Phoebe bournei PbGRF1 gene and application thereof in promoting growth of plant leaves

Abstract

The invention discloses a phoebe bournei PbGRF1 gene, and the phoebe bournei PbGRF1 gene has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence which is obtained by deleting, adding and / or substituting one or more nucleotides of the sequence and has an unchanged function. The invention also discloses a protein coded by the phoebe bournei PbGRF1 gene. The invention further discloses a method for promoting plant leaf growth. The invention also discloses an application of the phoebe bournei PbGRF1 gene in promoting the growth of plant leaves. The phoebe bournei PbGRF1 gene is cloned, arabidopsis thaliana is transformed through an agrobacterium tumefaciens mediated inflorescence infection method, a phoebe bournei PbGRF1 gene overexpressed plant is obtained, phenotypic observation shows that the leaf area of the arabidopsis thaliana with the overexpressed phoebe bournei PbGRF1 gene is larger than that of a wild type Col, and meanwhile the fresh weight and the dry weight of the overexpressed plant are larger than that of the wild type Col.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Phoebe bournei PbGRF1 gene and an application thereof in promoting the growth of plant leaves. Background Art

[0002] Phoebe bournei, an evergreen large tree of the genus Phoebe in the Lauraceae family, is mainly distributed in the subtropical regions of China and is a rare tree species unique to my country. Phoebe bournei prefers warm and humid climates, and most of them grow in scattered forms in valleys or shady slopes. Phoebe bournei wood has a pleasant aroma, a straight trunk, beautiful texture, and good corrosion and moisture resistance. It is often used in construction, high-end furniture, shipbuilding, carving and other purposes. It is deeply loved by the general public and enjoys the reputation of "golden nanmu". As an evergreen large tree with a straight trunk and beautiful crown shape, Phoebe bournei is used for modern landscaping. In recent years, studies have shown that the essential oils extracted from the leaves and wood of Phoebe bournei can inhibit fungal activity and also have a certain effect in anti-tumor. Therefore, Phoebe bournei has important economic and ecological value and is a rare timber and ornamental tree species unique to China. However, the natural forests of Phoebe fujian are growing slowly at present, and over-logging of wild resources has caused a sharp decrease in the number of Phoebe fujian germplasm resources, a decrease in the area of ​​natural pure forests, a large distance between groups, and a fragmented habitat. It has been listed as a national protected plant.

[0003] Growth-regulating-factor (GRF) genes are a highly conserved transcription factor family in plants, which play an important regulatory role in plant growth and development, signal transduction, stress response and regeneration efficiency. Most GRFs are target genes of miR396 and are negatively regulated by it. GRFs often interact with another type of protein factor GIF (GRF-interacting-factor) to co-regulate the expression of target genes, thereby regulating the growth and development of plants. The GRF family usually contains two conserved domains, WRC (Trp, Arg, Cys) and QLQ (Gln, Leu, Gln), which are important bases for the classification of the GRF family. Many studies in recent years have shown that GRFs are involved in many aspects of plant development, and their signaling pathways mediate important life processes such as plant leaf development, root growth, and flower development, reflecting a powerful regulatory function. Substantial progress has also been made in improving plant regeneration. The expression of GRF not only solves the shortcomings of low regeneration efficiency, time-consuming and labor-intensive transgenic lines in traditional transformation schemes, but also eliminates the defects of other developmental regulatory factors (such as BBM, WUS) that damage the development of plant reproductive organs, providing a new method for improving regeneration efficiency.

[0004] Transcription factors (TFs) are a large class of proteins in eukaryotic organisms that can specifically bind to DNA and regulate gene expression in that region. The regulation of target genes involves all levels of plant and animal development. The composition of their groups is very complex, with some existing as single members and others as family members. Among them, the GRF family is a unique class of transcription factors in plants. In the process of studying GRF, it was initially determined that it only had a regulatory role in the development of leaves and stems, but subsequent studies have found that GRF also plays an important role in regulating life processes such as leaf growth, flowering, seed and root development, plant lifespan, and growth regulation under adversity.

