Cbulbd9 gene for regulating nitrogen absorption and utilization of catalpa and application thereof

Abstract

This invention provides a method for regulating nitrogen absorption and utilization in catalpa trees. CbuLBD9 This invention relates to genes and their applications, belonging to the field of forest tree genetic engineering breeding technology. CbuLBD9 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence it encodes is shown in SEQ ID No. 2. This invention utilizes silencing... CbuLBD9 This gene significantly promotes the elongation and growth of the Catalpa tree root system and enhances the absorption and utilization of nitrogen by the plant roots. This invention provides an important candidate gene for genetic improvement related to efficient nitrogen utilization in Catalpa trees, and has broad application prospects in forest tree genetic engineering breeding and the development of Catalpa tree clones.

Description

Technical Field

[0001] This invention relates to the field of forest tree genetic engineering breeding technology, and in particular to a CbuLBD9 gene that regulates nitrogen absorption and utilization in catalpa trees and its application. Background Technology

[0002] Catalpa bungei (CA Mey) is a plant belonging to the genus Catalpa in the family Bignoniaceae. It is an important and valuable timber and landscaping tree species in my country, possessing significant economic and ecological value. However, its limited root system's absorption and utilization of nitrogen during growth is a key factor restricting its rapid growth and high yield. Nitrogen is an essential macronutrient for plant growth and development, and the root system, as the primary organ for nutrient absorption, directly influences the plant's nitrogen acquisition capacity. Therefore, regulating root development and optimizing root architecture through genetic means is an important approach to improving nitrogen utilization efficiency in forest trees.

[0003] Currently, domestic and international researchers' studies on the growth and development of Catalpa trees mainly focus on the above-ground parts, with insufficient attention paid to root growth and development, resulting in a relative lag in research on Catalpa root systems. As people's understanding of the role of forest tree roots increases, a systematic study of Catalpa root development and the molecular mechanisms of nitrogen absorption and utilization has become urgently needed. Summary of the Invention

[0004] In view of this, the present invention provides a CbuLBD9 gene that regulates the root architecture and nitrogen absorption and utilization of Catalpa macrophylla, and its application. This invention is the first to identify and functionally validate a key gene, CbuLBD9, in Catalpa macrophylla that can significantly affect root architecture and nitrogen absorption. Silencing this gene can effectively promote root elongation, enhance the plant's ability to absorb nitrate nitrogen and related enzyme activities, providing a new gene resource and technical means for high-efficiency nitrogen breeding of Catalpa macrophylla, and solving the problem of low nitrogen absorption and utilization efficiency in Catalpa macrophylla.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a CbuLBD9 gene for regulating nitrogen absorption and utilization in Catalpa bungei, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides the protein encoded by the CbuLBD9 gene that regulates nitrogen absorption and utilization in Catalpa bungei, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0008] This invention also provides the use of silencing or knocking down the CbuLBD9 gene as described in claim 1 in any of the following:

[0009] (1) Application in promoting the elongation of the root system of Catalpa trees;

[0010] (2) Application in improving the nitrogen absorption rate of Catalpa tree roots;

[0011] (3) Application in increasing the nitrate content and nitrate reductase activity in the roots of Catalpa trees;

[0012] (4) Application in the germplasm improvement of Catalpa trees.

[0013] The present invention also provides a CbuLBD9 gene silencing expression vector.

[0014] This invention also provides a method for constructing the CbuLBD9 gene silencing expression vector, comprising the following steps:

[0015] S1. The CbuLBD9 gene was amplified using specific primers, and the CbuLBD9 gene was ligated with pMD19-T to obtain the recombinant plasmid pMD19-T-CbuLBD9;

[0016] S2. Using plasmid pMD19-T-CbuLBD9 as a template, PCR amplification was performed using primers CbuLBD9-Cis-F / R to obtain a 214 bp forward fragment;

[0017] S3. The pFGC5941RNAi vector was double-digested with restriction endonucleases Asc I and Swa I. The forward fragment was then ligated into the pFGC5941RNAi linearized vector via homologous recombination to construct the intermediate recombination vector pM-CbuLBD9-C.

