A clpap7 gene encoding a cypress purple acid phosphatase and application thereof

Abstract

This invention belongs to the field of plant molecular breeding technology and provides a method for encoding purple acid phosphatase in Cunninghamia lanceolata. ClPAP7 Genes and their applications, the aforementioned ClPAP7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1. This invention will... ClPAP7 Gene overexpression in poplar trees was observed under low phosphorus stress. ClPAP7 The genetically modified poplar trees exhibited greater height, root surface area, root volume, and total root length than their wild counterparts; this demonstrates... ClPAP7 This is a key functional gene in the low phosphorus stress response of Cunninghamia lanceolata. This invention also systematically analyzed the expression regulation, enzymatic activity, and physiological and molecular functions of this gene in heterologous overexpression. The function and molecular mechanism of ClPAP7 purple acid phosphatase synthesis were clarified, providing an effective gene resource for improving low phosphorus tolerance in forest trees, and offering a reference for promoting genetic breeding improvement of forest trees and increasing production efficiency.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a method for encoding purple acid phosphatase in Cunninghamia lanceolata. ClPAP7 Genes and their applications. Background Technology

[0002] Phosphorus (P) is a key limiting factor for productivity in subtropical forest ecosystems. Soil phosphorus can only be absorbed and utilized by plants after being converted into water-soluble or weakly acidic inorganic forms, thus phosphorus bioavailability largely controls forest productivity. (Chinese fir (…)) Cunninghamia lanceolata Chinese fir (Cunninghamia lanceolata) is an important fast-growing coniferous timber species, possessing advantages such as high-quality timber, high yield, and wide range of uses, holding a significant position in my country's commercial timber production. However, due to the strong chemical fixation of phosphorus by abundant iron, aluminum, and soil colloids in the acidic red soils of southern China, the total phosphorus content of the soil is high, while the available phosphorus content that forest trees can absorb and utilize is scarce. Furthermore, the long-standing practice of multi-generational continuous planting of Chinese fir has further depleted the limited phosphorus resources in the soil, exacerbating the limitation of available phosphorus on Chinese fir productivity.

[0003] Therefore, identifying and characterizing key functional genes in forest trees that respond to low phosphorus stress can provide important theoretical basis and technical support for improving forest tree germplasm and cultivating new varieties of forest trees resistant to low phosphorus stress using molecular breeding techniques. Summary of the Invention

[0004] The purpose of this invention is to provide a method for encoding purple acid phosphatase of cedarwood. ClPAP7 Genes and their applications, overexpression under low phosphorus stress ClPAP7 Genetically modified poplars exhibit greater plant height, root surface area, root volume, and total root length than wild-type plants, proving... ClPAP7 These are key functional genes for low phosphorus stress response, providing important theoretical basis and technical support for breeding low phosphorus stress-resistant tree varieties.

[0005] This invention provides a method for encoding purple acid phosphatase from Chinese fir. ClPAP7 Genes, the ones mentioned ClPAP7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1.

[0006] As a preferred embodiment, the ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0007] The present invention also provides the aforementioned ClPAP7 Application of genes in improving the tolerance of trees to low phosphorus stress and / or in breeding low phosphorus stress tolerant tree varieties.

[0008] This invention also provides overexpression ClPAP7The application of genetically modified biomaterials in improving the tolerance of trees to low phosphorus stress and / or breeding low phosphorus stress-tolerant tree varieties, the aforementioned ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0009] As a preferred embodiment, the overexpression ClPAP7 Biological materials for gene overexpression include: ClPAP7 Gene amplification primers, overexpression... ClPAP7 Recombinant vectors of genes, overexpression of the aforementioned ClPAP7 Recombinant microorganisms of genes, overexpression of the aforementioned ClPAP7 Transgenic cell lines and overexpression of the gene ClPAP7 Any one or more of the genes in the transgenic plant tissue.

[0010] As a preferred embodiment, the amplification primers include a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:4.

[0011] As a preferred embodiment, the basic framework of the recombinant vector includes the pCambia3301 vector; the basic microorganism of the recombinant microorganism includes Agrobacterium.

[0012] This invention also provides a method for improving the tolerance of forest trees to low phosphorus stress, comprising the following steps: overexpressing the [specific phosphorus] in target forest trees. ClPAP7 Gene.

[0013] This invention also provides a method for breeding low-phosphorus stress tolerant tree varieties, comprising the following steps: overexpressing the [specific expression name] in target trees. ClPAP7 Gene.

[0014] The present invention also provides a low-phosphorus-stress-tolerant poplar, wherein the genome of the low-phosphorus-stress-tolerant poplar contains ClPAP7 The gene is overexpressed; ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0015] Beneficial effects: This invention provides a method for encoding purple acid phosphatase from Chinese fir. ClPAP7 Genes and their applications, the aforementioned ClPAP7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1. This invention cloned the purple acid phosphatase gene from *Cunninghamia lanceolata*. ClPAP7The expression regulation pattern, enzymatic characteristics, and physiological and molecular functions of heterologous overexpression of this gene were systematically analyzed. Enzymatic analysis showed that ClPAP7 is a typical acid phosphatase, capable of efficiently hydrolyzing nucleotide substrates under acidic conditions, participating in the catabolism of high-energy phosphate compounds and the recycling of phosphorus in plant cells. Functional validation through heterologous overexpression showed that under low phosphorus stress, overexpression... ClPAP7 The poplar plants exhibited significantly superior growth indicators compared to wild-type plants, including plant height, total root length, root surface area, and root volume, confirming that... ClPAP7 These are key functional genes involved in the low phosphorus stress response of Chinese fir. This study clarified... ClPAP7 The study of the functions and molecular mechanisms regulating the tolerance of forest trees to low phosphorus stress not only provides excellent functional gene resources for the improvement of low phosphorus-tolerant forest tree germplasm, but also provides important theoretical basis and technical reference for the genetic breeding of forest trees to resist stress and the efficient utilization of phosphorus in forest land and the improvement of productivity.

