Paulownia fortunei PfLAZY1 gene as well as expression protein and application thereof

Abstract

The invention discloses a paulownia fortunei PfLAZY1 gene as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the paulownia fortunei PfLAZY1 gene disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the paulownia fortunei PfLAZY1 gene is as shown in SEQ ID NO.2. An expression vector is constructed and converted into arabidopsis thaliana, and the result of the embodiment shows that the PfLAZY1 gene can promote the increase of the number of plant branches, the branching angle is increased, and the leaf included angle dispersion degree is increased. New gene resources and technical means are provided for paulownia plant type improvement, the breeding period can be shortened, the wood yield and quality can be improved, and the method has important significance on theoretical research and molecular breeding practice of plant branch form regulation and control; genetic engineering improvement is carried out by utilizing the gene, accurate regulation and control can be carried out according to specific characters such as branching angles, the breeding period is shortened, the breeding efficiency is improved, and the breeding process of high-stem good-talent varieties is accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically relates to a Paulownia candidum PfLAZY1 gene, an expression protein thereof, and an application thereof. Background Art

[0002] Paulownia fortunei is an important fast-growing timber species, widely used in furniture manufacturing, wood production, ecological restoration and other fields. Paulownia fortunei grows fast and has good wood properties, but its growth morphology, especially the branch angle and number of branches, has a significant impact on wood quality and production efficiency. Too many branches or too large an angle will make the tree morphology unsuitable for dense planting, thus affecting the quality of the wood; too few branches or too small an angle may not be conducive to the full utilization of light by the Paulownia, thus affecting its growth rate. Therefore, studying and regulating the branch morphology of Paulownia is of great economic significance for improving its wood yield and quality.

[0003] In plants, the regulation of branch angle and branch number is influenced by multiple factors, including genetic control and external environmental factors such as light and gravity. Numerous studies have shown that the PfLAZY1 gene is one of the key genes controlling plant branch morphology. The PfLAZY1 gene was first discovered in rice, where mutants exhibit abnormal branch angles and lodging. Subsequently, in other plant species, such as Arabidopsis, maize, and tomato, PfLAZY1 has also been shown to play an important role in regulating branch angle and gravity response. The primary function of the PfLAZY1 gene is related to the regulation of polar auxin transport in plants, regulating the distribution of auxin, thereby affecting the angle and direction of plant branches. However, research on the function of the PfLAZY1 gene in Paulownia is still in its infancy, and its regulatory mechanism is not yet fully understood.

[0004] At present, traditional hybrid breeding and selective breeding methods are widely used in Paulownia breeding. However, these methods often have long cycles, low efficiency, and it is difficult to effectively improve specific traits (such as branch angle). With the advancement of molecular biology technology, it has become possible to improve important trait genes through genetic engineering. In particular, regulating the expression of key functional genes through transgenic or gene editing technology can effectively shorten the breeding cycle and improve breeding efficiency. However, research on genes related to branch angle regulation in Paulownia is relatively limited, and there are no specific reports on the application of the Paulownia branching regulatory gene LAZY1. Summary of the Invention

[0005] In response to the aforementioned problems with the existing technologies, the present invention aims to provide the PfLAZY1 gene from Paulownia truncatum. Another technical problem addressed by the present invention is to provide an expression protein of the PfLAZY1 gene from Paulownia truncatum. A further technical problem addressed by the present invention is to provide applications of the PfLAZY1 gene from Paulownia truncatum to regulate plant branching angles, improve plant morphology, and accelerate the breeding of high-stem, high-quality varieties.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A PfLAZY1 gene from Paulownia candidum, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The amino acid sequence of the expressed protein of the Paulownia alba PfLAZY1 gene is shown in SEQ ID NO.2.

[0009] A vector, recombinant bacteria or host cell containing the Paulownia alba PfLAZY1 gene.

[0010] The application of the PfLAZY1 gene in regulating the branching angle of Paulownia truncatula includes:

[0011] 1) Constructing an expression vector for the Paulownia alba PfLAZY1 gene;

[0012] 2) Transforming the constructed Paulownia alba PfLAZY1 gene expression vector into Arabidopsis thaliana;

[0013] 3) Cultivate, screen and obtain transgenic Arabidopsis plants with increased branching angles.

[0014] The expression vector is a plant expression vector.

[0015] The plant expression vector is pBWA(V)BS-LAZY1.

