Method for improving properties of poplar by using NAC73 gene

Abstract

The invention discloses a method for improving the character of a poplar, which comprises the following steps: highly expressing NAC73 gene in the poplar through a genetic engineering means; poplar character improvement includes: increasing poplar height; the number of poplar internodes is increased; the internode length of the poplar is increased; the poplar leaf area is increased; the xylem width of poplar is increased; and the poplar xylem catheter aperture is improved. The fast-growing poplar is cultivated, and a foundation is laid for cultivating fast-growing high-quality and high-yield new forest species through molecular breeding.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering and relates to a method for improving poplar traits by using NAC73 gene. Background Art

[0002] Forestry is an important industry in the national economy. Forests are a major strategic resource for the country, providing important ecological and economic benefits for the sustainable development of mankind. At present, my country is vigorously promoting the construction of ecological civilization, and the ecological benefits of forestry are highly valued. At the same time, the economic benefits of forestry, that is, wood products, are closely related to all aspects of human daily production and life. The development of forestry can increase farmers' income.

[0003] my country ranks second in the world in terms of wood consumption and is the largest importer of wood. Its dependence on foreign wood continues to grow. In consideration of ecological protection, my country's main source of wood, in addition to imports, is plantations, which only account for 36.45% of the forest area. The contradiction between wood supply and demand is prominent. With severe global climate change and increasing attention to the ecological environment, my country should vigorously develop plantations and improve its independent supply of wood. In order to improve wood quality, make up for the shortage of raw materials and reduce production costs, cultivating new varieties of high-quality and high-yield trees is an important goal of my country's high-quality forestry development.

[0004] Poplars are an important timber species in my country, with straight trunks, high yield, rapid growth, short rotation period, and wide distribution. However, due to the low yield of poplar plantations and the lack of breakthrough varieties, it is difficult to meet the market demand of the industry, which seriously restricts the development of my country's poplar industry. In order to solve the problems of low wood quality and insufficient high-quality varieties, the use of genetic engineering to create new poplar materials with excellent traits is of great significance for the cultivation of fast-growing, high-quality and high-yield new forest varieties.

[0005] Transcription factors are important regulatory factors for plant growth and development. Among them, NAC transcription factors are one of the largest transcription factor families in plants, and are involved in a variety of biological processes such as plant abiotic stress, biotic stress, growth and development, and accumulation of secondary metabolites. In addition, more and more studies have shown that multiple NAC transcription factor members are involved in the hierarchical regulation of cell wall synthesis, playing an important role in plant growth and development, especially in the development of forest wood. Therefore, NAC transcription factors are important genetic resources for the creation of new fast-growing and high-quality forest varieties. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention found that the NAC73 gene can significantly affect the growth of trees by high expression and dominant inhibition of the poplar NAC73 gene. High expression plants have more significant high growth, while the growth of dominant inhibition plants is inhibited. This provides a theoretical basis and technical guarantee for the precise breeding of fast-growing, high-quality and high-yield poplars.

[0007] The present invention aims to solve the problems of low quality of forest trees and insufficient varieties, cultivates fast-growing, high-quality and high-yield new poplar materials, and lays a foundation for cultivating fast-growing, high-quality and high-yield new varieties through molecular breeding.

[0008] The first aspect of the present invention provides a method for improving the traits of poplars, the method comprising: using genetic engineering means to highly express the NAC73 gene in the poplars;

[0009] The improved poplar traits include:

[0010] Increase the height of poplar trees;

[0011] Increase the number of internodes in poplar trees;

[0012] Increase internode length of poplar trees;

[0013] Increase the leaf area of ​​poplars;

[0014] Increase the width of poplar xylem;

[0015] Increase the pore size of poplar xylem vessels.

[0016] In some embodiments, the method comprises the steps of:

[0017] S1: transferring the coding sequence of the NAC73 gene into an expression vector to obtain a recombinant vector containing the NAC73 gene;

[0018] S2: transforming Agrobacterium with the recombinant vector containing the NAC73 gene to obtain recombinant Agrobacterium containing the NAC73 gene;

[0019] S3: infecting a poplar leaf with a wound with the recombinant Agrobacterium containing the NAC73 gene to obtain an infected poplar leaf;

[0020] S4: placing the infected poplar leaves on a co-culture medium to obtain co-cultured leaves;

[0021] The co-culture medium is based on WPM, and the co-culture medium also contains 15-25 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 80-120 μM acetosyringone, and pH 5.5-6.5;

[0022] S5: placing the co-cultivated leaves on a callus induction medium to obtain poplar callus tissue;

[0023] The callus induction medium is based on WPM as a basic medium, and the callus induction medium further contains 15-25 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 0.8-1.2 mg / L 2,4-D, 0.8-1.2 mg / L KT, 150-250 mg / L cephalosporin, 150-250 mg / L timentin, 25-60 mg / L kanamycin, and a pH of 5.5-6.5;

[0024] S6: placing the poplar callus on a differentiation medium to obtain differentiated poplar buds;

[0025] The differentiation medium is based on WPM, and the differentiation medium further contains 15-25 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 0.04-0.06 mg / L NAA, 0.4-0.6 mg / L 6-BA, 150-250 mg / L cephalosporin, 150-250 mg / L timentin, 25-60 mg / L kanamycin, and pH is 5.5-6.5;

[0026] S7: inserting the differentiated poplar buds into a rooting screening medium to obtain rooted poplar plants;

[0027] The rooting screening medium is based on WPM as a basic medium, and further contains 8-12 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 150-250 mg / L cephalosporin, 150-250 mg / L timentin, 25-60 mg / L kanamycin, and has a pH of 5.5-6.5.