[0016] This invention is the first to screen and clone key functional genes for low phosphorus stress response from Chinese fir. ClPAP7 This study systematically elucidated the basic characteristics, enzymatic functions, and low-phosphorus tolerance regulatory molecular mechanisms of this gene and its encoded protein, effectively filling a gap in the research field of low-phosphorus tolerance functional genes in Chinese fir, and possessing significant theoretical and industrial application value. This invention identified highly conserved genes... ClPAP7 The gene encodes a hydrophilic acid phosphatase located in the cell nucleus and cytoplasm, which specifically responds to low phosphorus stress and exhibits significantly upregulated expression, providing a novel endogenous gene resource for molecular improvement of low phosphorus tolerance in forest trees. Enzymatic characterization confirmed that this protein can efficiently hydrolyze nucleotide substrates under acidic conditions, participating in the intracellular phosphate decomposition, metabolism, and recycling of plants, thus clarifying its functional mechanism of phosphorus activation and utilization. Heterologous overexpression functional experiments confirmed that overexpression... ClPAP7 This invention can significantly improve the growth status of trees under low phosphorus stress, effectively increasing core root indicators such as total root length, root surface area, root volume, and root biomass, alleviating the inhibitory effect of low phosphorus on plant growth, maintaining the photosynthetic physiological stability of plants, significantly improving the net photosynthetic rate of trees under low phosphorus conditions, and enhancing the adaptability of trees to barren soils. Furthermore, this invention includes the development of specific amplification primers and overexpression vectors, forming a complete gene cloning and application technology system. This provides mature technical support for cultivating new low-phosphorus tolerant tree germplasm and offers important theoretical basis and practical pathways for efficient soil phosphorus utilization, forest productivity improvement, and stress-resistance breeding in Southern Chinese fir plantations. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1The result of the hydrophobicity curve of ClPAP7 protein is shown in the figure. Figure 2 This is a diagram showing the multiple sequence alignment results of ClPAP7 with PAP amino acid sequences from other species; where AtPAP is Arabidopsis PAP, PtPAP is Populus tomentosa PAP, OsPAP is rice PAP, ZmPAP is maize PAP, ClPAP is Chinese fir PAP, SlPAP is Stylosanthes stylosum PAP, SgPAP is tomato PAP, GmPAP is soybean PAP, and CaPAP is chickpea PAP. Figure 3 A phylogenetic tree diagram of PAP (Phyllanthus alpinus) and other species; Figure 4 fir ClPAP7 Image showing the results of gene cloning; Figure 5 To express ClPAP7 Image of PCR detection of vector bacterial culture; Figure 6 Subcellular localization map of ClPAP7 protein; GFP autofluorescence under laser; cell state under bright field; Merged superposition effect showing the location of GFP fluorescence in the cell; Figure 7 for ClPAP7 Image showing the results of DNA level identification in poplar trees that overexpressed DNA; Figure 8 Overexpression under different phosphorus conditions ClPAP7 The results of the effect on poplar growth are shown in the figure; where A is the WT vs OE after 4 weeks of NP treatment; and B is the WT vs OE after 4 weeks of LP treatment. Figure 9 Figures show the results of plant height and leaf area of ​​different genotypes of 84K poplar under different phosphorus treatments; where A represents the plant height of different genotypes of 84K poplar under different phosphorus treatments; B represents the leaf area of ​​different genotypes of 84K poplar under different phosphorus treatments; * in the figures represents... p <0.05; ** represents p <0.01; *** represents p <0.001; Figure 10 Figure 1 shows the biomass results of different genotypes of Populus 84K under different phosphorus treatments; where A represents the fresh weight of the aboveground parts; B represents the dry weight of the aboveground parts; C represents the fresh weight of the underground parts; and D represents the dry weight of the underground parts. * in the figure represents... p <0.05; ** represents p <0.01; *** represents p <0.001; Figure 11The graphs show the root system parameters of different genotypes of Populus 84K under different phosphorus treatments; where A is the total root length, B is the root surface area, and C is the root volume. Figure 12 Figure 1 shows the net photosynthetic rate of different genotypes of 84K poplar under different phosphorus treatments; * in the figure represents p <0.05; ** represents p <0.01; *** represents p <0.001; Figure 13 Figure 1 shows the phosphorus content (P) of different genotypes of Populus 84K under different phosphorus treatments; where A represents the phosphorus content in the aboveground parts and B represents the phosphorus content in the underground parts; * in the figure represents... p <0.05; ** represents p <0.01; *** represents p <0.001; Figure 14 Figure 1 shows the phosphorus utilization efficiency of different genotypes of 84K poplar under different phosphorus treatments; * in the figure represents p <0.05; ** represents p <0.01; *** represents p <0.001; Figure 15 This is a PCR detection image of the prokaryotic expression vector pCold::ClPAP7-GFP bacterial culture; Figure 16 Image of SDS-PAGE detection results: Figure 17 This is a graph showing the results of Western blot analysis. Figure 18 The graph shows the effect of pH on ClPAP7 enzyme activity. Figure 19 Figure showing the effect of different temperatures on ClPAP7 enzyme activity; Figure 20 The graph shows the decomposition capabilities of ClPAP7 for different substrates. Detailed Implementation

[0019] This invention provides a method for encoding purple acid phosphatase from Chinese fir. ClPAP7 Genes, the ones mentioned ClPAP7The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:1: MIALLVILCFPIGRSDCSLAKHVINRKKYGSSLNFLVVGDWSRKELYNQSQVAAQMGKVGEKLQIDFIISVGDNFYDNGLNGTDDSSFEQSFSQVYTAKSLQKQWYAVLGNHDYLGNTLAQFSSTLRRRDRRWC*. The ClPAP7 protein of this invention is an unstable protein molecule with an instability coefficient of 48.68. It is a hydrophilic protein composed of 134 amino acids, with a molecular weight of approximately 15253.32 Da and an isoelectric point of 9.05.

[0020] This invention predicts the function of the ClPAP7 protein and finds that the ClPAP7 protein sequence contains the MPP_superfamily conserved domain. By comparing the protein sequence with homologous genes of other species, it was found that Chinese fir has a high degree of similarity with homologous genes of other species.

[0021] As one specific implementation method, the ClPAP7 The nucleotide sequence of the gene such as SEQ ID NO:2 shows: 5'-ATGATTGCTTTGTTGGTTATATTGTGCTTTCCAATAGGGAGATCTGATTGTTCACTCGCCAAACATGTTATAAATAGAAAGAAGTATGGGAGTTCTCTCAATTTCTTAGTTGTGGGAGATTGGAGCAGGAAAGAATTGTACAACCAGTCCCAAGTTGCTGCTCAGATGGGAAAAGTAGGAGAAAAGTTGCAGATTGACT TCATTATTTCTGTGGGAGATAATTTTTATGACAATGGATTGAATGGCACAGATGATTCAAGTTTTGAACAATCCTTTTCTCAAGTATACACGGCTAAAAGCCTACAAAAACAGTGGTATGCTGTCCTAGGAAACCATGATTACCTGGGAAATACTTTGGCTCAGTTCAGCTCCACTCTAAGAAGGCGGGATCGCAGATGGTGTTAG-3'.

[0022] The present invention also provides the aforementioned ClPAP7 Application of genes in improving the tolerance of trees to low phosphorus stress and / or in breeding low phosphorus stress tolerant tree varieties.

[0023] The present invention will describe ClPAP7 Gene overexpression in poplar trees, overexpression ClPAP7 The root growth of the overexpressing poplar gene was not significantly affected by low phosphorus treatment, and all indicators were significantly higher than those of the wild type under low phosphorus treatment, with no significant difference from the levels under normal phosphorus supply conditions. Under low phosphorus stress, the aboveground parts of the WT plant were poor, showing thin stems, small and yellow leaves, but the overexpression of the gene was not significantly affected. ClPAP7 The root biomass, root surface area, and root volume of the gene-expressing poplar were significantly higher than those of the wild type. Therefore, overexpression... ClPAP7 The gene promoted root growth and improved the poplar's tolerance to low phosphorus stress.