[0018] S4. Using pMD19-T-CbuLBD9 as a template, the reverse complementary fragment was amplified using primers CbuLBD9-Anti-F / R;

[0019] S5. The constructed intermediate vector pM-CbuLBD9-C was double-digested with BamHI and XbaI, and the reverse complementary fragment was ligated into the vector through homologous recombination to obtain the CbuLBD9 gene silencing expression vector.

[0020] Preferred specific primers for amplifying the CbuLBD9 gene are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0021] The CbuLBD9-cis-F / R primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6;

[0022] The CbuLBD9-Anti-F / R primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0023] A method for improving nitrogen absorption and utilization in plants is characterized by transforming plants with the CbuLBD9 gene silencing expression vector to obtain CbuLBD9 silent transgenic plants, thereby improving root elongation and nitrogen absorption and utilization.

[0024] Preferably, the transformation is mediated by Agrobacterium tumefaciens.

[0025] Preferably, the plant includes the catalpa tree.

[0026] By adopting the above technical solution, the present invention has the following beneficial effects: The nucleotide sequence of the CbuLBD9 gene described in the present invention is shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2. The present invention, by silencing the CbuLBD9 gene, significantly promotes the elongation growth of the Catalpa tree root system and improves the plant's nitrogen absorption and utilization efficiency. The present invention provides an important candidate gene for genetic improvement related to efficient nitrogen utilization in Catalpa trees, and has broad application prospects in forest tree genetic engineering breeding and the development of Catalpa tree clones. Attached Figure Description

[0027] Figure 1 The image shows the cloning results of the full-length cDNA of the CbuLBD9 gene. M: DL5000 marker; Lane 1 is a diagram showing the cloning results of the full-length cDNA of the CbuLBD9 gene using Catalpa tree DNA as a template.

[0028] Figure 2 The figures show the identification results of DNA and RNA in CbuLBD9 gene silencing and overexpression lines; where A represents the DNA identification results of CbuLBD9 gene silencing lines; B represents the DNA identification results of CbuLBD9 gene overexpression lines; C represents the RNA expression level identification results of CbuLBD9 gene silencing lines; and D represents the RNA expression level identification results of CbuLBD9 gene overexpression lines.

[0029] Figure 3 The image shows the root morphology of CbuLBD9 transgenic and wild-type (WT) Catalpa trees; where A represents the root phenotypes of Catalpa trees with CbuLBD9 gene silence, WT, and CbuLBD9 gene overexpression; B represents the root length statistics of Catalpa trees with CbuLBD9 gene silence, WT, and CbuLBD9 gene overexpression; and C represents the root fresh weight statistics of Catalpa trees with CbuLBD9 gene silence, WT, and CbuLBD9 gene overexpression.

[0030] Figure 4 Figure 1 shows the results of the determination of the net nitrate uptake rate of CbuLBD9 transgenic and WT Catalpa root systems.

[0031] Figure 5 The graph shows the results of nitrate content determination in the roots of CbuLBD9 transgenic and WT catalpa trees.

[0032] Figure 6 The figure shows the results of NR activity assays for CbuLBD9 transgenic and WT Catalpa trees. Detailed Implementation

[0033] The nucleotide sequence of the CbuLBD9 gene described in this invention is shown in SEQ ID No. 1, and the amino acid sequence it encodes is shown in SEQ ID No. 2.