[0005] Most GRFs promote leaf growth. Studies have shown that overexpression of AtGRF1, AtGRF2, AtGRF3 and AtGRF5 in Arabidopsis will lead to an increase in leaf area, indicating that these four genes can regulate leaf development. The larger leaf area shown by the overexpression lines of AtGRF1, AtGRF2, AtGRF3 and AtGRF5 is due to the increase in cell volume rather than the increase in cell number. The single gene loss-of-function mutants of these three genes do not show abnormal phenotypes, while the atgrf1 / 2 / 3 triple mutant is similar to the atgrf1 / 2 / 3 / 4 quadruple mutant, both of which lead to smaller leaf area and shorter petioles. Unlike AtGRF1, AtGRF2, AtGRF3 and AtGRF5, the larger leaf area shown by the overexpression lines of AtGRF5 is due to the increase in cell number, and its single gene mutants will also develop narrower leaves than the wild type due to the reduction in cell number. Not all Arabidopsis GRFs play a positive regulatory role in leaf growth. The AtGRF9 gene can directly activate the expression of the ORG3 (OBP3-RESPONSIVE GENE3) gene to reduce cell cycle activity, resulting in a decrease in leaf area. Vice versa, its functional loss will enhance cell cycle activity, leading to an increase in leaf area, indicating that the AtGRF9 gene regulates leaf growth by inhibiting leaf primordium cell proliferation. However, no studies have reported the function of the PbGRF1 gene in Phoebe bournei.

[0006] Based on transcriptome data and qRT-PCR data, the applicant screened and cloned the PbGRF1 gene of Phoebe bournei, verified its biological function through heterologous transformation into Arabidopsis thaliana, and revealed the molecular mechanism of the PbGRF1 gene in the regulation of Phoebe bournei leaves, which will help regulate the leaf growth of Phoebe bournei through molecular biological means and provide new strategies for its breeding and protection. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a PbGRF1 gene of Phoebe bournei and its application in promoting the growth of plant leaves.

[0008] The present invention provides a PbGRF1 gene of Phoebe bournei, which has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence in which one or more nucleotides are deleted, added and / or substituted but the function remains unchanged. Preferably, the nucleotide sequence of the PbGRF1 gene of Phoebe bournei is SEQ ID No: 1.

[0009] The present invention also provides a protein encoded by the PbGRF1 gene of Phoebe bournei, which is composed of the amino acids shown in SEQ ID No.2.

[0010] The present invention also provides a method for promoting plant leaf growth, the method comprising preparing a modified plant containing the PbGRF1 gene of Phoebe bournei, wherein the expression of the PbGRF1 gene of Phoebe bournei is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the PbGRF1 gene of Phoebe bournei is shown in SEQ ID NO: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the product obtained by transcription and the product obtained by translation can be increased.

[0011] The present invention also provides the use of Phoebe bournei PbGRF1 gene in promoting plant leaf growth, and prepares a modified plant containing Phoebe bournei PbGRF1 gene, wherein the expression of Phoebe bournei PbGRF1 gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of Phoebe bournei PbGRF1 gene is shown in SEQ ID NO: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the increase in the product obtained by transcription and the product obtained by translation can be an increase.

[0012] In one embodiment, the plant is Phoebe chinensis or Arabidopsis thaliana.

[0013] In one embodiment, the plant is Arabidopsis thaliana.

[0014] In one embodiment, the PbGRF1 gene of Phoebe thaliana is connected to a vector, transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation, and screened, cultured, and transgenic strains are obtained. Compared with the wild type, overexpression of the PbGRF1 gene in transgenic Arabidopsis thaliana can promote the enlargement of Arabidopsis leaves and increase the biomass of the seedlings, such as dry weight and fresh weight.

[0015] The present application cloned the PbGRF1 gene of Phoebe bournei and transformed Arabidopsis thaliana through the Agrobacterium tumefaciens-mediated inflorescence infection method to obtain Phoebe bournei PbGRF1 gene overexpressing plants. Phenotypic observation showed that the Arabidopsis thaliana overexpressing the PbGRF1 gene of Phoebe bournei had a larger leaf area than the wild-type Col. At the same time, the fresh weight and dry weight of the overexpressing plants were also heavier than those of the wild-type. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The total RNA electrophoresis diagram of the leaves of Phoebe bournei and transgenic Arabidopsis thaliana of the present invention.

[0017] Figure 2 This is the electrophoresis diagram of the PCR amplified PbGRF1 gene of Phoebe bournei of the present invention.