[0024] This invention demonstrated that, under different phosphorus supply levels, the net photosynthetic rate of overexpressing poplar was significantly higher than that of wild-type poplar. Under normal phosphorus supply, there was no significant difference in net photosynthetic rate between transgenic and wild-type lines. However, under low phosphorus stress, the net photosynthetic rate of transgenic lines was significantly higher than that of wild-type lines. This indicates that overexpression... ClPAP7 It can alleviate the inhibitory effect of low phosphorus stress on the photosynthetic capacity of plants and maintain the photosynthetic efficiency of leaves. This invention, to a certain extent, reveals the characteristics of Chinese fir. ClPAP7 Genes can respond to low phosphorus stress in plants and can provide effective gene resources for improving low phosphorus tolerance in forest trees in the future.

[0025] This invention also provides overexpression ClPAP7 The application of genetically modified biomaterials in improving the tolerance of trees to low phosphorus stress and / or breeding low phosphorus stress-tolerant tree varieties, the aforementioned ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0026] The overexpression described in this invention ClPAP7 Biological materials for gene overexpression include: ClPAP7 Gene amplification primers, overexpression... ClPAP7 Recombinant vectors of genes, overexpression of the aforementioned ClPAP7 Recombinant microorganisms of genes, overexpression of the aforementioned ClPAP7 Transgenic cell lines and overexpression of the gene ClPAP7 Any one or more of the genes in the transgenic plant tissue.

[0027] The present invention overexpressed the above ClPAP7 The gene amplification primers include a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:4. The primer sequences of the present invention are as follows: Forward primer (SEQ ID NO:3): 5'-ATGATTGCTTTGTTGGTTATATTGTGCT-3'; Reverse primer (SEQ ID NO:4): 5'-ACACCATCTGCGATCCCGC-3'.

[0028] In this invention, when using the primer pair for amplification, a mixed cDNA from multiple tissues of *Cunninghamia lanceolata* is used as a template. A high-fidelity polymerase is used to prepare the amplification system. The amplification system, in 50 μL increments, includes: 25 μL of 2×Hieff Canace® AdvanceFast PCR Master Mix, 1 μL of DNA template, 2 μL of forward primer, 2 μL of reverse primer, and the remainder ddH2O. The prepared amplification system is placed in a PCR instrument for PCR reaction. The program includes: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 10 s, 35 cycles; and a final extension at 72℃ for 2 min. Using the primer pair, amplification system, and amplification method described in this invention, the product obtained is as described above. ClPAP7 CDS sequence of the gene.

[0029] The basic framework of the recombinant vector of the present invention includes the pCambia3301 vector; the basic microorganism of the recombinant microorganism includes Agrobacterium. In a specific embodiment of the present invention, the recombinant vector is a 35S::ClPAP7-GFP vector; the recombinant microorganism is Agrobacterium GV3101 containing the 35S::ClPAP7-GFP vector; overexpression of the... ClPAP7 Transgenic plant tissues are ClPAP7 Overexpressing poplar trees.

[0030] This invention also provides a method for improving the tolerance of forest trees to low phosphorus stress, comprising the following steps: overexpressing the [specific phosphorus] in target forest trees. ClPAP7 Gene.

[0031] This invention also provides a method for breeding low-phosphorus stress tolerant tree varieties, comprising the following steps: overexpressing the [specific expression name] in target trees. ClPAP7 Gene.

[0032] The present invention also provides a low-phosphorus-stress-tolerant poplar, wherein the genome of the low-phosphorus-stress-tolerant poplar contains ClPAP7 The gene is overexpressed; ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0033] In a specific embodiment of the present invention, Agrobacterium GV3101 containing the 35S::ClPAP7-GFP vector was transformed into poplar 84K using the leaf disc transformation method to obtain... ClPAP7 Overexpressing poplar trees.

[0034] This invention analyzes the enzymatic properties of the purified recombinant protein ClPAP7 by setting IPTG concentration gradients (0 mmol / L, 0.25 mmol / L, 0.5 mmol / L, 0.75 mmol / L, 1 mmol / L), temperature gradients (15℃, 26℃, 37℃), and time gradients (0 h, 6 h, 12 h, 18 h, 24 h). The results show that the recombinant plasmid does not express the target protein without IPTG; increasing the IPTG concentration at lower concentrations increases expression levels, but the expression level no longer increases significantly at a concentration of 1 mmol / L. Temperature gradient results show that the target protein expression level is highest at 16℃ overnight induction, and gradually decreases with increasing temperature. Time gradient results show that the expression level reaches its maximum at 18 h of induction, and then decreases. In summary, the optimal induction conditions are: IPTG concentration 1 mmol / L, induction temperature 16℃, and induction time 18 h.

[0035] To further illustrate the present invention, the following examples demonstrate the application of the present invention to encode a purple acid phosphatase from cedarwood. ClPAP7 The genes and their applications are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0036] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0037] (1) The test materials included: 84K (Populus alba×Populus glandulosa) poplar and Nicotiana benthamiana; (2) Test vectors and strains: The overexpression vector was Pcambia3301 (Beijing Cooler Master Technology Co., Ltd.; catalog number VT308-10μg); the prokaryotic expression vectors were pCold-TF (TaKaRa; catalog number Code No. 3365) and pCold-TF-eGFP (eGFP inserted into the pCold-TF vector); the Escherichia coli strains were DH5α and BL21(DE3); the Agrobacterium strain was GV3101; (3) Test culture medium and formulation: Bacterial culture medium LB: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride; 84K rooting medium: 4.4 g / L WPM519, 30 g / L sucrose, 0.1 mg / L IBA, 0.01 mg / L NAA, 8 g / L agar powder; 84K co-culture medium: 4.4 g / L WPM519, 30 g / L sucrose, 0.01 mg / L TDZ, 0.2 mg / L 6-BA, 0.1 mg / L NAA, 8.3 g / L agar; 84K callus induction medium: 4.4 g / L WPM519, 30 g / L sucrose, 0.01 mg / L TDZ, 0.2 mg / L 6-BA, 0.1 mg / L NAA, 8.3 g / L agar; 84K differentiation screening medium: 2.4 g / L WPM449, 0.5 g / L MES, 0.05 mg / L NAA, 0.25 mg / L 6-BA, 20 g / L sucrose, 3.5 g / L gel, 200 mg / L cephalosporin, 2 mg / L hygromycin.

