Poplar ptonac43 transcription factor gene and application thereof

Abstract

The application belongs to the technical fields of plant molecular biology and genetic engineering, and relates to a poplar PtoNAC43 transcription factor gene and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO:1, and the gene encodes a transcription factor protein with a NAC domain. A transgenic plant is obtained by constructing an overexpression vector and introducing the vector into a poplar. The results show that overexpression of the gene can reduce plant height, biomass and growth potential, and affect photosynthesis parameters, endogenous hormone levels and tissue structure. The application can be used for plant growth regulation and dwarf plant cultivation.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and genetic engineering technology, specifically relating to a poplar PtoNAC43 transcription factor gene and its application, particularly the application of this gene in regulating plant growth. Background Technology

[0002] In the fields of plant molecular biology and genetic breeding, transcription factors, as important regulatory elements for gene expression, play a crucial role in plant growth, development, and environmental responses. Among them, the NAC transcription factor family (NAM, ATAF1 / 2, and CUC2) is one of the largest transcription factor families in plants, widely distributed across various terrestrial plants. With the completion of genome sequencing of multiple plants, the NAC gene family has been identified in various species, including Arabidopsis thaliana, wheat, and woody plants. These transcription factors typically possess a highly conserved NAC domain at the N-terminus of their proteins, used for DNA binding and protein-protein interactions, while the C-terminus is a structurally variable transcriptional regulatory region, thus endowing them with diverse biological functions.

[0003] Existing research indicates that NAC transcription factors are widely involved in the regulation of plant growth and development, including the development of vegetative and reproductive organs, secondary cell wall and xylem formation, root growth, and leaf senescence. For example, in Arabidopsis thaliana, some NAC transcription factors can regulate the expression of downstream genes, thereby affecting leaf development and reproductive organ formation; in woody plants, related NAC genes have been shown to participate in secondary cell wall formation and xylem development; in addition, studies have shown that some NAC transcription factors are closely related to plant stress resistance and yield traits.

[0004] However, due to the large number of members in the NAC transcription factor family, and the significant differences in expression patterns and functions among different species and tissues, their specific functions exhibit considerable diversity and complexity. This is especially true in perennial woody plants, where the growth and development regulation mechanisms are more complex than those of model plants like Arabidopsis thaliana or crops. Current techniques cannot directly infer the specific functions of particular NAC genes in woody plants based on existing research results. Therefore, for NAC transcription factors in specific plant species, functional identification through systematic experiments is still necessary.

[0005] Poplar, as an important fast-growing timber species and model woody plant, has wide application value in landscaping and forestry production. Improving its growth regulation capabilities, optimizing its plant structure, and achieving controlled growth while maintaining its superior traits are important research directions in the field of molecular breeding of forest trees. However, the resources of key functional genes regulating poplar growth traits in current technologies are still relatively limited, especially the transcriptional regulatory factors that can significantly regulate plant vigor and height remain unclear.

[0006] In summary, while existing technologies have investigated the functions of some members of the NAC transcription factor family, systematic research on the specific roles of NAC genes in poplar growth regulation is still lacking, and key genes capable of effectively inhibiting or regulating poplar growth have not yet been identified. Therefore, it is necessary to discover and identify new poplar NAC transcription factor genes and clarify their functions in plant growth regulation, providing new gene resources and technical means for the genetic improvement of poplar and other plants. Furthermore, there are currently no reports on the role of the poplar PtoNAC43 transcription factor in inhibiting plant growth. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a poplar PtoNAC43 transcription factor gene and its application, so as to solve the problem of lack of key functional genes for poplar growth regulation in the prior art, which makes it difficult to effectively regulate the growth traits of the plant.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In one embodiment of the present invention, an isolated nucleic acid molecule is provided, the nucleic acid molecule encoding poplar PtoNAC43 transcription factor protein, and the nucleic acid molecule is selected from any of the following: a nucleotide sequence as shown in SEQ ID NO:1; a nucleotide sequence having at least 80% homology with SEQ ID NO:1 and encoding a protein that has the function of inhibiting plant growth; or a nucleotide sequence complementary to the above nucleotide sequence.

[0009] Furthermore, the nucleic acid molecule has at least 85% or at least 90% homology with SEQ ID NO:1.

[0010] Furthermore, the protein encoded by the nucleic acid molecule can reduce one or more of the following in plants: plant height, internode spacing, biomass, or growth potential.

[0011] In one embodiment of the present invention, a recombinant expression vector is provided, the vector comprising a functional promoter and the nucleic acid molecule operatively linked to the promoter.

[0012] Preferably, the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0013] In one embodiment of the present invention, a recombinant cell is provided, the recombinant cell containing the nucleic acid molecule or the recombinant expression vector.

[0014] Optionally, the recombinant cells are Agrobacterium cells or plant cells.

[0015] In one embodiment of the present invention, a transgenic plant is provided in which the nucleic acid molecule is introduced and overexpressed.

[0016] Preferably, the plant is a woody plant.

[0017] Furthermore, the plant is preferably a poplar.

[0018] In one embodiment of the present invention, a method for inhibiting plant growth is provided, comprising overexpressing the nucleic acid molecule in the plant, thereby reducing the plant height, biomass, or growth potential.

[0019] In one embodiment of the present invention, a poplar PtoNAC43 transcription factor protein is provided, which contains the amino acid sequence shown in SEQ ID NO:2, or a protein that has at least 80% homology with it and has the function of inhibiting plant growth.

