A PagMUR1a protein and its applications

Abstract

The present invention discloses the application of PagMUR1a protein in promoting tree breeding, belonging to the field of forestry molecular bioscience; the PagMUR1a protein is a MUR1 homologous protein found in woody plants. After highly expressing the PagMUR1a protein in trees, the plant height increases and the growth rate improves. Therefore, obtaining new high-yield tree varieties by expressing the PagMUR1a gene has important value for improving forestry productivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of forestry molecular bioscience, and particularly relates to a PagMUR1a protein and its application. Background Art

[0002] Poplar, a tree of the genus Populus (Latin scientific name: Populus L.), is a tree species with ecological and economic significance. 84K poplar (Populus alba×Populus glandulosa), introduced into China in 1984, grows vigorously in arid and semi-arid regions, is an important fast-growing tree species, and has made great contributions to the restoration of shelter forests and the paper industry.

[0003] The cell wall is a unique structure of plant cells and is closely related to the growth and development of plant cells. The cell wall mainly provides mechanical support for plants, can strengthen cells and maintain cell integrity. The exertion of its functions requires the participation of multiple genes. Among them, MUR1, namely GDP-d-mannose 4,6-dehydratase, catalyzes the first step of de novo synthesis of GDP-L-fucose. GDP-L-fucose is the activated form of L-fucose and is a component of various structural polysaccharides and glycoproteins in plants. Its existence is crucial for maintaining the integrity of the cell wall and enhancing the mechanical properties of the cell wall. However, currently, MUR1 has been studied more in the model plant Arabidopsis thaliana, and there are few studies on the function and mechanism of action of the MUR1 gene in woody plants. Summary of the Invention

[0004] In this application, a homologous gene of the MUR1 gene was discovered in poplar, named PagMUR1a. It was found that its overexpression can increase the plant height of poplar and improve its growth rate, which provides a new method for cultivating new poplar varieties with high quality and high yield. Specifically:

[0005] In the first aspect of the present invention, an isolated PagMUR1a protein is provided. The PagMUR1a protein is derived from poplar, and the PagMUR1a protein has the activity of MUR1.

[0006] Preferably, the PagMUR1a protein has identity with the Arabidopsis thaliana MUR1 protein, and the identity does not exceed 90%, for example, about 89%, 88%, 87%, 86.10%, 85%, 83%, 80%, etc.

[0007] More preferably, the amino acid sequence of the PagMUR1a protein has the amino acid sequence shown in SEQ ID NO.2, or a sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.2.

[0008] Preferably, the sequence with at least 90% identity includes any value between 90% and 100%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

[0009] In a second aspect of the present invention, there is provided an isolated PagMUR1a gene, and the PagMUR1a gene encodes the PagMUR1a protein as described above.

[0010] Preferably, the nucleotide sequence of the PagMUR1a gene includes:

[0011] 1) The nucleotide sequence shown in SEQ ID NO.1,

[0012] 2) A degenerate sequence of the nucleotide sequence shown in SEQ ID NO.1, or,

[0013] 3) A sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO.1 and having the function of encoding the PagMUR1a protein.

[0014] Preferably, the sequence with at least 90% identity includes any value between 90% and 100%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

[0015] In a third aspect of the present invention, there is provided an application of the PagMUR1a protein or the PagMUR1a gene in promoting tree breeding, and the trees include those with exogenous introduction of the PagMUR1a protein or the PagMUR1a gene.

[0016] Preferably, the trees highly express the PagMUR1a protein.

[0017] Preferably, the PagMUR1a protein or the PagMUR1a gene is as defined above.

[0018] Based on the codon optimization strategy for different tree species, codon optimization can be carried out on the basis of the nucleotide sequence shown in SEQ ID NO.1 so that the nucleotide sequence is suitable for application in different tree species.

[0019] Preferably, the promotion of tree breeding is manifested as an increase in tree height and / or an improvement in growth rate in transgenic trees after exogenous introduction of the PagMUR1a protein or the PagMUR1a gene, as compared with wild-type trees without exogenous introduction of the PagMUR1a protein or the PagMUR1a gene.

