CbuLBD19 gene for regulating and controlling root system development and nitrogen absorption and utilization of catalpa bungei and application of CbuLBD19 gene

Abstract

The invention belongs to the technical field of forest tree genetic engineering breeding, and particularly relates to a CbuLBD19 gene for regulating and controlling root system development and nitrogen absorption and utilization of catalpa bungei and application of the CbuLBD19 gene. The nucleotide sequence of the CbuLBD19 gene is as shown in SEQ ID No.7, and the amino acid sequence coded by the CbuLBD19 gene is as shown in SEQ ID No.8. It is found that overexpression of the CbuLBD19 gene can cause reduction of the total root length and the root biomass of catalpa bungei. Meanwhile, the net absorption rate of nitrate ions in the root system, the activity of nitrate reductase (NR) and the content of amino acid are reduced, and the comprehensive expression is that nitrogen metabolism is reduced. The negative regulation and control function of the CbuLBD19 gene on the nitrogen absorption and utilization of the catalpa bungei is defined, and a technical route for improving the nitrogen absorption and utilization of the catalpa bungei by reducing the expression of the CbuLBD19 gene is provided.

Description

Technical Field

[0001] This invention belongs to the field of forest tree genetic engineering breeding technology, and more specifically, relates to a CbuLBD19 gene that regulates the root development and nitrogen absorption and utilization of Catalpa trees and its application. Background Technology

[0002] Plant root system architecture is a key agronomic trait determining its anchorage, nutrient and water absorption efficiency. For catalpa trees, which have high economic and ecological value, root development is directly related to their growth rate and biomass accumulation. In actual production, low nitrogen use efficiency is one of the main bottlenecks limiting the rapid growth and high yield of catalpa trees; therefore, analyzing and improving the regulatory mechanisms of root development from a genetic perspective has become an important topic in forest tree breeding.

[0003] In the complex regulatory network of plant root systematization, the LBD transcription factor family is recognized as a key class of regulators. Members of this family are functionally diverse and exhibit significant functional differentiation among species. For example, in the herbaceous model plant Arabidopsis thaliana, AtLBD37 / 38 / 39 has been reported to function as a negative regulator of nitrogen metabolism, while TaLBD41 in the gramineous crop wheat has been shown to positively regulate root growth and nitrogen uptake efficiency; its silencing leads to shorter roots and reduced nitrogen assimilation capacity. Currently, most related research focuses on annual herbaceous crops, while functional studies of LBD genes in perennial trees, especially Catalpa bungei, are almost nonexistent, and whether and how they participate in root morphogenesis and nitrogen response remains unclear. Summary of the Invention

[0004] The purpose of this invention is to provide a CbuLBD19 gene that regulates the root development and nitrogen absorption and utilization of Catalpa bungei and its application, so as to solve the above-mentioned technical problems.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a CbuLBD19 gene that regulates root development and nitrogen absorption and utilization in Catalpa bungei trees. The nucleotide sequence of the CbuLBD19 gene is shown in SEQ ID NO.7.

[0006] This invention successfully constructed an overexpression vector by cloning the CbuLBD19 gene of Catalpa bungei and obtained transgenic plants using Agrobacterium-mediated transformation. Functional verification results showed that, compared with the wild type, the total root length, root biomass, net nitrate uptake rate, NR activity, and amino acid content of the CbuLBD19 overexpression lines were significantly reduced. This fully demonstrates the key role of CbuLBD19 in negatively regulating root development and nitrogen uptake and assimilation in Catalpa bungei, providing conclusive experimental evidence and feasible technical solutions for improving nitrogen efficiency in Catalpa bungei by regulating the expression of this gene.

[0007] The present invention also provides a recombinant expression vector comprising the Catalpa tree CbuLBD19 gene.

[0008] Furthermore, the vector is formed by inserting the Catalpa tree CbuLBD19 gene into the pCAMBIA1302 vector. Nco I Endonuclease sites were obtained.

[0009] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the above-described recombinant expression vector.

[0010] Furthermore, the recombinant engineered bacteria are obtained by transferring the recombinant expression vector into Agrobacterium competent cells EHA105.

[0011] This invention provides the application of the Catalpa tree CbuLBD19 gene, the recombinant expression vector, or the recombinant engineered bacteria in regulating plant root development.

[0012] Furthermore, the regulation of plant root development is achieved by increasing the expression of the Catalpa tree CbuLBD19 gene to reduce plant root length.