[0028] In some embodiments, the method is selected from any one or a combination of the following situations C1, C2, C3, C4, C5, C6 and C7;

[0029] C1: In S1, the backbone of the expression vector is pK2GW7 vector;

[0030] C2: In S2, the Agrobacterium is Agrobacterium GV3101; or

[0031] The Agrobacterium resuspension solution is 1 / 2MS culture solution containing 25-35g / L sucrose;

[0032] C3: In S3, the poplar leaves are leaves of poplar tissue culture seedlings;

[0033] C4: In S4, the culture conditions are: dark culture for 1.5-2.5 days;

[0034] C5: In S5, the culture conditions are: dark culture for 14-30 days;

[0035] C6: In S6, the culture conditions are: culture temperature 20-28°C, light intensity 40-60 μmol·m -2 ·s -1 , the photoperiod is 14-18h light / 6-10h dark per day, and the cultivation time is 45-65 days;

[0036] C7: In S7, the culture conditions are: culture temperature 20-28°C, light intensity 40-60 μmol·m -2 ·s -1 , the photoperiod is 14-18h light / 6-10h dark every day, and the cultivation time is 5-10 days.

[0037] In some embodiments, the poplar tree is of the 84K variety.

[0038] The second aspect of the present invention provides a use of a biomaterial in preparing a formulation for poplar breeding for improving poplar traits;

[0039] The improved poplar traits include:

[0040] Increase the height of poplar trees;

[0041] Increase the number of internodes in poplar trees;

[0042] Increase internode length of poplar trees;

[0043] Increase the leaf area of ​​poplars;

[0044] Increase the width of poplar wood xylem;

[0045] Increase the pore size of poplar xylem vessels;

[0046] The biological material is selected from any one of the following P1, P2, P3, P4, P5, P6, P7 and P8:

[0047] P1: Protein

[0048] The protein is NAC73 protein;

[0049] P2: Fusion protein

[0050] The amino acid sequence of the fusion protein contains the amino acid sequence of the NAC73 protein described in P1 and the amino acid sequence of a functional protein fragment or an inert protein fragment;

[0051] P3: RNA

[0052] The RNA can be translated to obtain the NAC73 protein described in P1 or the fusion protein described in P2;

[0053] P4: Gene

[0054] The coding sequence of the gene can encode the NAC73 protein described in P1 or the fusion protein described in P2;

[0055] P5: Gene expression cassette

[0056] The gene expression product in the gene expression cassette is the RNA described in P3;

[0057] P6: Genetic Engineering Vector

[0058] The genetic engineering vector contains the gene expression cassette described in P5;

[0059] P7: Cells

[0060] The cell contains the genetic engineering vector described in P6;

[0061] The encoded protein in the gene expression cassette of the genetic engineering vector is expressed constitutively, tissue-specifically, or artificially induced; and

[0062] P8: Composition

[0063] The composition contains the RNA described in P3, the genetic engineering vector described in P6 or the cell described in P7.

[0064] In some embodiments, the amino acid sequence of the NAC73 protein is as shown in SEQ ID NO.2 or SEQ ID NO.4.

[0065] In some embodiments, the poplar tree is of the 84K variety.

[0066] In some embodiments, the backbone of the genetic engineering vector is a pK2GW7 vector, and / or

[0067] The cells are Agrobacterium GV3101 cells.

[0068] In some embodiments, the functional protein fragment is a tag peptide and / or a signal peptide used to separate and purify the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The plant height morphological results of seven poplar germplasm plants are shown, where the scale bar is 10 cm.

[0070] Figure 2 The statistical results of plant height of seven poplar germplasms are shown.

[0071] Figure 3The internode and leaf morphology results of seven poplar accessions are shown, where the scale bar is 10 cm.

[0072] Figure 4 The statistical results of internode numbers of seven poplar germplasm plants are shown.

[0073] Figure 5 The statistical results of internode length of seven poplar germplasm plants are shown.

[0074] Figure 6 Leaf area statistics of seven poplar accessions are shown.

[0075] Figure 7 The morphological results of stem sections and xylem of seven poplar accessions are shown. The scale bars in the figures are: A: 200 μm, B: 100 μm, C: 100 μm, D: 100 μm, and E: 10 μm.

