A method for improving the traits of poplar using the MYB75 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 MYB75 gene in poplar trees is highly expressed; improving the traits of poplar trees includes: increasing the stem diameter of poplar trees; promoting the growth of poplar tree leaves; promoting the growth of poplar tree roots; increasing the width of the xylem in the poplar tree stem; increasing the lengths of wood fibers and phloem fibers in the poplar tree stem. It lays a foundation for cultivating new materials of fast-growing, high-quality and high-yield poplar wood for plywood by molecular breeding to cultivate new varieties of high-quality and high-yield poplar wood for plywood.

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 the MYB75 gene. Background Art

[0002] With deteriorating environmental conditions, shrinking forestland, and declining timber quality, my country's timber resources can no longer meet the growing demands of the industry. Meanwhile, large-scale timber imports are increasing costs for Chinese companies. To improve timber quality, offset raw material shortages, and reduce production costs, cultivating new varieties of high-quality, high-yield plywood is a key goal for the high-quality development of my country's wood-based panel industry.

[0003] Poplars (Populus L.), with their straight trunks, high yield, rapid growth, short rotation period, and wide distribution, are important plywood species in my country. However, due to the generally low yields of poplar plantations, the lack of breakthrough improved varieties, and the lack of new high-quality, high-yield poplar varieties specifically designed for plywood, it is difficult to meet the diversified timber market demand, hindering the development of my country's poplar industry. To address the problems of low plywood quality and a shortage of varieties, creating new poplar materials with excellent comprehensive traits such as high biomass and long fibers is a key scientific issue in cultivating new fast-growing, high-quality, and high-yield plywood varieties.

[0004] Transcription factors are crucial regulators of plant growth and development. MYB transcription factors, one of the largest families of transcription factors in plants, are involved in a variety of biological processes, including abiotic and biotic stresses, growth and development, and the accumulation of secondary metabolites. Furthermore, a growing body of research indicates that multiple MYB transcription factor members participate in the hierarchical regulation of cell wall synthesis, playing a crucial role in plant growth and development, particularly in wood development in forest trees. Therefore, MYB transcription factors are crucial genetic resources for the development of new forest tree varieties. Summary of the Invention

[0005] To address the challenges of existing technologies, this study overexpressed and edited the MYB75 gene in poplar trees, demonstrating that it influences wood quality. Overexpression in plants resulted in longer fibers. This provides a theoretical foundation and technical support for the precise breeding of fast-growing, high-quality, and high-yield poplar plywood.

[0006] The present invention aims to solve the problems of low quality and insufficient varieties of plywood, cultivates fast-growing, high-quality and high-yield poplar new materials for plywood, and lays a foundation for cultivating new varieties of high-quality and high-yield plywood through molecular breeding.

[0007] The first aspect of the present invention provides a method for improving the traits of poplar trees, the method comprising: using genetic engineering means to highly express the MYB75 gene in the poplar trees;

[0008] The improved poplar traits include:

[0009] Increase the stem diameter of poplar trees;

[0010] Promote the growth of poplar leaves;

[0011] Promote poplar root growth;

[0012] Increase the width of the xylem in poplar stems;

[0013] Increase the length of wood fibers and bast fibers in poplar stems.

[0014] In some embodiments, the method comprises the steps of:

[0015] S1: transferring the coding sequence of the MYB75 gene into an expression vector to obtain a recombinant vector containing the MYB75 gene;

[0016] S2: Transforming Agrobacterium with the recombinant vector containing the MYB75 gene to obtain recombinant Agrobacterium containing the MYB75 gene;

[0017] S3: infecting a poplar leaf with a wound using the recombinant Agrobacterium containing the MYB75 gene to obtain an infected poplar leaf;

[0018] S4: placing the infected poplar leaves on a co-culture medium to obtain co-cultured leaves;

[0019] The co-culture medium is based on WPM, and the co-culture medium further 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 a pH of 5.5-6.5;

[0020] S5: placing the co-cultivated leaves on a callus induction medium to obtain poplar callus tissue;

[0021] The callus induction medium is based on WPM, and 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, and 30-70 mg / L kanamycin, with a pH of 5.5-6.5;

[0022] S6: placing the poplar callus on a differentiation medium to obtain differentiated poplar shoots;

[0023] The differentiation medium is based on WPM, and 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, and 30-70 mg / L kanamycin, with a pH of 5.5-6.5;

[0024] S7: inserting the differentiated poplar shoots into a rooting screening medium to obtain rooted poplar plants;

[0025] The rooting screening medium uses 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, 30-70 mg / L kanamycin, and has a pH of 5.5-6.5.