[0020] Based on the above technical solution, the poplar PtoNAC43 transcription factor gene and its application of the present invention are obtained by isolating a specific nucleic acid molecule encoding the PtoNAC43 transcription factor and constructing a recombinant expression vector containing the nucleic acid molecule, so that it can be stably expressed or overexpressed in plants; further, the nucleic acid molecule is introduced into plant cells through genetic transformation technology to obtain transgenic plants, thereby regulating the plant growth and development process at the molecular level.

[0021] This invention addresses the problems in existing technologies regarding plant growth regulation, such as reliance on complex regulatory networks, limited key functional gene resources, and difficulty in achieving precise regulation of growth traits in woody plants, especially poplar. It provides a novel transcription factor gene resource and its application pathway. Experimental studies have shown that overexpression of the PtoNAC43 transcription factor in plants significantly reduces plant height, internode spacing, biomass, and overall growth potential, and regulates photosynthetic parameters, endogenous hormone levels, and tissue structure, thereby effectively inhibiting plant growth.

[0022] Compared with existing technologies, the technical solution of this invention has at least the following beneficial effects: First, this invention is the first to identify and clone the poplar PtoNAC43 transcription factor gene and clarify its function in plant growth regulation, providing new experimental evidence for the functional study of the NAC transcription factor family; second, this invention found that overexpression of this gene has a significant growth-inhibiting effect, and this regulatory effect has clear phenotypic characteristics and stability, and can serve as an important molecular tool for regulating plant architecture; third, the nucleic acid molecules, recombinant vectors, and transgenic systems provided by this invention can be widely applied to the genetic improvement of woody plants and even other plants, providing new technical means for cultivating dwarf plants, improving population structure, and increasing cultivation management efficiency; in addition, through systematic analysis of changes in plant photosynthesis, hormone levels, and tissue structure, this invention further reveals the mechanism of action of PtoNAC43 in plant growth regulation, providing an important basis for theoretical research in related fields.

[0023] In summary, this invention not only provides a new key gene for negative regulation of plant growth, but also constructs a complete application system, which has important application prospects and promotional value in the fields of plant molecular breeding and trait improvement. Attached Figure Description

[0024] Figure 1 for PtoNAC43 The gene cloning results are shown in the figure, where (a) is the PCR amplification result and (b) is the colony PCR detection result.

[0025] Figure 2 For 741 Yang PtoNAC43 Genes and hairy poplar PtrNAC43 Image showing the CDS sequence alignment results of gene (LOC7487740).

[0026] Figure 3 for PtoNAC43 A diagram of the amino acid sequence encoded by the gene.

[0027] Figure 4 This is a diagram showing the domain structure analysis of the poplar PtoNAC43 protein.

[0028] Figure 5 This is a sequence alignment diagram of PtoNAC43 with NAC proteins from other species.

[0029] Figure 6 This is a phylogenetic tree constructed based on PtoNAC43 and its homologous proteins.

[0030] Figure 7 The image shows the predicted transmembrane structure and N-terminal signal peptide of the PtoNAC43 protein.

[0031] Figure 8This is a predicted diagram of the secondary structure of the poplar PtoNAC43 protein.

[0032] Figure 9 This is a diagram showing the secondary structure distribution of the poplar PtoNAC43 protein.

[0033] Figure 10 A diagram of the predicted tertiary structure of the poplar PtoNAC43 protein.

[0034] Figure 11 Confocal microscopy image for subcellular localization of PtoNAC43 protein.

[0035] Figure 12 for PtoNAC43 Figure showing the construction of the overexpression vector and the results of colony PCR detection.

[0036] Figure 13 This diagram illustrates the process of obtaining transgenic poplar plants.

[0037] Figure 14 This is a graph showing the PCR detection results of a transgenic poplar line.

[0038] Figure 15 for PtoNAC43 A graph showing the relative expression levels of genes in different transgenic lines.

[0039] Figure 16 for PtoNAC43 Comparison of growth status between overexpression lines and control plants.

[0040] Figure 17 for PtoNAC43 Comparison of leaf and root growth of overexpression lines.

[0041] Figure 18 for PtoNAC43 Comparison of growth indicators of overexpression lines.

[0042] Figure 19 for PtoNAC43 Comparison of photosynthetic pigment content in leaves of overexpression strains.

[0043] Figure 20 for PtoNAC43 Comparative images of stem and leaf sections of the overexpressing strain. Detailed Implementation

[0044] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can more clearly understand and implement the present invention.

[0045] It should be noted that the following embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Without departing from the technical concept of the present invention, those skilled in the art can make various modifications or equivalent substitutions to the embodiments, and all such modifications or equivalent substitutions should fall within the scope of protection of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the description of this invention are conventional methods in the art, and the reagents, materials and equipment used are all from conventional sources in the art or can be obtained through commercial means.

[0047] Furthermore, the technical features involved in each embodiment can be combined with each other without conflicting with each other.

[0048] I. General Description In embodiments of the present invention, the poplar PtoNAC43 transcription factor gene is used as the research object, and a systematic study is conducted on its molecular characteristics and biological functions. Specifically, firstly, the aforementioned... PtoNAC43 The gene was cloned to obtain its complete coding sequence; then, bioinformatics analysis was performed on the protein encoded by the gene, including domain analysis, physicochemical property analysis, homology sequence alignment and phylogenetic analysis, to clarify its classification and potential functional characteristics in the NAC transcription factor family.