[0034] SEQ ID No. 1:

[0035] ATGCGAATAAGTTGTAATGGATGTCGAGTGCTGCGCAAAGGGTGCAGCGATAACTGCAGTATCAGACCGTGTTTGGAATGGATCAAAAACCCCGATTCACAAGCCAACGCCACCGTCTTCCTCGCTAAGTTCTACGGCCGCGCCGGCCTCATGAACCTCATCAATGCTGGGCCGCACCACCTCCGCCCTGCTATTTTTAGGTCTTTACTATACGAGGCTTGCGGGAGGATTGTGAACCCGATATACGGTTCGGTCGGGTTAATGTGGTCGGGAAGCTGGCAGCTCTGCCAGAACGCTGTGGAGGCGGTTCTGAAAGGGGCTCCGATTACCCAAATAGCGAGTGATATTGCTGAGGCAAAAAATGGGCCTCCTCTCAAGGCGTATGATATCAGGCATGTTAACAAGGAAGATAACTCATCCGGGTCGAACGATTTGCACCGGGTCCAAACCCGGTGCCGGTTCAAGCGATCCGGACCGAAGTCGAAAAAGAGTCGGGTCTGTGACGGGTCGGCTGAAGAGACTAGTCACGAGGAGGTGAACCGGTCGCCGAGCCACGAGTCTTCTCTGAGCCACCAGTCTGAGACGGTGGAGCCGGTGGTGGAGAGGGTGAGCCAACAGAGCGAGAGTCTGGGTTCCGCCGATGTGGAAGGTGAACATCTCGTCGGAGTTGAGCCGAATTCGGACTACCGGCGAGCCGATGGTGATGGGATTGAGCTGGATCTCACTCTCGGCTTCGAGCCAATTAAACGCTGTGTGAAGAGGAAAGAATTGAAGCGGAGGGAAGAAGAAGGTGGATTTTGTGGAATGGAGCTATGGCTTGATTATTCGGCTGAGAGATTGATTGTCGGCTCAGATCAATCGCTTCATTGA.

[0036] SEQ ID No.2:

[0037] MRISCNGCRVLRKGCSDNCSIRPCLEWIKNPDSQANATVFLAKFYGRAGLMNLINAGPHHLRPAIFRSLLYEACGRIVNPIYGSVGLMWSGSWQLCQNAVEAVLKGAPITQIASDIAEAKNGPPLKAYDIRHVNKEDNSSGSNDL HRVQTRCRFKRSGPKKSKKSRVCDGSAEETSHEEVNRSPSHESSLSHQSETVEPVVERVSQQSESLGSADVEGEHLVGVEPNSDYRRADGDGIELDLTLGFEPIKRCVKRKELKRREEEGGFCGMELWLDYSAERLIVGSDQSLH.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1. Cloning of the Catalpa tree CbuLBD9 gene and construction of its expression vector

[0040] 1. Cloning of the CbuLBD9 gene in Catalpa bungei

[0041] Total RNA was extracted from Catalpa root tissue, and cDNA was synthesized by reverse transcription using the extracted RNA as a template. PCR amplification was then performed using the cDNA as a template with specific primers CbuLBD9-F / R.

[0042] CbuLBD9-F: 5'-ATGCGAATAAGTTGTAATGGATGTCGA-3' (SEQ ID No. 3);

[0043] CbuLBD9-R: 5'-TCAATGAAGCGATTGATCTGAGC-3' (SEQ ID No. 4);

[0044] The product was detected by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown. Figure 1 The results showed that a specific band (lane 1) was obtained at approximately 900 bp, which was consistent with the expected target fragment size.

[0045] 2. Construction of CbuLBD9 gene knockout vector

[0046] The amplification products were recovered and ligated into the pMD19-T vector using a cloning kit (Baori Biotechnology Co., Ltd., catalog number: 6013). The ligation method was performed according to the instructions. The positive plasmid pMD19-T-CbuLBD9 was obtained by sequencing verification. The specific steps are as follows:

[0047] Specific primers for homologous recombination cloning were designed and synthesized. The primer sequences are as follows:

[0048] CbuLBD9-Cis-F:

[0049] 5'-ATTTACAATTACCATGGGGCGCCATGCGAATAAGTTGTAATGGATGTCG-3' (SEQ IDNo. ​​5);

[0050] CbuLBD9-Cis-R:

[0051] 5'-ACATAAGAAATTCTTACACATTTAAATCGTATAGTAAAGACCTAAAAATAGCAGGG-3' (SEQ ID No. 6);

[0052] CbuLBD9-Anti-F:

[0053] 5'-GTCAAATTTGCAGGTATTTGGATCCCGTATAGTAAAGACCTAAAAATAGCAGGG-3' (SEQ ID No. 7);

[0054] CbuLBD9-Anti-R:

[0055] 5'-CGGGTCTTAATTAACTCTCTAGAATGCGAATAAGTTGTAATGGATGTCG-3' (SEQ ID No. 8);

[0056] Using plasmid pMD19-T-CbuLBD9 as a template, PCR amplification was performed using primers CbuLBD9-Cis-F / R to obtain a 214 bp forward fragment, which was detected by agarose gel electrophoresis and then purified.

[0057] The vector pFGC5941RNAi (Wuhan Miaoling Biotechnology Co., Ltd., catalog number: P0659) was double-digested with restriction endonucleases Asc I and Swa I at 37℃ for 50 min in a 10 μL volume. Then, using a one-step directional cloning kit (Nearshore Protein Technology Co., Ltd., catalog number: NR005), the forward fragment was ligated into the enzyme-digested linearized vector according to the manufacturer's instructions to construct the intermediate recombinant vector pM-CbuLBD9-C. Using pMD19-T-CbuLBD9 as a template, the reverse complementary fragment was amplified using primers CbuLBD9-Anti-F / R, and the amplified fragment was detected and purified by agarose gel electrophoresis.

[0058] The constructed intermediate vector pM-CbuLBD9-C was double-digested with BamHI and XbaI, and the reverse complementary fragment was ligated into the vector via homologous recombination to finally obtain the CbuLBD9 gene silencing expression vector.

[0059] The silenced plasmid containing the target gene CbuLBD9 was transformed into Agrobacterium EHA105 using the freeze-thaw method. The bacterial culture was then spread on LB solid medium (containing 50 mg / L kanamycin and 25 mg / L rifampin). Single colonies were picked and the bacterial culture was verified by PCR using vector primers. After detection by agarose gel electrophoresis, bacterial cultures with the correct band size were selected, glycerol was added, and the cultures were stored at -80 °C for later use.

[0060] 3. Construction of CbuLBD9 gene overexpression vector

[0061] Specific amplification primers containing the homologous arm of the pCAMBIA1302 vector (Wuhan Miaoling Biotechnology Co., Ltd., catalog number: P0654) were designed and synthesized. The nucleotide sequence is as follows:

[0062] CbuLBD9-OE-F:

[0063] 5'-CACGGGGGACTCTTGACATGCGAATAAGTTGTAATGGATGTCG-3' (SEQ ID No. 9);

[0064] CbuLBD9-OE-R:

[0065] 5'-TCTCCTTTACTAGTCAGATCTACCATGCCATGAAGCGATTGATCTGAGC-3' (SEQ ID No. 10);

[0066] Using the correctly sequenced recombinant plasmid pMD19-T-CbuLBD9 as a template, PCR amplification was performed using the primers CbuLBD9-OE-F / R to obtain the CbuLBD9 gene coding region fragment with homologous arms. The target band was recovered and purified after verification by agarose gel electrophoresis.

[0067] Then, the pCAMBIA1302 empty vector was linearized by single-enzyme digestion with QuickCut™ Nco I restriction enzyme at 37 ℃ for 40 min in a 10 μL volume. Further, the purified CbuLBD9 fragment was ligated to the linearized vector using a one-step directional cloning kit (Nearshore Protein Technology Co., Ltd., catalog number: NR005) via homologous recombination to construct a recombinant overexpression plasmid.

[0068] The overexpression plasmid containing the target gene CbuLBD9 was transformed into Agrobacterium EHA105 using the freeze-thaw method. The bacterial culture was then spread on LB solid medium (containing 50 mg / L kanamycin and 25 mg / L rifampin). Single clones were picked and the bacterial culture was verified by PCR using vector primers. After detection by agarose gel electrophoresis, the recombinant Agrobacterium culture with the correct band size was selected, glycerol was added, and the culture was stored at -80 ℃ for later use.