[0026] In some embodiments, the method is selected from any one of the following cases C1, C2, C3, C4, C5, C6 and C7 or a combination thereof;

[0027] C1: In S1, the backbone of the expression vector is pK2GW7 vector;

[0028] C2: In S2, the Agrobacterium is Agrobacterium GV3101; or

[0029] The Agrobacterium resuspension solution is 1 / 2MS culture solution containing 25-35g / L sucrose;

[0030] C3: In S3, the poplar leaves are leaves of poplar tissue culture seedlings;

[0031] C4: In S4, the culture conditions are: dark culture for 1.5-2.5 days;

[0032] C5: In S5, the culture conditions are: dark culture for 14-30 days;

[0033] 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;

[0034] 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 per day, and the culture time is 5-10 days.

[0035] In some embodiments, the poplar variety is 84K Poplar.

[0036] A second aspect of the present invention provides a use of a biomaterial in preparing a formulation for poplar breeding for improving poplar traits;

[0037] The improved poplar traits include:

[0038] Increase the stem diameter of poplar trees;

[0039] Promote the growth of poplar leaves;

[0040] Promote poplar root growth;

[0041] Increase the width of the xylem in poplar stems;

[0042] Increase the length of wood fibers and bast fibers in poplar stems;

[0043] The biological material is selected from any one of the following P1, P2, P3, P4, P5, P6, P7 and P8:

[0044] P1: Protein

[0045] The protein is MYB75 protein;

[0046] P2: fusion protein

[0047] The amino acid sequence of the fusion protein contains the amino acid sequence of the MYB75 protein described in P1 and the amino acid sequence of the functional protein fragment or the inert protein fragment;

[0048] P3: RNA

[0049] The RNA can be translated to produce the MYB75 protein described in P1 or the fusion protein described in P2;

[0050] P4: Gene

[0051] The coding sequence of the gene can encode the MYB75 protein described in P1 or the fusion protein described in P2;

[0052] P5: Gene expression cassette

[0053] The gene expression product in the gene expression cassette is the RNA described in P3;

[0054] P6: Genetic Engineering Vectors

[0055] The genetic engineering vector contains the gene expression cassette described in P5;

[0056] P7: Cells

[0057] The cells contain the genetic engineering vector described in P6;

[0058] The encoded protein in the gene expression cassette of the genetic engineering vector is expressed constitutively, tissue-specifically, or artificially induced; and

[0059] P8: Composition

[0060] The composition contains the RNA described in P3, the genetic engineering vector described in P6 or the cells described in P7.

[0061] In some embodiments, the amino acid sequence of the MYB75 protein is shown as SEQ ID NO.2 or SEQ ID NO.4.

[0062] In some embodiments, the poplar variety is 84K Poplar.

[0063] In some embodiments, the backbone of the genetic engineering vector is a pK2GW7 vector, and / or

[0064] The cells are Agrobacterium GV3101 cells.

[0065] In some embodiments, the functional protein fragment is a tag peptide and / or signal peptide used to separate and purify the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The PagMYB75 gene knockout target and knockout identification results are shown.

[0067] Figure 2 The effects of the PagMYB75 gene on plant height, leaf length, root length and stem diameter of poplar trees are shown.

[0068] Figure 3 The figures show the xylem morphology and statistical results of poplar stem sections. The scale bars in the figures are: A: 500 μm, B: 100 μm, and C: 500 μm.

[0069] Figure 4 The figures show the statistical results of the xylem fiber morphology and fiber length of poplar stem slices. A shows the results of the xylem fiber morphology of poplar stem slices, where the scale bar in the lower left corner represents 400 μm, and B shows the statistical results of the fiber length of poplar stem. DETAILED DESCRIPTION

[0070] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0071] Materials and instruments not described herein are conventional in the art, and operational details not described herein are conventional in the art. Unless otherwise specified, nucleic acid sequences shown herein are written from left to right in the 5' to 3' direction.

[0072] Example 1: Preparation of 84K poplar MYB75 gene knockout plants

[0073] 1. Plant species

[0074] The poplar used in the present invention is Populus alba×P. glandulosa '84K' (referred to as "84K poplar").

[0075] 2. Genetic information

[0076] According to the gene homology naming rules, the 84K poplar MYB75 gene (called PagMYB75 gene) was cloned.