[0020] Preferably, the trees are broad-leaved trees, including deciduous broad-leaved trees and evergreen broad-leaved trees, for example, trees of the genera Salix, Populus and Salix in the Salicaceae family. More preferably, the trees are poplar trees, including poplars of the Tacamahaca, Leuce, Aigeiros, Turanga and Leucoides schools. Further preferably, the trees are Populus tomentosa, Populus alba, Populus tremella, Populus hebei, Populus rapa, Populus tomentosa, triploid Populus tomentosa and 84k Populus in the Populus school.

[0021] According to a fourth aspect of the present invention, a cell is provided, wherein the cell comprises an exogenously introduced PagMUR1a protein or PagMUR1a gene, and the cell is derived from a tree cell.

[0022] Preferably, the cells highly express PagMUR1a protein.

[0023] Preferably, the cell comprises an exogenously introduced PagMUR1a gene.

[0024] Preferably, the PagMUR1a protein or PagMUR1a gene is as defined above.

[0025] Preferably, the trees are broad-leaved trees, including deciduous broad-leaved trees and evergreen broad-leaved trees, for example, trees of the genera Salix, Populus and Salix in the Salicaceae family. More preferably, the trees are poplar trees, including poplars of the Tacamahaca, Leuce, Aigeiros, Turanga and Leucoides schools. Further preferably, the trees are Populus tomentosa, Populus alba, Populus tremella, Populus hebei, Populus rapa, Populus tomentosa, triploid Populus tomentosa and 84k Populus in the Populus school.

[0026] Preferably, the cell may be a cell derived from any tree, for example, a cell derived from a root, stem, trunk, leaf, flower, callus, etc. of a tree.

[0027] Preferably, the cell does not develop into a plant.

[0028] A fifth aspect of the present invention provides a method for promoting tree reproduction, wherein the method comprises exogenously introducing PagMUR1a protein or PagMUR1a gene into trees.

[0029] Preferably, the promoting of tree reproduction includes promoting an increase in plant height and / or an increase in growth rate of the trees.

[0030] Preferably, the trees highly express PagMUR1a protein.

[0031] Preferably, the PagMUR1a protein or PagMUR1a gene is as defined above.

[0032] Based on the codon optimization strategies for different tree species, codon optimization can be carried out on the basis of the nucleotide sequence shown in SEQ ID NO.1 to make the nucleotide sequence suitable for the application of different tree species.

[0033] Preferably, the step of introducing the PagMUR1a gene into trees includes:

[0034] S1 Gene cloning: including obtaining the coding region sequence of the PagMUR1a gene, synthesizing the coding region sequence of the PagMUR1a gene and constructing a plasmid, and then amplifying the coding region sequence of the PagMUR1a gene using the obtained plasmid as a template;

[0035] S2 Vector construction: connecting the coding region sequence of the PagMUR1a gene to an expression vector by double digestion to construct a PagMUR1a overexpression vector; preferably, the overexpression vector is a 35S::PagMUR1a overexpression vector;

[0036] S3 Genetic transformation: using the Agrobacterium-mediated genetic transformation method to introduce the expression vector into trees to obtain transgenic plants with overexpression.

[0037] Preferably, the step further includes: S4 Identification of positive seedlings: extracting the genome of the plants to identify positive transgenic plants.

[0038] Preferably, the trees are suitable for the range of trees defined above. More preferably, the trees are Populus sect. Populus trees, and in a specific embodiment, the trees are 84K poplar.

[0039] The sixth aspect of the present invention provides a tree prepared by the method for promoting tree breeding as described above, wherein the tree is exogenously introduced with any of the above PagMUR1a proteins or any of the above PagMUR1a genes.

[0040] Preferably, the tree highly expresses the PagMUR1a gene.

[0041] Preferably, compared with wild-type trees, the plant height of transgenic plant trees highly expressing PagMUR1a increases and / or the growth rate increases.

[0042] Preferably, the trees are suitable for the range of trees defined above. More preferably, the trees are Populus sect. Populus trees, and in a specific embodiment, the trees are 84K poplar.

[0043] The seventh aspect of the present invention provides an application of the above trees, and the application includes:

[0044] (1) As an energy source;

[0045] (2) Pulp manufacturing;

[0046] (3) Building decoration; or,

[0047] (4) Environmental protection.