[0069] Example 2: Obtaining and Molecular Identification of CbuLBD9 Transgenic Catalpa Trees

[0070] 1. Genetic transformation of transgenic catalpa trees

[0071] Using the CbuLBD9 silent and overexpressing Agrobacterium strains prepared in Example 1 as transformation materials, the bacterial culture was first activated and resuspended. Single clones were then picked and inoculated into LB liquid medium containing 50 mg / L kanamycin, 25 mg / L rifampin, and 10 μM acetylsylgenin (AS), and cultured at 28°C with shaking until OD. 600 The value was 1.2. The bacterial cells were collected by centrifugation and the concentration was adjusted to OD using a resuspension (containing 10 mM MgCl2, 10 mM MES, 10 μM AS, pH 5.6). 600 The value was 0.8, and the activation was carried out at room temperature in the dark for 3 hours.

[0072] Then, infection and co-culture were carried out. Catalpa callus tissue was immersed in activated bacterial solution and infected at 28℃ and 120 rpm for 15 min with shaking. After removal, excess bacterial solution was absorbed with sterile filter paper and transferred to co-culture medium (MS + 30 g / L sucrose + 3 g / L gel, pH 5.8) and incubated in the dark at 25℃ for 2 days. Then, screening and regeneration were carried out. The co-cultured callus tissue was transferred to the corresponding screening medium. Silent plants were supplemented with glufosinate-ammonia (1 mg / L), and overexpression plants were supplemented with hygromycin (1.5 mg / L). The medium was changed every three weeks. After the resistant shoots grew, they were cut and transferred to rooting medium to induce the regeneration of complete plants.

[0073] 2. Molecular identification of transgenic plants

[0074] Genomic DNA was extracted from transgenic Catalpa trees using the SDS method, and total RNA was extracted using the Trizol method and reverse transcribed into cDNA. PCR amplification was performed using vector-specific primers, with wild-type plants as negative controls and the corresponding recombinant plasmids as positive controls. Electrophoresis was then performed for detection.

[0075] Primers for silent strains:

[0076] pFGC5941-jd-F: 5'-AGAGGGGAGGACTTTTGTTATACT-3' (SEQ ID No. 11),

[0077] pFGC5941-jd-R: 5'-CACATGTCAGAAACATTCTGATGTAC-3' (SEQ ID No. 12);

[0078] Overexpression strain primers:

[0079] pCAMBIA1302-jd-F: 5'-CTGACGTAAGGGATGACGCA-3' (SEQ ID No. 13),

[0080] pCAMBIA1302-jd-R:5'-CTTAGCGAGGAAGACGGTGG-3' (SEQ ID No. 14).

[0081] The results are as follows Figure 2 A and Figure 2 As shown in B, specific bands of the same size as the positive control were amplified in some strains, which preliminarily proves that the exogenous gene has been integrated into the Catalpa genome.

[0082] Using CbuActin as an internal reference gene, the expression of CbuLBD9 gene at the transcriptional level was detected by qRT-PCR using the following primers;

[0083] CbuActin upstream primer (internal control): 5'-GATGATGCTCCAAGGGCTGT-3' (SEQ ID No. 15);

[0084] The downstream primer for the internal control CbuActin was 5'-TCCATATCATCCCAGTTGCT-3' (SEQ ID No. 16). The results are as follows: Figure 2 C and Figure 2 As shown in D, compared with wild type (WT), three silenced lines with significantly reduced CbuLBD9 expression (CbuLBD9-RNAi2, 3, 5) and three overexpression lines with significantly increased CbuLBD9 expression (CbuLBD9-OE10, 12, 16) were successfully screened for subsequent phenotypic and functional analysis.