[0077] The coding sequence of the 84K poplar PagMYB75 allele from the first parent (Populus alba, referred to as parent A) is as follows (SEQ ID NO. 1):

[0078] ATGGGTAGACAACCTTGTTGCGACAAACTTGGTGTGAAGAAGGGGCCTTGGACAGCTGAGGAAGACAAGAAGTTGGTCAGTTTTATTCTCTCACACGGCCAATGTTGTTGGCGTGCTGTACCAAAGCTCGCCGGACTCCGCCGATGTGGCAAGAGCTGCCGTCTTCGCTGGACTAATTACCTCCGGCCAGACTTGAAGAGAGGCCTTCTTAACGAGGATGAGGAAAAACTTGTCATTGATCTCCATGCCCGCCTTGGCAATAGGTGGTCCAAAATCGCTGCAAGATTGCCGGGAAGAACAGATAATGAGATCAAGAATCATTGGAATACTCACATTAAGAAAAAGCTTATTAAGATGGGCATCGATCCTGTTACACATGGGTCTCTCAGTAGACAAGTGAGCCCACAAGAAAGCACAGTATCTTCTCACACTAATTATGATCAACCCATTATTAATGCTGATAATCAGCAGATTCTTCCCAAAATTTGCGCCCACGCCTCCTCTCGTACTGATAATTCAAGCACTACGACTACACCGACAGAAAATTCCTCAGTGGATGAATGCGTAGGGTCGTCAGAACCTAATAATGACAATGATCCATCAATGAGTTTCATATGGTCAGATGCATTTCTTGATGACTCATCTTGGGACTTCCAAGCCACGAGAGAAGATTACAGTGAATTTGGGGTATCTAATTCTTCGTCAGAGGATAGTAACTCTACATGGTTTTTAGACTGTAAGGATCTTGGAGATGAATTCTTTGGGCTTAGTTGCTTCAGTGACGTGGACTTGAGCATTCTAGACATGGTTGGCAAGCATTAA

[0079] The amino acid sequence of the protein encoded by the PagMYB75 allele from the first parent (parent A) is as follows (SEQ ID NO.2):

[0080] MGRQPCCDKLGVKKGPWTAEEDKKLVSFILSHGQCCWRAVPKLAGLRRCGKSCRLRWTNYLRPDLKRGLLNEDEEKLVIDLHARLGNRWSKIAARLPGRTDNEIKNHWNTHIKKKLIKMGIDPVTHGSLSRQVSPQ ESTVSSHTNYDQPIINADNQQILPKICAHASSRTDNSSTTTTPTENSSVDECVGSSEPNNDNDPSMSFIWSDAFLDDSSWDFQATREDYSEFGVSNSSSEDSNSTWFLDCKDLGDEFFGLSCFSDVDLSILDMVGKH

[0081] The coding sequence of the 84K poplar PagMYB75 allele from the second parent (Populus glandulosa, referred to as G parent) is as follows (SEQ ID NO.3):

[0082] ATGGGTAGACAACCTTGTTGCGACAAACTTGGTGTGAAGAAGGGGCCCTGGACAGCTGAGGAAGACAAGAAGTTGGTCAGTTTTATTCTCTCACACGGCCAATGTTGTTGGCGTGCTGTACCAAAGCTCGCCGGACTCCGCCGATGTGGCAAGAGCTGCCGTCTTCGCTGGACTAATTACCTCCGGCCTGACTTAAAGAGAGGCCTTCTTAATGAGGAGGAGGAAAAACTTGTCATTGATCTCCATGCCCGCCTTGGCAATAGGTGGTCCAAAATCGCTGCAAGATTGCCGGGAAGAACAGATAATGAGATCAAGAATCATTGGAATACTCACATTAAGAAAAAGCTTATTAAGATGGGCATCGATCCTGTTACACATGGTTCTCTCAGTAGACAAGTGAGCCCACAAGAAAGCACAGTATCTTGTCACACTAATTATGATCAACCCATTATTAATGCTGATAATCAGCAGGTTCTTCCCAAAATTTGCGCCCACGCCTCCTCTCGTACTGATAATTCAAGCACTACGACTACACCGACAGAAAATTCCTCAGTGGATGAATGCGTAGGGTCGTCAGAACCTAATAATGACAATGATCCATCAATGAGTTTCATATGGTCAGAGGCATTTCTTGATGACTCGTCTTGGAACTTCCAAGCCACGAGAGAAGATTATAGTGAATTTGGGGTATCTAATTCTTCGTCAGAGGATAGTAACTCTACATGGTTTTTAGACTGTAAGGATCTTGGAGATGAATTCTTTGGGCTTAGTTGCTTCAGTGACGTGGACTTGAGTATCCTAGACATGGTTGGCAAGCATTAA