[0013] This invention provides the application of the Catalpa tree CbuLBD19 gene, the recombinant expression vector, or the recombinant engineered bacteria in regulating nitrogen absorption and utilization in plant roots.

[0014] Furthermore, the regulation of nitrogen absorption and utilization in plant roots is achieved by increasing the expression of the Catalpa tree CbuLBD19 gene to reduce the net absorption rate of nitrate ions, NR activity, and amino acid content in plant roots.

[0015] The present invention has at least the following beneficial effects: New discovery of gene function: This invention is the first experimental demonstration that CbuLBD19 is a key gene in catalpa trees that is induced by high nitrogen levels and negatively regulates root development and nitrogen absorption and utilization. This provides a new perspective for understanding the molecular mechanism of nitrogen response in catalpa trees.

[0016] This invention provides a clear technical pathway: By creating CbuLBD19 overexpressing lines and combining systematic phenotypic and physiological index analysis (including root morphology, net nitrate uptake rate, NR activity, and amino acid content), the negative regulatory function of this gene has been conclusively demonstrated. This discovery directly suggests a feasible genetic improvement technical route: by reducing the expression of endogenous CbuLBD19 in Catalpa bungei through antisense RNA, RNA interference, or gene editing, it is expected to cultivate new Catalpa bungei germplasm with more developed root systems and stronger nitrogen uptake capacity.

[0017] Huge application potential: This invention provides valuable genetic resources and clear operational targets for molecular breeding of catalpa trees, which is of great significance for solving the bottleneck problem of low nitrogen utilization efficiency in catalpa production and achieving rapid growth and high yield. Attached Figure Description

[0018] Figure 1 A plot showing the transcript levels of CbuLBD19 in the roots of wild-type Catalpa trees under different nitrogen supply levels.

[0019] Figure 2 This is a diagram showing the results of the full-length cDNA cloning of the CbuLBD19 gene.

[0020] Figure 3 DNA and RNA were identified in CbuLBD9 overexpressing lines. In this study, A represents the DNA identification results of CbuLBD9 overexpressing lines, and B represents the RNA expression level identification results of CbuLBD9 overexpressing lines.

[0021] Figure 4 The figures show a comparison of the morphological characteristics of Catalpa WT and CbuLBD19 overexpressing species. In the figures, A is the phenotypic diagram of Catalpa WT overexpressing species, B is the total root length statistics, and C is the root biomass statistics.

[0022] Figure 5 This is a comparative analysis of the net nitrate ion uptake rates of Catalpa macrocarpa overexpressed with WT and CbuLBD19.

[0023] Figure 6 The graphs show a comparative analysis of NR activity and AA content in WT and CbuLBD19-overexpressing Catalpa bungei. In the graphs, A represents the statistical analysis of NR activity and B represents the statistical analysis of AA content. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0025] The transgenic material used in the following examples was a half-sib family of Catalpa bungei, collected from the Catalpa bungei germplasm resource nursery of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The plant overexpression vector pCAMBIA1302 was purchased from Wuhan Miaoling Biotechnology Co., Ltd., and pMD19-T was purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0026] Catalpa co-culture medium: MS medium (M519, PhytoTech, Lenexa, USA) 4.43 g / L + sucrose 30 g / L + gel 3 g / L, pH 5.8, autoclaved at 121℃ for 20 min.

[0027] Selective medium for CbuLBD19 overexpression in Catalpa bungei: DKW medium (D2470, PhytoTech, Lenexa, USA) 5.32 g / L + 6-benzylaminopurine 0.6 mg / L + 1-naphthaleneacetic acid 0.15 mg / L + sucrose 30 g / L + Gelzan™ CM gellan gum (G1910, Sigma) 3 g / L, pH 5.8, autoclaved at 121℃ for 20 min, then 0.2 mg / L zeatin, 200 mg / L Timentin (SL4080, purchased from Beijing Cooler Master Technology Co., Ltd.) and 1 mg / L hygromycin were added.

[0028] Catalpa tree proliferation medium: DKW medium 5.32g / L + 6-benzylaminopurine 0.6mg / L + 1-naphthaleneacetic acid 0.15mg / L + sucrose 30g / L + Gelzan™CM 3g / L, pH 5.8, autoclaved at 121℃ for 20min, then 0.2mg / L of zeatin was added.