[0038] (4) Main reagents and equipment for the experiment Reagents: Steadp Pure Plant Genomic DNA Extraction Kit (Aikerui Biotechnology), RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen), Reverse Transcription Kit (11141ES10) (Yisheng), qPCR Kit (11201ES03) (Yisheng), Plasmid Mini-Prep Kit (Classic Type) (Meiji), SDS-PAGE AB Solution Rapid Gel Preparation Kit 10% (Lanbolide), His Tag Protein Purification Kit (Reduction-Resistant Chelating Type) (Beyotime Biotechnology), Gel Recovery Kit (Meiji), 2× Rapid Taq Super Mix (Baimeng), 2x HieffCanace AdvanceFast PCR Master Mix (Yisheng), PBS phosphate buffer (Nanjing Weisente Biotechnology Co., Ltd.), trichromatographic pre-stained protein marker (Shanghai Tianneng Technology Co., Ltd.), DNA molecular weight standard marker (Shanghai Yisheng Biotechnology Co., Ltd.), ECL chemiluminescence solution (Shanghai Tianneng Technology Co., Ltd.), β-mercaptoethanol, 1 / 2MS, MS, Silwet L-77, acetylsuccinone, MES, DMSO, rifampicin antibiotic, kanamycin antibiotic, hygromycin antibiotic, ITPG, EDTA, Tris-HCl, DEPC, sucrose, chloroform, TAE electrophoresis buffer, agarose, nucleic acid dye, LB broth, etc.

[0039] Equipment: Cryogenic grinder (Eppendorf, Germany), pure water system (WP-UP-LH-40, China), turbine oscillator (SCILOGEX, USA), PCR instrument (Eppendorf, Germany), micro-volume nucleic acid concentration analyzer (Thermo Fisher Scientific, USA), real-time PCR instrument (BIO-RAD iQ5, USA), pipette (Eppendorf, Germany), high-temperature sterilizer, gel imaging system (Eppendorf, Germany), benchtop high-speed refrigerated centrifuge (Eppendorf, Germany), constant temperature metal bath (TIANGENBIOTECH), UV-Vis spectrophotometer (NanoDrop One), constant temperature shaker (Xudong Bantian Electronics Co., Ltd.), etc.

[0040] Example 1: Fir wood ClPAP7 Gene bioinformatics analysis This invention constructed a transcriptional regulatory network of Cunninghamia lanceolata under nitrogen and phosphorus stress and screened out key purple acid phosphatase genes in the low phosphorus stress response module. ClPAP7 Under low phosphorus stress, the roots and leaves of Chinese fir... ClPAP7 Expression levels were significantly increased, and its promoter sequence contained a P1BS structure that could bind to PHR, a core transcription factor in the phosphorus signaling regulatory network. This indicates that... ClPAP7 It may play an important role in the way that Chinese fir copes with low phosphorus stress.

[0041] This invention utilizes the ProtScale tool combined with the Kyte-Doolittle algorithm to analyze the hydrophilicity and hydrophobicity of the Cunninghamia lanceolata ClPAP7 protein. Figure 1 The results showed that the protein is 134 amino acids in length, with a strongly hydrophobic domain at the N-terminus (0-15 aa) and a hydrophobicity peak of 3.5. The rest of the sequence is mainly composed of hydrophilic amino acids, with no other typical transmembrane structures. The overall protein is a soluble hydrophilic protein.

[0042] To further verify ClPAP7 Gene function was assessed using BLAST comparison from the NCBI database. ClPAP7 Homologous protein sequences of the conserved ClPAP7 domain from different species were obtained. Multiple sequence alignment analysis was performed on PAP family proteins. Figure 2A study found that using Cunninghamia lanceolata ClPAP7 (as shown in SEQ ID NO:1) as the core, its sequence was compared with PAPs sequences from species such as Arabidopsis thaliana and rice, showing a similarity of 37.4%. ClPAP7 and other PAPs sequences exhibit some discontinuously distributed conserved regions (amino acid sites marked with the same color blocks or symbols in the figure). These regions belong to characteristic conserved domains of the PAPs family, suggesting that ClPAP7 possesses the core functional characteristics of this family of proteins. Furthermore, ClPAP7 exhibits significant species specificity, with a relatively short sequence length (numerous deletion sites indicated by "-"), and some segments [such as the "GSSLNFLVVGDWS" region (SEQ ID NO:5)] are unique to Cunninghamia lanceolata, showing significant differences from corresponding regions in PAPs sequences from other species. These analytical results reflect both the family conservation of PAPs genes during evolution and suggest that ClPAP7 may have undergone functional differentiation in Cunninghamia lanceolata, potentially undertaking unique physiological functions, such as adaptive phosphorus metabolism regulation.

[0043] To investigate the evolutionary relationships of ClPAP7, homologous genes to ClPAP7 were screened using NCBI. A phylogenetic tree was constructed by combining 29 PAP genes from the model plant Arabidopsis thaliana with homologous genes from maize (ZmPAP7a-g, ZmPAP7), poplar (PtPAP31, PtPAP30, PtPAP28, PtPAP29, PtPAP17, PtPAP32, PtPAP33), Stylosanthes styrax (SlPAP7), tomato (SgPAP7), soybean (GmPAP7c, GmPAP7d), chickpea (CaPAP7), and rice (OsPAP7, OsPAP3a, OsPAP3b). Figure 3 The results showed that ClPAP7 was most closely related to soybean GmPAP7c, followed by maize ZmPAP7, rice OsPAP3 and OsPAP7, and poplar PtPAP30, PtPAP31, PtPAP29, PtPAP17, and PtPAP28.

[0044] Example 2: Fir wood ClPAP7 Cloning and overexpression vector construction 1. ClPAP7 Full-length gene cloning according to ClPAP7 The CDS sequence (as shown in SEQ ID NO:2) was obtained. Specific primers were designed using snapgene, and high-fidelity polymerase was used to amplify the mixed cDNA from multiple tissues of Cunninghamia lanceolata as a template. ClPAP7The full-length CDS region of the gene was amplified. High-fidelity polymerase was used to amplify the target gene using 2×HieffCanace AdvanceFast PCR Master Mix from Yisheng Company.

[0045] CDS-ClPAP7-F (SEQ ID NO:3): 5'-ATGATTGCTTTGTTGGTTATATTGTGCT-3'; CDS-ClPAP7-R (SEQ ID NO:4): 5'-ACACCATCTGCGATCCCGC-3'.

[0046] The amplification system consisted of 25 μL of 2×Hieff Canace® AdvanceFast PCR Master Mix, 1 μL of DNA template, 2 μL of forward primer (10 μmol / L), 2 μL of reverse primer (10 μmol / L), and ddH2O added to a final volume of 50 μL. The PCR reaction program was 98℃ for 30 s; 98℃ for 10 s; 60℃ for 5 s; 72℃ for 10 s, for 35 cycles; followed by 72℃ for 2 min. Detection and recovery were performed using 1% agarose gel electrophoresis (Solarbio, Beijing, China).

[0047] Using a mixture of multiple tissue parts of Chinese fir as experimental material, specific primers were used to amplify the sample via PCR. ClPAP7 The full-length CDS sequence of the gene, as determined by 0.8% agarose gel electrophoresis, was between 250 and 500 bp in length, consistent with the target gene. ClPAP7 The lengths are basically the same at 402bp, indicating that the target gene has been successfully cloned. Figure 4 ).