[0083] The amino acid sequence of the protein encoded by the PagMYB75 allele from the second parent (G parent), Populus alba × Populus glandulosa 84K, is as follows (SEQ ID NO.4):

[0084] MGRQPCCDKLGVKKGPWTAEEDKKLVSFILSHGQCCWRAVPKLAGLRRCGKSCRLRWTNYLRPDLKRGLLNEEEEKLVIDLHARLGNRWSKIAARLPGRTDNEIKNHWNTHIKKKLIKMGIDPVTHGSLSRQVSPQ ESTVSCHTNYDQPIINADNQQVLPKICAHASSRTDNSSTTTTPTENSSVDECVGSSEPNNDNDPSMSFIWSEAFLDDSSWNFQATREDYSEFGVSNSSSEDSNSTWFLDCKDLGDEFFGLSCFSDVDLSILDMVGKH

[0085] 3. Gene Target Selection

[0086] In order to ensure that both PagMYB75 alleles can be edited simultaneously and improve the efficiency of biallelic gene knockout, the identical sequences in exon 1 of the two PagMYB75 alleles were detected (see the design idea for details). Figure 1 The following target sequences were designed:

[0087] T1(SEQ ID NO:5):TTCGCTGGACTAATTACCTC

[0088] Oligonucleotides gRNA1_MYB75_F and gRNA1_MYB75_R were synthesized for the target sequence and annealed to form a double-stranded CT.

[0089] gRNA1_MYB75_F(SEQ ID NO:6):attgTTCGCTGGACTAATTACCTCgRNA1_MYB75_R(SEQ ID NO:7):aaacGAGGTAATTAGTCCAGCGA A

[0090] 4. Preparation of Gene Editing Vectors

[0091] The CRISPR / Cas9 system used in the present invention (for the usage method of this version, see the literature Yi An, Yangyan Zhou, Xiao Han, Chao Shen, Shu Wang, Chao Liu, Weilun Yin, Xinli Xia. The GATA transcription factor GNC plays an important role in photosynthesis and growth in poplar. J Exp Bot. 2020 Mar 25; 71(6): 1969-1984.) was donated by Andrew Groover's laboratory. Its entry vector is pEn-Chimera, which contains a target sequence insertion site and a guide RNA (gRNA) expression element; the final vector (also called the destination vector) is pDe-Cas9, which has a Cas9 protein coding sequence and expression elements; after the target sequence expression element in the entry vector is inserted into the final vector through the Gateway reaction, the target receptor is transformed by Agrobacterium.

[0092] Under the action of restriction endonuclease BbsI, the pEn-Chimera vector was linearized by enzyme digestion, and the linearized vector was verified by electrophoresis and purified and recovered. The recovered product was connected to the double-stranded CT under the action of T4 DNA ligase. The recombinant vector was transformed into Escherichia coli DH5α competent cells and screened and cultured using LB solid medium containing 50 mg / L ampicillin. Monoclonal colonies were picked and monoclonal detection PCR was performed using gRNA1_MYB75_F and M13_R as primers. After the successful recombinant positive clone was confirmed by electrophoresis, the correctness of the recombinant sequence was verified by Sanger sequencing using SS42 primers to obtain the recombinant entry vector EVCT.

[0093] M13_R(SEQ ID NO:8):CACAGGAAACAGCTATGAC

[0094] SS42 (SEQ ID NO:9):TCCCAGGATTAGAATGATTAGG

[0095] The recombinant entry vector EVCT was mixed with the final vector plasmid and a Gateway reaction was performed using the LR clonase II enzyme mix kit to form a recombinant product. The recombinant product was transformed into Escherichia coli DH5α competent cells and screened and cultured using LB solid medium containing 50 mg / L spectinomycin. Monoclonal colonies were selected and single-clone detection PCR was performed using Crispr_F and gRNA1_MYB75_R as primers. After confirming the success of the recombinant positive clone by electrophoresis, the correctness of the recombinant sequence was verified by Sanger sequencing using Crispr_F primers. Positive recombinant plasmids were selected and transformed into Agrobacterium GV3101 competent cells. Screening and culture were performed using YEP solid medium containing spectinomycin and rifamycin. Single-clone detection PCR was performed using Crispr_F and gRNA1_MYB75_R as primers. Successful transformation of single clones was confirmed by electrophoresis. Positive single clones were expanded to obtain recombinant Agrobacterium culture 1.

