CbuLBD9 gene for regulating nitrogen absorption and utilization of catalpa bungei and application of CbuLBD9 gene

Abstract

The invention provides a CbuLBD9 gene for regulating nitrogen absorption and utilization of catalpa bungei and application of the CbuLBD9 gene, and belongs to the technical field of forest tree genetic engineering breeding. The nucleotide sequence of the CbuLBD9 gene disclosed by the invention is as shown in SEQ ID No. 1, and the amino acid sequence coded by the CbuLBD9 gene is as shown in SEQ ID No. 2. By silencing the CbuLBD9 gene, the elongation and growth of the root system of the catalpa bungei can be obviously promoted, and the absorption and utilization of the plant root system to nitrogen can be improved. The invention provides an important candidate gene for genetic improvement related to efficient nitrogen utilization of catalpa bungei, and has a wide application prospect in the aspects of forest tree genetic engineering breeding and catalpa bungei clonal development.

Description

Technical Field

[0001] This invention relates to the field of forest tree genetic engineering breeding technology, and more particularly to a method for regulating nitrogen absorption and utilization in catalpa trees. CbuLBD9 Genes and their applications. Background Technology

[0002] Catalpa ( Catalpa bungei *Catalpa macrocarpa* (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 nitrogen absorption and utilization by its root system during growth is one of the key factors restricting its rapid growth and high yield. Nitrogen is a macronutrient essential for plant growth and development, and the root system, as the main organ for nutrient absorption, directly affects the plant's ability to acquire nitrogen. Therefore, regulating root development and optimizing root architecture through genetic means is an important way to improve the nitrogen utilization efficiency of 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 method for regulating the root system architecture and nitrogen absorption and utilization of Catalpa trees. CbuLBD9 This invention, for the first time, identifies and functionally validates a key gene from Catalpa trees that can significantly affect root architecture and nitrogen uptake. CbuLBD9 Silencing this gene can effectively promote the elongation of the catalpa root system, enhance the plant's ability to absorb nitrate nitrogen and the activity of related enzymes, providing new gene resources and technical means for high-efficiency nitrogen breeding of catalpa, and solving the problem of low nitrogen absorption and utilization efficiency of catalpa.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for regulating nitrogen absorption and utilization in catalpa trees. CbuLBD9 Genes, the ones mentioned CbuLBD9 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] This invention also provides the aforementioned method for regulating nitrogen absorption and utilization in catalpa trees. CbuLBD9 A gene-encoded protein, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0007] The present invention also provides a method for silencing or knocking down the device as described in 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.

[0008] The present invention also provides CbuLBD9 Gene silencing expression vector.

[0009] The present invention also provides the aforementioned 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 214 bp 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 vector.

[0010] Preferred, amplification CbuLBD9 Gene-specific primers are shown in SEQ ID NO.3 and SEQ ID NO.4; The PagPB-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.

[0011] A method for improving nitrogen absorption and utilization in plants, characterized in that the aforementioned CbuLBD9 Transform plants with gene silencing expression vectors to obtainCbuLBD9 Silent transgenic plants are used to improve root elongation and nitrogen absorption and utilization.

[0012] Preferably, the transformation is mediated by Agrobacterium tumefaciens.

[0013] Preferably, the plant includes the catalpa tree.

[0014] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention described 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 invention significantly promotes root elongation in Catalpa bungei and improves the plant's nitrogen absorption and utilization efficiency. It provides an important candidate gene for genetic improvement related to efficient nitrogen utilization in Catalpa bungei and has broad application prospects in forest tree genetic engineering breeding and the development of Catalpa bungei clones. Attached Figure Description

[0015] Figure 1 for CbuLBD9 Full-length cDNA cloning results, M: DL5000 marker; lane 1 uses catalpa DNA as a template. CbuLBD9 Image showing the cloning results of the full-length cDNA of the gene.

[0016] Figure 2 for CbuLBD9 DNA and RNA identification results of gene silencing and overexpression lines; where A is... CbuLBD9 DNA identification results of gene silencing lines; B is CbuLBD9 DNA identification results of gene overexpression lines; C is CbuLBD9 Results of RNA expression level identification in gene silencing lines; D is CbuLBD9 Figure showing the results of RNA expression level identification in gene overexpression lines.

[0017] Figure 3 for CbuLBD9 Morphological characteristics of root systems in transgenic and wild-type (WT) Catalpa trees; where A represents... CbuLBD9 Gene silencing, WT and CbuLBD9 Gene overexpression in Catalpa root phenotype; B is CbuLBD9 Gene silencing, WT and CbuLBD9 Statistical results of root length in Catalpa bungei trees with overexpressed genes; C represents... CbuLBD9 Gene silencing, WT and CbuLBD9 Statistical results of fresh weight of Catalpa tree roots.