[0076] Figure 8 Statistics of xylem width of seven poplar accessions are shown;

[0077] Fig. 9 Statistics of vessel diameters for seven poplar accessions are shown. DETAILED DESCRIPTION

[0078] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0079] Materials and instruments not described in the present invention are conventional materials and instruments in the art, and operation details not described in the present invention are conventional operations in the art. Unless otherwise specified, the nucleic acid sequences shown in the present invention are written from left to right in the direction of 5' to 3'.

[0080] Example 1: Preparation of 84K poplar NAC73 gene high expression plants

[0081] 1. Plant species

[0082] The poplar used in the present invention is Populus alba×P. glandulosa '84K' (referred to as "84K poplar").

[0083] 2. Genetic information

[0084] According to the gene homology naming rules, the 84K poplar NAC73 gene (called PagNAC73 gene) was cloned.

[0085] The coding sequence of the 84K poplar PagNAC73 allele from the first parent (Populus alba, referred to as parent A) is as follows (SEQ ID NO.1):

[0086] ATGACTTGGTGCAATGACTGCAACGATGTCCAAACAATCGAAAGAAGC

[0087] TCTCCTCCACCTTGCAATGCAAGTGTTATTGCTCAAAGACACAAAGAAT

[0088] GCTTGATTCGAAGTTGCCCGTCATGTGGACACCAAATCAAATGCCAAG

[0089] ACCAGGCAAGAATCCATGACTTGCCAGGGCTACCGGCCGGAGTGAAGT

[0090] TTGATCCGACTGATCAAGAGCTGCTTGAGCATTTGGAGGGGAAGGTGA

[0091] AGTCTGATACCCGCAAGGTCCACCCTCTAATTGATGAGTTCATCCCTAC

[0092] AATCGATGGAGAGAATGGGATTTGCTATACACACCCAGAAAAGTTACCA

[0093] GGGGTGAGCAAAGATGGGCTAATTCGCCACTTCTTCCATCGGCCATCGA

[0094] AGGCATACACGACCGGAACAAGGAAGAGAAGAAAGGTACACACGGAC

[0095] ACAGAAGGTGGTGAGACGAGATGGCACAAAACAGGCAAGACCAGACC

[0096] GGTTCTTGCTGGTGGAAAAGTGAAAGGTTACAAGAAGATACTAGTGCT

[0097] TTATACCAACTATGGGAAGCAAAGGAAGCCAGAGAAAACAAATTGGGT

[0098] GATGCATCAATACCATCTTGGAAACAATGAAGAAGAGAAAGATGGAGA

[0099] GCTTGTGGTTTCTAAAGTCTTCTACCAAACACAACCTAGACAGTGTGGT

[0100] TCTCTCATCAAGGATGCTGCTCCTGCTCCTTCAAAATTAAAGGTACCAA

[0101] GTAGTGGGCATGAGGGCTCTAACCTTAAGAATAGTACCCATCTTGCTGA

[0102] TCAGTACTATCACCCTTCTTCTTTTATATCCTTTGACCAAAGTGGACAGA

[0103] ATAGATCAACAAACCCTAACCCTCCTCAACAGCTACTCTCCCATTTTGC

[0104] AGTTCATGATGGATCTTCTTTTATCCCCTGA

[0105] The amino acid sequence of the protein encoded by the 84K poplar PagNAC73 allele from the first parent (parent A) is as follows (SEQ ID NO.2):

[0106] MTWCNDCNDVQTIERSSPPPCNASVIAQRHKECLIRSCPSCGHQIKCQDQ

[0107] ARIHDLPGLPAGVKFDPTDQELLEHLEGKVKSDTRKVHPLIDEFIPTIDGENGICYTHPEKLPGVSKDGLIRHFFHRPSKAYTTGTRKRRKVHTDTEGGETRWHKTGKTRPVLAGGKVKGYKKIL VLYTNYGKQRKPEKTNWVMHQYHLGNNEEEKDGELVVSKVFYQTQPRQCGSLIKDAAPAPSKLKVPSSGHEGSNLKNSTHLADQYYHPSSFISFDQSGQNRSTNPNPPQQLLSHFAVHDGSSFIP