[0096] Crispr_F(SEQ ID NO:10):CTCCCTAGGCCTGTTATCCCT

[0097] 5. Poplar Leaf Disc Infection Transformation

[0098] (1) Explant Treatment: Young leaves of 84K poplar tissue culture seedlings, 4-6 weeks old, were used as explant transformation materials. After collection, the leaves were washed with clean water, then sterilized in a clean bench with a 20% (w / v) sodium hypochlorite solution for 20 min. The leaves were then rinsed at least five times with sterile distilled water to ensure that no sodium hypochlorite residue remained on the surface of the material. Excess distilled water was then removed with sterile filter paper.

[0099] (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 amplification in the logarithmic phase, 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 600 It is about 0.4, and the infected bacterial solution 1 is obtained for use.

[0100] (3) Receptor infection: Use the tip of a knife to cut the main veins of the sterilized explant leaves, remove the leaf margins, and cut them into approximately 0.5 × 2.0 cm squares. Then, infect them in infection solution 1 for 10-20 minutes, gently rotating and shaking them to ensure that each leaf is in close contact with Agrobacterium.

[0101] (4) Co-cultivation: The infected leaves were dried with filter paper and spread flat on a co-culture medium. The co-culture medium was kept in the dark for 2 days. The co-culture medium was WPM as a basal medium, and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES (2-morpholinoethanesulfonic acid), and 100 μM acetosyringone, with a pH of 5.9.

[0102] (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 / 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.

[0103] (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 16 hours of light / 8 hours of darkness. During this period, the callus will turn green, harden, and produce buds. This stage lasts approximately two months. The differentiation medium is based on WPM and also contains 20g / L sucrose, 7.8g / L agar, 0.5g / L MES, 0.05mg / L NAA, 0.5mg / L 6-BA, 200mg / L cephalosporin, 200mg / L timentin, and 50mg / L kanamycin, with a pH of 5.9.

[0104] (7) Rooting culture: After the seedlings grow to about 2 cm, they are cut and placed in rooting medium for about a week to take root. 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 WPM basal medium, 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, and 50 mg / L kanamycin, with a pH of 5.9.

[0105] (8) Positive detection: After 2 weeks of rooting, plant DNA was extracted and PCR detection was performed using specific primers to obtain positive transgenic plants for subsequent analysis.

[0106] 6. Mutation Identification of Transgenic Plants

[0107] The genomic DNA of the positive transgenic poplars was extracted and amplified by PCR using the target site detection primers MYB75_F1 and MYB75_R1. The amplified product was ligated into the cloning vector. E. coli DH5α competent cells were transformed to obtain single colonies, which were used for Sanger sequencing of the target site amplicons to analyze the mutation events. The corresponding target sequences of plants #7 and #8 were compared with those of the wild-type control plant (CK). Figure 1 Bottom half. The two sequences in each plant represent two alleles. This indicates that both PagMYB75 alleles in this plant underwent frameshift mutations, successfully knocking out the target gene. The two knockout lines were named crispr-PagMYB75#7 and crispr-PagMYB75#8.

[0108] MYB75_F1(SEQ ID NO:11):GGTAGACAACCTTGTTGCGAC

[0109] MYB75_R1(SEQ ID NO:12):CTGTCGGTGTAGTCGTAGTGC

[0110] Example 2: Preparation of 84K poplar MYB75 gene high expression plants

[0111] 1. Plant species

[0112] Same as Example 1.

[0113] 2. Genetic information

[0114] Same as Example 1.

[0115] 3. Cloning of the PagMYB75 gene

[0116] Based on the 84K poplar PagMYB75 gene sequence, cloning primers PagMYB75_F and PagMYB75_R were designed.

[0117] PagMYB75_F(SEQ ID NO:13):ATGGGTAGACAACCTTGTTGCG

[0118] PagMYB75_R(SEQ ID NO:14):ATGCTTGCCAACCATGTCTAG

[0119] RNA was extracted from 84K poplar stems using the TRIZOL method and then reverse-transcribed into cDNA for use as a template for gene cloning. PCR products were amplified using primers PagMYB75_F and PagMYB75_R and the high-fidelity enzyme Phanta Max Master Mix. The amplification reaction was set in a PCR thermocycler as follows: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 15 seconds, 58°C annealing for 30 seconds, and 72°C extension for 2 minutes. The denaturation-annealing-extension cycle was repeated 29 times, followed by a final extension at 72°C for 10 minutes. The PCR product was purified, recovered, ligated into the cloning vector, and sequenced. Two PagMYB75 alleles were obtained, and the G parent allele, PagMYB75g, was selected for subsequent gene expression vector construction.