[0018] Figure 3 This is an electrophoresis diagram for semi-quantitative identification of Phoebe bournei PbGRF1 gene-positive plants of the present invention.

[0019] Figure 4 This is a comparison diagram of leaf sizes between Arabidopsis thaliana plants expressing the PbGRF1 gene of the present invention and the wild type.

[0020] Figure 5 This is a comparison chart of leaf areas between Arabidopsis thaliana plants expressing the PbGRF1 gene of the present invention and the wild type.

[0021] Figure 6 The figure is a comparison of the dry weight and fresh weight of the Arabidopsis thaliana plant expressing the PbGRF1 gene of the present invention and the wild type. DETAILED DESCRIPTION

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] 1 Materials

[0026] 1.1 Experimental Materials

[0027] The Phoebe chinensis material selected was 3-month-old container seedlings from the “Wuyuan No. 8” family.

[0028] Arabidopsis thaliana used the Columbia-0 wild type and was cultured in the growth room of the Intelligent Laboratory Building of Zhejiang Agriculture and Forestry University under the growth conditions of 25°C and 16 h / d of light.

[0029] 1.2 Experimental reagents and instruments

[0030] Experimental reagents: DL2000 DNA Marker and 10× Loading Buffer were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; DNA gel recovery kit was purchased from Shanghai Huiling Biotechnology Co., Ltd.; M5 Hipure Next III Gelred nucleic acid dye was purchased from Beijing Poly-Mei Biotechnology Co., Ltd.; 2×TransStart FastPfu PCR SuperMix, M5Hiper ultra-light speed mix, and pEASY-Blunt Zero Cloning Kit were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; DH5α, GV3101, INVSc1 and other chemical competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.; FastPurePlant Total RNA Isolation Kit, reverse transcription kit, quantitative PCR detection kit, and seamless cloning kit were purchased from Nanjing Novogene Biotechnology Co., Ltd.; 2×Easy Taq PCR SuperMix was purchased from Zhejiang Easyside Biotechnology Co., Ltd.; restriction endonucleases were purchased from NEB (Beijing) Co., Ltd.

[0031] Experimental instruments: NanoDrop2000 ultra-micro spectrophotometer, micro centrifuge, basic electrophoresis instrument, Bio-RadGel Doc XR gel imager, Bio-Rad S1000 PCR thermal cycler, CFX96 real-time fluorescence quantitative PCR system, micropipette gun (Eppendorf), clean bench, water bath, ice maker, constant temperature culture shaker and incubator, handheld centrifuge, high-speed centrifuge, electronic balance, etc.

[0032] 1.3 Primer synthesis and sequencing

[0033] Primer synthesis and sequencing were completed by Zhejiang Qingke Biotechnology Co., Ltd.

[0034] 2 Methods

[0035] 2.1 Extraction of total RNA from Phoebe bournei and Arabidopsis thaliana

[0036] The total RNA of Phoebe bournei was extracted using FastPure Plant Total RNA Isolation Kit. The steps are as follows:

[0037] (1) Determine the sample input amount and appropriate lysis buffer according to the 08-2 / lysis buffer selection scheme. Take an appropriate amount of plant tissue ground by liquid nitrogen and immediately add 600 μL Buffer EL or 600 μL Buffer PSL. Vortex vigorously for 30 seconds to mix the sample and lysis buffer thoroughly. Centrifuge at 12,000 rpm (13,400 × g) for 5 minutes and immediately proceed to the subsequent operation.

[0038] (2) Take about 500 μL of the supernatant and transfer it to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III have been placed in the collection tube), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate;

[0039] (3) Add 0.5 times the volume of filtrate anhydrous ethanol (about 250 μL, adjusted according to the actual situation of the supernatant) to the collection tube and shake and mix for 15 seconds;

[0040] (4) Transfer the above mixed solution to FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate;

[0041] (5) Add 700 μl Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm (13,400 × g) for 30 sec, and discard the filtrate;

[0042] (6) Add 500 μL Buffer RWB to FastPure RNA Columns V (please check whether 48 ml of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate;

[0043] (7) Repeat step 6;

[0044] (8) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 2 min.