[0016] Application of the Paulownia alba PfLAZY1 gene in regulating the number of plant branches or the degree of leaf angle dispersion.

[0017] The regulating the number of plant branches is to promote the increase of the number of branches.

[0018] The regulating of the degree of dispersion of plant leaf angles is to promote an increase in the degree of dispersion of leaf angles.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The present invention discloses for the first time the nucleotide sequence (SEQ ID NO.1) of the Paulownia alba PfLAZY1 gene and the amino acid sequence (SEQ ID NO.2) of its expressed protein, providing a new genetic resource for the study of plant branch morphology regulation.

[0021] 2) By constructing an expression vector and transforming it into Arabidopsis thaliana, the present invention demonstrated that this gene can significantly increase the branching angle of transgenic plants, helping to optimize the spatial structure of plants and making them more adaptable to different growth environments and cultivation requirements. The results of the examples show that the PfLAZY1 gene can promote an increase in the number of plant branches, which has positive implications for improving plant photosynthesis efficiency, enhancing their ecological adaptability, or achieving specific agricultural production goals (such as increasing biomass). The gene can also increase the degree of leaf angle spread, which helps leaves receive more light, improves light energy utilization, and thus promotes plant growth.

[0022] 3) This invention provides a new technical approach for molecular breeding of plants such as Paulownia. While traditional hybrid breeding processes are long and inefficient, genetic engineering using this gene allows for precise regulation of specific traits such as branching angle, shortening the breeding cycle, improving breeding efficiency, and accelerating the selection of high-stem, high-quality varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 1 is a diagram of the PCR amplification product of the PfLAZY1 gene (M is Marker DL5000; a is the PCR product of the PfLAZY1 gene);

[0024] Figure 2 This is the plasmid map of the PfLAZY1 gene overexpression vector;

[0025] Figure 3 PCR identification of positive plants of Arabidopsis thaliana transgenic with PfLAZY1 gene (M is Marker; + is positive control; - is negative control; O is water control; 1-22 are PCR products of transgenic plants)

[0026] Figure 4 The phenotypes of wild-type Arabidopsis and transgenic Arabidopsis with PfLAZY1 gene are shown in Figure 2. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0028] The plant materials used in this application, Paulownia alba leaves, were all collected from the Mengzhou Experimental Base of the Economic Forestry Research Institute, Chinese Academy of Forestry.

[0029] Example 1

[0030] 1. Total RNA extraction and cDNA acquisition

[0031] Using the leaf tissue of Paulownia truncatum as the material, RNA was extracted according to the operating procedures of the FastPure Plant Total RNA Isolation Kit (Vazyme). The results of 1% agarose gel electrophoresis of the total RNA of the leaf tissue of Paulownia truncatum showed that the bands were complete and clear, the RNA integrity was good, and the concentration was high, which was suitable for reverse transcription. Using the extracted RNA as a template, reverse transcription was performed to obtain cDNA (Vazyme's Ⅲ1st Strand cDNA Synthesis Kit (+gDNA wiper)).

[0032] 2. Cloning of target genes

[0033] Based on the genome sequence information of Paulownia alba (PRJNA624264), the following primers were designed:

[0034] LAZY1-F: 5'-ATGAAGCTACTAGGTTGGATGC-3',

[0035] LAZY1-R:5'-TTAGAGCTCCAACACCAGTAG-3'.

[0036] PCR reaction system: ddH2O 18μL, 2×Phanta Max Buffer a 25 μL, dNTP Mix (10 mM each) 1 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, cDNA 1 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL.

[0037] The PCR reaction program was as follows: 95°C for 3 min; 95°C for 15 sec, 56°C for 15 sec, 72°C for 30 sec, 35 cycles; 72°C for 5 min, 4°C∞.

[0038] The PCR products of PfLAZY1 gene were detected by 1% agarose gel electrophoresis. Figure 1 The target fragment was recovered and purified using the DNA gel recovery kit from TSINGKE, and the target gene fragment was ligated with the cloning vector pClone007 Blunt Vector Kit (TSINGKE TSV-007B).