[0085] Primers used for silent strains:

[0086] pFGC5941-CbuLBD9-RT-F: 5'-CTATACGAGGCTTGCGGGAG-3' (SEQ ID No. 17),

[0087] pFGC5941-CbuLBD9-RT-R: 5'-AATCGTTCGACCCGGATGAG-3' (SEQ ID No. 18);

[0088] Primers used for overexpression lines:

[0089] pCAMBIA1302-CbuLBD9-RT-F: 5'-AAAACCCCGATTCACAAGCC-3' (SEQ ID No. 19),

[0090] pCAMBIA1302-CbuLBD9-RT-R: 5'-AATCGGAGCCCTTTCAGAA-3' (SEQ ID No. 20).

[0091] Ultimately, we successfully obtained a silent strain with significantly suppressed CbuLBD9 expression and an overexpression strain with significantly increased expression, providing a material basis for subsequent phenotypic and functional analysis.

[0092] Example 3: Functional verification of CbuLBD9 gene regulating root architecture and nitrogen uptake and utilization in Catalpa bungei

[0093] 1. Material handling and sample collection

[0094] Wild-type (WT) Catalpa trees, CbuLBD9 silent lines (RNAi2, RNAi3, RNAi5), and overexpression lines (OE10, OE12, OE16) with consistent growth and complete rooting were selected and hydroponically cultured in LA nutrient solution in an artificial climate chamber. After three weeks of culture, root samples were collected from each line, and their fresh weight was measured and recorded. The samples were quickly sealed in aluminum foil bags, flash-frozen in liquid nitrogen, ground into a fine powder using a ball mill, and stored at -80℃ for later use.

[0095] 2. Root morphology analysis

[0096] Root length and biomass were measured in transgenic and wild-type plants, and the results are as follows: Figure 3 As shown in A, compared to WT, the root length of the CbuLBD9 silenced lines was significantly increased, while the root length of the CbuLBD9 overexpression lines was significantly decreased. Figure 3 (B in the text). Compared to WT, the root fresh weight of CbuLBD9-silenced lines was significantly increased, while the root fresh weight of CbuLBD9-overexpressing lines was significantly decreased. Figure 3 (C in the middle).

[0097] 3. Determine the net nitrate nitrogen uptake rate in the roots of transgenic and wild-type plants.

[0098] Non-invasive micrometer measurement (NMT) was used. White fine roots with uniform growth and a diameter of approximately 1.5 mm were selected and placed in a petri dish containing 10 mL of assay buffer (0.1 mM CaCl2, 2 mM KNO3, pH 6.0) for equilibration for 20 minutes. Afterward, they were transferred to fresh assay solution for detection. Each sample was measured three times. Results are as follows: Figure 4 As shown, NO3 in the CbuLBD9 silent strain - The net ion uptake rate was significantly higher than that of WT, while the net nitrate uptake rate of the CbuLBD9 overexpression line was significantly lower than that of WT.

[0099] (4) To determine the nitrate content in the roots of transgenic and wild-type plants, 0.1 g of sample powder was weighed, 1 mL of deionized water was added, and the mixture was extracted in a water bath at 45℃ for 1 hour. After centrifugation at 5000 × g for 10 minutes, 0.2 mL of the supernatant was taken and measured at a wavelength of 410 nm using the salicylic acid-sulfuric acid method. The results are as follows: Figure 5 As shown, compared to WT, the nitrate content of the three transgenic lines with CbuLBD9 silencing increased by 25.4%, 34.9%, and 36.3%, respectively. The NO3 content of the three transgenic lines with CbuLBD9 overexpression also increased. - The content reduction rates were 37.4%, 32.1%, and 30.6%.

[0100] (5) Using a nitrate reductase activity assay kit, the nitrate reductase activity in the roots of transgenic and wild-type plants was determined, and the results are as follows: Figure 6 As shown in the figure. The results showed that the NR activity of the roots of CbuLBD9-silenced lines was higher than that of WT lines, while the NR activity of the roots of CbuLBD9-overexpressing lines was lower than that of WT lines.