[0108] The coding sequence of the PagNAC73 allele from the second parent (Populus glandulosa, referred to as the G parent), i.e., 84K poplar, is as follows (SEQ ID NO.3): ATGACTTGGTGCAATGACTGCAACGATGTCCAAACAATTGAAAGAAGCTCTCCTCCACCTTGTAATGCAAGTGTTATTGCTCAAAGACACAGAGAATGCTTGATTCGAAGCTGCCCTTCATGTGGACACCAAATCAAATGCCAAGACCAGGCAAGAATCCATGACTTGCCAGGGCTACCGGCCGGAGTGAAGTTTGATCCGACTGATCAAGAGCTGCTTGAGCATTTGGAGGGGAAGGTGAAGTCTGATACCCGCAAGGTCCACCCTCTAATTGATGAGTTCATCCCTACAATCGATGGAGAGAATGGGATTTGCTATACACACCCAGAAAAGTTACCAGGGGTGAGCAAAGATGGGCTAATTCGCCACTTCTTCCATCGGCCATCGAAGGCATACACGACTGGAACAAGGAAGAGAAGAAAGGTACACACGGACACAGAAGGTGGTGAGACGAGATGGCACAAAACAGGCAAGACTAGACCGGTTCTTGCTGGTGGAAAAGTGAAAGGGTACAAGAAGATACTAGTGCTTTATACCAACTATGGGAAGCAAAGGAAGCCAGAGAAAACAAATTGGGTGATGCATCAATACCATCTTGGAAACAATGAAGAAGAGAAAGATGGAGAGCTTGTGGTTTCTAAAGTTTTCTACCAAACACAACCTAGACAGTGTGGTTCTCTCATCAAGGATTCTGTTCCTGCTCCTTCAAAATTAAAGGTACAAAGTAGTGAGCATGAGAGCTCTAACCTTAAGAATAGTACCACTCTTGTTGAGTACTATCACCCTTCTTCTTTTATATCCTTTGACCAAAGTGGACAGAATAGATCAACAAACCCTAACCCTCCTCAACAGCTACTCTCCCATTTTGCAGTTCATGATGGATCTTCTTTTATCCCCTGA

[0109] The amino acid sequence of the protein encoded by the 84K poplar PagNAC73 allele from the second parent (G parent) is as follows (SEQ ID NO.4):

[0110] MTWCNDCNDVQTIERSSPPPCNASVIAQRHRECLIRSCPSCGHQIKCQDQARIHDLPGLPAGVKFDPTDQELLEHLEGKVKSDTRKVHPLIDEFIPTIDGENGICYTHPEKLPGVSKDGLIRHFFHRPSKAYTTGTRKRRKVHTDTEGGETR

[0111] WHKTGKTRPVLAGGKVKGYKKILVLYTNYGKQRKPEKTNWVMHQYHL

[0112] GNNEEEKDGELVVSKVFYQTQPRQCGSLIKDSVPAPSKLKVQSSEHESSNL

[0113] KNSTTLVEYYHPSSFISFDQSGQNRSTNPNPPQQLLSHFAVHDGSSFIP

[0114] 3. Cloning of PagNAC73 gene

[0115] Based on the 84K poplar PagNAC73 gene sequence, cloning primers PagNAC73_F and PagNAC73_R were designed.

[0116] PagNAC73_F (SEQ ID NO:5): ATGACTTGGTGCAATGACTGCAACPagNAC73_R (SEQ ID NO:6): TCAGGGGATAAAAGAAGATCCATC

[0117] RNA was extracted from 84K poplar stems using the TRIZOL method and then reverse transcribed into cDNA for gene cloning templates. Primers PagNAC73_F and PagNAC73_R were used to amplify PCR products using the high-fidelity enzyme Phanta Max Master Mix. The amplification reaction program was set in the PCR thermal cycler as 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 58℃ annealing for 30s, and 72℃ extension for 2min, including 29 cycles of denaturation-annealing-extension, and finally 72℃ full extension for 10min. The PCR product was purified, recovered, connected to the cloning vector, and sequenced to obtain two PagNAC73 allele sequences. The A parent allele PagNAC73a was selected for subsequent gene expression vector construction.

[0118] 4. Construction of PagNAC73 gene expression vector

[0119] The pK2GW7 vector (https: / / www.sciencedirect.com / science / article / pii / S1360138502022513) was double-digested with SpeI and PmeI restriction endonucleases (purchased from NEB), and the target band was detected by agarose gel electrophoresis, and the linearized vector fragment was obtained by gel recovery. The target gene recombination primers PagNAC73-pK-F and PagNAC73-pK-R were designed, and the recombinant cloning vector containing the target gene (A parent allele PagNAC73 gene) obtained in step 3 was used as a template to PCR amplify the product. The aforementioned PCR amplification product was connected to the linearized vector fragment using the homologous recombinase Exnase II (purchased from Nanjing Vazyme), cultured at 37°C for 30 minutes, and placed on ice for 5 minutes. Transform Escherichia coli DH5α competent cells and screen and culture using LB solid medium containing 50 mg / L spectinomycin. Use PK-F and PagNAC73-pK-R as primers for bacterial detection, the target band is 1000bp, and the amplified product is sequenced and verified after amplification with primers PK-F and PK-R. Sequencing verification was used to transform the recombinant pK2GW7 vector into the PagNAC73 gene sequence into Agrobacterium GV3101 competent cells. Use PK-F and PagNAC73-pK-R as primers for monoclonal detection PCR, and determine the successfully transformed monoclonal by electrophoresis. The positive monoclonal was expanded to obtain the recombinant Agrobacterium bacterial solution containing the PagNAC73 gene (bacterial solution 1), which was stored at -80°C for future use.