[0039] The ligation reaction system is as follows: 2 μL of the recovered target fragment, 1 μL of pClone007 Blunt Vector, 1 μL of 10×TopoMix, and 6 μL of ddH2O. After all the solutions are added directly to the tube, mix them by pipetting, and react at room temperature (22-30°C) for 5 minutes to complete the reaction. The ligation product is transformed into the Escherichia coli strain TreliefTM5α (TSINGKE TSC01). The recombinant plasmid is detected, and the positive clones obtained are sequenced and analyzed. The final sequencing result shows that the PfLAZY1 gene coding region is 1170 bp in length and contains a complete open reading frame (ORF). The sequence is shown in SEQ ID NO.1. The amino acid sequence of the expressed protein is shown in SEQ ID NO.2, which contains 389 amino acids.

[0040] Example 2

[0041] 1. Construction of PfLAZY1 gene overexpression vector of Paulownia davidiana

[0042] Design amplification primers, the primer sequences are as follows:

[0043] Forward:

[0044] 5'-AACACGGGGGACTTTGCAACatgaagctactaggttggatgcatcgg-3';

[0045] Reverse:

[0046] 5'-TGAAGACAGAGCTAGTTACAttagagctccaacaccaagtagtctgc-3'.

[0047] The gene fragment obtained above was used as a template for PCR amplification. The 50μL reaction system is as follows: 2×Phanta MaxBuffera 25μL; dNTP Mix (10mM each) 1μL; upstream primer (10μM) 2μL; downstream primer (10μM) 2μL; plasmid containing the target fragment 1μL; Phanta Max Super-Fidelity DNA Polymerase 1μL; ddH2O 18μL. Reaction procedure: pre-denaturation 95℃3min; 95℃15s, 56℃15s, 72℃1min, 35 cycles; 72℃5min; 4℃∞. The amplified product was detected by 1% agarose gel electrophoresis to obtain the target fragment. Vazyme was used. Perform the recombination reaction using the II OneStep Cloning Kit. Set up the following reaction mixture on ice: 4 μL of the linearized PBWA(V)BS plasmid fragment, 1 μL of the PfLAZY1 gene recovery fragment, 4 μL of 5× CE II Buffer, 2 μL of Exnase II, and 9 μL of ddH2O. Gently pipette to mix thoroughly, then briefly centrifuge to collect the bottom of the tube. Incubate at 37°C for 30 minutes (PCR instrument); upon completion, cool the tube to 4°C. Transformation was performed using TIANGEN DH5α competent cells: 1) Place the competent cells in an ice bath. 2) Add 10 μL of the recombinant product to the competent cell suspension, gently flick to mix, and incubate on ice for 30 minutes. 3) Place the centrifuge tube in a 42°C water bath for 90 seconds, then quickly transfer the tube to an ice bath to cool the cells for 3 minutes. Do not shake the centrifuge tube during this process. 4) Add 900 μL of sterile LB culture medium (without antibiotics) to the centrifuge tube, mix well, and place in a shaker at 37°C and 150 rpm for 45 minutes to allow the bacteria to recover. 5) Mix the contents of the centrifuge tube, centrifuge at 4000 rpm for 2 minutes, discard part of the culture solution, suspend the bacterial solution, and spread it on LB solid agar medium containing kanamycin. Place the plate at room temperature until the liquid is absorbed, invert the plate, and culture at 37°C for 16 hours. Pick a single colony from the screening plate for PCR detection. The PCR reaction uses Vazyme's 2×Rapid TaqMaster Mix reagent. The single clone that is positive for bacterial solution PCR detection is sent to Qingke Biotechnology Company for sequencing and identification. If the sequencing is correct, the expression vector pBWA(V)BS-LAZY1( Figure 2 ).

[0048] 2. Agrobacterium infection of Arabidopsis

[0049] The constructed LAZY1 overexpression vector was transformed into Agrobacterium using the liquid nitrogen freeze-thaw method. The specific process is as follows: 1) Thaw the competent Agrobacterium cells in an ice bath, add 1-5 μL (100 ng) to each tube to recover the purified recombinant plasmid, gently pipette to mix, place on ice, and let it stand in an ice bath for 5 minutes. 2) Quickly freeze in liquid nitrogen for 5 minutes, heat shock at 37°C for 5 minutes, and quickly place on ice for 5 minutes. 3) Add 700 μL of antibiotic-free LB liquid culture medium and culture at 28°C with shaking for 2-4 hours. 4) Centrifuge at 6000 rpm for 1 minute to collect the bacteria, and remove part of the supernatant. 5) Keep an appropriate amount of bacterial solution, mix gently, and spread it on LB solid culture medium containing kanamycin. 6) Invert and culture at 28°C for 30-48 hours until a single colony grows. 7) Detect positive clones by PCR of the bacterial solution and store at 4°C for later use.