[0029] Catalpa rooting medium: DKW medium 5.32 g / L + 3-indolebutyric acid 0.15 mg / L + 1-naphthaleneacetic acid 0.15 mg / L + sucrose 20 g / L + Gelzan™ CM 3 g / L, pH 5.8, autoclaved at 121℃ for 20 min, then add 0.2 mg / L zeatin and 200 mg / L Timentin.

[0030] Example 1: Changes in CbuLBD19 expression levels under different nitrogen supply levels.

[0031] 1. Nitrogen treatment: In an artificial climate chamber (day / night temperature: 25 / 18℃; relative humidity: 50%~60%; 14 hours of light per day; photosynthetic photon flux: 150 μmol·m⁻¹), nitrogen was applied. -2 ·s -1 In this study, wild-type Catalpa trees were subjected to hydroponic treatment with normal nitrogen (NN, 2 mM KNO3) and low nitrogen (LN, 0.2 mM KNO3) for 21 days.

[0032] 2. Total RNA extraction and reverse transcription: Using the treated young roots of Catalpa trees as material, total RNA was extracted by the Trizol method, and then reverse transcribed into cDNA using the PrimeScript™ 1st Strand cDNA Synthesis Kit.

[0033] 3. Real-time quantitative PCR (RT-qPCR): Design qPCR-specific primers for the CbuLBD19 gene and the internal reference gene. The sequence of the upstream primer CbuLBD19-qPCR-F is shown in SEQ ID NO.1, the sequence of the downstream primer CbuLBD19-qPCR-R is shown in SEQ ID NO.2, the sequence of the upstream primer CbuActin-F is shown in SEQ ID NO.3, and the sequence of the downstream primer CbuActin-R is shown in SEQ ID NO.4.

[0034] SEQ ID NO. 1: TGCTATTTGAAGCTGCCGGA.

[0035] SEQ ID NO. 2: TGTGCACTCAGAAGCATCGT.

[0036] SEQ ID NO. 3:GATGATGCTCCAAGGGCTGT.

[0037] SEQ ID NO. 4: TCCATATCATCCCAGTTGCT.

[0038] The cDNA obtained from reverse transcription was diluted 5-fold with sterile water, and then RT-qPCR was performed according to the instructions of the RT-qPCR kit (MF787-NR-01, Beijing Polymer Biotechnology Co., Ltd.). Figure 1 The results showed that under low nitrogen concentration culture conditions, the transcript level of the gene CbuLBD19 was significantly reduced in wild-type Catalpa trees.

[0039] Example 2: Cloning of the Catalpa CbuLBD19 gene and construction of gene overexpression plasmid and recombinant Agrobacterium.

[0040] 1. Cloning of the Catalpa 'CbuLBD19' gene and construction of the pMD19-T vector: Using young roots of Catalpa 'CbuLBD19' as material, total RNA was extracted using the Trizol method, and then reverse transcribed into cDNA using the PrimeScript™ 1st Strand cDNA Synthesis Kit. Specific primers for the CbuLBD19 gene were designed. The sequence of the upstream primer CbuLBD19-F is shown in SEQ ID NO. 5, and the sequence of the downstream primer CbuLBD19-R is shown in SEQ ID NO. 6.

[0041] SEQ ID NO. 5: ATGAGTTGCAATGGCTGCCGT.

[0042] SEQ ID NO. 6: AGCGATGGGCTTCCGCGT.

[0043] PCR amplification was performed using cDNA as a template, such as Figure 2 As shown, the amplified target fragment of approximately 560 bp was recovered via gel electrophoresis. Following the instructions of the cloning kit (6013, Takara, Beijing, China), the recovered product was ligated into the pMD19-T vector, and gene sequencing was performed. The sequenced sequence was compared with the genome sequence. The coding region of the cloned CbuLBD19 gene is 561 bp in length, as shown in SEQ ID NO.7. The amino acid sequence of the expressed protein is shown in SEQ ID NO.8, and its molecular weight is 20.4 kDa. The successfully constructed positive plasmid was named pMD19-T-CbuLBD19.

[0044] SEQ ID NO.7:

[0045] SEQ ID NO. 8: MSCNGCRVLRKGCSENCILRACLQWIESAEAQGHATIFVAKFFGRAGLMSFISAVPENQRPALFQSLLFEAAGRTVNPVNGAAGLLWTGNWHVCKAAVEAVLRGGTLKPIPEFLGDPSEPDDASECTDMFELQDPDLSPRQKRRRFPDEPAKIMQLADLDLSLTPGFSWQEKEPFTGEAATRKPIA.