[0048] Compared with the prior art, the present invention has the following beneficial technical effects:

[0049] The present invention discovers for the first time a homologous protein of MUR1 or its coding gene in woody plants and determines its function.

[0050] The present invention discovers that after introducing the PagMUR1a gene into trees and the PagMUR1a protein is highly expressed, it can promote the growth of trees, increase the plant height of plants, and improve the growth rate. Therefore, obtaining a new high-yield poplar variety by expressing the PagMUR1a gene has important value for improving forestry productivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0052] Figure 1 It is the vector map of pCAMBIA2300;

[0053] Figure 2 It is the phenotypes of 35Spro::PagMUR1a / mur1 transgenic plants and Arabidopsis mur1 mutants;

[0054] Figure 3 It is the DNA identification of 35Spro::PagMUR1a-GFP transgenic poplars;

[0055] Figure 4 It is the detection of the transcriptional level expression of 35Spro::PagMUR1a-GFP transgenic poplars;

[0056] Figure 5 It is that overexpression of PagMUR1a increases the plant height of poplars;

[0057] Figure 6 It is that overexpression of PagMUR1a improves the growth rate of poplars. DETAILED DESCRIPTION OF THE INVENTION

[0058] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0059] The following materials or reagents are all purchased unless otherwise specified.

[0060] Example 1: Cloning of the coding region sequence of the PagMUR1a gene of Populus

[0061] I. Cloning of the target gene:

[0062] 1. According to the sequence of the AtMUR1 protein (Arabidopsis thaliana MUR1 protein) queried on the TAIR website, and aligning its sequence on the Phytozome website of the Populus trichocarpa genome, the coding sequence of its homologous gene (gene number: Potri.007G013900.1) was obtained, and primers were designed based on the coding sequence. The gene and protein sequences are as follows:

[0063] CDS sequence:

[0064] ATGGCATCCGAATCAAACCAACCCGGATCCGACTCCACTATTCCTGTCAACGGC

[0065] GAAATCACACCCAAAATCGCCCTGATCACCGGTATAACCGGCCAAGATGGTTCTTA

[0066] TCTTACCGAATTCCTCCTGAACAAAGGCTATGAAGTTCATGGCTTGATCCGTCGGT

[0067] CATCCAATTTCAATACGCAGCGGATTAACCATATTTATATAGATCCACACAATGCTCA

[0068] TAAGGCCCGAATGAAACTCCACTATGCAGATCTAAGCGACGCGTCGTCTCTCCGTC

[0069] GTTGGCTCGACACCATCAACCCCGACGAAGTCTATAACCTGGCGGCCCAATCACAC

[0070] GTGGCTGTCTCTTTCGAAATCCCTGATTACACGGCGGATGTCGTCGCCACCGGAGC

[0071] CCTCCGCCTTCTTGAGGCGGTGAGATCTCATATTGCCGCGACAGGGCGGAGCCATA

[0072] TCAAGTATTACCAAGCGGGATCTTCGGAGATGTTTGGATCTACGCCGCCTCCGCAA

[0073] TCAGAGACCACCCCGTTTCATCCAAGATCCCCATACGCGGCGTCAAAATGCGCGGC

[0074] GCATTGGTATACTGTCAATTACAGGGAAGCTTATGGGTTGTATGCTTGCAATGGGAT

[0075] TCTTTTCAACCATGAATCTCCACGTAGGGGAGAGAATTTTGTGACCCGAAAGATTA

[0076] CGAGAGCTGTTGGGAGAATTAAGGTGGGGTTGCAGAATAAGCTGTTTTTAGGGAA

[0077] TTTGCAGGCGTCGAGGGATTGGGGTTTCGCAGGGGATTACGTGGAAGCAATGTGG

[0078] ATGATGTTGCAGCAGGAAAAGCCTGATGATTACGTGGTTGCGACGGAGGAGTCGC

[0079] ATACGGTGGAGGAGTTTTTGGATGTGGCATTTGGATATGTTGGATTGAATTGGAAG

[0080] GATCATGTTGTGATTGATAAGAGGTATTTTAGGCCTGCTGAAGTGGATAATTTGAAA

[0081] GGGGATTCGAGTAAGACGAGGAAAGTGCTTGGTTGGAAGCCTAAAGTTGGGTTTG

[0082] AGCAATTGGTGAAGATGATGGTTGATGAAGATATTGATTTAGCCAAGAGGGAGAAGGTTCTTGTTGATGCTGGCTATATGGATGCCCAGCAGCAACCTTGA(SEQ ID No.1) Amino acid sequence:

[0083] MASESNQPGSDSTIPVNGEITPKIALITGITGQDGSYLTEFLLNKGYEVHGLIRRSSN

[0084] FNTQRINHIYIDPHNAHKARMKLHYADLSDASSLRRWLDTINPDEVYNLAAQSHVAV

[0085] SFEIPDYTADVVATGALRLLEAVRSHIAATGRSHIKYYQAGSSEMFGSTPPPQSETTPFH

[0086] PRSPYAASKCAAHWYTVNYREAYGLYACNGILFNHESPRRGENFVTRKITRAVGRIKV

[0087] GLQNKLFLGNLQASRDWGFAGDYVEAMWMMLQQEKPDDYVVATEESHTVEEFLDV

[0088] AFGYVGLNWKDHVVIDKRYFRPAEVDNLKGDSSKTRKVLGWKPKVGFEQLVKMMVDEDIDLAKREKVLVDAGYMDAQQQP(SEQ ID No.2)

[0089] 2. Amplification of the target gene

[0090] 1) Design double digestion primers according to the CDS sequence, as shown in Table 1:

[0091] Table 1: Primers for PagMUR1a gene amplification

[0092]

[0093] 2) Using the cDNA of Populus alba×Populus glandulosa as a template, amplify the gene sequence according to the PCR reaction system in Table 2, and name the gene obtained from Populus alba×Populus glandulosa as PagMUR1a.

[0094] Table 2: PCR amplification system

[0095]

[0096] Reaction procedure: 95°C for 2 min; (denaturation: 95°C for 20 s, annealing: 67°C for 20 s, extension: 72°C for 1 min) × 33 cycles; extension: 72°C for 2:30 s; incubation at 16°C.

[0097] 3) Agarose gel electrophoresis and gel extraction

[0098] Gel preparation: 0.5 g of agarose, 50 mL of 1× TAE, dissolved by microwave heating, cooled to about 50°C, added with 5 μL of GoldView (purchased from Zhongke Ruite Biotechnology Co., Ltd.), shaken well and poured into the gel plate and inserted with a comb for standby.

[0099] Gel running: Add 10× Loading buffer to the PCR product and then add it to the gel wells for electrophoresis detection. Cut the gel strip of PagMUR1a and recover the cDNA fragment of PagMUR1a using a gel extraction kit.

[0100] 2. Construction of PagMUR1a expression vector

[0101] 1) Cloning and ligation: Use restriction enzymes Kpn I and XbaI I to digest the PagMUR1a product and the pCAMBIA2300 vector (with a 35S promoter, which can achieve overexpression of the transformed gene, and the map is as Figure 1 shown). The restriction enzymes Kpn I and XbaII are purchased from New England Biolabs (NEB) company. The specific operation methods and dosages refer to the instructions. Recover the digested products respectively, and use T4-DNA ligase (purchased from Baori Biotechnology Beijing Co., Ltd., and the specific operation methods and dosages refer to the instructions) to ligate the target gene fragment and the expression vector.

[0102] 2) Transformation of Escherichia coli: Transfer the ligation product into Escherichia coli competent TOP 10 (purchased from Shanghai Weidi Biotechnology Co., Ltd., and the specific operation methods and dosages refer to the instructions), add 700 μL of LB culture medium, and then incubate in a shaker at 37°C at 180 rpm for 1 h. Then centrifuge at 5000 rpm for 1 min, retain 100 μL of the supernatant, pipette and mix well, and evenly coat it on the LB solid medium containing 50 μg / mL kanamycin with a disposable spreader, and then invert and place it in a 37°C constant temperature incubator for 12 - 16 h. After the colonies grow, pick a single colony into 700 μL of LB liquid medium (containing 50 μg / mL kanamycin), culture at 37°C for 1 h, and extract the plasmid (kit The Plasmid MiniPrep Kit was purchased from Beijing TransGen Biotech Co., Ltd. The specific operation steps were referred to the instruction manual. Then it was sent to the company for sequencing to screen positive clones, and the recombinant vector was named pCAMBIA2300-35Spro::PagMUR1a-GFP.

