A method for improving the traits of poplar using the NAC73 gene

Abstract

The present invention discloses a method for improving the traits of poplar trees. The method is as follows: by means of genetic engineering, the NAC73 gene in poplar trees is highly expressed; improving the traits of poplar trees includes: increasing the height of poplar trees; increasing the number of internodes of poplar trees; increasing the length of internodes of poplar trees; increasing the leaf area of poplar trees; increasing the width of the xylem of poplar trees; increasing the pore diameter of the xylem vessels of poplar trees. Cultivating fast-growing poplar trees lays a foundation for cultivating new varieties of fast-growing, high-quality and high-yield forest trees through molecular breeding.

Description

Technical Field

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

[0002] Forestry is an important industry in the national economy. Forests are major strategic resources of the country, providing important ecological and economic benefits for the sustainable development of humanity. Currently, China is vigorously promoting the construction of ecological civilization, and the ecological benefits of forestry have been highly emphasized. At the same time, the economic benefits of forestry, namely wood products, are closely related to all aspects of human daily production and life. Developing forestry can increase farmers' income.

[0003] China's wood consumption ranks second in the world, and it is the largest wood-importing country, with the dependence on imported wood continuously increasing. Due to ecological protection considerations in China, in addition to imports, the main source of wood is artificial forests, which only account for 36.45% of the forest area. The contradiction between wood supply and demand is prominent. With the severe global climate change and increasing attention to the ecological environment, China should vigorously develop the construction of artificial forests to improve the self-supply capacity of wood. In order to improve the quality of wood, make up for the shortage of raw materials, and reduce production costs, cultivating new varieties of high-quality and high-yield forest trees is an important goal for the high-quality development of China's forestry.

[0004] Poplar has a straight trunk shape, a high timber yield, rapid growth, a short rotation period, and a wide distribution range, and it is an important timber tree species in China. However, due to the generally low yield level of poplar plantations and insufficient creation of breakthrough improved varieties, it is difficult to meet the market demand of the industry, seriously restricting the development of China's poplar industry. In view of problems such as low wood quality and insufficient high-quality varieties, using genetic engineering means to create new poplar materials with excellent traits is of great significance for cultivating new varieties of fast-growing, high-quality, and high-yield forest trees.

[0005] Transcription factors are important regulatory factors in plant growth and development. Among them, NAC transcription factors are one of the largest transcription factor families in plants, participating in various biological processes such as plant abiotic stress, biotic stress, growth and development, and the accumulation of secondary metabolites. Moreover, more and more studies have shown that multiple NAC transcription factor members participate in the hierarchical regulation process of cell wall synthesis and play an important role in plant growth and development, especially in the process of wood development in forest trees. Therefore, NAC transcription factors are important gene resources for creating new varieties of fast-growing and high-quality forest trees. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention overexpresses and dominantly inhibits the poplar NAC73 gene and finds that the NAC73 gene can significantly affect the growth of forest trees. The overexpressing plants have more significant height growth, while the growth of the dominantly inhibited plants is inhibited. This provides a theoretical basis and technical guarantee for the precise breeding of fast-growing, high-quality, and high-yield poplar.

[0007] In view of problems such as low forest tree quality and insufficient varieties, the present invention cultivates new materials of fast-growing, high-quality and high-yield poplars, laying a foundation for cultivating new fast-growing, high-quality and high-yield varieties through molecular breeding.

[0008] In the first aspect of the present invention, a method for improving poplar traits is provided, and the method is: by means of genetic engineering, making the NAC73 gene highly expressed in poplars;

[0009] The improvement of poplar traits includes:

[0010] Increasing the height of poplars;

[0011] Increasing the number of internodes of poplars;

[0012] Increasing the internode length of poplars;

[0013] Increasing the leaf area of poplars;

[0014] Increasing the width of the xylem of poplars;

[0015] Increasing the pore diameter of the xylem vessels of poplars.