[0046] 2. Construction of CbuLBD19 gene overexpression plasmid and Agrobacterium transformation: CbuLBD19 gene amplification primers containing homologous arms of pCAMBIA1302 vector were designed. The sequence of the upstream primer CbuLBD19-OE-F is shown in SEQ ID NO.9, and the sequence of the downstream primer CbuLBD19-OE-R is shown in SEQ ID NO.10.

[0047] SEQ ID NO.9: CACGGGGGACTCTTGACATGAGTTGCAATGGCTGCCGT.

[0048] SEQ ID NO. 10: TCTCCTTTACTAGTCAGATCTACCATGCCAGCGATGGGCTTCCGCGT.

[0049] Using pMD19-T-CbuLBD19 plasmid as a template, PCR was performed using primers CbuLBD19-OE-F and CbuLBD19-OE-R. The target fragment was recovered after detection by agarose gel electrophoresis. QuickCut™ was used as the template. Nco The pCAMBIA1302 vector was digested with an I (1620, Takara, Beijing, China) restriction enzyme. Then, the target fragment was ligated into the digested pCAMBIA1302 vector using homologous recombination. The successfully constructed vector was transformed into DH5α *E. coli* via heat shock, and single colonies with normal growth were selected for sequencing. The overexpression plasmid containing the target gene CbuLBD19 was transformed into *Agrobacterium* EHA105 using a freeze-thaw method. The bacterial culture was then plated on LB agar (containing 50 mg / L kanamycin and 25 mg / L rifampin), and single colonies were selected. PCR verification of the bacterial culture was performed using the vector primers. After detection by agarose gel electrophoresis, bacterial cultures with correct bands were added to glycerol and stored at -80°C for later use.

[0050] Example 3: Obtaining Catalpa 'CbuLBD19 overexpressing plants.

[0051] 1. Genetic transformation steps for CbuLBD19 overexpression in Catalpa bungei: The obtained CbuLBD19 overexpressing recombinant Agrobacterium EHA105 was activated, single colonies were picked, and cultured in LB liquid medium (containing 50 mg / L kanamycin and 25 mg / L rifampin) and 10 μM acetylsyl syringone (As) until OD600 = 1.25. Resuspension solution was prepared: 100 mL sterile water + 10 mM MgCl2 + 10 mM MES + 10 μM As solution.

[0052] The bacterial cells were resuspended in a resuspension solution, and the OD600 concentration of the resuspension solution was adjusted to 0.65. Activation was carried out at room temperature in the dark for 3 hours. Catalpa callus tissue was placed in the activated resuspension solution and incubated at 28°C and 120 rpm for 15 minutes. The bacterial suspension on the callus surface was then blotted dry with sterile paper. The callus was cultured on co-culture medium for 2 days, and then transferred to CbuLBD19 overexpression Catalpa selective medium for further culture. The medium was changed every three weeks until shoots appeared. After shoots appeared, the callus was transferred to rooting medium for rooting.

[0053] 2. Identification and screening of CbuLBD19-overexpressing Catalpa trees: DNA and total RNA were extracted from leaves of CbuLBD19-overexpressing and wild-type Catalpa trees using SDS and Trizol methods, respectively. Primers for identifying CbuLBD19-overexpressing Catalpa trees were designed. The sequence of pCAMBIA1302-CbuLBD19-jd-F is shown in SEQ ID NO.11, and the sequence of pCAMBIA1302-CbuLBD19-jd-R is shown in SEQ ID NO.12.

[0054] SEQ ID NO. 11: CTGACGTAAGGGATGACGCA.

[0055] SEQ ID NO. 12: CTTAGCGAGGAAGACGGTGG.

[0056] Using leaf DNA as a template, PCR was performed using the identification primers pCAMBIA1302-CbuLBD19-jd-F and pCAMBIA1302-CbuLBD19-jd-R to identify CbuLBD19 overexpressing transgenic plants. The positive control was the CbuLBD19 overexpression recombinant vector plasmid, the negative control was wild-type Catalpa trees (WT), and the blank was water. Figure 3 As shown in Figure A, a total of five CbuLBD19 overexpression positive lines were identified at the DNA level: CbuLBD19-OE1, CbuLBD19-OE3, CbuLBD19-OE5, CbuLBD19-OE6, and CbuLBD19-OE8.