[0103] 3) Agrobacterium transformation: 1 μL of the recombinant plasmid was transferred into Agrobacterium tumefaciens GV3101 (purchased from Shanghai Sangon Biotech Co., Ltd. The specific operation steps were referred to the instruction manual) to obtain Agrobacterium tumefaciens containing the vector pCAMBIA2300-35Spro::PagMUR1a-GFP.

[0104] 4) Preparation of LB liquid medium: 5 g of tryptone, 5 g of NaCl, 2.5 g of yeast extract, 500 mL of ddH2O. Autoclaved at 121 °C for 20 min.

[0105] 5) The preparation method of LB solid medium was referred to that of LB liquid medium. 7.5 g of agar powder was added before sterilization and autoclaved at 121 °C for 20 min.

[0106] Example 2: Verification of PagMUR1a function

[0107] Obtaining Arabidopsis thaliana transgenic plants: The obtained Agrobacterium tumefaciens 35Spro::PagMUR1a-GFP was transferred into the Arabidopsis thaliana mur1 mutant by the floral dip method to obtain the 35Spro::PagMUR1a / mur1 transgenic material.

[0108] Observation of Arabidopsis thaliana transgenic phenotype: After Arabidopsis thaliana seeds grew on 1 / 2 MS solid medium containing 0.1% sucrose and pH 5.8 for 7 days, the seedlings were planted in nutrient soil and cultured in an incubator (16 h light / 8 h dark, temperature 25 °C). During this period, the transgenic phenotype was continuously observed. As Figure 2 shown, the upper figure is the 35Spro::PagMUR1a / mur1 transgenic plant, and the lower figure is the mur1 mutant. Due to the MUR1 mutation in the mur1 mutant, after the function was lost, the leaves were round without serrated structures, the leaves were small, and the plants were weak. The leaves of the 35Spro::PagMUR1a / mur1 transgenic plants had serrated structures, the seedlings were significantly larger than the mur1 mutant, and the leaves were enlarged, approaching the phenotype of the wild type WT. This indicates that PagMUR1a can significantly complement the defective phenotypes of leaf roundness and small plants in the mur1 mutant, suggesting that PagMUR1a has the function of MUR1.

[0109] Example 3: Poplar gene transformation

[0110] Transgenic transformation: The overexpression vector containing the PagMUR1a protein coding sequence prepared in Example 1 was transferred into Populus alba×Populus glandulosa (Latin name) by Agrobacterium-mediated method. Specifically,

[0111] 1. Pre-culture of leaves: Select aseptic seedlings that have grown vigorously in the tissue culture room for about one month. Cut the 4th to 6th leaves from the top in the ultra-clean bench, and use a sterile scalpel to make a transverse cut on the main vein of the leaf. Place the leaf with the back side up on the pre-culture medium for 1 - 2 days.

[0112] 2. Preparation of Agrobacterium infection solution: Pipette 100 μL of Agrobacterium containing pCAMBIA2300 - 35Spro::PagMUR1a and add it to 100 mL of LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin as antibiotics). After mixing, incubate at 28°C with shaking at 180 rpm / min for 24 h. When the OD600 is measured to be 0.6 - 0.8, perform Agrobacterium transformation.

[0113] 3. Infection and co-culture: In the ultra-clean bench, place the pre-treated leaves into the Agrobacterium solution and soak for 15 min, shaking once every 5 min to ensure that the wounded parts of the leaves are fully in contact with the solution. Use sterile forceps to take out the leaves and place them on sterile filter paper to absorb excess solution. Then place the leaves with the back side up on the pre-culture medium and incubate in the dark at 25°C for 3 days.

[0114] 4. Resistance culture: In the ultra-clean bench, place the leaves that have been incubated in the dark for 3 days on sterile filter paper to absorb the excess solution, and then place them on the differentiation medium containing antibiotics (30 mg / L kanamycin, 0.2 g / L ticarcillin) and incubate at 25°C with a 16 h light / 8 h dark cycle for 2 - 3 weeks.