[0045] (9) Transfer FastPure RNA Columns V to new RNase-free Collection Tubes 1.5 mL centrifuge tubes, add 30-100 μL of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12,000 rpm (13,400 × g) for 1 min;

[0046] (10) Use the first eluate to add back to the adsorption column and repeat step 9.

[0047] 2.2 Synthesis of the first strand of reverse transcribed cDNA

[0048] cDNA was reverse transcribed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing).

[0049] (1) Removal of genomic DNA

[0050]

[0051] Reaction procedure: 42°C, 2 min;

[0052] (2) Reverse transcription

[0053]

[0054] Reaction program: 37°C, 15 min; 85°C, 5 s.

[0055] 2.3 Target gene cloning

[0056] 2.3.1 Gene cloning

[0057] Specific primers for PbGRF1 were designed (Table 1), and the amplification system and procedure are shown in Table 2.

[0058] Table 1 Cloning primers of PbGRF1

[0059]

[0060] Table 2 Cloning system

[0061]

[0062]

[0063] Reaction program: 95°C, 2 min; 95°C, 20 s, 50°C-60°C, 20 s, 72°C, 4 kb / min, 35 cycles; 72°C, 5 min, 12°C, hold.

[0064] 2.3.2 Target fragment recovery

[0065] Prepare 1% agarose gel and detect the PCR product by agarose gel electrophoresis. If the band is correct, refer to the instruction manual (HLingene, Shanghai) to excise the gel and recover the target fragment.

[0066] (1) Prepare a sterilized 2 mL centrifuge tube and weigh the empty tube;

[0067] (2) Use a clean scalpel to cut out the gel block containing the target fragment under ultraviolet light, chop the gel block into pieces and put it into a 2 mL centrifuge tube, weigh and calculate the volume of the gel block (with 1 mg = 1 μL as the standard);

[0068] (3) Add sol solution DD 3 times the volume of gel to the gel block, dissolve the gel block at 56°C, and shake intermittently to mix;

[0069] (4) Add the solution from the previous step to the EC adsorption column, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0070] (5) Add 600 μL of rinse solution WB to the adsorption column, incubate at room temperature at 12000 rpm for 30 s, and discard the waste solution;

[0071] (6) Repeat step (5);

[0072] (6) Idle: 12000 rpm, 2 min;

[0073] (7) Place the EC adsorption column in a sterilized 1.5 mL centrifuge tube, add 30 μL of sterile water (preheated to 65°C) to the adsorption membrane, let stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, and store the collected solution at -20°C.

[0074] 2.3.3 Target fragment ligation and transformation

[0075] (1) Ligation: Use pEASY Blunt Zero vector ( -Blunt Zero Cloning Kit, Transgene) and the target gene, mix the following solutions, mix gently, and centrifuge briefly. PCR: 25℃, 30min.

[0076]

[0077] (2) Transformation: Take competent DH5α Escherichia coli (Weidi Biotechnology, Shanghai; CAT#: DL1001), put it in ice to melt, add 5 μL of the above ligation product, and let it stand in ice for 25 minutes. Heat shock at 42°C for 45 seconds, then move it to ice and let it stand for 2 minutes, add 700 μL of antibody-free LB, and resuscitate at 37°C, 200 rpm for 1 hour. Centrifuge at 5000 rpm for 2 minutes, retain about 100 μL of supernatant, blow and resuspend the bacterial block, and spread it on LB+Kan (50 mg / L) medium. Culture at 37°C for about 12 hours until a single colony grows.

[0078] (3) Bacterial test: Pick a single white colony on the plate, add 500 μL of liquid LB medium (containing 50 mg / L Kana), and culture at 37°C with a shaker at 200 rpm for 3-5 hours. Take 1 μL of bacterial solution as a template for PCR detection. The primers are gene cloning primers. The system and procedure are as follows:

[0079]

[0080]

[0081] The reaction program was: 94°C, 5 min; 94°C, 30 s, 50°C-60°C, 30 s, 72°C, 1 kb / min, 35 cycles; 72°C, 10 min, 12°C, hold.

[0082] (4) Detect the PCR products of the bacterial culture by agarose gel electrophoresis, and select the positive clones and send them to Zhejiang Qingke Biotechnology Co., Ltd. for sequencing.