[0120] PagNAC73-pK-F(SEQ ID NO:7):CCGCCCCCTTCACCGAATTCATG ACTTGGTGCAATGACTG

[0121] PagNAC73-pK-R(SEQ ID NO:8):TCATCCTTGTAATCGAATTCTCAGGGGATAAAAGAAGATCCATC

[0122] PK-F(SEQ ID NO:9):GGACTCCGGTATTTTTACAACAA

[0123] PK-R(SEQ ID NO:10):GTTTACCGCCAATATATCCTGTCA

[0124] 5. Preparation of Poplars with High Expression of PagNAC73 Gene

[0125] (1) Explant treatment: Tender leaves of 84K poplar tissue culture seedlings grown for 4-6 weeks were used as explant transformation materials. After collection, the leaves were first washed with clean water, then disinfected with 20% (w / v) sodium hypochlorite aqueous solution in a clean bench for 20 minutes, and washed with sterile distilled water at least 5 times to ensure that there was no sodium hypochlorite residue on the surface of the material, and excess distilled water was absorbed with sterile filter paper.

[0126] (2) Agrobacterium culture: The recombinant Agrobacterium culture liquid 1 was cultured in YEP liquid medium (200 mL) containing 100 mg / L kanamycin and 50 mg / L rifamycin at 28°C and 180 rpm overnight. After logarithmic phase amplification, the culture liquid was centrifuged at 3600 rpm and 4°C for 10-15 min. The cells were resuspended in sterile 1 / 2MS solution (containing 30 g / L sucrose) to an OD of 1. 600 It is about 0.4, and the infected bacterial solution 2 is obtained for standby use.

[0127] (3) Receptor infection: Use the tip of a knife to make an incision on the main vein of the explant leaves sterilized above, remove the leaf margins, and cut them into approximately 0.5 × 2.0 cm squares. Then infect them in infection solution 2 for 10-20 min, gently rotating and shaking them so that each leaf can be in close contact with Agrobacterium.

[0128] (4) Co-culture: The infected leaves were dried with filter paper and spread on co-culture medium and kept away from light for 2 days. The co-culture medium was based on WPM and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES (2-morpholineethanesulfonic acid), 100 μM acetosyringone, pH 5.9.

[0129] (5) Callus induction culture: The leaves were transferred to callus induction medium and continued to be cultured in the dark for about two weeks. After callus grew, they were transferred to new callus induction medium. The medium was changed every two weeks, and callus grew in about 2-4 weeks. The screening medium was based on WPM and also contained 20g / L sucrose, 7.8g / L agar, 0.5g / LMES, 1.0mg / L 2,4-D, 0.1mg / L KT (kinetin), 200mg / L cephalosporin, 200mg / L timentin, 50mg / L kanamycin, pH 5.9.

[0130] (6) Differentiation culture: When the callus grows to the size of a rice grain, it is transferred to the differentiation medium. The medium is changed every three weeks. The culture temperature is 25°C and the light intensity is 50 μmol·m -2 ·s -1, the photoperiod is 16h light / 8h dark. During this period, the callus will turn green, harden, and sprout. This stage lasts for about two months. The differentiation medium is based on WPM, and also contains 20g / L sucrose, 7.8g / L agar, 0.5g / LMES, 0.05mg / L NAA, 0.5mg / L6-BA, 200mg / L cephalosporin, 200mg / L timentin, 50mg / L kanamycin, pH 5.9.

[0131] (7) Rooting culture: When the seedlings grow to about 2 cm, they are cut and placed in rooting medium for about a week to grow roots. The culture temperature is 25°C and the light intensity is 50 μmol·m -2 ·s -1 The photoperiod was 16 h light / 8 h dark. The rooting medium was based on WPM and also contained 10 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 200 mg / L cephalosporin, 200 mg / L timentin, 50 mg / L kanamycin, and pH 5.9.

[0132] 6. Identification of transgenic poplars

[0133] The DNA of the plants to be tested was extracted, and PCR detection was performed using specific primers PK-F and PagNAC73-pK-R. Among them, the four transgenic plants #6, #12, #14 and #26 (oxPagNAC73a#6, #12, #14, #26) showed positive amplification results, and it was inferred that the PagNAC73 gene had been transferred into the recipient genome, and positive transgenic plants (oxPagNAC73) were obtained.

[0134] Example 2: Preparation of 84K poplar NAC73 gene dominant suppression plants

[0135] 1. Plant species

[0136] Same as Example 1.

[0137] 2. Genetic information

[0138] Same as Example 1.