[0115] 5. Adventitious bud culture: After the leaves have been cultured on the differentiation medium for 2 - 3 weeks, a cluster of adventitious buds will grow at the wound. In the ultra-clean bench, use sterile forceps to separate the adventitious buds and place them on a new differentiation medium for continued culture for about one week.

[0116] 6. Rooting culture: Cut individual adventitious buds about 2 cm in length and transfer them to the resistant rooting medium (containing 30 mg / L kanamycin, 0.2 g / L ticarcillin) for root induction. Roots can be seen to grow after 2 weeks, while the buds that do not root will turn albino and die. After growing for one month, the leaves can be cut for DNA identification, and the plants identified as positive seedlings can be subcultured for propagation.

[0117] 7. Medium formula is shown in Table 3: Prepare 1 L (adjust the pH to 5.8 - 6.0 with NaOH):

[0118] Table 3: Medium formula

[0119]

[0120]

[0121] Example 4: Identification of Transgenic Plants

[0122] 1. Design identification primers: Design primer F from the middle of the sequence of PagMUR1a and primer R from the vector to ensure that PagMUR1a is transferred into the vector. The primers are as follows, with a total fragment length of 871 bp:

[0123] PagMUR1a-F: GTGACCCGAAAGATTACGAGAGCT (SEQ ID No.5)

[0124] 2300-R: GTTATTTGTATAGTTCATCCATGCCAT (SEQ ID No.6)

[0125] 2. Crude extraction of genomic DNA:

[0126] 1) Put 100 mg of poplar leaves into a centrifuge tube, add small steel beads, and freeze in liquid nitrogen for later use;

[0127] 2) Use a grinder, set it for 1 min, place the centrifuge tubes symmetrically to obtain a powder sample;

[0128] 3) Add 500 μL of DNA crude extraction solution and mix well up and down;

[0129] 4) Centrifuge at 12000 g for 5 min at room temperature, take the supernatant and transfer it to a new 1.5 ml centrifuge tube;

[0130] 5) Add an equal volume of isopropanol and mix well, place on ice for 10 min;

[0131] 6) Centrifuge at 12000 g for 5 min at room temperature, discard the supernatant;

[0132] 7) Add 1 ml of 70% ethanol to wash once, centrifuge at 12000 g for 2 min, discard the supernatant, and let the ethanol evaporate completely for several hours;

[0133] 8) Add 30 μL of ddH2O to dissolve the residue at 4 degrees, and the resulting liquid is genomic DNA.

[0134] Preparation of genomic DNA crude extraction solution (Edwards):

[0135] 100 ml: 1.464 g of NaCl, 5 ml of 10% SDS, 5 ml of 0.5 M EDTA (pH = 8.0), 20 ml of 1 M Tris-HCl (pH = 8.0), 70 ml of ddH2O

[0136] 3. PCR Identification

[0137] 1) Using the crudely extracted DNA as a template, amplification was performed using 2×Taq PCR MasterMix (purchased from Beijing Bomed Biotechnology Co., Ltd.).

[0138] 2) Reaction system: 20 μL

[0139] 2×Taq PCR MasterMix 10 μL

[0140] Forward Primmer 1 μL

[0141] Revers Primmer 1 μL

[0142] Template 1 μL

[0143] ddH2O 7 μL

[0144] 3) PCR Program:

[0145] Pre-denaturation: 95°C, 5 min;

[0146] Three-step amplification: 95°C, 10 s

[0147] 55°C, 30 s

[0148] 72°C, 5 min

[0149] Repeat the three-step amplification step 30 times;

[0150] Extension: 72°C, 5 min;

[0151] 16°C, keep warm.

[0152] 3) Electrophoresis detection: Take 10 μL of the PCR product for agarose gel electrophoresis to detect whether the size of the PCR fragment is correct. As shown, the band sizes in lanes 2 - 7, 9 - 16, 18 - 25, and 27 - 35 are correct, where WT is the blank control. Figure 3 Shown in lanes 2 - 7, 9 - 16, 18 - 25, and 27 - 35, the band sizes are correct, where WT is the blank control.

[0153] Example 5: Identification of the expression level of transgenic poplar PagMUR1a:

[0154] 1. Extraction of poplar RNA

[0155] Cut 100 mg of poplar leaves, grind them in liquid nitrogen, and use the EASYspin Plus Plant RNA Rapid Extraction Kit (purchased from Beijing Aidlab Biotechnologies Co., Ltd., and the specific operation steps refer to the instruction manual) to extract the RNA solution.