[0083] (5) After the SnapGene sequencing results are correct, the plasmid is extracted using the Transgene EasyPure PlasmidMiniPrep Kit, and the expression vector of the PbGRF1 gene is constructed using the obtained positive plasmid.

[0084] 2.3.4 Construction of expression vector using homologous recombination

[0085] according to IIOne Step Cloning Kit was used to construct the expression vector by homologous recombination.

[0086] (1) Design insert fragment primers, add the vector terminal homologous sequence before the gene-specific primer, and remove the stop codon from the reverse primer. Use the above positive plasmid as a template for amplification, the system is as follows:

[0087]

[0088] Reaction program: 95°C, 2 min; 95°C, 20 s, 50°C-60°C, 20 s, 72°C, 4 kb / min, 35 cycles; 72°C, 5 min, 12°C, hold.

[0089] (2) After ensuring that the product is a single band, the target fragment is excised and recovered according to the instructions of the gel recovery kit (HLingene, Shanghai).

[0090] (3) The overexpression vector pK2W7-eYGFPuv-3xFLAG was linearized by double restriction digestion, and the following PCR system was configured using the pK2W7-eYGFPuv-3xFLAG plasmid as a template:

[0091]

[0092] Reaction procedure: 37°C, 3h; 16°C, hold.

[0093] (4) Refer to the instructions of the gel recovery kit (HLingene, Shanghai) to excise and recover the target fragment.

[0094] (5) Vector usage: 240 ng, insert usage: 60 ng

[0095]

[0096]

[0097] After mixing by pipetting, incubate in PCR instrument at 37°C for 30 min.

[0098] The reaction product was transformed into DH5α E. coli, and the transformation method was referred to 2.3.3 (2), and the product was spread on LB+Spec (50 mg / L) medium. The bacterial test was referred to 2.3.3 (3).

[0099] After detecting the bands by agarose gel electrophoresis, 200 μL of bacterial solution was sent to Qingke Biotechnology Co., Ltd. for sequencing. Positive plasmids were extracted for subsequent Agrobacterium transformation.

[0100] 2.4 Transformation of Agrobacterium tumefaciens

[0101] (1) Take the competent Agrobacterium GV3101 (Weidi Biotechnology, Shanghai; CAT#: AC1001) stored at -80°C, melt it to an ice-water mixture at room temperature, and insert it into ice;

[0102] (2) Add 1 μg of plasmid per 100 μL competent medium, calculate the volume of plasmid added according to the plasmid concentration, and mix evenly by tapping the bottom of the EP tube;

[0103] (3) Place in ice for 5 min, liquid nitrogen for 5 min, 37°C water bath for 5 min, and ice bath for 5 min;

[0104] (4) Add 700 μL of antibody-free LB and culture at 28°C, 200 rpm for 2 h.

[0105] (5) Centrifuge at 5000 rpm for 2 min, collect about 100 μL of supernatant, resuspend the bacterial mass and spread it on LB+Spec (50 mg / L)+Rif (50 mg / L) medium. Incubate at 28°C for 2-3 days.

[0106] (6) Select a single clone for bacterial culture PCR verification, using the same reaction system and procedure as 2.3.3(3).

[0107] (7) Add an equal volume of 50% glycerol to the correctly tested bacterial solution and store at -80°C.

[0108] 2.5 Arabidopsis transformation by inflorescence infection

[0109] (1) WT seeds were sterilized in a clean bench (75% ethanol for 10 min, and anhydrous ethanol for three times), placed on filter paper and blown dry, then sown on MS medium. After vernalization at 4°C for 2 days, they were moved to the Arabidopsis culture room and cultured for 10-14 days. After growing four true leaves, they were transplanted to seedling medium for culture.