[0139] 3. Dominant Suppression Vector Preparation

[0140] The pBI121-SRDX vector (the GUS gene fragment in the pBI121 vector was replaced with the SRDX coding sequence fragment using SmaI and Eco53kI restriction endonucleases (purchased from NEB) was double-digested, and the target band was detected by agarose gel electrophoresis, and the linearized vector fragment was obtained by gel recovery. The target gene recombination primers PagNAC73-SRDX-F and PagNAC73-SRDX-R were designed, and the recombinant cloning vector containing the target gene (A parent allele PagNAC73 gene) obtained in step 3 of Example 1 was used as a template to PCR amplify the product. The aforementioned PCR amplification product was connected to the linearized vector fragment using the homologous recombinase Exnase II (purchased from Nanjing Vazyme), cultured at 37°C for 30 minutes, and placed on ice for 5 minutes. Transform Escherichia coli DH5α competent cells and screen and culture using LB solid medium containing 50 mg / L kanamycin. Use SRDX-F and PagNAC73-SRDX-R as primers for bacterial detection, and the target band is 950bp. After amplification with primers SRDX-F and SRDX-R, the amplified product is sequenced and verified. The recombinant pBI121-SRDX vector with the PagNAC73 gene sequence (A parent) was transformed into Agrobacterium GV3101 competent cells by sequencing verification. Use SRDX-F and PagNAC73-SRDX-R as primers for single clone detection PCR, and determine the single clone that has been successfully transformed by electrophoresis. The positive single clone is expanded and cultured to obtain the recombinant Agrobacterium culture containing the PagNAC73-SRDX gene (culture solution 3), which is stored at -80℃ for future use.

[0141] SRDX coding sequence fragment (SEQ ID NO: 11): CTGGATCTGGACCTAGAACTCCGTTTGGGTTTCGCTTAA

[0142] PagNAC73-SRDX-F (SEQ ID NO:12): AGAACACGGGGGACTCTAGAATGACTTGGTGCAATGACTG

[0143] PagNAC73-SRDX-R (SEQ ID NO:13): AGATCCAGCCCGGGGATCCCGGGGATAAAAGAAGATCCAT

[0144] SRDX-F(SEQ ID NO:14):TTGAAGATGCCTCTGCCGAC

[0145] SRDX-R(SEQ ID NO:15):GACCGGCAACAGGATTCAAT

[0146] 4. Poplar leaf disc infiltration transformation

[0147] (1) Explant treatment: Tender leaves of 84K poplar tissue culture seedlings grown for 4-6 weeks were used as explant transformation materials. After collection, the leaves were first washed with clean water, then disinfected with 20% (w / v) sodium hypochlorite aqueous solution in a clean bench for 20 minutes, and washed with sterile distilled water at least 5 times to ensure that there was no sodium hypochlorite residue on the surface of the material, and excess distilled water was absorbed with sterile filter paper.

[0148] (2) Agrobacterium culture: The recombinant Agrobacterium culture liquid 3 was cultured in YEP liquid medium (200 mL) containing 100 mg / L kanamycin and 50 mg / L rifamycin at 28°C and 180 rpm overnight. After logarithmic phase amplification, the culture liquid was centrifuged at 3600 rpm and 4°C for 10-15 min. The cells were resuspended in sterile 1 / 2MS solution (containing 30 g / L sucrose) to an OD of 1. 600 It is about 0.4, and the infected bacterial solution 4 is obtained for standby use.

[0149] (3) Receptor infection: Use the tip of a knife to make cuts on the main veins of the explant leaves sterilized above, remove the leaf margins, and cut into cubes of approximately 0.5 × 2.0 cm. Then infect in infection solution 4 for 10-20 min, gently rotating and oscillating during the process so that each leaf can be in close contact with Agrobacterium.

[0150] (4) Co-culture: The infected leaves were dried with filter paper and spread on co-culture medium and kept away from light for 2 days. The co-culture medium was based on WPM and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES (2-morpholineethanesulfonic acid), 100 μM acetosyringone, pH 5.9.

[0151] (5) Callus induction culture: The leaves were transferred to callus induction medium and continued to be cultured in the dark for about two weeks. After callus grew out, they were transferred to new callus induction medium. The medium was changed every two weeks, and callus grew out in about 2-4 weeks. The screening medium was based on WPM and also contained 20g / L sucrose, 7.8g / L agar, 0.5g / L MES, 1.0mg / L 2,4-D, 0.1mg / L KT (kinetin), 200mg / L cephalosporin, 200mg / L timentin, 50mg / L kanamycin, pH 5.9.

[0152] (6) Differentiation culture: When the callus grows to the size of a rice grain, it is transferred to the differentiation medium. The medium is changed every three weeks. The culture temperature is 25°C and the light intensity is 50 μmol·m -2 ·s -1, the photoperiod is 16h light / 8h dark. During this period, the callus will turn green, harden, and sprout. This stage is about two months. The differentiation medium is based on WPM, and also contains 20g / L sucrose, 7.8g / L agar, 0.5g / LMES, 0.05mg / L NAA, 0.5mg / L 6-BA, 200mg / L cephalosporin, 200mg / L timentin, 50mg / L kanamycin, pH 5.9.

[0153] (7) Rooting culture: When the seedlings grow to about 2 cm, they are cut and placed in rooting medium for about a week to grow roots. The culture temperature is 25°C and the light intensity is 50 μmol·m -2 ·s -1 The photoperiod was 16 h light / 8 h dark. The rooting medium was based on WPM and also contained 10 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 200 mg / L cephalosporin, 200 mg / L timentin, 50 mg / L kanamycin, and pH 5.9.