[0018] Figure 4 for CbuLBD9 Figure showing the results of the determination of the net nitrate nitrogen uptake rate in the roots of transgenic and WT catalpa trees.

[0019] Figure 5 for CbuLBD9 Figure showing the results of nitrate content determination in the roots of genetically modified and WT catalpa trees.

[0020] Figure 6 for CbuLBD9 Figure showing the results of NR activity assays for transgenic and WT Catalpa trees. Detailed Implementation

[0021] The present invention 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.

[0022] SEQ ID No. 1: ATGCGAATAAGTTGTAATGGATGTCGAGTGCTGCGCAAAGGGTGCAGCGATAACTGCAGTATCAGACCGTGTTTGGAATGGATCAAAAACCCCGATTCACAAGCCAACGCCACCGTCTTCCTCGCTAAGTTCTACGGCCGCGCCGGCCTCATGAACCTCATCAATGCTGGGCCGCACCACCTCCGCCCTGCTATTTTTAGGTCTTTACTATACGAGGCTTGCGGGAGGATTGTGAACCCGATATACGGTTCGGTCGGGTTAATGTGGTCGGGAAGCTGGCAGCTCTGCCAGAACGCTGTGGAGGCGGTTCTGAAAGGGGCTCCGATTACCCAAATAGCGAGTGATATTGCTGAGGCAAAAAATGGGCCTCCTCTCAAGGCGTATGATATCAGGCATGTTAACAAGGAAGATAACTCATCCGGGTCGAACGATTTGCACCGGGTCCAAACCCGGTGCCGGTTCAAGCGATCCGGACCGAAGTCGAAAAAGAGTCGGGTCTGTGACGGGTCGGCTGAAGAGACTAGTCACGAGGAGGTGAACCGGTCGCCGAGCCACGAGTCTTCTCTGAGCCACCAGTCTGAGACGGTGGAGCCGGTGGTGGAGAGGGTGAGCCAACAGAGCGAGAGTCTGGGTTCCGCCGATGTGGAAGGTGAACATCTCGTCGGAGTTGAGCCGAATTCGGACTACCGGCGAGCCGATGGTGATGGGATTGAGCTGGATCTCACTCTCGGCTTCGAGCCAATTAAACGCTGTGTGAAGAGGAAAGAATTGAAGCGGAGGGAAGAAGAAGGTGGATTTTGTGGAATGGAGCTATGGCTTGATTATTCGGCTGAGAGATTGATTGTCGGCTCAGATCAATCGCTTCATTGA. SEQ ID No.2: MRISCNGCRVLRKGCSDNCSIRPCLEWIKNPDSQANATVFLAKFYGRAGLMNLINAGPHHLRPAIFRSLLYEACGRIVNPIYGSVGLMWSGSWQLCQNAVEAVLKGAPITQIASDIAEAKNGPPLKAYDIRHVNKEDNSSGSNDL HRVQTRCRFKRSGPKKSKKSRVCDGSAEETSHEEVNRSPSHESSLSHQSETVEPVVERVSQQSESLGSADVEGEHLVGVEPNSDYRRADGDGIELDLTLGFEPIKRCVKRKELKRREEEGGFCGMELWLDYSAERLIVGSDQSLH. 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.

[0023] Example 1. Catalpa tree CbuLBD9 Gene cloning and expression vector construction 1. Catalpa tree CbuLBD9 Cloning of genes 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.

[0024] CbuLBD9-F: 5'-ATGCGAATAAGTTGTAATGGATGTCGA-3' (SEQ ID No. 3); CbuLBD9-R: 5'-TCAATGAAGCGATTGATCTGAGC-3' (SEQ ID No. 4); 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.

[0025] 2. CbuLBD9 Construction of gene knockout vector 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: Specific primers for homologous recombination cloning were designed and synthesized. The primer sequences are as follows: CbuLBD9-Cis-F: 5'-ATTTACAATTACCATGGGGCGCCATGCGAATAAGTTGTAATGGATGTCG-3' (SEQ ID No. 5); CbuLBD9-Cis-R: 5'-ACATAAGAAATTCTTACACATTTAAATCGTATAGTAAAGACCTAAAAATAGCAGGG-3' (SEQ ID No. 6); CbuLBD9-Anti-F: 5'-GTCAAATTTGCAGGTATTTGGATCCCGTATAGTAAAGACCTAAAAATAGCAGGG-3' (SEQ ID No. 7); CbuLBD9-Anti-R: 5'-CGGGTCTTAATTAACTCTCTAGAATGCGAATAAGTTGTAATGGATGTCG-3' (SEQ ID No. 8); 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.