[0049] Furthermore, by constructing a fusion expression vector and combining it with an Agrobacterium-mediated transient expression system, subcellular localization analysis of the PtoNAC43 protein was performed to determine its intracellular distribution, thereby providing a basis for studying its functional mechanism. Based on this, a system containing the aforementioned... PtoNAC43 A plant overexpression vector for the gene was used, and the vector was introduced into poplar trees through Agrobacterium-mediated genetic transformation to obtain stably inherited transgenic plants.

[0050] After obtaining transgenic lines, their growth phenotypes and physiological characteristics are systematically analyzed, including but not limited to the determination of growth indicators such as plant height, internode spacing, and biomass, as well as physiological indicators such as photosynthetic parameters, endogenous hormone content, and soluble substance content. Combined with tissue structure observation, the growth regulation effect of transgenic plants is comprehensively evaluated.

[0051] The above experimental system systematically verified the function of PtoNAC43 transcription factor in plant growth regulation, providing experimental evidence for revealing its mechanism of action and its application in plant genetic improvement.

[0052] It should be noted that the "nucleotide sequence homologous to SEQ ID NO:1" described in this invention is not limited to completely identical sequences, but also includes sequence variants formed by substitution, deletion, or insertion of one or more nucleotides while retaining the function of the PtoNAC43 transcription factor. These sequence variants can still exert an inhibitory effect on plant growth without altering the basic structure and biological function of the encoded protein.

[0053] Those skilled in the art will understand that, due to the degeneracy of codons, different nucleotide sequences can encode the same or functionally equivalent proteins. Furthermore, homologous substitutions (including but not limited to conserved substitutions) of nucleotide sequences to a certain extent, without affecting the protein's spatial structure and functional activity, are a conventional technique in this field.

[0054] Furthermore, the nucleic acid or protein described in this invention as having the function of inhibiting plant growth refers to a functional limitation that, when overexpressed in plants, can cause at least one phenotypic change, such as reduced plant height, decreased biomass, or weakened growth potential. For homologous sequences having the above-mentioned function, even if their nucleotide or amino acid sequences differ to some extent from SEQ ID NO:1 or SEQ ID NO:2, they should still be considered to fall within the protection scope of this invention.

[0055] Therefore, within the scope of protection of this invention, the homologous sequence includes not only sequences with high sequence identity to SEQ ID NO:1 or SEQ ID NO:2, but also sequence variants that are functionally equivalent and capable of achieving the same or similar biological effects.

[0056] II. Example 1: PtoNAC43 Gene cloning and expression vector construction 2.1 PCR amplification of the target gene In this embodiment, the PCR amplification of the poplar PtoNAC43 transcription factor gene was performed using 741 poplar cDNA preserved in the laboratory as a template.

[0057] Based on previous transcriptome sequencing results, the design was performed using Primer 6.0 software. PtoNAC43 Gene-specific primers were used, with the upstream primer being NAC-F1 (ATGACAGAAAACATGAGTATATCTGT) and the downstream primer being NAC-R1 (CTATACACTAGTGTTTGGCAAGTG).

[0058] The PCR reaction system is shown in Table 1.

[0059] Table 1 PtoNAC43 Gene PCR amplification system Element Dosage cDNA <1 μg 2×M5 HiPer plus Tap HiFi PCR mix (with blue dye) 10 μL NAC-F1 (10 mM) 1 μL NAC-R1 (10 mM) 1 μL <![CDATA[ddH2O]]> Make up to 20 μL The PCR reaction program was set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 25 s; 58℃ annealing for 25 s; 72℃ extension for 11 s, for 35 cycles; and finally 72℃ extension for 5 min.

[0060] After the PCR reaction, the amplification products were detected by agarose gel electrophoresis, and clear bands were obtained at the expected size and location (see [link]). Figure 1 (a)). The target band was then excised and purified using a gel extraction kit to obtain high-purity PCR products.

[0061] 2.2 Cloning Vector Construction The purified PCR product was ligated into the pTOPO-TA / Blunt simple Vector to construct... PtoNAC43 Gene cloning vector. The ligation reaction system is shown in Table 2.

[0062] Table 2 Cloning vector construction system Element Dosage Purified PCR products 3 μL pTOPO-TA / Blunt Simple Vector 2 μL 10×Enhancer 1 μL <![CDATA[ddH2O]]> Make up to 10 μL The ligation reaction was carried out at 25°C for 5 min. After ligation, the reaction product was transformed into E. coli DH5α competent cells and screened using a plate culture method.

[0063] After culturing, single colonies were picked for colony PCR to verify successful insertion of the target gene. Colony PCR results showed that some clones exhibited specific bands at the expected size and location (see [link to relevant documentation]). Figure 1 (b) indicates that the target fragment has been successfully ligated into the vector.

[0064] Further, plasmid DNA was extracted from positive clones using a plasmid mini-extraction kit, following the kit's instructions. The obtained plasmid was validated by sequencing, and the results showed that the inserted fragment sequence matched the target sequence. PtoNAC43 The gene sequences are identical, thus obtaining a recombinant cloning vector containing the complete CDS sequence.

[0065] 2.3 Analysis of Gene Cloning Results Using the above method, it was successfully cloned from Yang 741. PtoNAC43 The gene has a complete CDS sequence length of 1146 bp, encoding 381 amino acids.

[0066] The PtoNAC43 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0067] The amino acid sequence of the protein encoded by PtoNAC43 is shown in SEQ ID NO:2.