[0110] (2) Select wild-type Arabidopsis thaliana that is in good growth and in full bloom, cut off the fruit pods and set aside;

[0111] (3) Take 200 μL of the Agrobacterium tumefaciens culture medium with the correct overexpression vector from the bacterial test strip, add it to 200 mL of YEP medium with resistance (50 mg / L Spec + 50 mg / L Rif), and culture it in a shaking incubator at 28°C and 200 rpm until OD600 = 0.6-0.8;

[0112] (4) Centrifuge the bacterial solution at 5000 rpm for 10 min, discard the supernatant, resuspend with an equal volume of 6% sucrose solution, and add 0.02% Sliwet L-77;

[0113] (5) Immerse the wild-type Arabidopsis inflorescence completely in the infection solution for 45 seconds, absorb the excess liquid with a paper towel, place it flat on a tray, keep it moist, and culture it in the dark for 24 hours;

[0114] (6) After new inflorescences grow out after 7-10 days of normal light culture, a second infection is carried out according to the above method to increase the success rate of infection;

[0115] (7) The collected seeds are T0 generation seeds. Sow them in MS+Kan (50 mg / L) medium according to the method in (1). Positive plants are T1 generation. Continue to collect seeds and screen positive plants until T3 generation for subsequent experiments.

[0116] 2.6 Identification of positive transgenic Arabidopsis

[0117] Use M5 Hiper ultrafast mix reagent to identify Arabidopsis positive plants as follows:

[0118] (1) Add 20 μL of lysis buffer of Polymerase Mix MF848 (Mei5bio, Beijing) into a PCR tube, cut leaves of about 2 mm2, and grind them with a grinding rod;

[0119] (2) Place in a PCR instrument and heat at 95°C for 10 min;

[0120] (3) Centrifuge at 12000 rpm for 2 min and take 2 μL of supernatant as DNA template for later use;

[0121] (4) Configure the PCR reaction system as follows:

[0122]

[0123] The reaction program was: 95°C, 3 min; 94°C, 25 s, 55°C-64°C, 25 s, 72°C, 1 kb / min, 35 cycles; 72°C, 5 min, 12°C, hold.

[0124] 2.7 Semi-quantitative PCR identification of transgenic Arabidopsis

[0125] (1) Extract RNA from wild-type Col and overexpressing plants of Arabidopsis thaliana and reverse transcribe it into cDNA for later use, using the same method as 2.1 and 2.2;

[0126] (2) Dilute the cDNA concentration to make the brightness of the internal reference gene bands of all cDNAs consistent. The internal reference is AtActin (Table 3).

[0127] Table 3 Semi-quantitative PCR primers

[0128]

[0129] 2.8 Phenotypic determination of T3 transgenic Arabidopsis

[0130] (1) Arabidopsis leaf area determination

[0131] Wild-type Arabidopsis and T3 generation overexpressing Arabidopsis were sown at the same time. After 20 days of growth, the rosette leaves were cut before bolting, sorted from large to small and each leaf was numbered. The pictures were imported into Image J software (download website: https: / / imagej.net.) to measure the leaf area and record the data. It was found that the leaves of overexpressing transgenic Arabidopsis were significantly larger than those of the wild type.

[0132] (2) Determination of fresh weight and dry weight of Arabidopsis

[0133] Wild-type Arabidopsis and T3 generation overexpressing Arabidopsis were sown at the same time. After 20 days of growth, the Arabidopsis was washed with clean water and the excess water was wiped off with a paper towel before it bolted. At this time, its fresh weight was weighed with a 1 / 10,000 balance and the data was recorded. It was then placed in a 75°C oven for drying until it reached a constant weight, and its dry weight was taken out and weighed and the data was recorded.

[0134] 3. Experimental results

[0135] 3.1 Total RNA extraction and analysis from Phoebe bournei leaves and Arabidopsis thaliana

[0136] FastPure Plant Total RNA Isolation Kit was used to extract total RNA from Phoebe bournei ( Figure 1 ), and the OD260 / 280 ratio of the extracted RNA was determined by UV spectrophotometer to be between 1.8 and 2.1, indicating that the total RNA purity was good; the agarose gel electrophoresis results showed that the 18s and 28s bands of the RNA sample were very clear, which could be inferred that the RNA was not degraded and met the requirements of the next experiment.

[0137] 3.2 Cloning and vector construction of Phoebe bournei PbGRF1 gene sequence

[0138] Using the cDNA of Phoebe as template and the Phoebe PbGRF1 gene-specific primers, PCR amplification was performed, and the target band of 1808 bp was obtained ( Figure 2 ). The specific primers of the vector linker were PCR amplified using the homologous recombination method and connected to the expression vector pK2W7-eYGFPuv-3xFLAG and transformed into Escherichia coli. After sequencing by Zhejiang Youkang Biotechnology Co., Ltd., a single clone with the correct sequence was selected to extract the plasmid and transformed into GV3101 Agrobacterium, and the cultured Agrobacterium was used to transform Phoebe bournei and Arabidopsis thaliana.