[0026] 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. 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... CbuLBD9 Gene silencing expression vector.

[0027] 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.

[0028] 3. CbuLBD9 Construction of gene overexpression vectors 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: CbuLBD9-OE-F: 5'-CACGGGGGACTCTTGACATGCGAATAAGTTGTAATGGATGTCG-3' (SEQ ID No. 9); CbuLBD9-OE-R: 5'-TCTCCTTTACTAGTCAGATCTACCATGCCATGAAGCGATTGATCTGAGC-3' (SEQ ID No. 10); Using the correctly sequenced recombinant plasmid pMD19-T-CbuLBD9 as a template, PCR amplification was performed using the primers CbuLBD9-OE-F / R described above, yielding plasmids with homologous arms. CbuLBD9 The gene coding region fragment was verified by agarose gel electrophoresis and then the target band was recovered and purified. 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 vector was linearized using a one-step directional cloning kit (Nearshore Protein Technology Co., Ltd., catalog number: NR005) via homologous recombination. CbuLBD9 The fragments were ligated to a linearized vector to construct a recombinant overexpression plasmid.

[0029] 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.

[0030] Example 2:CbuLBD9 Obtaining and molecularly identifying transgenic catalpa trees 1. Genetic transformation of transgenic catalpa trees Prepared in Example 1 CbuLBD9 Silent and overexpressing Agrobacterium strains were used as transformation materials. First, the bacterial culture was 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.

[0031] 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.

[0032] 2. Molecular identification of transgenic plants 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.

[0033] Primers for silent strains: pFGC5941-jd-F: 5'-AGAGGGGAGGACTTTTGTTATACT-3' (SEQ ID No. 11), pFGC5941-jd-R: 5'-CACATGTCAGAAACATTCTGATGTAC-3' (SEQ ID No. 12); Overexpression strain primers: pCAMBIA1302-jd-F: 5'-CTGACGTAAGGGATGACGCA-3' (SEQ ID No. 13), pCAMBIA1302-jd-R:5'-CTTAGCGAGGAAGACGGTGG-3' (SEQ ID No. 14).

[0034] 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.

[0035] Using CbuActin as an internal reference gene, the following primers were used to detect it by qRT-PCR. CbuLBD9 Gene expression at the transcriptional level; CbuActin upstream primer (internal control): 5'-GATGATGCTCCAAGGGCTGT-3' (SEQ ID No. 15); 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, three strains were successfully screened compared to the wild type (WT). CbuLBD9 Silent lines with significantly reduced expression (CbuLBD9-RNAi2, 3, 5) and 3 CbuLBD9 The overexpression lines with significantly elevated expression (CbuLBD9-OE10, 12, 16) were used for subsequent phenotypic and functional analyses.

[0036] Primers used for silent strains: pFGC5941-CbuLBD9-RT-F: 5'-CTATACGAGGCTTGCGGGAG-3' (SEQ ID No. 17), pFGC5941-CbuLBD9-RT-R: 5'-AATCGTTCGACCCGGATGAG-3' (SEQ ID No. 18); Primers used for overexpression lines: pCAMBIA1302-CbuLBD9-RT-F: 5'-AAAACCCCGATTCACAAGCC-3' (SEQ ID No. 19), pCAMBIA1302-CbuLBD9-RT-R: 5'-AATCGGAGCCCTTTCAGAA-3' (SEQ ID No. 20).

[0037] Ultimately succeeded CbuLBD9The silenced lines with significantly suppressed expression and the overexpression lines with significantly increased expression provide a material basis for subsequent phenotypic and functional analyses.

[0038] Example 3: CbuLBD9 Functional verification of gene regulation of root architecture and nitrogen absorption and utilization in Catalpa bungei 1. Material handling and sample collection Select wild-type (WT) catalpa trees with consistent growth and complete rooting. CbuLBD9 Silent lines (RNAi2, RNAi3, RNAi5) and overexpression lines (OE10, OE12, OE16) were 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 foil bags, flash-frozen in liquid nitrogen, ground into a fine powder using a ball mill, and stored at -80°C for later use.