[0068] MTENMSISVNGQSQVPPGFRFHPTEEELLDYYLRKKVSYEKIDLEVIRDVDLNKLEPWDIQERCKIGTAPQNDWYFFSHKDKKYPAGTRTNRATAAGFWKATGRDKVIYGTGKRVGMRKTLVFYKGRAPHGQKSDWIMHEYRLDDNPTDTTVFNVMGEAAQEDGWVVCRIFKKKNLNKTLDRAMSSSPVT Edit Furthermore, the aforementioned PtoNAC43 Gene sequence and hairy poplar PtrNAC43 Alignment analysis of the CDS sequence of gene (LOC7487740) revealed a difference between the two, specifically a difference of 27 bases (see [link to relevant documentation]). Figure 2 This describes the cloned product of the present invention. PtoNAC43 Genes are species- or strain-specific.

[0069] III. Example 2: Subcellular localization of PtoNAC43 3.1 Construction of subcellular localization vector and Agrobacterium-mediated transformation In this embodiment, to determine the intracellular localization of the PtoNAC43 protein, a fusion expression vector of PtoNAC43 and a fluorescent protein was constructed. The vector contained... PtoNAC43 The gene expression vector was transformed into Agrobacterium and then screened and cultured on a medium containing the corresponding antibiotics.

[0070] Single colonies were picked and inoculated into liquid culture medium and incubated at 28°C and 150 rpm until the optical density (OD) of the bacterial culture was reached. 600 The concentration was then increased to approximately 0.6. The bacterial culture was then centrifuged to collect the bacterial cells, and resuspended in a suitable buffer to obtain an Agrobacterium suspension for infection.

[0071] 3.2 Transient expression in tobacco leaves Tobacco plant leaves in good growth condition were selected as recipient materials, and the Agrobacterium suspension prepared above was injected into the tobacco leaves using a syringe to allow Agrobacterium to invade the leaf mesophyll tissue.

[0072] After infection, the treated tobacco plants were placed under suitable conditions and covered with open cardboard boxes to maintain a suitable humidity environment for 3 days.

[0073] 3.3 Observation of fluorescence signals After cultivation, tobacco leaves from the injection area were used to prepare temporary slides, which were then observed under a confocal laser scanning microscope, and the distribution of fluorescence signals was recorded (see [reference]). Figure 11 ).

[0074] 3.4 Results Analysis The observation results show that the PtoNAC43 fusion protein exhibits a specific distribution pattern in cells, with its fluorescence signal mainly concentrated in the nuclear region and co-localizing with nuclear localization marker signals; meanwhile, weak fluorescence signals can also be observed in some cellular regions.

[0075] In contrast, the control group showed a more uniform fluorescence signal distribution and no obvious specific localization characteristics. These results indicate that the PtoNAC43 protein is mainly located in the cell nucleus, consistent with the functional characteristics of transcription factors playing a regulatory role in the cell nucleus. However, the possibility of its expression in cell membrane or cell wall-related regions cannot be ruled out.

[0076] IV. Example 3: PtoNAC43 Gene bioinformatics analysis 4.1 PtoNAC43 Gene structure analysis In this embodiment, the cloned... PtoNAC43 Open Reading Frame (ORF) analysis was performed on the gene sequence. The CDS sequence of the gene was analyzed using the ORFfinder tool, and the amino acid sequence of the encoded protein was deduced based on the correspondence between codons and amino acids.

[0077] The analysis results show that PtoNAC43 The complete CDS sequence of the gene is 1146 bp in length, with the stop codon being TAG, and encodes 381 amino acids. The amino acid sequence of the encoded protein is shown in SEQ ID NO:2. Figure 3 Its amino acid sequence is shown. Further, domain analysis of the protein sequence using the InterPro database revealed a typical NAC domain in the region of amino acids 15 to 173 (see [link to relevant documentation]). Figure 4 This indicates that the protein belongs to the NAC transcription factor family.

[0078] 4.2 Homologous sequence alignment and phylogenetic analysis To analyze the conservation and evolutionary relationships of PtoNAC43 protein in different species, its amino acid sequence was compared with Protein BLAST in the NCBI database, and several protein sequences with high homology were obtained.

[0079] Furthermore, multiple sequence alignment analysis of the screened protein sequences was performed using DNAMAN software. The results showed that the corresponding proteins of each species all possessed a highly conserved NAC domain in the amino acid region from position 15 to 173 (see [link to DNAMAN software]). Figure 5 This indicates that the domain is highly conserved across different species.

[0080] Based on this, MEGA 11.0 software was used to construct a phylogenetic tree using the Neighbor-Joining (NJ) method (see...). Figure 6 The results showed that the PtoNAC43 protein is closely related to NAC proteins in some woody plants, indicating that it is somewhat conserved in evolution and may have similar biological functions.

[0081] 4.3 Physicochemical Properties Analysis of PtoNAC43 Protein The physicochemical properties of the PtoNAC43 protein were analyzed using the ExPASy online tool. The results showed that the protein has a theoretical molecular weight of approximately 43 kDa, an isoelectric point of 5.57, and is acidic.

[0082] Further analysis of the amino acid composition revealed a high content of serine (Ser) and a low content of cysteine ​​(Cys), and the absence of pyrrolidone (Pyl) and selenocysteine ​​(Sec). Furthermore, the protein's instability coefficient was greater than 40, indicating that it is an unstable protein; its overall average hydrophilicity was negative, suggesting that it is a hydrophilic protein.