[0120] 4. Construction of PagMYB75 gene expression vector

[0121] 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. The linearized vector fragment was recovered from the gel. The target gene recombination primers PagMYB75-pK-F and PagMYB75-pK-R were designed, and the recombinant cloning vector containing the target gene (G parent allele PagMYB75 gene) obtained in step 3 was used as a template to PCR amplify the product. The aforementioned PCR amplification product and the linearized vector fragment were connected using the homologous recombinase Exnase II (purchased from Nanjing Vazyme), cultured at 37°C for 30 minutes, and placed on ice for 5 minutes. Escherichia coli DH5α competent cells were transformed and screened and cultured using LB solid medium containing 50 mg / L spectinomycin. Using primers PK-F and PagMYB75-pK-R for bacterial testing, the target band was 820 bp. After amplification with primers PK-F and PK-R, the amplified product was sequenced. Sequencing confirmed that the recombinant pK2GW7 vector, containing the PagMYB75 gene sequence, was transformed into Agrobacterium GV3101 competent cells. Single-clone detection PCR was performed using primers PK-F and PagMYB75-pK-R, and successful transformation clones were identified by electrophoresis. Positive single clones were expanded to obtain a recombinant Agrobacterium culture containing the PagMYB75 gene (culture solution 2), which was stored at -80°C until further use.

[0122] PagMYB75-pK-F(SEQ ID NO:15):TCGACCTGCAGGCGGCCGCAATGGGTAGACAACCTTGTTG

[0123] PagMYB75-pK-R(SEQ ID NO:16):TCCTTGTAATCGTTTGTTTGATGCTTGCCAACCATGTCTA

[0124] PK-F(SEQ ID NO:17):GGACTCCGGTATTTTTACAACAA

[0125] PK-R(SEQ ID NO:18):GTTTACCGCCAATATATCCTGTCA

[0126] 5. Preparation of Poplars with High Expression of PagMYB75 Gene

[0127] (1) Explant Treatment: Young leaves of 84K poplar tissue culture seedlings, 4-6 weeks old, were used as explant transformation materials. After collection, the leaves were washed with clean water, then sterilized in a clean bench with a 20% (w / v) sodium hypochlorite solution for 20 min. The leaves were then rinsed at least five times with sterile distilled water to ensure that no sodium hypochlorite residue remained on the surface of the material. Excess distilled water was then removed with sterile filter paper.

[0128] (2) Agrobacterium culture: The recombinant Agrobacterium culture liquid 2 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 amplification in the logarithmic phase, 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 600 It is about 0.4, and the infected bacterial solution 2 is obtained for use.

[0129] (3) Receptor infection: Use the tip of a knife to cut the main veins of the sterilized explant leaves, 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 minutes, gently rotating and shaking them to ensure that each leaf is in close contact with Agrobacterium.

[0130] (4) Co-cultivation: The infected leaves were dried with filter paper and spread flat on a co-culture medium. The co-culture medium was kept in the dark for 2 days. The co-culture medium was WPM as a basal medium, and also contained 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES (2-morpholinoethanesulfonic acid), and 100 μM acetosyringone, with a pH of 5.9.

[0131] (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 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 cephalosporin, 200 mg / L timentin, 50 mg / L kanamycin, pH 5.9.

[0132] (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 16 hours of light / 8 hours of darkness. During this period, the callus will turn green, harden, and produce buds. This stage lasts approximately two months. The differentiation medium is based on WPM and also contains 20g / L sucrose, 7.8g / L agar, 0.5g / L MES, 0.05mg / L NAA, 0.5mg / L 6-BA, 200mg / L cephalosporin, 200mg / L timentin, and 50mg / L kanamycin, with a pH of 5.9.

[0133] (7) Rooting culture: After the seedlings grow to about 2 cm, they are cut and placed in rooting medium for about a week to take root. 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 WPM basal medium, 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, and 50 mg / L kanamycin, with a pH of 5.9.

[0134] 6. Identification of Transgenic Poplars

[0135] The DNA of the plants to be tested was extracted, and PCR detection was performed using specific primers PK-F and PagMYB75-pK-R. Among them, six transgenic plants #3, #9, #12, #16, #26 and #30 (oxPagMYB75#3, #9, #12, #16, #26, #30) showed positive amplification results, suggesting that the PagMYB75 gene had been transferred into the recipient genome, and positive transgenic plants (oxPagMYB75) were obtained.