[0050] The positive monoclonal bacterial solution detected by PCR was expanded and cultured, and the bacteria were shaken to OD 600=0.6-0.8, the target gene was transferred into wild-type Arabidopsis using the inflorescence soaking method. The specific process is as follows: 1) Centrifuge the bacterial solution at 5000 rpm for 5 minutes, collect the cells, and suspend them in a 1 / 2 MS solution containing 5% sucrose; 2) Before soaking, add 0.05% Silwet L-77 and shake out the foam; 3) Soak the unopened inflorescence of the aerial part of the Arabidopsis in the Agrobacterium suspension for 15-30 seconds, gently shaking during the soaking; 4) Place the soaked Arabidopsis flat on a tray, cover with plastic wrap to keep it moist, and protect from light for 24 hours; 5) Remove the plastic wrap and culture under normal conditions until the seeds mature. Once mature, stop watering.

[0051] 3. Obtaining T2 transgenic lines

[0052] After the T1 generation Arabidopsis matured, the seeds were collected and numbered. 1) The T1 generation seeds and wild-type Arabidopsis seeds were placed in sterilized centrifuge tubes, first washed several times by immersion in sterile water, and then disinfected with 1% sodium hypochlorite for 15 minutes; 2) After standing, the supernatant was removed, and the tubes were washed 4-5 times with sterile water. 0.1% agarose was added, and the resuspended Arabidopsis seeds were spotted on 1 / 2MS solid culture medium containing kanamycin using a pipette for culture; 3) The tubes were vernalized at 4°C for 72 hours, and then transferred to a light incubator for culture for 10 days. The growth of the transgenic plants and wild-type plants was observed; 4) Transgenic PfLAZY1 seedlings and wild-type Arabidopsis seedlings with dark green leaves and well-developed root systems were selected and transplanted into nutrient soil to obtain T2 generation transgenic plants, and genetic testing was performed on the T2 generation transgenic plants.

[0053] The results are as follows Figure 3 As shown, the target gene was not detected in lanes 1, 7, and 15, while the target gene was detected in other lanes, corresponding to the T2 generation transgenic positive plants. The T2 generation transgenic plants corresponding to lanes 5, 6, and 8 were numbered PfLAZY1-OE5, PfLAZY1-OE6, and PfLAZY1-OE8.

[0054] 4. Phenotype of T2 transgenic Arabidopsis thaliana overexpressing the PfLAZY1 gene

[0055] Three transgenic lines (numbered: PfLAZY1-OE5, PfLAZY1-OE6, PfLAZY1-OE8) were selected and cultured in a light incubator under the same growth environment as the wild-type plants (23°C, 16h light / 8h dark), and the phenotypes of the transgenic plants and wild-type Arabidopsis plants were observed.

[0056] The results are as follows Figure 4As shown, transgenic Arabidopsis plants overexpressing the LAZY1 gene had increased branch angles, increased branch numbers, and increased leaf angle dispersion. The results showed that the LAZY1 gene can regulate the number and angle of plant branches, and has important application value in plant genetic engineering research.

[0057] The above description is only illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all of them will fall within the scope of protection of the present invention.

Claims

1. A PfLAZY1 gene from Paulownia candidum, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein of the Paulownia candidum PfLAZY1 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A vector, recombinant bacteria or host cell containing the Paulownia candidum PfLAZY1 gene according to claim 1.

4. Use of the Paulownia white flower PfLAZY1 gene according to claim 1 in regulating the branching angle of plants.

5. The use according to claim 4, characterized in that include: 1) Constructing an expression vector for the Paulownia alba PfLAZY1 gene; 2) Transforming the constructed Paulownia alba PfLAZY1 gene expression vector into Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis plants with increased branching angles.

6. The use according to claim 5, characterized in that The expression vector is a plant expression vector.

7. The use according to claim 5, characterized in that The plant expression vector is pBWA(V)BS-LAZY1.

8. Use of the PfLAZY1 gene of Paulownia tomentosa according to claim 1 in regulating the number of branches or the degree of leaf angle spread of a plant.

9. The use according to claim 8, characterized in that The regulating the number of plant branches is to promote the increase of the number of branches.

10. The use according to claim 8, characterized in that The regulating of the degree of dispersion of plant leaf angles is to promote an increase in the degree of dispersion of leaf angles.

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