[0083] 4.4 Prediction of transmembrane structures and signal peptides The transmembrane structure of the PtoNAC43 protein was predicted using the TMHMM Server v.2.0 tool. The results showed that the protein does not contain a transmembrane helical structure, suggesting that it is not a membrane protein.

[0084] Furthermore, the SignalP tool was used to predict the signal peptide of the protein, and the results showed that its N-terminus did not contain a signal peptide sequence (see [link to relevant documentation]). Figure 7 This indicates that the protein is not a secretory protein.

[0085] 4.5 Prediction of protein secondary and tertiary structures The secondary structure of the PtoNAC43 protein was predicted using SOPMA software. The results showed that the protein is mainly composed of α-helices, β-turns, extended strands, and random coils, with random coils accounting for a relatively high proportion (see [link to SOPMA software]). Figure 8 and Figure 9 ).

[0086] Furthermore, the tertiary structure of the PtoNAC43 protein was predicted using the SWISS-MODEL online tool, obtaining its three-dimensional structural model (see [link to SWISS-MODEL]). Figure 10 The results showed that the protein possesses the spatial structural characteristics typical of transcription factor proteins.

[0087] 4.6 Results Analysis The above analysis results indicate that the PtoNAC43 protein exhibits typical structural characteristics of NAC transcription factors, with its N-terminus containing a highly conserved NAC domain and showing high homology across different species. Furthermore, this protein is hydrophilic, non-membrane-bound, and primarily located in the cell nucleus (see Example 2), consistent with the functional characteristics of transcription factors.

[0088] The above results confirm the rationale for PtoNAC43 as an NAC transcription factor from multiple aspects, including sequence structure, physicochemical properties, and evolutionary relationships, and provide a theoretical basis for its functional study in plant growth regulation.

[0089] V. Example 4: Construction of overexpression vector 5.1 PCR amplification of the overexpressed fragment In this embodiment, for the construction PtoNAC43 The plant overexpression vector of the gene was used as a template to amplify the target gene by PCR.

[0090] Amplification was performed using specific primers with seamless cloning adapter sequences, with the upstream primer being NAC-F2 (gagaacacgggggactctagaATGACAGAAAACATGAGTATATCTGT) and the downstream primer being NAC-R2 (cgatcggggaaattcgagctcCTATACACTAGTGTTTGGCAAGTG).

[0091] The PCR reaction system is shown in Table 3.

[0092] Table 3. PCR amplification system for cloning vector plasmids Element Dosage Prime STAR Max Premix 25 μL NAC-F2 (10 mM) 1.5 μL NAC-R2 (10 mM) 1.5 μL plasmid 5 μL <![CDATA[ddH2O]]> Make up to 50 μL The PCR reaction program was set as follows: denaturation at 98℃ for 10 s; annealing at 55℃ for 15 s; extension at 72℃ for 60 s, for a total of 35 cycles.

[0093] After PCR amplification, the product was detected by agarose gel electrophoresis, and clear bands were obtained at the expected size and location (see [link]). Figure 12 The target fragment is then cut and recovered for use in subsequent vector construction.

[0094] 5.2 Construction of overexpression vectors The purified PCR product was ligated into the plant expression vector pNC-Cam2304-35S to construct the PtoNAC43 overexpression vector. The ligation reaction system is shown in Table 4.

[0095] Table 4 NC Cloning System Element Dosage PCR products 3 μL pNC-Cam2304-35S 1 μL <![CDATA[ddH2O]]> 1 μL Nimble Mix Make up to 10 μL The ligation reaction was carried out at 50°C for 60 min. After ligation, the reaction product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing the appropriate antibiotics, and incubated overnight at 37°C.

[0096] After cultivation, single colonies were picked for colony PCR to verify the successful construction of the recombinant vector. The results showed that the amplified band size was consistent with expectations (see [link to relevant documentation]). Figure 12 This indicates that the target gene has been successfully inserted into the expression vector.

[0097] Furthermore, the positive clones were cultured in a shake culture and then sent for sequencing verification. Sequencing results showed that the inserted fragment sequence was correct and consistent with the target sequence. PtoNAC43 The gene sequences were identical, thus successfully obtaining the recombinant expression vector pNC-Cam2304-PtoNAC43.

[0098] 5.3 Agrobacterium-mediated transformation The recombinant expression vector, which was constructed and verified to be correct by sequencing, was transformed into Agrobacterium competent cells EHA105.

[0099] The specific procedure is as follows: the recombinant plasmid is added to Agrobacterium competent cells, transformed by methods such as ice bath, heat shock or electroporation, then revived and cultured, and plated on a selection medium containing the corresponding antibiotics and cultured at 28°C.

[0100] After the culture was completed, single colonies were picked for colony PCR identification to confirm positive clones containing the target gene.

[0101] Furthermore, the Agrobacterium tumefaciens bacterial suspension that was identified as positive was mixed with an equal volume of 70% glycerol to prepare a glycerol strain, which was stored at -80°C for use in subsequent genetic transformation experiments.

[0102] 5.4 Results Analysis Through the above steps, a system containing [data / information] was successfully constructed. PtoNAC43The plant overexpression vector pNC-Cam2304-PtoNAC43 was used to obtain stably transformed Agrobacterium strains. This vector enables the expression of the target gene in plant cells, providing a foundation for the subsequent construction of transgenic plants.

[0103] VI. Example 5: Construction and Identification of Transgenic Poplar Plants 6.1 Agrobacterium-mediated genetic transformation In this embodiment, Agrobacterium-mediated leaf disc method was used to genetically transform Populus 741 to obtain overexpression. PtoNAC43 Genetically modified plants.