[0136] Example 3. Characterization of genetically engineered poplar plant traits

[0137] The following parallel test experiments were conducted on two-month-old seedlings (two months after conventional tissue culture transplanting) of six poplar plants (referred to as six poplar germplasms), namely the parent 84K poplar (abbreviated as CK), oxPagMYB75#9, oxPagMYB75#26, oxPagMYB75#30, crispr-PagMYB75#7, and crispr-PagMYB75#8.

[0138] (1) Effects of the PagMYB75 gene on poplar stem and root morphology

[0139] Conventional tissue culture seedlings were prepared in parallel for the six aforementioned poplar germplasms. Plant height and morphology were observed two months after transplantation. The results are shown in Figure 2 A; observe the root morphology, the results are shown in Figure 2 A; observe the morphology of the 7th internode leaves. Figure 2 B. Observe the morphology of all internodes from the top to the base of the stem at the same height (see the results for details). Figure 2 C) and the stem diameter of the 7th internode was measured (≥6 plants were used for each accession, and the results are shown in Figure 3 B).

[0140] It can be seen that phenotypic analysis found that there was no significant difference in height between PagMYB75 overexpression, gene knockout and control plants; the stems of the overexpression plants were significantly thicker, while the gene knockout plants were similar to the control; root system comparison found that the roots of PagMYB75 overexpression plants were significantly denser and had more fine roots than the control, while the gene knockout plants had relatively fewer; the leaf area of ​​PagMYB75 overexpression was larger than that of the control.

[0141] These results indicate that high expression of PagMYB75 can make poplar trees stronger.

[0142] (2) Effects of PagMYB75 gene on poplar xylem

[0143] The seventh internode of the stem of two-month-old seedlings of the six aforementioned poplar germplasms was sectioned using a vibrating microtome (VT1200S, Leica) with a thickness of 50 μm. The fresh sections were stained with 0.01% toluidine blue O (TBO) for 1 min, washed three times with water to remove excess staining solution on the surface, and covered with a coverslip. The TBO-stained sections were observed and photographed using an optical microscope (Leica DM6B) to analyze the morphological differences of the stem cross-sections.

[0144] Calcifluor white (CFW) staining was used to stain the seventh internode stem sections of the six aforementioned poplar accessions to reveal cellulose.

[0145] Phloroglucinol staining was used to stain the seventh internode stem sections of the six poplar accessions mentioned above to reveal the distribution of cellulose and lignin.

[0146] Results see Figure 3 , where A shows the morphology of the stem cross section (upper part) and the morphology of the xylem (lower part); B shows the statistical data of the stem diameter; C shows the statistical data of the cambium width; D shows the statistical data of the vessel width; E shows the statistical data of the xylem width; F shows the distribution of the stem cellulose (upper part) and lignin (lower part) contents.

[0147] The results showed that the diameter and xylem width of the PagMYB75 overexpression lines were significantly increased, while the xylem width of the knockout plants was reduced compared to the control. There was no significant difference in cambium width between the overexpression plants and the control, but the cambium in the knockout plants was significantly reduced. Compared to the control, the overexpression plants had fewer cambium layers but larger cambium cells. There was no significant difference in cellulose content between the transgenic plants and the control. The lignin distribution range was larger in the overexpression plants, while the lignin distribution range was smaller in the knockout plants, indicating that the lignin content in the knockout plants was significantly reduced.

[0148] These results indicate that high expression of PagMYB75 promotes the overall cell development and elongation of the plant, thereby promoting the widening of the xylem.

[0149] (3) Effects of the PagMYB75 gene on the size of poplar fiber cells and vessels

[0150] A vibrating microtome (VT1200S, Leica) was used to section the seventh internode of two-month-old seedlings from each of the six aforementioned poplar accessions to a thickness of 50 μm. Double-sided carbon conductive tape (Nissin NEM) was first applied to the observation area of ​​the scanning electron microscope stage. The sections were then dried and attached to the tape in the observation area. The stage was then placed in the scanning electron microscope for observation, and the images were saved.

[0151] The results are as follows Figure 3 Figure 2 shows the fiber cell pore size and cell wall thickness. Scanning electron microscopy results showed that the fiber cell pore size of PagMYB75 overexpressing plants was significantly increased, and the fiber cell corner (CC) of the overexpressing transgenic plants was significantly reduced. Compared with the control, the CC of the gene knockout plants increased, so the overall fiber cell outline tended to be more rounded ( Figure 3 G). Comparison of vessel pore diameters showed that the vessel pore diameters of plants with high expression of PagMYB75 were significantly increased ( Figure 3 G).