[0057] Using CbuLBD19-overexpressing Catalpa trees (CbuLBD19-OE1, CbuLBD19-OE3, CbuLBD19-OE5, CbuLBD19-OE6, and CbuLBD19-OE8) and wild-type Catalpa (WT) cDNA as templates, quantitative real-time PCR was performed using CbuLBD19-qPCR-F and CbuLBD19-qPCR-R primers to identify the relative expression levels of CbuLBD19 in different transgenic Catalpa lines overexpressing CbuLBD19. CbuActin was used as an internal control gene. Figure 3 As shown in Figure B, at the RNA level, three CbuLBD19 overexpression lines with high expression levels were identified by gene expression levels: CbuLBD19-OE3, CbuLBD19-OE5, and CbuLBD19-OE6. The selected transgenic lines were subcultured into antibiotic-free proliferation medium for propagation.

[0058] Wild-type Catalpa trees and Catalpa trees overexpressing CbuLBD19 with similar root growth were selected and hydroponically cultured in an artificial climate chamber using LA nutrient solution. After six weeks of culture, root samples were harvested, weighed, and their fresh weight recorded. The harvested samples were placed in foil bags and immediately frozen in liquid nitrogen. The frozen samples were then ground into a fine powder using a ball mill and stored at -80°C for further analysis.

[0059] Example 4: Functional verification of the Catalpa tree CbuLBD19 gene.

[0060] Root morphology characteristics determination: 1g root samples were collected and root scanning analysis was performed using the WinRHIZO root analysis system (WinRHIZO version 2012b, Regent Instruments Canada, Montreal, Canada). Figure 4 It can be seen that, compared with WT, the total root length and root biomass of the overexpression lines were significantly reduced.

[0061] Net nitrate ion uptake rate determination: White fine roots with a diameter of 1.5 mm were selected and measured using a non-destructive microelectrode technique (NMT-YG-100, Younger USA LLC, Amherst, MA, USA). A fine root was transferred to a petri dish containing 10 ml of measurement solution (0.1 mM CaCl2, pH 6.0) and 2 mM KNO3 was added, followed by equilibration for 20 min. Before measurement, the equilibrated root was transferred to a new petri dish containing fresh measurement solution. Recordings were taken for 5 min at each site. Figure 5It can be seen that, compared with WT, the net nitrate uptake rate of CbuLBD19 overexpression lines was significantly reduced, indicating that CbuLBD19 inhibits nitrogen uptake in Catalpa bungei.

[0062] NR activity assay: Assay was performed using a nitrate reductase (NR) kit (BC0085, Solarbio, Beijing, China). Figure 6 As can be seen from A, CbuLBD19 overexpression inhibits NR activity in plants.

[0063] Amino acid (AA) concentration determination: The concentration was determined according to the instructions of the amino acid content determination kit (AA-1-W, Comin, Suzhou, China). AA content can reflect changes in nitrogen metabolism in plants. Figure 6 The results of the B assay showed that CbuLBD19 overexpression also inhibited the AA content in the plant.

[0064] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0065] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0066] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A CbuLBD19 gene for regulating Catalpa bungei root development and nitrogen absorption and utilization, characterized in that, The nucleotide sequence of the CbuLBD19 gene is shown as SEQ ID NO.

7.

2. A recombinant expression vector, characterized in that, The CbuLBD19 gene of Catalpa bungeana is used.

3. The recombinant expression vector of claim 2, wherein, is the pCAMBIA1302 vector into which the Schisandra CbuLBD19 gene is inserted Nco I endonuclease sites are obtained.

4. A recombinant engineered bacterium, characterized in that, The recombinant engineering bacteria comprise the recombinant expression vector of claim 3.

5. The recombineering bacteria of claim 4, wherein, The recombinant expression vector is transformed into Agrobacterium competent cell EHA105.

6. The Catalpa bungeana CbuLBD19 gene of claim 1, the recombinant expression vector of claim 2 or the recombinant engineering bacteria of claim 4 are used for regulating the development of plant root system.

7. Use according to claim 6, characterized in that, The regulation of the development of plant root system is to reduce the plant root length and root biomass by increasing the expression of the Catalpa bungeana CbuLBD19 gene.

8. The Catalpa bungeana CbuLBD19 gene of claim 1, the recombinant expression vector of claim 2 or the recombinant engineering bacteria of claim 4 are used for regulating the nitrogen absorption and utilization of plant root system.

9. Use according to claim 8, characterized in that, The regulation of the nitrogen absorption and utilization of plant root system is to reduce the net absorption rate of nitrate ion, NR activity and amino acid content of plant root system by increasing the expression of the Catalpa bungeana CbuLBD19 gene.

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