[0104] (1) Pre-culture Select robust 741 poplar tissue culture seedlings, choose tender green and flat leaves, and make 3-4 transverse cuts along the main vein of the leaf. Lay the treated leaves flat on the pre-culture medium with the underside facing down for further culture.

[0105] The pre-culture medium was MS medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA, and the culture time was 2-3 days.

[0106] (2) Agrobacterium culture After thawing the Agrobacterium glycerol obtained in Example 4 from -80°C, it was inoculated onto solid LB medium containing antibiotics (containing 50 mg / L Kan and 20 mg / L Rif) and cultured at 28°C for 3 days.

[0107] Single colonies were picked and inoculated into 40 mL of liquid LB medium (containing 50 mg / L Kan and 20 mg / L Rif), and cultured with shaking at 28°C and 150 rpm for 12–16 h until the bacterial culture OD reached its maximum. 600 It is approximately 0.6.

[0108] The cultured bacterial solution was mixed with a 5% sucrose solution at a 1:1 volume ratio for subsequent infection.

[0109] (3) Infection and co-cultivation Place the pre-cultured leaves in the above Agrobacterium infection solution and gently shake for 10-15 minutes to ensure that the leaves are in full contact with Agrobacterium.

[0110] After removing the leaves, use sterile filter paper to absorb excess bacterial solution from the surface, and then spread them flat on a co-culture medium for cultivation.

[0111] The co-culture medium was: MS medium + 1.0 mg / L 6-BA + 0.05 mg / L IAA + 0.01 mg / L TDZ + 100 μM AS, and the culture time was 2-3 days.

[0112] (4) Screening and differentiation culture The co-cultured leaves were transferred to a selection medium for further culture.

[0113] The initial screening medium was: MS medium + 1.0 mg / L 6-BA + 0.05 mg / L IAA + 0.01 mg / LTDZ + 35 mg / L Kan + 300 mg / L Tim, and cultured for about 7 days.

[0114] The leaves were then transferred to another selection medium: MS medium + 1.0 mg / L 6-BA + 0.05 mg / L IIA + 50 mg / L Kan + 300 mg / L Tim, and cultured for another period of time, with the medium being changed every 5 to 6 days.

[0115] After the resistant buds have differentiated and formed, they are transferred to a new culture container for further cultivation (see [link]). Figure 13 ).

[0116] (5) Rooting culture When the resistant shoots grow to about 3 cm, they are cut off and transferred to a selection rooting medium for rooting culture.

[0117] The rooting medium was: 1 / 2 MS medium + 0.5 mg / L IBA + 50 mg / L Kan + 300 mg / L LTim.

[0118] After about one week of cultivation, the plants developed radial root systems and grew well, thus obtaining preliminary transgenic positive plants.

[0119] 6.2 Propagation and Transplantation of Transgenic Plants The obtained positive plants were propagated on the screening differentiation medium and then further transferred to the rooting medium for culture.

[0120] Select seedlings with uniform growth and adventitious roots of about 2 cm in length, loosen the caps of the culture bottles and harden them off for about 3 days.

[0121] The seedlings were then transplanted into flowerpots containing a substrate prepared by mixing garden soil, vermiculite, sand and nutrient soil in a volume ratio of 1:1:1:1.

[0122] After transplanting, water the plant and cover it with a thin film to maintain humidity. Gradually remove the film as the plant adapts to the external environment until it is fully adapted to the growing environment.

[0123] 6.3 Molecular identification of transgenic plants (1) PCR detection Transgenic lines and wild-type Populus 741 plants that were cultured in an artificial climate chamber for 50 days after transplanting were selected, and their genomic DNA was extracted for PCR detection.

[0124] Wild-type plants were used as a negative control (CK-), with plants containing PtoNAC43 The bacterial culture containing the gene was used as a positive control (CK+).

[0125] The test results showed that specific bands consistent with the positive control were amplified in all transgenic lines (see...). Figure 14 This indicates that the target gene has been successfully integrated into the poplar genome.

[0126] (2) qRT-PCR detection For further analysis PtoNAC43 Gene expression in transgenic lines was investigated. Total RNA was extracted from each transgenic line and wild-type plant and reverse transcribed into cDNA.

[0127] The expression level of the target gene was detected by real-time quantitative PCR (qRT-PCR), and the relative expression level was analyzed by the 2^-ΔΔCt method.

[0128] The results showed that, compared with the wild-type control, each transgenic line had... PtoNAC43 Gene expression levels were significantly increased (see...) Figure 15 This indicates that the target gene was successfully overexpressed in the constructed transgenic plant.

[0129] 6.4 Results Analysis Multiple overexpression strains were successfully obtained using Agrobacterium-mediated genetic transformation. PtoNAC43 Transgenic poplar lines containing the gene were obtained. PCR detection and qRT-PCR analysis confirmed that the target gene in the transgenic lines was stably integrated and efficiently expressed, providing a reliable material basis for subsequent functional analysis.

[0130] VII. Example 6: Analysis of the growth and physiological characteristics of transgenic lines In this embodiment, the obtained transgenic poplar lines were systematically analyzed for growth phenotype and physiological characteristics to evaluate... PtoNAC43 The role of genes in plant growth regulation.