[0039] 2. Root morphology analysis 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, CbuLBD9 The root length of the silent strains increased significantly. CbuLBD9 The root length of the overexpression lines was significantly reduced. Figure 3 (B) Compared to WT, CbuLBD9 The root fresh weight of the silent strains increased significantly. CbuLBD9 The root fresh weight of the overexpression lines was significantly reduced. Figure 3 (C in the middle).

[0040] 3. Determine the net nitrate nitrogen uptake rate in the roots of transgenic and wild-type plants. 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, CbuLBD9 Silent strain NO3 - The net ion absorption rate is significantly higher than that of WT, while CbuLBD9 The net nitrate uptake rate of the overexpression strain was significantly lower than that of the WT strain.

[0041] (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, CbuLBD9 The nitrate content increases in the three transgenic silent lines were 25.4%, 34.9%, and 36.3%, respectively. CbuLBD9 Overexpression of NO3 in three transgenic lines - The content reduction rates were 37.4%, 32.1%, and 30.6%.

[0042] (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. The results show that, CbuLBD9 The root NR activity of the silent strain was higher than that of the WT strain. CbuLBD9 The NR activity in the roots of overexpressing lines was lower than that in WT lines.

[0043] As can be seen from the above embodiments, the present invention provides a method for regulating nitrogen absorption and utilization in catalpa trees. CbuLBD9 Genes and their applications. Through silencing CbuLBD9 The gene significantly promotes root elongation and increases root biomass in Catalpa bungei, as well as enhancing nitrate uptake rate, nitrate content, and nitrate reductase activity. This confirms... CbuLBD9 This is a key gene that negatively regulates 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.

[0044] 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 gene for regulating nitrogen absorption and utilization of Catalpa bungeana CbuLBD9 characterized in that, The CbuLBD9 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The protein encoded by the gene of claim 1, wherein the protein is capable of regulating nitrogen uptake and utilization in Catalpa bungeana. CbuLBD9 The amino acid sequence of the protein is shown as SEQ ID NO.

2. ​ 3. Silencing or knocking down the claim 1 CbuLBD9 The application of genes in any of the following: (1) in promoting the root elongation growth of the plant of Sorbus pohuashanensis; (2) in improving the nitrogen absorption rate of the root system of Sorbus pohuashanensis; (3) in improving the nitrate content and nitrate reductase activity of the root system of Sorbus pohuashanensis; (4) in the improvement of the germplasm of Sorbus pohuashanensis.

4. A method according to any one of claims 1 to 3 wherein the gene silencing expression vector is a siRNA expression vector. CbuLBD9 gene silencing expression 5. The method of claim 4 wherein the CbuLBD9 A method of constructing a gene silencing expression vector, comprising the steps of: The method comprises the following steps: S1. Amplification using specific primers CbuLBD9 Genes will CbuLBD9 The gene was ligated with pMD19-T to obtain the recombinant plasmid pMD19-T-CbuLBD9; S2. Using the plasmid pMD19-T-CbuLBD9 as a template, a forward fragment of 214 bp is obtained by PCR amplification using primers CbuLBD9-Cis-F / R; S3. The pFGC5941 RNAi vector is double-digested with restriction endonucleases Asc I and Swa I, and the forward fragment is connected to the pFGC5941 RNAi linearized vector through homologous recombination to construct an intermediate recombination vector pM-CbuLBD9-C; S4. Using the pMD19-T-CbuLBD9 as a template, an anti-complementary fragment is amplified using primers CbuLBD9-Anti-F / R; S5. The constructed intermediate vector pM-CbuLBD9-C was double-digested with BamH I and Xba I, and the reverse complementary fragment was ligated into the vector by homologous recombination, i.e. CbuLBD9 Gene silencing expression vector.

6. The method of claim 5, wherein, amplification CbuLBD9 Specific primers for the gene are shown as SEQ ID NO. 3 and SEQ ID NO. 4; The primer sequence of the PagPB-cis-F / R is shown as SEQ ID NO. 5 and SEQ ID NO. 6; The primer sequence of the CbuLBD9-Anti-F / R is shown as SEQ ID NO. 7 and SEQ ID NO.

8.

7. A method for improving nitrogen uptake and utilization in plants, characterized by, The plant of claim 4 is transformed with the gene silencing expression vector CbuLBD9 The plant is transformed with the gene silencing expression vector CbuLBD9 The transgenic plant is silenced to achieve the elongation of root system and the improvement of nitrogen absorption and utilization.

8. The method of claim 7, wherein, The transformation is an Agrobacterium tumefaciens-mediated genetic transformation.

9. The method of claim 7, wherein, The plant comprises Sorbus pohuashanensis.

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