[0016] In some embodiments, the method includes the following steps:

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

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

[0019] S3: Infect the wounded poplar leaves with the recombinant Agrobacterium containing the NAC73 gene to obtain infected poplar leaves;

[0020] S4: Place 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-morpholinoethanesulfonic acid, 80 - 120 μM acetosyringone, and the pH is 5.5 - 6.5;

[0022] S5: Place the co-cultured leaves on a callus induction medium to obtain poplar callus;

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

[0024] S6: Place the poplar callus on the differentiation medium to obtain differentiated poplar young shoots;

[0025] The differentiation medium is based on WPM medium, and the differentiation medium also contains 15 - 25 g / L sucrose, 6 - 10 g / L agar, 0.4 - 0.6 g / L 2 - morpholinoethanesulfonic acid, 0.04 - 0.06 mg / L NAA, 0.4 - 0.6 mg / L 6 - BA, 150 - 250 mg / L cefamycin, 150 - 250 mg / L ticarcillin, 25 - 60 mg / L kanamycin, with a pH of 5.5 - 6.5;

[0026] S7: Insert the differentiated poplar young shoots on the rooting screening medium to obtain rooted poplar plants;

[0027] The rooting screening medium is based on WPM medium, and the rooting screening medium also contains 8 - 12 g / L sucrose, 6 - 10 g / L agar, 0.4 - 0.6 g / L 2 - morpholinoethanesulfonic acid, 150 - 250 mg / L cefamycin, 150 - 250 mg / L ticarcillin, 25 - 60 mg / L kanamycin, with a pH of 5.5 - 6.5.

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

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

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

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

[0032] C3: In S3, the poplar leaf is a leaf of a poplar tissue culture seedling;

[0033] C4: In S4, the culture condition is: 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: the culture temperature is 20 - 28 °C, the light intensity is 40 - 60 μmol·m -2 ·s -1 , the photoperiod is 14 - 18 h light / 6 - 10 h dark per day, and the culture time is 45 - 65 days;

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

[0037] In some embodiments, the variety of the poplar is Populus alba×Populus glandulosa '84K'.

[0038] The second aspect of the present invention provides the use of a biological material in the preparation of a preparation for poplar breeding for improving poplar traits;

[0039] The improvement of poplar traits includes:

[0040] Increasing the height of the poplar;

[0041] Increasing the number of internodes of the poplar;

[0042] Increasing the internode length of the poplar;

[0043] Increasing the leaf area of the poplar;

[0044] Increasing the xylem width of the poplar;

[0045] Increasing the xylem vessel pore diameter of the poplar;

[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: Cell

[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 constitutively expressed, tissue-specifically expressed or artificially induced to express; 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 variety of the poplar is Populus alba×Populus glandulosa 84K.

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

[0067] The cell is Agrobacterium tumefaciens GV3101 cell.

[0068] In some embodiments, the functional protein fragment is a tag peptide and / or a signal peptide for protein isolation and purification. Description of the Drawings

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

[0070] Figure 2 Shows the plant height statistical results of seven poplar germplasm plants.

[0071] Figure 3The morphological results of internodes and leaves of seven poplar germplasm plants are shown, where the scale bar is 10 cm.

[0072] Figure 4 The statistical results of the number of internodes of seven poplar germplasm plants are shown.

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

[0074] Figure 6 The statistical results of the leaf areas of seven poplar germplasm plants are shown.

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

[0076] Figure 8 The statistical data of the xylem widths of seven poplar germplasms are shown;

[0077] Figure 9 The statistical data of the vessel diameters of seven poplar germplasms are shown. Detailed implementation mode

[0078] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0079] The materials and instruments not described in the present invention are conventional materials and instruments in the art, and the 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 5' to 3' direction.

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

[0081] 1. Plant species

[0082] The poplar used in the present invention is Populus alba×P. glandulosa‘84K’ (abbreviated as “84K poplar”).