[0131] Transgenic lines and wild-type (CK) plants that had been cultured in an artificial climate chamber for 50 days and showed uniform growth were selected and transplanted into large pots for uniform cultivation. Ten replicates were set up for each line, and the plants of each line were placed randomly. At the same time, the soil weight of each pot was kept consistent, and the placement and watering were carried out regularly to minimize the influence of environmental factors on the experimental results.

[0132] When there are significant differences in growth among different strains, the following indicators are measured.

[0133] 7.1 Measurement of growth indicators Use a ruler to measure the plant height, internode distance, and length and width of the third functional leaf; use a vernier caliper to measure the plant diameter at ground level; at the same time, count the number of leaves for each line, with 10 replicates for each line.

[0134] The results showed that, compared with the wild-type control, overexpression PtoNAC43 The transgenic lines of the gene showed significant reductions in plant height, internode distance, and ground diameter, and exhibited weakened growth vigor (see [link to relevant documentation]). Figure 18 ).

[0135] 7.2 Biomass determination Three complete plants from each strain were randomly selected, washed and dried, and then divided into three parts: root, stem, and leaf, which were placed in envelopes respectively.

[0136] The samples were dried in an oven at 105℃ to constant weight. The biomass of each part was calculated by measuring the dry weight. Each strain was replicated three times.

[0137] The results showed that the total biomass and dry weight of each tissue in the transgenic lines were lower than those in the control group, further indicating that... PtoNAC43 Overexpression has an inhibitory effect on plant growth.

[0138] 7.3 Determination of photosynthetic pigment content in leaves The third functional leaf of each strain was selected, and about 0.1 g of sample was weighed and chopped. The sample was then placed in 10 mL of 95% ethanol and extracted in the dark for 24 h.

[0139] After extraction, the extract was mixed and sampled for colorimetric analysis to determine the contents of chlorophyll a (Ca), chlorophyll b (Cb), total chlorophyll (CT), and carotenoids (Car).

[0140] The results showed that the content of photosynthetic pigments in some transgenic lines was significantly higher than that in the control group (see...). Figure 19 ).

[0141] 7.4 Measurement of photosynthetic parameters The third functional leaf of each strain cultured in an artificial climate chamber for 50 days was selected, and photosynthetic parameters, including net photosynthetic rate (...), were measured using a Li-6400XT portable photosynthetic measurement system. P n ), intercellular CO2 concentration ( C i ), transpiration rate ( T r ) and porosity ( G s The measurement results are shown in Table 5.

[0142] Table 5 Comparison of photosynthetic parameters in leaves of PtoNAC43 overexpression lines strain <![CDATA[ P n ]]> <![CDATA[ G s ]]> <![CDATA[ C i ]]> <![CDATA[ T r ]]> CK 7.00±0.36a 0.10±0.00a 266.13±3.71d 1.50±0.10c T1 5.03±0.45c 0.17±0.06a 398.10±2.11a 2.87±0.31a T2 6.17±0.45b 0.10±0.00a 304.90±3.65c 1.83±0.12b T3 5.80±0.10b 0.13±0.06a 361.47±3.59b 2.83±0.06a Note: Different lowercase letters in the same column of the figure indicate significant differences. P <0.05) The results showed that, compared with the control group, the net photosynthetic rate of the transgenic lines was significantly reduced, while the intercellular CO2 concentration and transpiration rate were significantly increased, and the stomatal conductance did not change significantly.

[0143] 7.5 Determination of Plant Hormone Content The 3rd to 4th functional leaves of each strain were selected, and the content of endogenous plant hormones, including indoleacetic acid (IAA), abscisic acid (ABA), zeatin (ZT), and gibberellin (GA3), was determined using a kit method. The results are shown in Table 6.

[0144] Table 6 Comparison of endogenous hormone levels in PtoNAC43 overexpression lines strain IAA (μmol / g) ABA(ng / g) ZT(pg / g) <![CDATA[GA3(pmol / g)]]> CK 0.076±0.003c 272.502±18.074d 1446.598±42.681c 372.554±39.284b T1 0.073±0.003c 423.745±15.269a 1355.669±80.727c 339.270±8.092b T2 0.113±0.009a 308.935±6.490c 1970.138±105.146b 497.140±57.811a T3 0.093±0.003b 349.167±22.446b 2402.927±56.333a 477.121±10.040a Note: Different lowercase letters in the same column of the figure indicate significant differences. P <0.05) The results showed that the ABA content in the transgenic lines was significantly higher than that in the control group. At the same time, the contents of IAA, ZT and GA3 in some lines also showed significant changes, indicating that PtoNAC43 may affect plant growth by regulating plant hormone levels.

[0145] 7.6 Determination of soluble substance content The 3rd to 4th functional leaves of each strain were selected, and the contents of soluble sugar, soluble starch and soluble protein were determined using a kit method.

[0146] The measurement results are shown in Table 7.

[0147] Table 7 PtoNAC43 Comparative analysis of soluble sugar, starch, and protein content in overexpression lines strain Soluble sugars (mg / g) Soluble starch (mg / g) Soluble protein (mg / g) CK 95.73±0.88c 26.69±0.26a 77.73±1.63a T1 79.53±0.46d 19.71±0.18d 71.72±0.11b T2 104.28±1.27b 23.63±0.48b 72.92±0.22b T3 120.16±0.23a 22.30±0.34c 72.75±1.09b Note: Different lowercase letters in the same column of the figure indicate significant differences.P <0.05) The results showed that the soluble starch and protein content in the transgenic lines was significantly lower than that in the control group, while the soluble sugar content varied among different lines.