[0048] 2. Construction of overexpression vectors and plasmid extraction Using *Cunninghamia lanceolata* cDNA as a template, the target fragment CDS-ClPAP7-F / CDS-ClPAP7-R was amplified using specific primers ClPAP7-F / R. After electrophoresis, the fragment was purified and recovered for later use. The plant expression vector pCambia3301, with its promoter modified, was used. The promoter in pCambia3301 was replaced with the CaMV 35S promoter (labeled 35S::DsRed). This vector contains a BamHI restriction endonuclease recognition site, and linearization was performed using BamHI (Lambolid, Beijing). The digestion products were separated by 1% agarose gel electrophoresis (SolarBio, Beijing), and the target band was excised and purified.

[0049] Using in-fusion ligase (TransGen), the PCR product containing restriction enzyme sites, recovered from gel electrophoresis, was ligated with the linear pCambia3301 vector containing the BamHI site to obtain the 35S::ClPAP7-GFP overexpression vector. The ligation product of the constructed 35S::ClPAP7-GFP overexpression vector was transformed into *E. coli* DH5α competent cells for amplification. Positive clones were selected using kanamycin, and single colonies were selected for PCR verification. Figure 5 Deletion and sequencing were performed. Strains with correct sequencing results were selected for amplification culture, and then recombinant plasmids were extracted.

[0050] CaMV 35S promoter sequence (SEQ ID NO:6): 5’-AGAGATAGATTTGTAGAGAGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCAATACGC-3’.

[0051] Example 3: Subcellular localization of ClPAP7 protein 1. The plasmid extracted in step 2 of Example 1 was transferred into Agrobacterium GV3101. 1 μg of plasmid DNA was added to every 100 μL of competent Agrobacterium cells. After mixing, the cells were sequentially placed on ice for 5 min, flash-frozen in liquid nitrogen for 60 s, and then incubated in a 37°C metal bath for 90 s. YEB liquid medium containing rifampicin and kanamycin was then added, and the cells were incubated at 28°C and 220 rpm for 4 h. The bacterial culture was centrifuged at 4°C and 5000 rpm for 10 min to collect the cells. After resuspending in sterile water, 50 μL was plated onto YEB solid plates containing antibiotics and cultured overnight at 28°C inverted mode to obtain Agrobacterium GV3101 containing the 35S::ClPAP7-GFP overexpression vector.

[0052] 2. Select single colonies for colony PCR verification. Inoculate positive clones into 1 mL of YEB liquid medium containing double antibodies and incubate at 28°C and 200 rpm for 8 h until OD500. 600 The OD value reached 0.6, and the culture was transferred to 7 mL of liquid medium containing YEB anti-YEB. The culture was incubated at 28°C with shaking for 16 h until the bacterial suspension turned bright orange-yellow. The cells were collected by centrifugation at 5000 rpm for 10 min. The cells were washed once with 1 M MgCl2 solution, and then resuspended in tobacco infection solution. An equal volume of infection solution was used as a control. 600 Adjust the pH to 0.6-0.8, and let stand at room temperature in the dark for 3 hours to induce gene expression.

[0053] 3. Select healthy young tobacco leaves, make a cut on the back of the leaf with a syringe needle, and then use a needleless syringe to inject the wound... ClPAP7 Agrobacterium GV3101 bacterial suspension containing the target gene vector was injected into the wound and the injection area was labeled. The mixture was first incubated in the dark for 24 hours, then transferred to a light incubator for another 24 hours. Leaf sections approximately 1 cm × 1 cm from the injection site were prepared, and the GFP fluorescence signal was observed using a laser confocal microscope.

[0054] 4. Observe the leaves of tobacco infected with Agrobacterium GV3101 under a laser confocal microscope. The results are as follows: Figure 6 As shown, the unloaded GFP has a fluorescent signal throughout the cell, while ClPAP7-GFP only shows a strong fluorescent signal in the cytoplasm and nucleus, indicating that the ClPAP7 protein is localized in the nucleus and cytoplasm.

[0055] Example 4: Poplar genetic transformation and screening identification Agrobacterium GV3101 containing the 35S::ClPAP7-GFP overexpression vector obtained in Example 3 was used to transform 35S::ClPAP7-GFP into Populus tomentosa 84K using the leaf disc transformation method. The specific steps are as follows: 1. Plant material preparation: Select tender leaves of tissue culture seedlings that have been subcultured for one month as explants. Use a sterile blade in a clean bench to remove the edges and petioles, cut into small pieces, make small wounds along the veins, and then place them in co-culture medium for pre-culture for 2 days.

[0056] 2. Genetic transformation: Agrobacterium glycerol containing recombinant plasmids was taken from -80℃ and streaked onto agar plates containing rifampicin and kanamycin. After incubation upside down, single colonies were picked and shaken, then centrifuged to collect the bacterial resuspension. Pre-cultured leaves were immersed in the bacterial solution for 15-20 minutes, then removed and excess solution was absorbed with sterile filter paper. The leaves were then placed face down on co-culture medium and incubated in the dark for 2-3 days. After co-culture, the leaves were transferred to differentiation and selection medium under low light conditions, with fresh medium changed every 10 days until resistant adventitious shoots appeared. When the adventitious shoots reached approximately 2 cm in length, they were transferred to rooting medium containing Tim and Hyg to induce rooting.

[0057] 3. Screening and Identification: After obtaining resistant regenerated plants, fresh leaves from wild-type and overexpressing poplar lines were cut, labeled, wrapped in aluminum foil, and stored in liquid nitrogen. RNA was extracted from the thoroughly ground samples and reverse transcribed into cDNA. PCR was used for molecular identification of transgenic plants to screen for positively transformed seedlings. Figure 7 ) Analysis of different strains by qRT-PCR ClPAP7 Gene expression levels. (Selection) ClPAP7 The strain with the highest expression level was used for subsequent subculture. During subculture, the leaf tips were selected, and 1-2 leaves were retained. The strains were then cultured in rooting medium supplemented with Tim and Hyg.

[0058] After positive identification screening, selected ClPAP7 The transgenic Populus tomentosa line with the highest expression level and wild-type plants were asexually propagated and used as materials for subsequent experiments.

[0059] Example 5: ClPAP7 Low phosphorus treatment of poplar overexpression Select transgenic plants with consistent growth status (obtained in Example 4) ClPAP7 Poplar (overexpressing) and wild-type plants were treated for 30 days in two different phosphorus-containing liquid culture media with normal phosphorus supply (NP, 1.25 mmol / L) and low phosphorus (LP, 0.125 mmol / L), respectively, and then the relevant physiological and biochemical indicators of the plants were measured.

[0060] 1. ClPAP7Biomass and morphological parameters of overexpressed poplar trees were determined as follows: Wild-type and transgenic Populus tomentosa plants treated for 30 days were removed from the culture medium, their roots were washed, and samples were collected from both aboveground and underground parts. Fresh weight was measured using an analytical balance. The samples were blanched at 105℃ for 30 min, then dried in a 65℃ forced-air drying oven to constant weight. Dry weight of both aboveground and underground parts was measured. For morphological parameters, leaf area of ​​the third functional leaf and plant height were measured using a leaf scanner, and root length and root surface area were analyzed using a root scanner.