[0139] 3.3 Identification of transiently transformed Phoebe bournei leaves and quantitative analysis by qRT-PCR

[0140] After the injection of Agrobacterium tumefaciens, it can be detected whether it has been transferred into the leaves in the dark for 88 hours. After the successful injection of the bacterial solution, a handheld fluorescence instrument is used to illuminate the leaves in the dark for fluorescence detection. Take the fluorescent leaf samples and extract RNA for qRT-PCR analysis. Compared with the empty EV, the expression level of the corresponding gene of the overexpression plant OE increased by about 10 times, and the expression level of the PbGRF1 gene of the overexpression plant Phoebe bournei increased significantly.

[0141] 3.4 Identification of positive plants of PbGRF1 transgenic Arabidopsis

[0142] After the PbGRF1 gene of Phoebe bournei was transformed into Arabidopsis thaliana, the transgenic positive lines were obtained by PCR identification. The RNA of Col and transgenic Arabidopsis thaliana was extracted and reverse transcribed for semi-quantitative PCR analysis ( Figure 3 ), the expression levels of PbGRF1 genes OE1, OE2 and OE3 in transgenic Arabidopsis thaliana were significantly higher than those in the wild type Col.

[0143] 3.5 Phenotypic observation of transgenic Arabidopsis

[0144] (1) Comparison of Arabidopsis leaf area

[0145] The leaf growth of wild-type Col and transgenic Arabidopsis sown at the same time was observed. It was found that the leaves of transgenic Arabidopsis tended to be larger and greener than those of Col wild-type plants when they grew for the same number of days. The leaf area was measured using Image J software. The leaves of transgenic Arabidopsis were larger and greener than those of Col wild-type plants at 10, 15, and 20 days of growth. The leaves of transgenic Arabidopsis were significantly larger than those of Col wild-type plants ( Figure 4 and Figure 5 )

[0146] (2) Comparison of fresh weight and dry weight of Arabidopsis

[0147] By observing the growth of wild-type Col and transgenic Arabidopsis sown at the same time, it was found that the fresh weight and dry weight of transgenic Arabidopsis showed a significant upward trend compared with the wild-type Col ( Figure 6 ).

[0148] In summary, the leaf size of Arabidopsis thaliana overexpressing the PbGRF1 gene of Phoebe bournei was larger and greener than that of the wild-type Col. At the same time, the fresh weight and dry weight of the overexpressing plants were also heavier than those of the wild-type.

[0149] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Phoebe bournei PbGRF1 gene, characterized in that: The PbGRF1 gene of Phoebe bournei has the nucleotide sequence shown in SEQ ID No: 1, or a sequence in which one or several nucleotides are deleted, added and / or substituted but the function remains unchanged.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene is SEQ ID No:

1.

3. The protein encoded by the gene according to claim 1 or 2, characterized in that The protein consists of the amino acids shown in SEQ ID No.

2.

4. A method for promoting plant leaf growth, the method comprising preparing a modified plant containing the PbGRF1 gene of Phoebe bournei, characterized in that: Compared with the wild-type plant, in the modified plant, the expression of the Phoebe bournei PbGRF1 gene is increased; wherein the base sequence of the Phoebe bournei PbGRF1 gene is shown in SEQ ID NO:

1.

5. The use of Phoebe bournei PbGRF1 gene in promoting plant leaf growth, characterized in that: A modified plant containing the Phoebe bournei PbGRF1 gene is prepared. Compared with a wild-type plant, in the modified plant, the expression of the Phoebe bournei PbGRF1 gene is increased; wherein the base sequence of the Phoebe bournei PbGRF1 gene is shown in SEQ ID NO:

1.

6. The use according to claim 5, characterized in that: The plant is Phoebe bournei or Arabidopsis thaliana.

7. The use according to claim 6, characterized in that: The plant is Arabidopsis thaliana.

8. The use according to claim 7, characterized in that: The Phoebe bournei PbGRF1 gene is connected to a vector, transformed into Arabidopsis thaliana through Agrobacterium-mediated transformation, and then screened, cultured, and a transgenic strain is obtained.

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