[0148] 7.7 Organizational Structure Observation A stem segment about 1 cm from the soil surface and the third functional leaf were selected, and then fixed, dehydrated, embedded, and sliced. The slice thickness was about 10 μm.

[0149] After staining, the tissue sections were observed under a microscope to observe changes in their tissue structure (see [reference]). Figure 20 ).

[0150] The observation results showed that, compared with the control group, the stem tissue cell morphology of the transgenic line underwent significant changes, including increased cell volume, looser arrangement, and enlarged intercellular spaces; at the same time, the leaf tissue structure also showed certain changes.

[0151] 7.8 Results Analysis Based on the above analysis of growth indicators, physiological indicators, and tissue structure, it can be concluded that overexpression PtoNAC43 The transgenic poplar lines were significantly lower than the wild-type control in terms of plant height, biomass, and growth vigor.

[0152] Meanwhile, the transgenic lines showed significant changes in photosynthetic parameters, endogenous hormone levels, and tissue structure, indicating that... PtoNAC43 Genes participate in regulating plant growth processes through a variety of physiological pathways.

[0153] The above results indicate that the PtoNAC43 transcription factor plays a negative regulatory role in the growth regulation of poplar trees, and its overexpression can significantly inhibit plant growth.

[0154] Through the systematic study of the above embodiments, the structural characteristics, expression properties, and biological functions of the poplar PtoNAC43 transcription factor gene were comprehensively analyzed. Firstly, molecular cloning was used to obtain... PtoNAC43 The complete coding sequence of the gene was obtained, and bioinformatics analysis confirmed that the encoded protein has typical NAC transcription factor structural features and is highly conserved across different species, indicating that it may play an important regulatory role in plant growth and development.

[0155] Furthermore, subcellular localization experiments showed that the PtoNAC43 protein is mainly located in the cell nucleus, which is consistent with the functional characteristics of transcription factors regulating gene expression in the cell nucleus, providing experimental evidence for its participation in transcriptional regulation.

[0156] Based on this, multiple overexpression vectors were successfully obtained by constructing overexpression vectors and using Agrobacterium-mediated genetic transformation. PtoNAC43 Transgenic poplar lines containing the gene. PCR and qRT-PCR tests confirmed that the target gene was stably integrated and expressed at high levels in all transgenic lines.

[0157] Systematic analysis of the growth and physiological characteristics of transgenic lines showed that, compared with wild-type controls, overexpression of [the transgenic lines] significantly improved the growth and physiological characteristics of the transgenic lines. PtoNAC43 The transgenic plants exhibited significantly reduced growth indicators such as plant height, internode spacing, and biomass, displaying phenotypic characteristics of growth inhibition. Simultaneously, the transgenic lines showed varying degrees of change in photosynthetic parameters, photosynthetic pigment content, endogenous hormone levels, and soluble substance content, and their stem and leaf tissue structures also showed significant differences.

[0158] Based on the above results, it can be concluded that the PtoNAC43 transcription factor plays a negative regulatory role in the growth regulation of poplar trees. Its overexpression can significantly inhibit the growth and development of plants through multiple pathways, such as affecting photosynthesis, physiological metabolism and hormone balance.

[0159] Therefore, this invention successfully identified and verified a NAC transcription factor gene associated with negative regulation of plant growth, which not only enriches the functional research content of the plant NAC transcription factor family, but also provides new gene resources and theoretical basis for growth regulation and trait improvement of poplar and other plants, and has good application prospects.

[0160] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For those skilled in the art, various modifications, substitutions, or equivalent transformations can be made to the above embodiments without departing from the spirit and essence of the technical solution of the present invention, and all such modifications, substitutions, or equivalent transformations should fall within the scope of protection of the present invention.

[0161] Furthermore, the technical features described in the various embodiments of this invention can be used in any combination without conflict. Any modifications, equivalent substitutions, and improvements made within the concept and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the poplar PtoNAC43 transcription factor protein, and the nucleic acid molecule is selected from any of the following: (1) The nucleotide sequence shown in SEQ ID NO:1; (2) A nucleotide sequence that has at least 80% homology with SEQ ID NO:1 and encodes a protein that has the function of inhibiting plant growth; (3) A nucleotide sequence complementary to the nucleotide sequence described in (1) or (2).

2. The nucleic acid molecule according to claim 1, characterized in that, The nucleic acid molecule has at least 85% or at least 90% homology with SEQ ID NO:

1.

3. The nucleic acid molecule according to claim 1 or 2, characterized in that, The proteins encoded by the nucleic acid molecules can reduce one or more of the following in plants: plant height, internode spacing, biomass, or growth potential.

4. A recombinant expression vector, characterized in that, The vector comprises a functional promoter and a nucleic acid molecule according to any one of claims 1-3 operatively linked to the promoter.

5. The recombinant expression vector according to claim 4, characterized in that, The promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter, preferably the CaMV 35S promoter.

6. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecules according to any one of claims 1-3, or the recombinant expression vector according to claim 4 or 5.

7. The recombinant cell according to claim 6, characterized in that, The recombinant cells are Agrobacterium cells or plant cells.

8. A transgenic plant, characterized in that, The plant is introduced and overexpressed with the nucleic acid molecule described in any one of claims 1-3.

9. The transgenic plant according to claim 8, characterized in that, The plant in question is a woody plant, preferably a poplar.

10. A method for inhibiting plant growth, characterized in that, This includes overexpressing the nucleic acid molecules of any one of claims 1-3 in plants, thereby reducing plant height, biomass, or growth potential.

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