[0083] 2. Gene information

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

[0085] The coding sequence of the 84K poplar PagNAC73 allele from the first parent (Populus alba, abbreviated as the A parent) 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 PagNAC73 allele from the first parent (parent A) of Populus alba × Populus glandulosa is as follows (SEQ ID NO.2):

[0106] MTWCNDCNDVQTIERSSPPPCNASVIAQRHKECLIRSCPSCGHQIKCQDQ

[0107] ARIHDLPGLPAGVKFDPTDQELLEHLEGKVKSDTRKVHPLIDEFIPTIDGENGICYTHPEKLPGVSKDGLIRHFFHRPSKAYTTGTRKRRKVHTDTEGGETRWHKTGKTRPVLAGGKVKGYKKILVLYTNYGKQRKPEKTNWVMHQYHLGNNEEEKDGELVVSKVFYQTQPRQCGSLIKDAAPAPSKLKVPSSGHEGSNLKNSTHLADQYYHPSSFISFDQSGQNRSTNPNPPQQLLSHFAVHDGSSFIP

[0108] The coding sequence of the PagNAC73 allele from the second parent (Populus glandulosa, abbreviated 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 PagNAC73 allele from the second parent (G parent), Populus alba × Populus glandulosa 84K, is as follows (SEQ ID NO.4):

[0110] MTWCNDCNDVQTIERSSPPPCNASVIAQRHRECLIRSCPSCGHQIKCQDQARIHDLPGLPAGVKFDPTDQELLEHLEGKVKSDTRKVHPLIDEFIPTIDGENGICYTHPEKLPGVSKDGLIRHFFHRPSKAYTTGTRKRRKVHTDTEGGETR

[0111] WHKTGKTRPVLAGGKVKGYKKILVLYTNYGKQRKPEKTNWVMHQYHL

[0112] GNNEEEKDGELVVSKVFYQTQPRQCGSLIKDSVPAPSKLKVQSSEHESSNL

[0113] KNSTTLVEYYHPSSFISFDQSGQNRSTNPNPPQQLLSHFAVHDGSSFIP

[0114] 3. Cloning of the PagNAC73 gene

[0115] Based on the PagNAC73 gene sequence of Populus alba × Populus glandulosa 84K, cloning primers PagNAC73_F and PagNAC73_R were designed.

[0116] PagNAC73_F (SEQ ID NO:5): ATGACTTGGTGCAATGACTGCAAC PagNAC73_R (SEQ ID NO:6): TCAGGGGATAAAAGAAGATCCATC

[0117] RNA was extracted from the stems of Populus alba × Populus glandulosa 84K using the TRIZOL method and then reverse-transcribed into cDNA for use as a template for gene cloning. Using primers PagNAC73_F and PagNAC73_R, PCR product amplification was performed using the high-fidelity enzyme Phanta Max Master Mix. The amplification reaction program was set in a PCR thermal cycler: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, with 29 cycles of denaturation-annealing-extension, and finally complete extension at 72°C for 10 min. The PCR products were purified, recovered, ligated to a cloning vector, and sequenced to obtain two PagNAC73 allele sequences. The A parent allele PagNAC73a was selected for subsequent construction of the gene expression vector.

[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). Agarose gel electrophoresis was used to detect the target band, and the linearized vector fragment was obtained by gel extraction. The recombinant primers PagNAC73-pK-F and PagNAC73-pK-R for the target gene were designed. Using the recombinant cloning vector containing the target gene (A parental allele PagNAC73 gene) obtained in step 3 as a template, the PCR amplification product was obtained. The above-mentioned PCR amplification product was ligated with the linearized vector fragment using the homologous recombinase Exnase II (purchased from Vazyme, Nanjing), cultured at 37 °C for 30 min, and placed on ice for 5 min. The competent cells of Escherichia coli DH5α were transformed, and the LB solid medium containing 50 mg / L spectinomycin was used for screening and culturing. The bacteria were detected using PK-F and PagNAC73-pK-R as primers. The target band was 1000 bp, and the amplified product was sequenced and verified after amplification with primers PK-F and PK-R. The recombinant pK2GW7 vector into which the PagNAC73 gene sequence was transferred was transformed into the competent cells of Agrobacterium tumefaciens GV3101. The monoclonal detection PCR was carried out using PK-F and PagNAC73-pK-R as primers, and the successfully transformed monoclonal was determined by electrophoresis. The positive monoclonal was amplified and cultured to obtain a recombinant Agrobacterium tumefaciens solution (bacterial solution 1) containing the PagNAC73 gene, which was stored at -80 °C for later 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): GTTTACCCGCCAATATATCCTGTCA

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

[0125] (1) Explant treatment: Young leaves of tissue-cultured seedlings of Populus alba×Populus glandulosa '84K' that had grown for 4 - 6 weeks were used as explant transformation materials. After sampling, the leaves were first washed with clear water, and then disinfected with 20% (w / v) sodium hypochlorite aqueous solution for 20 min in a laminar flow hood, and washed with sterile distilled water at least 5 times to ensure that there was no residual sodium hypochlorite on the material surface, and the excess distilled water was blotted with sterile filter paper.