[0156] 2. Reverse transcription into cDNA: Use a reverse transcription kit (purchased from Beijing TransGen Biotech Co., Ltd.) to reverse transcribe the RNA solution into cDNA. The reverse transcription system of 20 μL is as follows:

[0157]

[0158] After mixing, put it into a PCR instrument, incubate at 42 °C for 30 min, and heat at 85 °C for 5 s.

[0159] 3. Detect the expression level of PagMUR1a in transgenic poplar by fluorescence real-time quantitative PCR method:

[0160] After diluting the reverse-transcribed cDNA to an appropriate concentration, use the quantitative PCR kit TB Premix Ex Taq TM (Tli RNaseH Plus) (brand Taraka) for quantitative determination of gene expression.

[0161] The amplification primers are:

[0162] qRT-F: ACTCCACTATTCCTGTCAACGGC (SEQ ID No.7)

[0163] qRT-R: GCTTAGATCTGCATAGTGGAGTTTCA (SEQ ID No.8)

[0164] The Real-time PCR reaction system is shown in Table 4:

[0165] Table 4: Real-time PCR reaction system

[0166]

[0167] Fluorescence real-time quantitative PCR amplification conditions:

[0168] Pre-denaturation: 95 °C, 15 min;

[0169] Three-step amplification: 95 °C, 10 s

[0170] 50 - 60 °C, 20 s

[0171] 72 °C, 30 s

[0172] Repeat the three-step amplification procedure 40 times, and collect fluorescence signals after each cycle; raise the temperature by 0.5 °C every 5 s from 65 - 95 °C to plot the melting curve. Select samples OE 22, 23, 24, 27, 34 for illustrative purposes. The expression level of transgenic poplar PagMUR1a is as Figure 4 shown. WT is the wild-type 84K poplar, and OE 22, 23, 24, 27, 34 are different transgenic positive lines obtained, respectively. The expression level of their PagMUR1a protein is significantly higher than that of WT. Two lines, OE23 and OE24, were selected for subsequent phenotypic analysis.

[0173] Example 6: Transgenic Phenotypic Analysis

[0174] In a laminar flow hood, cut the apical buds of WT, OE23, and OE24 poplars of the same length and place them in a common rooting medium to grow. After about 1 month of growth, transplant them into nutrient soil and place them in a greenhouse (16 h of light / 8 h of darkness, temperature 25 °C) to measure the plant height after 42 days of growth, and measure the plant height again after 137 days of growth.

[0175] Formula for the rooting medium: 2.215 g of MS powder, 30 g of sucrose, 5 g of agar.

[0176] The results are as Figure 5 shown. The left figure is the actual growth effect diagram, and the right figure is the plant height statistical chart. The average plant height of the wild-type WT is 31.3 cm, and the average plant heights of the overexpression lines OE23 and OE24 are 34.93 cm and 39.025 cm, respectively. The results show that the plant heights of the overexpression lines are significantly higher than those of the wild-type.

[0177] And according to the formula, growth rate = plant height difference / number of days between measurements, calculate the growth rate of the poplar, as Figure 6 shown. The average growth rate of the wild-type WT is 0.119 cm / day, and the average growth rates of the overexpression lines OE23 and OE24 are 0.146 cm / day and 0.166 cm / day, respectively. The results show that the growth rates of the overexpression lines are significantly higher than those of the wild-type.

[0178] After transferring PagMUR1a into Populus alba × Populus glandulosa '84K' by molecular biology means in the present invention, it promotes tree breeding, increases biomass, improves the plant height and growth rate of the trees, thus obtaining a tree species with improved breeding performance.

[0179] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and changes can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A PagMUR1a The use of a gene in promoting the increase of plant height and / or growth rate of poplars is characterized in that: The poplar includes exogenously introduced PagMUR1a Gene, the PagMUR1a The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for promoting the increase of plant height and / or growth rate of poplars, characterized in that: The method comprises placing a PagMUR1a Genes are introduced into poplar trees, PagMUR1a The nucleotide sequence of the gene is shown in SEQ ID NO.1.

Images