key flowering genes in shoot tip tissue were analyzed at different time points after treatment. MdAFL1 (MD06G1129500, AB056158) MdAFL2 (MD14G1146700) MdFT1 (MD12G1262000) MdFUL (MD14G1215700) MdSOC1 (MD02G1197400) MdTFL1 (MD12G1023900) and MdNAC29 Gene expression dynamics; and on MdNAC29 Pearson correlation analysis was performed on gene expression levels and the relative expression levels of flowering genes.

[0038] 2. Test Results: Transcriptome sequencing was performed on the red-fleshed apple tissue after girdling, and a gene encoding an NAC transcription factor whose expression pattern was highly positively correlated with the key stage of flowering was identified. This gene was named... MdNAC29 The gene, whose nucleotide sequence is shown in SEQ ID NO.2, encodes the MdNAC29 protein, whose amino acid sequence is shown in SEQ ID NO.1.

[0039] Expression of key flowering genes in shoot tip tissue Figure 1 As shown, the results indicate that girdling treatment can systematically activate multiple flowering pathway genes. (Flowering integrators) MdAFL2 The expression level of flowering integrator began to increase significantly on day 16 after severe girdling treatment and peaked on day 29. MdFUL The expression levels of these genes began to increase significantly on day 16 after heavy girdling treatment and peaked on day 44. In contrast, the expression of these genes did not change significantly throughout the observation period in the control group without girdling treatment.

[0040] MdNAC29 Its own expression responded rapidly to severe girdling treatment, with its transcript levels significantly increasing after treatment, peaking on day 16, preceding [previous treatment]. MdAFL2 and MdFUL peak of expression ( Figure 2 ).

[0041] The results of Pearson correlation analysis are as follows: Figure 3 As shown, the results indicate that MdNAC29 The expression levels are respectively with MdAFL2 (R = 0.4592, p < 0.0001) and MdFUL The expression of (R = 0.317, p < 0.0001) showed a highly significant positive correlation. This strong correlation, combined with the chronological order of their expression, suggests... MdNAC29 Most likely located in MdAFL2 and MdFUL Upstream of the girdling-induced flowering signaling pathway, it plays a key early regulatory role.

[0042] Example 2: MdNAC29 Functional verification of genes 1. Overexpression MdNAC29 Construction of transgenic Arabidopsis thaliana: Using homologous recombination linking method MdNAC29 The gene (SEQ ID NO.2) was cloned downstream of the CaMV35S promoter in the plant binary expression vector (pPZP211-3Flag) to construct the recombinant vector (35S:MdNAC29). The recombinant vector was introduced into wild-type Arabidopsis using the Agrobacterium-mediated flower-dipping method to obtain transgenic plants.

[0043] In transgenic plants MdNAC29 The relative expression levels of genes were measured, and the results were as follows: Figure 4-5 As shown, three overexpression strains were successfully constructed. MdNAC29 Transgenic Arabidopsis thaliana (N1, N2, and N3).

[0044] 2. Overexpression MdNAC29 Flowering phenotype investigation of transgenic Arabidopsis thaliana overexpression MdNAC29 Transgenic Arabidopsis thaliana (N1, N2, and N3) and wild-type Arabidopsis thaliana seeds were planted under the same conditions, and the flowering time of Arabidopsis thaliana was recorded; the early flowering rate of Arabidopsis thaliana was also statistically analyzed.

[0045] Flowering time refers to the number of days from sowing to the appearance of the first flower bud (bolting and flowering) at the top of the main stem.

[0046] "Early flowering rate" refers to the percentage of plants exhibiting the early flowering phenotype in transgenic lines or wild-type populations out of the total number of sown plants.

[0047] The criteria for judging the early flowering phenotype are: when the bolting height is 1 cm, the number of rosette leaves is ≤11; and the flowering time is less than 25 days.

[0048] overexpression MdNAC29 Flowering behavior of transgenic Arabidopsis and wild-type Arabidopsis at bolting stage is as follows: Figure 6 As shown; the statistical results of flowering time and early flowering rate of Arabidopsis thaliana are as follows. Figure 7 As shown; overexpression MdNAC29 A total of 47 independent lines of transgenic Arabidopsis thaliana were sown, of which 46 showed early flowering, with an early flowering rate of 98%; a total of 47 independent lines of wild-type were sown, of which 3 showed early flowering, with an early flowering rate of 6%.

[0049] The results showed that overexpression MdNAC29 Genes enable Arabidopsis thaliana to flower much earlier than the wild type.

[0050] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of overexpression of MdNAC29 protein in promoting early flowering in plants, characterized by, The amino acid sequence of the MdNAC29 protein is shown in SEQ ID NO.1; The plant in question is either apple or Arabidopsis thaliana.

2. The application of the gene encoding the MdNAC29 protein overexpression in promoting early flowering in plants, characterized by: The gene encoding the MdNAC29 protein is MdNAC29 A gene is a nucleic acid molecule as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1; The plant in question is either apple or Arabidopsis thaliana.

3. The application according to claim 2, characterized in that, Overexpression in plants MdNAC29 Genes are generated using any of the following substances: C1) contains MdNAC29 Gene expression cassettes; C2) contains MdNAC29 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains MdNAC29 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains MdNAC29 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains MdNAC29 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1).

4. The application according to claim 3, characterized in that, Constructing a plant binary expression vector containing MdNAC29 Gene recombination vectors.

5. The application according to claim 4, characterized in that, The plant binary expression vector is the pPZP211 plasmid.

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