[0126] (2) Agrobacterium culture: Recombinant Agrobacterium liquid 1 was cultured overnight in a shaker at 28 °C and 180 rpm in 200 mL of YEP liquid medium containing 100 mg / L kanamycin and 50 mg / L rifamycin. After amplification in the logarithmic phase, it was centrifuged at 3600 rmp and 4 °C for 10 - 15 min, and the cells were resuspended in sterile 1 / 2 MS (containing 30 g / L sucrose) solution to an OD 600 of approximately 0.4 to obtain the infection liquid 2 for use.

[0127] (3) Receptor infection: The previously disinfected explant leaves were incised along the main vein with a scalpel tip, the leaf margins were excised, and cut into squares of about 0.5×2.0 cm size, and then infected in the infection liquid 2 for 10 - 20 min, during which it was gently rotated and shaken to ensure that each leaf could be in close contact with Agrobacterium.

[0128] (4) Co-culture: The infected leaves were blotted dry with filter paper and laid flat on the co-culture medium, and placed in the dark for 2 days. The co-culture medium was based on WPM medium and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES (2-morpholinoethanesulfonic acid), 100 μM acetosyringone, pH 5.9.

[0129] (5) Callus induction culture: The leaves were transferred to the callus induction medium and continued to be cultured in the dark for about two weeks. After the callus grew, it was transferred to a 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 medium and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 1.0 mg / L 2,4-D, 0.1 mg / L KT (kinetin), 200 mg / L cefotaxime, 200 mg / L ticarcillin, 50 mg / L kanamycin, pH 5.9.

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

[0131] (7) Rooting culture: After the small seedlings grow to about 2 cm, cut them off and place them in the rooting medium for about one week to root. The culture temperature is 25 °C, and the light intensity is 50 μmol·m -2 ·s -1 , and the photoperiod is 16 h light / 8 h dark. The rooting medium is based on WPM and also contains 10 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 200 mg / L cefotaxime, 200 mg / L ticarcillin, 50 mg / L kanamycin, and pH 5.9.

[0132] 6. Identification of transgenic poplars

[0133] Extract the DNA of the plants to be detected and perform PCR detection using the specific primers PK-F and PagNAC73-pK-R. Among them, the 4 transgenic plants #6, #12, #14, and #26 (oxPagNAC73a #6, #12, #14, #26) showed positive amplification results, indicating that the PagNAC73 gene has been transferred into the receptor genome, and positive transgenic plants (oxPagNAC73) were obtained.

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

[0135] 1. Plant species

[0136] The same as in Example 1.

[0137] 2. Gene information

[0138] The same as in Example 1.