[0061] 2. ClPAP7 Determination of phosphorus content in overexpressed poplar: The dried sample was ground and passed through a 100-mesh sieve. 0.2g of sample was placed in a microwave digestion tube, 7mL of nitric acid and 1mL of H2O2 were added, and microwave digestion was performed according to the following program: 120℃-5min, 140℃-5min, 160℃-20min, 170℃-20min, 180℃-20min. After digestion, the sample was left to stand in a fume hood with the lid open until no yellow fumes were emitted. After cooling, the sample was filtered through phosphorus-free filter paper into a 50mL volumetric flask and diluted to volume for analysis. Molybdenum-antimony colorimetric reagent: 3g of ascorbic acid was dissolved in 200mL of molybdenum-antimony reagent stock solution. Dinitrophenol indicator: 0.2g of 2,4-dinitrophenol was dissolved in 100mL of distilled water. Preparation of phosphorus standard stock solution: Dissolve 0.2195 g of KH₂PO₄ dried at 105℃ for 2 h in 200 mL of distilled water, add 5 mL of concentrated sulfuric acid, and dilute to 1 L (concentration 50 μg / mL) to obtain a 5 μg / mL standard working solution. Take 0-12 mL of the standard working solution sequentially into a 50 mL volumetric flask, add distilled water to half-full volume, add 2 drops of dinitrophenol indicator, adjust to a pale yellow color with 2 mol / L NaOH, add 2.5 mL of molybdenum antimony colorimetric reagent, dilute to volume, and let stand at room temperature for 30 min. Measure the absorbance at 710 nm to establish a standard curve. Treat samples using the same method and measure absorbance. Calculate the total phosphorus content of each tissue according to the standard curve.

[0062] 3. Results Analysis After 30 days of phosphorus treatment, under normal phosphorus supply (NP) conditions, there were no significant phenotypic differences between wild-type (WT) and overexpression lines (OE), and the plants exhibited vigorous growth and comparable vigor (see [link to article]). Figure 8 (A). When faced with low phosphorus (LP) stress, WT plants showed significant growth inhibition, with a substantial reduction in plant height and poor leaf development; while OE lines maintained higher plant height and vigorous leaf growth, with overall growth significantly better than WT (see A). Figure 8 (B) indicates overexpression ClPAP7 It can effectively alleviate the inhibitory effect of low phosphorus stress on plant growth.

[0063] Plant height measurement results are as follows Figure 9As shown in Figure A, under normal phosphorus supply (NP) conditions, there was no significant difference in plant height between wild-type (WT) and overexpression lines (OE). Low phosphorus (LP) treatment significantly inhibited the growth of WT plants, resulting in a substantial decrease in plant height compared to NP conditions. However, OE lines under LP treatment maintained a similar plant height to those under NP conditions, and were significantly taller than WT lines under LP treatment. Leaf area measurements are shown in... Figure 9 In the B group, under NP conditions, there was no significant difference in leaf area between WT and OE. LP treatment significantly reduced the leaf surface area of ​​WT, while the leaf area of ​​OE lines decreased slightly, but was not significantly different from that under NP conditions, and was significantly higher than that of WT under LP treatment. These results indicate that overexpression... ClPAP7 It can enhance the plant's tolerance to low phosphorus stress, maintain normal plant height growth, and provide the leaf area required for photosynthesis.

[0064] Results of fresh and dry weight measurements of aboveground parts are as follows: Figure 10 As shown in the figure. Under normal phosphorus supply (NP) conditions, there was no significant difference in aboveground fresh weight between wild-type (WT) and overexpression lines (OE). Under low phosphorus (LP) treatment, the aboveground fresh weight of WT was significantly lower than that under NP treatment, while OE maintained a higher aboveground fresh weight under LP treatment, which was significantly higher than that of WT under the same treatment. The aboveground dry weight of WT and OE did not differ significantly under NP conditions, but LP treatment significantly reduced the dry weight of both, although the reduction was greater for WT. In terms of belowground fresh and dry weight, LP treatment significantly promoted the accumulation of belowground biomass in both WT and OE, and the belowground fresh and dry weight of OE was significantly higher than that of WT under LP conditions. These results indicate that overexpression... ClPAP7 The plant can maintain a stronger biomass accumulation capacity under low phosphorus stress by optimizing its biomass allocation.

[0065] Root morphology index measurement results as follows Figure 11 As shown in the figure, under normal phosphorus supply (NP) conditions, there were no significant differences in root surface area, root volume, and total root length between wild-type (WT) and overexpression lines (OE). Low phosphorus (LP) treatment significantly inhibited root growth in WT, with root surface area, root volume, and total root length all significantly reduced compared to NP conditions; while root growth in OE lines was not significantly affected by LP treatment, with all indicators significantly higher than those of WT under LP treatment, and no significant difference compared to their own NP-condition levels. This indicates that overexpression... ClPAP7 It can effectively maintain root growth of plants under low phosphorus stress and enhance nutrient absorption capacity.

[0066] The results of the net photosynthetic rate measurement are as follows: Figure 12As shown, under normal phosphorus supply (NP) conditions, the net photosynthetic rates of wild-type (WT) and overexpression lines (OE) did not differ significantly and remained at a high level. Low phosphorus (LP) treatment reduced the net photosynthetic rates of both lines, but the net photosynthetic rate of OE was significantly higher than that of WT under the same treatment conditions, and the decrease was significantly smaller than that of WT. This indicates that overexpression... ClPAP7 It can alleviate the inhibitory effect of low phosphorus stress on the photosynthetic capacity of plants and maintain the photosynthetic efficiency of leaves.

[0067] Phosphorus content determination results are as follows Figure 13 As shown, under normal phosphorus supply (NP) conditions, the phosphorus content in both the aboveground and underground parts of the OE strain was significantly lower than that of the wild type (WT). Under low phosphorus (LP) treatment, the phosphorus content in the aboveground parts of the OE strain was significantly higher than that of the WT strain, while the phosphorus content in the underground parts was not significantly different from that of the WT strain. LP treatment significantly reduced the phosphorus content in both the aboveground and underground parts of the WT and OE strains, with a more significant reduction in the WT strain.

[0068] Phosphorus utilization efficiency (PUE) measurement results are as follows Figure 14 As shown in the figure, under normal phosphorus supply (NP) conditions, the PUE of the overexpression line (OE) was significantly higher than that of the wild type (WT). Under low phosphorus (LP) treatment, the PUE of both WT and OE was significantly increased compared to the NP conditions, and there was no significant difference between the two. The above results indicate that overexpression... ClPAP7 Under normal phosphorus supply, the OE strain tends to achieve higher biomass output with lower phosphorus accumulation, exhibiting higher phosphorus use efficiency; while under low phosphorus stress, the OE strain maintains a higher biomass accumulation level by enhancing phosphorus absorption and utilization.

