A method for improving poplar traits using superoxide dismutase 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, a gene expression cassette is transferred into the genome of poplar trees to highly express the superoxide dismutase gene in poplar tissues; the gene expression cassette contains the coding sequence of the superoxide dismutase gene; the promoter in the gene expression cassette is the promoter of the poplar EXPA1 gene, and the improvement of poplar traits includes: increasing the area of xylem vessels in the poplar stem; increasing the number of vessels per unit area in the poplar stem xylem; increasing the cross-sectional area of fiber cells in the poplar stem xylem; increasing the cell lumen of the poplar stem xylem; increasing the length of fiber cells in the poplar stem xylem; reducing the lignin content in the poplar stem; reducing the cell wall thickness of fiber cells. The new poplar tree strain obtained by this method has better application value in aspects such as papermaking.

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 trees by using superoxide dismutase genes. Background Art

[0002] Poplar (Latin scientific name: Populus L.) belongs to the Salicaceae family. Poplar trees grow fast, have strong adaptability, and mature early, and are widely used industrial and agricultural raw materials. Poplar wood is an excellent raw material for pulp and paper making. Poplar has the advantages of rich genetic background, easy rapid asexual propagation and genetic transformation. At the same time, with the completion of its genome sequencing work and the increasing maturity of gene engineering technology, modern molecular breeding techniques can be used to improve the wood properties of poplar trees, so as to cultivate poplar strains with better pulp and paper making performance.

[0003] Wood is an important renewable resource and can be used as raw material for pulp and paper making. The formation of wood includes a series of biological processes such as the division, differentiation and expansion of vascular cambium cells, the deposition of secondary cell walls, and programmed cell death. And the proliferation rate of vascular cambium cells, the shape, size, proportion, etc. of various types of cells formed by differentiation determine the wood yield, structure and properties. Research results show that there is a certain inevitable connection between the various wood properties and the pulp and paper making performance of wood. The pulp and paper making performance can be evaluated by studying the chemical composition analysis and wood anatomical structure of this tree species or strain. For example, the lower the lignin content in wood, the better its pulp and paper making performance; the longer the fiber, the larger the length-width ratio, the higher the tearing strength, breaking length and bursting strength of the paper; the thinner the fiber cell wall and the larger the lumen, the smaller the wall-lumen ratio, the higher the tensile strength and bursting index of the paper. Therefore, fully understanding the regulatory mechanism of vascular cambium cell proliferation and differentiation, and using this as a theoretical basis to promote modern molecular breeding techniques for improving the wood properties of forest tree varieties, and then improving the practical application value such as wood pulp and paper making performance.

[0004] Reactive oxygen species (ROS) are a class of natural by-products in the normal metabolism of plant cells, including superoxide anion (O2 ·- ) and hydrogen peroxide (H2O2), etc. Reactive oxygen species can participate in regulating the proliferation and differentiation of cells in the apical meristem of plants. There is an antioxidant system in plants, including enzymatic and non-enzymatic antioxidants, which can maintain the balance of ROS within a certain range. In the enzymatic antioxidant system, superoxide dismutase (SOD) can dismutate O2 ·- generated in plants into H2O2 and plays an important role in maintaining the balance of ROS. Summary of the Invention

[0005] In the present invention, the PagSOD2 gene in Populus alba×P. glandulosa‘84K’ was cloned, an overexpression vector was constructed, and PagSOD2 transgenic overexpressing poplars were obtained through transformation. Combining genetic engineering and molecular biology, the regulation of poplar xylem development by the PagSOD2 gene in poplar was analyzed, which is of great significance for molecular design to improve the wood properties of forest tree varieties and further enhance the practical application value such as the pulp and paper making performance of wood.

[0006] To solve the problems existing in the prior art, a first aspect of the present invention provides a method for improving poplar traits, and the method is as follows: by means of genetic engineering, a gene expression cassette is transferred into the poplar genome to highly express the superoxide dismutase gene in poplar tissues;

[0007] The gene expression cassette contains the coding sequence of the superoxide dismutase gene;

[0008] The improvement of poplar traits includes:

[0009] Increasing the area of xylem vessels in the poplar stem;

[0010] Increasing the number of vessels per unit area in the poplar stem xylem;

[0011] Increasing the cross-sectional area of fiber cells in the poplar stem xylem;

[0012] Increasing the cell lumen of the poplar stem xylem;

[0013] Increasing the length of fiber cells in the poplar stem xylem;

[0014] Reducing the lignin content in the poplar stem;

[0015] Reducing the cell wall thickness of poplar fiber cells;

[0016] In some embodiments, the superoxide dismutase gene is the superoxide dismutase 2 gene; or

[0017] The poplar variety is Populus alba×P. glandulosa‘84K’.