[0139] 3. Preparation of dominant inhibitory vector

[0140] The pBI121-SRDX vector (using SmaI and Eco53kI restriction endonucleases to replace the GUS gene fragment in the pBI121 vector with the SRDX coding sequence fragment, thereby constructing the pBI121-SRDX vector) was double digested with XbaI and BamHI restriction endonucleases (purchased from NEB). Agarose gel electrophoresis was used to detect the target band, and the linearized vector fragment was obtained by gel extraction. The recombinant primers PagNAC73-SRDX-F and PagNAC73-SRDX-R for the target gene were designed. Using the recombinant cloning vector containing the target gene (A parental allele PagNAC73 gene) obtained in step 3 of Example 1 as a template, the PCR amplification product was obtained. The above-mentioned PCR amplification product was ligated with the linearized vector fragment using the homologous recombinase Exnase II (purchased from Vazyme, Nanjing), cultured at 37 °C for 30 min, and placed on ice for 5 min. The competent cells of Escherichia coli DH5α were transformed, and screening culture was carried out using LB solid medium containing 50 mg / L kanamycin. SRDX-F and PagNAC73-SRDX-R were used as primers for bacterial detection, and the target band was 950 bp. After amplification with the primers SRDX-F and SRDX-R, the amplification product was sequenced and verified. The recombinant pBI121-SRDX vector into which the PagNAC73 gene sequence (A parental) was transferred as verified by sequencing was transformed into the competent cells of Agrobacterium tumefaciens GV3101. Monoclonal detection PCR was carried out using SRDX-F and PagNAC73-SRDX-R as primers, and the successfully transformed monoclonal was determined by electrophoresis. The positive monoclonal was amplified and cultured to obtain the recombinant Agrobacterium tumefaciens bacterial liquid containing the PagNAC73-SRDX gene (bacterial liquid 3), which was stored at -80 °C for later 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. Transformation of Poplar Leaf Disks by Infection

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

[0148] (2) Agrobacterium culture: Recombinant Agrobacterium liquid 3 was cultured overnight in a shaker at 28 °C and 180 rpm in 200 mL of YEP liquid medium containing 100 mg / L kanamycin and 50 mg / L rifamycin. After amplification in the logarithmic phase, it was centrifuged at 3600 rmp and 4 °C for 10 - 15 min, and the cells were resuspended in sterile 1 / 2MS (containing 30 g / L sucrose) solution to an OD 600 of approximately 0.4 to obtain the infection liquid 4 for use.

[0149] (3) Receptor infection: The main veins of the previously disinfected explant leaves were scratched with a scalpel tip, the leaf margins were excised, and the leaves were cut into squares approximately 0.5×2.0 cm in size, and then infected in the infection liquid 4 for 10 - 20 min, gently rotating and oscillating during this period to ensure that each leaf was in close contact with Agrobacterium.

[0150] (4) Co-culture: The infected leaves were blotted dry with filter paper and spread on the co-culture medium, and placed in the dark for 2 days. The co-culture medium was based on WPM medium and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES (2-morpholinoethanesulfonic acid), 100 μM acetosyringone, and pH 5.9.

[0151] (5) Callus induction culture: The leaves were transferred to the callus induction medium and continued to be cultured in the dark for about two weeks. After the callus grew, it was transferred to a 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 medium and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 1.0 mg / L 2,4-D, 0.1 mg / L KT (kinetin), 200 mg / L cefotaxime, 200 mg / L ticarcillin, 50 mg / L kanamycin, and pH 5.9.

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

[0153] (7) Rooting culture: After the small seedlings grow to about 2 cm, cut them off and place them in the rooting medium for about one week to root. The culture temperature is 25 °C, and the light intensity is 50 μmol·m -2 ·s -1 , the photoperiod is 16 h light / 8 h dark. The rooting medium is based on WPM and also contains 10 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 200 mg / L cefamycin, 200 mg / L timentin, 50 mg / L kanamycin, and pH 5.9.

[0154] 5. Identification of transgenic plants

[0155] Extract the DNA of the plants to be detected and perform PCR detection using the 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. It is speculated that the PagNAC73-SRDX gene has been transferred into the receptor genome, and positive transgenic plants (srdxPagNAC73) were obtained.

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

[0157] For the two-month-old seedlings of the parental 84K poplar (abbreviated as CK), oxPagNAC73a#6, oxPagNAC73a#12, oxPagNAC73a#14, oxPagNAC73a#26, srdxPagNAC73a#20, and srdxPagNAC73a#25 (abbreviated as 7 poplar germplasms), the following parallel test experiments were carried out respectively.

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

[0159] For the aforementioned 7 poplar germplasms, conventional tissue culture seedlings were prepared in parallel. After transplanting for two months, the plant morphology was observed (the results are shown in Figure 1 ), and the plant height was statistically analyzed (the results are shown in Figure 2 ). Observe the morphology of all internodes arranged from the top to the base of the stem at the same height (the results are shown inFigure 3 ) and the number of internodes (the results are shown in Figure 4 ) and measure the lengths of the 1st to 18th internodes (≥6 plants of each germplasm were used for statistics, and the results are shown in Figure 5 ). Observe the leaf morphology of the 4th, 5th, and 6th internodes (the results are shown in Figure 3 ) and count the leaf area (the results are shown in Figure 6 ).