[0069] Example 6: Expression and detection of ClPAP7 protein 1. Construction of ClPAP7 prokaryotic expression vector After removing the start and stop codons of the eGFP gene, restriction enzyme sites SacI and XhoI, which match the multiple cloning site of the pCold TF vector, were introduced at both ends of the gene. The PCR product and the vector were then treated with double enzyme digestion, ligated with ligase, transformed into competent E. coli cells, and recombinants were obtained by ampicillin resistance selection. Finally, colony PCR and sequencing verification were performed to ensure that the insertion direction and sequence of the eGFP gene were completely correct, thus successfully constructing the pCold-TF-eGFP vector.

[0070] The enhanced green fluorescent vector pCold-TF-eGFP with a GFP tag was selected as a template, linearized with NdeI as the restriction enzyme site, incubated at 37℃ for 2 h for restriction enzyme digestion, then inactivated at 80℃ for 20 min, purified and recovered to obtain the purified linearized vector.

[0071] Amplification of Chinese fir using specific primers ClPAP7 The gene, and the specific primers used preferably include PcoldTF-GFP:ClPAP7-F and PcoldTF-GFP:ClPAP7-R, wherein: PcoldTF-GFP:ClPAP7-F (SEQ ID NO:7): 5'-TGGTGGTATCGAAGGTAGGCATATGATTGCTTTGTTGGTTATATTGTGCT-3'; PcoldTF-GFP:ClPAP7-R (SEQ ID NO:8): 5'-TGAACAGCTCCTCGCCCTTGCTCACCATACACCATCTGCGATCCCGC-3'; after amplification, the following results are obtained. ClPAP7 Fragment. The purified linearized vector was then combined with... ClPAP7 The fragments were ligated using a one-step cloning method. The recombinant product was introduced into DH5α competent cells and plated on LB agar plates containing ampicillin. The plates were incubated overnight at 37°C with the plates inverted. Single colonies were picked and inoculated into LB liquid medium containing ampicillin, and cultured at 37°C and 220 rpm for 6 hours. Preliminary screening was performed by colony PCR. Figure 15 Positive clones were sent to Qingke Biotechnology for sequencing. After the sequence was confirmed to be correct, the recombinant plasmid was extracted.

[0072] 2. Expression and purification of ClPAP7 protein Escherichia coli BL21(DE3) containing the recombinant plasmid was inoculated at a 1:20 volume ratio into 10 mL of LB broth containing 50 mg / L ampicillin and cultured overnight at 37°C and 220 rpm. Subsequently, the culture was transferred at a 1:100 volume ratio to 200 mL of LB broth containing antibiotics and cultured at 37°C and 220 rpm until the OD reached 0.6–0.8. One mL of the bacterial culture was used as a pre-induction control. The remaining bacterial culture was pre-chilled on ice for 30 min and then induced with IPTG under optimized conditions. The bacterial culture was transferred to centrifuge tubes and centrifuged at 4°C and 4000 rpm for 20 min. The supernatant was discarded, and the bacterial pellet was collected. Resuspend the bacterial cells in 4 mL of non-denaturing lysis buffer per gram of precipitate, add lysozyme to a final concentration of 1 mg / mL, mix well, and incubate on an ice-water bath for 30 min. Then, sonicate on ice for lysis (200-300 W, 30 min). Centrifuge the lysis buffer at 4°C and 10,000 rpm for 30 min, collect the supernatant, and place it in an ice-water bath. Purify the supernatant using a His-tagged protein purification kit (Beyotime), collect the washing buffer and eluent, and store at -80°C for later use after SDS-PAGE and Western blot analysis.

[0073] 3. ClPAP7 protein detection The purity of the purified pCold::ClPAP7-GFP recombinant protein was analyzed by SDS-PAGE and Western blot.

[0074] 3.1 SDS-PAGE: Fix the SDS-PAGE gel glass plate containing 10% separating gel to the electrophoresis tank. Add electrophoresis buffer to both the inner and outer tanks, ensuring the liquid level in the inner tank exceeds the notch of the concave plate. Thaw the purified protein on ice, transfer 20 μL to a 1.5 mL centrifuge tube, add 4× loading buffer, mix well, and heat in a water bath for 10 min to denature. Cool before use. Add protein standards and samples sequentially to the bottom of the concave sample well of the gel, loading 16 μL of sample into each well. Fill the remaining wells with 4× loading buffer.

[0075] Connect the electrophoresis apparatus to the positive and negative electrodes and start electrophoresis. Stop when bromophenol blue reaches the bottom of the gel. After electrophoresis, remove the glass plate, pry it open, remove the gel, cut off the excess, mark the corners, and place it in Coomassie Brilliant Blue staining solution. Stain on a shaker for 1-2 hours, recover the staining solution, wash with destaining solution (250 mmol / L KCl), and then soak in destaining solution on a shaker overnight until the bands are clear. Observe on a gel electrophoresis apparatus. The products are detected by 10% SDS-PAGE. Figure 16 The results showed that the purified fusion protein was a single band located between 75-95 kDa, which was consistent with the target band size of 94 kDa.

[0076] 3.2 Western blot analysis: After electrophoresis, excess gel was removed and soaked in transfer buffer. The PVDF membrane was cut to the appropriate size according to the gel dimensions and activated with methanol for 2-3 min. The sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad were stacked from cathode to anode, and the transfer clamps were tightened. The membrane was then placed in a semi-dry transfer apparatus containing transfer buffer and transferred for 90 min at a constant voltage of 100V and a constant current of 200mA under ice bath conditions. After transfer, the PVDF membrane was removed, rinsed with 1×TBST for 15 min, blocked with 5% skim milk powder solution at room temperature for 3-4 h, and then washed with 1×TBST. A 1:1000 dilution of mouse GFP-tag monoclonal antibody was added, and the membrane was incubated overnight at 4℃ on a shaker, followed by washing with 1×TBST. The developing and fixing solutions were mixed 1:1 and added dropwise to the protein side of the PVDF membrane. The membrane was then exposed using a chemiluminescence imaging system, and the images were recorded. Western blot experiments were performed using GFP as a tag for detection. Figure 17 The results showed specific bands between 70-100 kDa, indicating that the purified protein was ClPAP7 with a GFP tag and had high purity, which could be used for subsequent enzymatic property analysis.

[0077] Example 7: Analysis of enzyme kinetic properties After thawing ClPAP7 protein stored at -80℃ on ice, the protein concentration was determined using a differential spectrophotometer (Nano-300).

[0078] (1) Optimal reaction pH and pH stability To determine the optimal pH of ClPAP7, different buffer systems and pH gradients were set up: Gly / HCl buffer (pH 3.0, 3.5, 4.0, 4.5), NaAc buffer (pH 4.5, 5.0, 5.5, 6.0), and Tris-HCl buffer (pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0).