[0152] These results indicate that high expression of PagMYB75 inhibits the accumulation of lignin in cell corners, making the fiber cell lumen larger.

[0153] (4) Effects of PagMYB75 gene on poplar xylem fiber morphology

[0154] Two-month-old internodes of the 8th to 10th internodes of seedlings from each of the six aforementioned poplar accessions were removed. The bark was peeled off, and the separated phloem and xylem were cut longitudinally into several small pieces using a knife and placed in centrifuge tubes. The separation solution (glacial acetic acid (AR) analytical grade: 30% hydrogen peroxide = 1:1) was placed in the centrifuge tubes and placed in a 65°C constant-temperature water bath. Vigorous shaking was required during the separation. After separation was complete, the separation solution containing the fibers was poured into the centrifuge tubes, which were then centrifuged and the supernatant discarded. RO water was added for centrifugal washing, and the process was repeated until the glacial acetic acid odor dissipated. The washed fibers were pipetted from the centrifuge tubes and observed and photographed using an optical microscope (Leica DM6B). Fiber length was measured using Image J software.

[0155] Figure 4 A shows the morphology of xylem and phloem fibers of six poplar accessions.

[0156] Figure 4 B shows the statistical graph of xylem fiber length of six poplar accessions.

[0157] Figure 4 C shows a statistical graph of phloem fiber lengths of six poplar accessions.

[0158] Comparison of xylem fiber size revealed that xylem fibers in poplars overexpressing PagMYB75 were longer and wider, while those in knockout plants were shorter. A comparison of phloem fibers revealed that poplars overexpressing PagMYB75 had more long fibers (>1.6 mm) but also more short fibers, with significant variation in fiber length. Phloem fibers in knockout plants were more uniform in length and smaller than those in controls. These results suggest that the fibers in poplars overexpressing PagMYB75 are longer and wider, which is beneficial for applications such as plywood production.

[0159] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A method for improving the characteristics of poplars, the method comprising: using genetic engineering to MYB75 High gene expression; The variety of the poplar is 84K poplar; Said MYB75 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4; The improved poplar traits include: It does not affect the height of poplar plants and increases the stem diameter of poplars; Promote the growth of poplar leaves; Promote poplar root growth; Increase the width of poplar stem xylem; and Increase the length of wood fibers and bast fibers in poplar stems.

2. The method according to claim 1, characterized in that The method comprises the following steps: S1: Put the MYB75 The coding sequence of the gene is transferred into the expression vector to obtain the MYB75 Recombinant vectors of genes; S2: containing the MYB75 The recombinant vector of the gene is transformed into Agrobacterium to obtain MYB75 Genetic recombinant Agrobacterium; S3: containing the MYB75 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, 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, 30-70 mg / L kanamycin, and pH 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, 30-70 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, 30-70 mg / L kanamycin, and has a pH of 5.5-6.

5.

3. The method according to claim 2, characterized in that In S1, the backbone of the expression vector is pK2GW7 vector.

4. The method according to claim 2, characterized in that In S2, the Agrobacterium is Agrobacterium GV3101.

5. The method according to claim 2, characterized in that In S3, the poplar leaves are leaves of poplar tissue culture seedlings.

6. The method according to claim 2, characterized in that In S4, the culture conditions are: dark culture for 1.5-2.5 days.

7. The method according to claim 2, characterized in that In S5, the culture conditions are: dark culture for 14-30 days.

8. The method according to claim 2, characterized in that In S6, the culture conditions were: 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 cultivation time is 45-65 days.

9. The method according to claim 2, characterized in that In S7, the culture conditions were: 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.

10. Use of a biomaterial in preparing a formulation for poplar breeding for improving poplar traits; The variety of the poplar is 84K poplar; The improved poplar traits include: It does not affect the height of poplar plants and increases the stem diameter of poplars; Promote the growth of poplar leaves; Promote poplar root growth; Increased stem xylem width in poplar trees; and Increase the length of wood fibers and bast fibers in poplar stems; The biological material is selected from any one of the following P3, P4, P5, P6, P7 and P8: P3: RNA The RNA can be translated to obtain MYB75 protein, and the amino acid sequence of the MYB75 protein is shown in SEQ ID NO.4; P4: Gene The coding sequence of the gene can encode MYB75 protein, and the amino acid sequence of the MYB75 protein is shown in SEQ ID NO.4; 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 cell is Agrobacterium GV3101 cell; 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.

11. The use according to claim 10, characterized in that The skeleton of the genetic engineering vector is a pK2GW7 vector.

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