[0160] It can be seen that the plant height of the overexpression lines is significantly higher than that of the control lines, while the dominant inhibitory plants are significantly lower than the control. The internode analysis results show that the number of internodes of the overexpression lines is significantly more than that of the control. And compared with the control, the internode lengths of the 1st - 18th internodes of the overexpression lines are all significantly increased. The leaf area results show that the leaves of the overexpression plants become larger, and the leaves of the dominant inhibitory lines are significantly smaller compared with the control. It can be seen that the high expression of PagNAC73 can promote the height growth and leaf area increase of poplar.

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

[0162] Use a vibratome (VT1200S, Leica) to make cross - section and longitudinal - section slices of the 10th internode stems of the 7 - month - old seedlings of the aforementioned 7 poplar germplasms respectively. The slice thickness is 50 μm. After the fresh slices are stained with 0.01% toluidine blue O (toluidine blue O, abbreviated as TBO) conventionally for 1 min, wash them three times with water to remove the excess staining solution on the surface, cover with a coverslip, and use an optical microscope (Leica DM6B) to observe and photograph the TBO - stained slices to analyze the morphological differences in the cross - section and longitudinal - section of the stem. Stick a double - sided carbon conductive tape (Nisshin NEM) on the observation area of the scanning electron microscope stage, and then stick the cross - section slices of the 7th internode stems of the aforementioned 7 poplar germplasms after blotting off the excess water on the tape in the observation area. Put the stage into the scanning electron microscope for observation. The results are shown in Figure 7 , where 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 cross - section of the stem under the scanning electron microscope; E shows the morphology of the vessel under the scanning electron microscope.

[0163] The result analysis shows that compared with the control, the xylem of the overexpression plants is wider ( Figure 8 ). The longitudinal - section results show that the cell length of the overexpression lines is shorter than that of the wild - type cells, and the cell number is more than that of the wild - type, indicating that the cell division of the overexpression plants is vigorous. The scanning electron microscope results show that the vessel diameter of the PagNAC73 high - expression lines is higher than that of the wild - type ( Figure 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 the characteristics of poplars, the method comprising: using genetic engineering to NAC73 Gene overexpression; The variety of the poplar is 84K poplar; Said NAC73 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; The improved poplar traits are: 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 diameter of poplar xylem vessels.

2. The method according to claim 1, characterized in that The method comprises the following steps: S1: Put the NAC73 The coding sequence of the gene is transferred into the expression vector to obtain the NAC73 Recombinant vectors of genes; S2: containing the NAC73 The recombinant vector of the gene is transformed into Agrobacterium to obtain NAC73 Genetic recombinant Agrobacterium; S3: containing the NAC73 The gene-recombinant Agrobacterium infects the poplar leaf with wounds to obtain 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 μΜ 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 also 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 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 the 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 / 2 MS culture solution containing 25-35 g / 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 14-18 h light / 6-10 h dark per day, and the culture 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 14-18 h light / 6-10 h dark per day, and the culture time is 5-10 days.

4. Use of a biomaterial in the preparation of a formulation for poplar breeding for improving poplar traits; The variety of the poplar is 84K poplar; The improved poplar traits are: 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 diameter of poplar xylem vessels; The biological material is selected from any one of the following P1, P2, P3, P4, P5, P6 and P7: P1: Protein The protein is NAC73 protein; NAC73 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; P2: Fusion protein The amino acid sequence of the fusion protein contains the amino acid sequence of the NAC73 protein described in P1 and a tag peptide and / or a signal peptide used for separating and purifying the protein; 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; and The encoded protein in the gene expression box of the genetic engineering vector is constitutively expressed, tissue-specifically expressed or artificially induced.

5. The use according to claim 4, characterized in that The backbone of the genetic engineering vector is a pK2GW7 vector, and / or The cells are Agrobacterium GV3101 cells.

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