[0079] Prepare a 50 mmol / L reaction buffer containing 5 mmol / L MgCl2 and 10 mmol / L p-NPP, and adjust to different pH values. Add 100 μL of the reaction buffer to each well of an ELISA plate, then add 10 ng of ClPAP7 protein, incubate at 37°C for 30 min, and terminate the reaction with 100 μL of 1 mol / L NaOH. Measure the OD using an ELISA reader. 410 Absorbance was used to calculate enzyme activity based on the p-NP standard curve, thereby determining the optimal pH. Results are as follows: Figure 18 As shown, the optimal pH for the ClPAP7 protein reaction is 4.5.

[0080] (2) Optimal reaction temperature and thermal stability The optimal reaction temperature for ClPAP7 was determined under optimal pH conditions. 100 μL of reaction mixture (50 mmol / L NaAc, 5 mmol / L MgCl2, 10 mmol / L p-NPP, pH 4.5) and 10 ng ClPAP7 protein (the blank control was replaced with an equal volume of elution buffer) were added to an eight-tube PCR apparatus. The mixture was incubated at 25℃, 30℃, 35℃, 37℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, and 75℃ for 30 min, respectively. The reaction was terminated by adding 100 μL of 1 mol / L NaOH. OD was measured using a microplate reader. 410 Absorbance was used to calculate enzyme activity based on the p-NP standard curve, thereby determining the optimal pH. Results are as follows: Figure 19 As shown, within the temperature range of 20℃-70℃, the activity of ClPAP7 enzyme gradually increases with increasing temperature, with the optimal reaction temperature being 70℃. As the temperature continues to rise, the enzyme activity decreases significantly; at 80℃, the enzyme activity is only 50% of that at 70℃.

[0081] (3) Substrate specificity assay The substrate specificity of ClPAP7 was evaluated by measuring the amount of inorganic phosphorus (Pi) released in the enzymatic reaction. Reaction solutions containing 10 mmol / L of different phosphorus-containing organic compounds—glucose-6-phosphate (G6P), fructose-6-phosphate (F-6-P), guanosine monophosphate (GMP), sodium pyrophosphate (Na4P2O7), p-nitrophenyl phosphate (p-NPP), adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP)—were prepared. The reaction system contained 50 mmol / L NaAc and 5 mmol / L MgCl2, and the pH was adjusted to 4.5. 100 μL of each reaction solution was added to the wells of an ELISA plate, along with 10 ng of ClPAP7 protein per well, and incubated at 37°C for 30 min. After the reaction, the product was diluted, reacted with malachite green chromogenic reagent, and the OD was measured using an ELISA reader. 605 The absorbance was used to calculate the amount of inorganic phosphorus released according to the standard curve, and then the enzyme activity of ClPAP7 for different substrates was calculated. Under optimal temperature (70℃) and optimal pH (4.5) conditions, the decomposition ability of ClPAP7 for different substrates was determined, and the relative enzyme activity was calculated with p-NPP substrate enzyme activity as 100%. Figure 20 The results showed that ClPAP7 exhibited high activity towards nucleotide substrates such as NADP, ADP, and ATP. Considering its optimal temperature of 70℃ and optimal pH of 4.5, it is speculated that ClPAP7 may participate in nucleotide-related metabolic processes (such as nucleotide hydrolysis and energy metabolism) under high-temperature and acidic conditions.

[0082] Therefore, this invention is the first to screen and clone key functional genes for low phosphorus stress response from Chinese fir. ClPAP7 This study systematically elucidates the basic characteristics, enzymatic functions, and low-phosphorus tolerance regulatory molecular mechanisms of this gene and its encoded protein, effectively filling a gap in the research field of low-phosphorus tolerance functional genes in Chinese fir, and possessing significant theoretical and industrial application value. This invention addresses the production challenges of acidic red soil with available phosphorus deficiency and soil phosphorus degradation due to multiple generations of continuous planting in the main Chinese fir producing areas of southern my country, identifying and obtaining highly conserved genes... ClPAP7 The gene encodes a hydrophilic acid phosphatase located in the cell nucleus and cytoplasm, which specifically responds to low phosphorus stress and exhibits significantly upregulated expression, providing a novel endogenous gene resource for molecular improvement of low phosphorus tolerance in forest trees. Enzymatic characterization confirmed that this protein can efficiently hydrolyze nucleotide substrates under acidic conditions, participating in the intracellular phosphate decomposition, metabolism, and recycling of plants, thus clarifying its functional mechanism of phosphorus activation and utilization. Heterologous overexpression functional experiments confirmed that overexpression... ClPAP7This invention can significantly improve the growth status of trees under low phosphorus stress, effectively increasing core root indicators such as total root length, root surface area, root volume, and root biomass, alleviating the inhibitory effect of low phosphorus on plant growth, maintaining the photosynthetic physiological stability of plants, significantly improving the net photosynthetic rate of trees under low phosphorus conditions, and enhancing the adaptability of trees to barren soils. Furthermore, this invention includes the development of specific amplification primers and overexpression vectors, forming a complete gene cloning and application technology system. This provides mature technical support for cultivating new low-phosphorus tolerant tree germplasm and offers important theoretical basis and practical pathways for efficient soil phosphorus utilization, forest productivity improvement, and stress-resistance breeding in Southern Chinese fir plantations.

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gene encoding purple acid phosphatase from Chinese fir. ClPAP7 Genes, characterized by, The ClPAP7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

1.

2. As described in claim 1 ClPAP7 Genes, characterized by, The ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. The claim 1 or 2 ClPAP7 Application of genes in improving the tolerance of trees to low phosphorus stress and / or in breeding low phosphorus stress tolerant tree varieties.

4. Overexpression ClPAP7 The application of gene-based biomaterials in improving the tolerance of forest trees to low phosphorus stress and / or cultivating low phosphorus stress-tolerant forest tree varieties is characterized by, The ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

5. The application according to claim 4, characterized in that, The overexpression ClPAP7 Biological materials for gene overexpression include: ClPAP7 Gene amplification primers, overexpression... ClPAP7 Recombinant vectors of genes, overexpression of the aforementioned ClPAP7 Recombinant microorganisms of genes, overexpression of the aforementioned ClPAP7 Transgenic cell lines and overexpression of the gene ClPAP7 Any one or more of the genes in the transgenic plant tissue.

6. The application according to claim 5, characterized in that, The amplification primers include forward primers and reverse primers; The nucleotide sequence of the forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

4.

7. The application according to claim 5, characterized in that, The basic framework of the recombinant vector includes the pCambia3301 vector; the basic microorganism of the recombinant microorganism includes Agrobacterium.

8. A method for improving the tolerance of forest trees to low phosphorus stress, characterized in that, Includes the following steps: Overexpression of the substance as described in claim 1 or 2 in target forest trees ClPAP7 Gene.

9. A method for cultivating tree varieties tolerant to low phosphorus stress, characterized in that, Includes the following steps: Overexpression of the substance as described in claim 1 or 2 in target forest trees ClPAP7 Gene.

10. A poplar tree resistant to low phosphorus stress, characterized in that, The genome of the low phosphorus stress-resistant poplar ClPAP7 The gene is overexpressed; The ClPAP7 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

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