[0154] 5. Identification of transgenic plants

[0155] The DNA of the plants to be tested was extracted, and PCR detection was performed using specific primers SRDX-F and PagNAC73-SRDX-R. Among them, the two transgenic plants #20 and #25 (srdxPagNAC73a#20 and srdxPagNAC73a#25) showed positive amplification results, which indicated that the PagNAC73-SRDX gene had been transferred into the recipient genome, and positive transgenic plants (srdxPagNAC73) were obtained.

[0156] Example 3. Characterization of genetically engineered poplar plant traits

[0157] The following parallel test experiments were conducted on two-month-old seedlings (two months after conventional tissue culture transplanting) of seven poplar plants (referred to as seven poplar germplasms), namely, parent 84K poplar (abbreviated as CK), oxPagNAC73a#6, oxPagNAC73a#12, oxPagNAC73a#14, oxPagNAC73a#26, srdxPagNAC73a#20 and srdxPagNAC73a#25.

[0158] (1) Effects of PagNAC73 gene on poplar morphology

[0159] Conventional tissue culture seedlings were prepared in parallel for the seven poplar germplasms mentioned above, and the plant morphology was observed two months after transplantation (the results are shown in Figure 1 ) and counted the plant height (see Figure 2 ). Observe the morphology of all internodes arranged from the top to the base of the stem at the same height (see the results for details). Figure 3 ) and internode number (see Figure 4 ) and measured the length of the 1st to 18th internodes (≥6 plants were used for each germplasm, and the results are shown in Figure 5 ). Observe the morphology of the 4th, 5th and 6th internodes (for results, see Figure 3 ) and counted the leaf area (see Figure 6 ).

[0160] It can be seen that the plant height of the overexpression strain was significantly higher than that of the control strain, while the dominant inhibition plant was significantly lower than that of the control group. The internode analysis results showed that the number of internodes in the overexpression strain was significantly more than that in the control group. And compared with the control group, the internode lengths of the 1st to 18th internodes of the overexpression strain increased significantly. The leaf area results showed that the leaves of the overexpression plant became larger, and the leaves of the dominant inhibition strain were significantly smaller than those of the control group. It can be seen that high expression of PagNAC73 can promote the height growth and increase the leaf area of ​​poplar.

[0161] (2) Effects of PagNAC73 gene on poplar xylem

[0162] A vibrating microtome (VT1200S, Leica) was used to slice cross sections and longitudinal sections of the 10th internode of the two-month-old seedlings of the seven poplar germplasms mentioned above. The slice thickness was 50 μm. The fresh slices were routinely stained with 0.01% toluidine blue O (TBO) for 1 min, washed three times with water, and the excess staining liquid on the surface was removed. The slices were covered with coverslips and observed and photographed using an optical microscope (Leica DM6B) to analyze the morphological differences between the cross sections and longitudinal sections of the stems. Double-sided carbon conductive tape (Nissin NEM) was adhered to the observation area of ​​the scanning electron microscope stage, and then the cross-sectional slices of the 7th internode of the stems of the seven poplar germplasms mentioned above were dried and adhered to the tape in the observation area. The stage was placed in a scanning electron microscope for observation. For results, see Figure 7 , wherein A shows the microscopic morphology of the stem cross section; B shows the microscopic morphology of the xylem; C shows the microscopic morphology of the stem cross section; D shows the stem cross section under a scanning electron microscope; and E shows the vessel morphology under a scanning electron microscope.

[0163] The results showed that compared with the control, the xylem of the overexpression plants was wider ( Figure 8 ). The longitudinal section results showed that the cell length of the overexpression strain was shorter than that of the wild type, and the number of cells was greater than that of the wild type, indicating that the cell division of the overexpression plant was vigorous. The scanning electron microscopy results showed that the diameter of the duct of the PagNAC73 high-expression strain was higher than that of the wild type ( Fig. 9). Therefore, it can be inferred that the larger proportion of vascular pore area per unit volume can make the overexpressing plants have a stronger ability to transport water and inorganic salts, so that water and inorganic salts can be transported from the roots to various parts of the plant faster, which is beneficial to the growth and development of the plant. It can be seen that high expression of PagNAC73 promotes cell division and thus promotes plant growth.

[0164] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A method for improving poplar traits, the method comprising: using genetic engineering to highly express the NAC73 gene in poplars; The improved poplar traits include: Increase the height of poplar trees; Increase the number of internodes in poplar trees; Increase internode length of poplar trees; Increase the leaf area of ​​poplars; Increase the width of poplar wood xylem; Increase the pore size of poplar xylem vessels.