[0101] As demonstrated by the above embodiments, this invention provides a CbuLBD9 gene for regulating nitrogen absorption and utilization in Catalpa bungei and its application. Silencing the CbuLBD9 gene significantly promotes root elongation, increases root biomass, and enhances nitrate uptake rate, nitrate content, and nitrate reductase activity in Catalpa bungei. This confirms that CbuLBD9 is a key gene negatively regulating nitrogen absorption and utilization in Catalpa bungei. This invention provides important gene resources and breeding materials for nitrogen-efficient molecular breeding of Catalpa bungei and other forest trees, and has broad application prospects for reducing fertilizer application and increasing forest yield.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for regulating nitrogen absorption and utilization in catalpa trees CbuLBD9 Genes, characterized by, The CbuLBD9 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method for regulating nitrogen absorption and utilization in catalpa trees as described in claim 1 CbuLBD9 Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Silencing or knocking down the claim 1 CbuLBD9 The application of genes in any of the following: (1) Application in promoting the elongation of the root system of Catalpa trees; (2) Application in improving the nitrogen absorption rate of Catalpa tree roots; (3) Application in increasing the nitrate content and nitrate reductase activity in the roots of Catalpa trees; (4) Application in the germplasm improvement of Catalpa trees.

4. A kind CbuLBD9 Gene silencing expression vector, characterized in that, The CbuLBD9 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CbuLBD9 The method for constructing a gene silencing expression vector includes the following steps: S1. Amplification using specific primers CbuLBD9 Genes will CbuLBD9 The gene was ligated to pMD19-T to obtain the recombinant plasmid pMD19-T-CbuLBD9; S2. Using plasmid pMD19-T-CbuLBD9 as a template, PCR amplification was performed using primers CbuLBD9-Cis-F / R to obtain a 214bp forward fragment; S3. The pFGC5941RNAi vector was double-digested with restriction endonucleases Asc I and Swa I. The forward fragment was then ligated into the pFGC5941RNAi linearized vector via homologous recombination to construct the intermediate recombination vector pM-CbuLBD9-C. S4. Using pMD19-T-CbuLBD9 as a template, the reverse complementary fragment was amplified using primers CbuLBD9-Anti-F / R; S5. The constructed intermediate vector pM-CbuLBD9-C was double-digested with BamHI and XbaI, and the reverse complementary fragment was ligated into the vector via homologous recombination to obtain the desired result. CbuLBD9 Gene silencing expression vectors; The CbuLBD9-cis-F / R primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6; The CbuLBD9-Anti-F / R primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.

8.

5. The claim 4 CbuLBD9 A method for constructing a gene silencing expression vector, characterized in that, Includes the following steps: S1. Amplification using specific primers CbuLBD9 Genes will CbuLBD9 The gene was ligated to pMD19-T to obtain the recombinant plasmid pMD19-T-CbuLBD9; S2. Using plasmid pMD19-T-CbuLBD9 as a template, PCR amplification was performed using primers CbuLBD9-Cis-F / R to obtain a 214bp forward fragment; S3. The pFGC5941RNAi vector was double-digested with restriction endonucleases Asc I and Swa I. The forward fragment was then ligated into the pFGC5941RNAi linearized vector via homologous recombination to construct the intermediate recombination vector pM-CbuLBD9-C. S4. Using pMD19-T-CbuLBD9 as a template, the reverse complementary fragment was amplified using primers CbuLBD9-Anti-F / R; S5. The constructed intermediate vector pM-CbuLBD9-C was double-digested with BamHI and XbaI, and the reverse complementary fragment was ligated into the vector via homologous recombination to obtain the desired result. CbuLBD9 Gene silencing expression vectors; The CbuLBD9-cis-F / R primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6; The CbuLBD9-Anti-F / R primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.

8.

6. The construction method according to claim 5, characterized in that, Amplification CbuLBD9 Gene-specific primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

7. A method for improving nitrogen absorption and utilization in plants, characterized in that, The claim 4 CbuLBD9 Transform plants with gene silencing expression vectors to obtain CbuLBD9 Silent transgenic plants, thereby achieving root elongation and improved root nitrogen absorption and utilization; the plant is Catalpa tree.

8. The method according to claim 7, characterized in that, The transformation was mediated by Agrobacterium tumefaciens.

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