[0018] In some embodiments, the method is as follows: the promoter in the gene expression cassette is the promoter of the poplar EXPA1 gene.

[0019] In some embodiments, it is selected from any one or a combination of the following cases M1, M2, and M3;

[0020] M1: The protein sequence encoded by the superoxide dismutase gene is as shown in SEQ ID NO.2;

[0021] M2: The coding sequence of the poplar EXPA1 gene is as shown in SEQ ID NO.3;

[0022] M3: The nucleotide sequence of the promoter of the poplar EXPA1 gene is as shown in SEQ ID NO.4.

[0023] In some embodiments, the method comprises the following steps:

[0024] S1: Transfer the gene expression cassette into an expression vector to obtain a recombinant vector containing the superoxide dismutase gene;

[0025] S2: Transform Agrobacterium with the recombinant vector containing the superoxide dismutase gene to obtain recombinant Agrobacterium containing the superoxide dismutase gene;

[0026] S3: Infect wounded poplar leaves with the recombinant Agrobacterium containing the superoxide dismutase gene to obtain infected poplar leaves;

[0027] S4: Place the infected poplar leaves on a co-culture medium to obtain co-cultured leaves;

[0028] 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-(N-morpholino)ethanesulfonic acid, 80 - 120 μM acetosyringone, with a pH of 5.5 - 6.5;

[0029] S5: Place the co-cultured leaves on a differentiation medium to obtain differentiated poplar adventitious buds;

[0030] 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-(N-morpholino)ethanesulfonic acid, 0.04 - 0.06 mg / L NAA, 0.4 - 0.6 mg / L 6-BA, 150 - 250 mg / L cefotaxime, 150 - 250 mg / L ticarcillin, 1.5 - 2.5 mg / L hygromycin, with a pH of 5.5 - 6.5;

[0031] S6: Insert the differentiated poplar adventitious buds on a rooting selection medium to obtain rooted poplar plants;

[0032] The rooting selection medium is based on WPM, and the rooting selection medium further contains 8 - 12 g / L sucrose, 6 - 10 g / L agar, 0.4 - 0.6 g / L 2-(N-morpholino)ethanesulfonic acid, 150 - 250 mg / L cefotaxime, 150 - 250 mg / L ticarcillin, 1.5 - 2.5 mg / L hygromycin, with a pH of 5.5 - 6.5.

[0033] In some embodiments, any one or a combination thereof is selected from the following cases A1, A2, and A3;

[0034] A1: In S4, the culture conditions are: dark culture for 2.5 - 3.5 days;

[0035] A2: In S5, 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 30 - 50 days;

[0036] A3: 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 5 - 10 days.

[0037] In some embodiments, any one or a combination thereof is selected from the following cases B1, B2, and B3;

[0038] B1: In S3, the poplar leaf is a leaf of a tissue culture seedling of poplar;

[0039] The backbone of the expression vector is the pMDC32 vector;

[0040] B2: In S2, the Agrobacterium is Agrobacterium tumefaciens GV3101;

[0041] B3: The resuspension of the Agrobacterium uses WPM as the basal medium, and the resuspension also contains 15 - 25 g / L sucrose, 0.4 - 0.6 g / L 2 - morpholinoethanesulfonic acid, 80 - 120 μΜ acetosyringone, 0.8 - 1.2 mg / L 2,4 - D, 0.08 - 0.12 mg / L KT, and pH 5.0 - 6.0.

[0042] 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;

[0043] The improvement of poplar traits includes:

[0044] Increasing the area of xylem vessels in the poplar stem;

[0045] Increasing the number of vessels per unit area in the poplar stem xylem;

[0046] Increasing the cross - sectional area of fiber cells in the poplar stem xylem;

[0047] Increasing the cell lumen of the poplar stem xylem;

[0048] Increase the length of xylem fiber cells in poplar stems;

[0049] Reduce the lignin content in poplar stems;

[0050] Reduce the cell wall thickness of poplar fiber cells.