2. The method according to claim 1, characterized in that The method comprises the following steps: S1: transferring the coding sequence of the NAC73 gene into an expression vector to obtain a recombinant vector containing the NAC73 gene; S2: transforming Agrobacterium with the recombinant vector containing the NAC73 gene to obtain recombinant Agrobacterium containing the NAC73 gene; S3: infecting a poplar leaf with a wound with the recombinant Agrobacterium containing the NAC73 gene to obtain an infected poplar leaf; S4: placing the infected poplar leaves on a co-culture medium to obtain co-cultured leaves; The co-culture medium is based on WPM, and the co-culture medium also contains 15-25 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 80-120 μM acetosyringone, and pH 5.5-6.5; S5: placing the co-cultivated leaves on a callus induction medium to obtain poplar callus tissue; The callus induction medium is based on WPM as a basic medium, and the callus induction medium further contains 15-25 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 0.8-1.2 mg / L 2,4-D, 0.8-1.2 mg / L KT, 150-250 mg / L cephalosporin, 150-250 mg / L timentin, 25-60 mg / L kanamycin, and a pH of 5.5-6.5; S6: placing the poplar callus on a differentiation medium to obtain differentiated poplar buds; The differentiation medium is based on WPM, and the differentiation medium further contains 15-25 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 0.04-0.06 mg / L NAA, 0.4-0.6 mg / L 6-BA, 150-250 mg / L cephalosporin, 150-250 mg / L timentin, 25-60 mg / L kanamycin, and pH is 5.5-6.5; S7: inserting the differentiated poplar buds into a rooting screening medium to obtain rooted poplar plants; The rooting screening medium is based on WPM as a basic medium, and further contains 8-12 g / L sucrose, 6-10 g / L agar, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 150-250 mg / L cephalosporin, 150-250 mg / L timentin, 25-60 mg / L kanamycin, and has a pH of 5.5-6.

5.

3. The method according to claim 2, characterized in that The method is selected from any one of the following cases C1, C2, C3, C4, C5, C6 and C7 or a combination thereof; C1: In S1, the backbone of the expression vector is pK2GW7 vector; C2: In S2, the Agrobacterium is Agrobacterium GV3101; or The Agrobacterium resuspension solution is 1 / 2MS culture solution containing 25-35g / L sucrose; C3: In S3, the poplar leaves are leaves of poplar tissue culture seedlings; C4: In S4, the culture conditions are: dark culture for 1.5-2.5 days; C5: In S5, the culture conditions are: dark culture for 14-30 days; C6: In S6, the culture conditions are: culture temperature 20-28°C, light intensity 40-60 μmol·m -2 ·s -1 , the photoperiod is 14-18h light / 6-10h dark per day, and the cultivation time is 45-65 days; C7: In S7, the culture conditions are: culture temperature 20-28°C, light intensity 40-60 μmol·m -2 ·s -1 , the photoperiod is 14-18h light / 6-10h dark every day, and the cultivation time is 5-10 days.

4. The method according to claim 1, characterized in that The amino acid sequence of the protein encoded by the NAC73 gene is shown in SEQ ID NO.2 or SEQ ID NO.

4.

5. The method according to claim 1, characterized in that The variety of the poplar is 84K poplar.

6. Use of a biomaterial in the preparation of a formulation for poplar breeding for improving poplar traits; The improved poplar traits include: Increase the height of poplar trees; Increase the number of internodes in poplar trees; Increase internode length of poplar trees; Increase the leaf area of ​​poplars; Increase the width of poplar wood xylem; Increase the pore size of poplar xylem vessels; The biological material is selected from any one of the following P1, P2, P3, P4, P5, P6, P7 and P8: P1: Protein The protein is NAC73 protein; P2: Fusion protein The amino acid sequence of the fusion protein contains the amino acid sequence of the NAC73 protein described in P1 and the amino acid sequence of a functional protein fragment or an inert protein fragment; P3: RNA The RNA can be translated to obtain the NAC73 protein described in P1 or the fusion protein described in P2; P4: Gene The coding sequence of the gene can encode the NAC73 protein described in P1 or the fusion protein described in P2; P5: Gene expression cassette The gene expression product in the gene expression cassette is the RNA described in P3; P6: Genetic Engineering Vector The genetic engineering vector contains the gene expression cassette described in P5; P7: Cells The cell contains the genetic engineering vector described in P6; The encoded protein in the gene expression cassette of the genetic engineering vector is expressed constitutively, tissue-specifically, or artificially induced; and P8: Composition The composition contains the RNA described in P3, the genetic engineering vector described in P6 or the cell described in P7.

7. The use according to claim 6, characterized in that The amino acid sequence of the NAC73 protein is shown in SEQ ID NO.2 or SEQ ID NO.

4.

8. The use according to claim 6 or 7, characterized in that The variety of the poplar is 84K poplar.

9. The use according to any one of claims 6 to 8, characterized in that The backbone of the genetic engineering vector is a pK2GW7 vector, and / or The cells are Agrobacterium GV3101 cells.

10. The use according to any one of claims 6 to 9, characterized in that The functional protein fragment is a tag peptide and / or a signal peptide used for separating and purifying the protein.

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