[0051] The biological material is selected from any one of the following P1, P2, P3, P4, P5, P6, P7, P8, and P9:

[0052] P1: Protein

[0053] The protein is superoxide dismutase protein;

[0054] P2: Fusion protein

[0055] The amino acid sequence of the fusion protein contains the amino acid sequence of the superoxide dismutase protein described in P1 and the amino acid sequence of a functional protein fragment or an inert protein fragment;

[0056] P3: RNA

[0057] The RNA can be translated to obtain the superoxide dismutase protein described in P1 or the fusion protein described in P2;

[0058] P4: Gene

[0059] The coding sequence of the gene can encode the superoxide dismutase protein described in P1 or the fusion protein described in P2;

[0060] P5: Gene expression cassette

[0061] The gene expression product in the gene expression cassette is the RNA described in P3;

[0062] P6: Genetic engineering vector

[0063] The genetic engineering vector contains the gene expression cassette described in P5;

[0064] P7: Cell

[0065] The cell contains the genetic engineering vector described in P6;

[0066] The coding proteins in the gene expression cassette of the genetic engineering vector are constitutively expressed, tissue-specifically expressed, or artificially inducedly expressed;

[0067] P8: Composition

[0068] The composition contains the protein described in P1, the fusion protein described in P2, the RNA described in P3, the genetic engineering vector described in P6, or the cell described in P7; and

[0069] P9: Kit

[0070] The kit contains the protein described in P1, the fusion protein described in P2, the RNA described in P3, the genetic engineering vector described in P6, or the cell described in P7.

[0071] In some embodiments, it is selected from any one or a combination of the following cases C1, C2, C3, C4, C5, and C6;

[0072] C1: The variety of the poplar is Populus alba×Populus glandulosa '84K';

[0073] C2: The superoxide dismutase gene is superoxide dismutase 2 gene;

[0074] C3: The functional protein fragment is a tag peptide and / or a signal peptide for protein isolation and purification;

[0075] C4: The backbone of the genetic engineering vector is pMDC32 vector;

[0076] C5: The cell is Agrobacterium tumefaciens GV3101 cell;

[0077] C6: The promoter in the gene expression cassette is the promoter of Populus alba×Populus glandulosa EXPA1 gene.

[0078] In some embodiments, it is selected from any one or a combination of the following cases K1, K2, and K3;

[0079] K1: The protein sequence encoded by the superoxide dismutase gene is as shown in SEQ ID NO.2;

[0080] K2: The coding sequence of the Populus alba×Populus glandulosa EXPA1 gene is as shown in SEQ ID NO.3;

[0081] K3: The nucleotide sequence of the promoter of the Populus alba×Populus glandulosa EXPA1 gene is as SEQ ID NO.4. Brief Description of the Drawings

[0082] Figure 1 It is a statistical chart of the tissue expression level of PagSOD2 gene.

[0083] Figure 2 It is the analysis of the overall morphology and PagSOD2 gene expression level of PagSOD2 gene overexpression transgenic lines (OE#2, OE#9) and wild-type plants.

[0084] Figure 3 It is the statistical results of the microscopic morphology of the xylem region of the stem cross-section of the 7th internode, the number of vessels per unit area, the vessel area ratio, and the cross-sectional area of fiber cells of PagSOD2 gene overexpression transgenic poplars (OE#2, OE#9) and wild-type.

[0085] Figure 4 The microscopic morphology of fiber cells and the statistical results of fiber cell length of PagSOD2 gene overexpressing transgenic poplars (OE#2, OE#9) and wild type.

[0086] Figure 5 The phloroglucinol histochemical staining results, Calcofluor White staining results of the stem vascular tissue, and the quantitative analysis results of the cell wall components of the stem vascular tissue cells of PagSOD2 gene overexpressing transgenic poplars (OE#2, OE#9) and wild type.

[0087] Figure 6 The scanning electron microscope images of fiber cells and the statistical results of the fiber cell wall thickness of PagSOD2 gene overexpressing transgenic poplars (OE#2, OE#9) and wild type. Detailed implementation manners

[0088] 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 with reference to the accompanying drawings.

[0089] 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.

[0090] Example 1: Preparation of 84K poplar plants with high expression of SOD2 gene

[0091] (I) Plant species

[0092] The poplar used in the present invention is Populus alba×P. glandulosa‘84K’ (abbreviated as "84K poplar").

[0093] (II) Gene information

[0094] The superoxide dismutase 2 (SOD2) gene of 84K poplar is called PagSOD2 gene, abbreviated as PagSOD2, and the Pag prefix represents poplar. The homologous gene accession number of PagSOD2 in the reference genome of Populus trichocarpa (cultivar name selected as Populustrichocarpa v3.1) on the plantgenie platform (website: https: / / plantgenie.org / ) is Potri.005G044400.

[0095] In the transgenic materials of the present invention, the PagSOD2 coding sequence is cloned from the female parent.

[0096] The coding sequence of the female parent PagSOD2 gene is as follows (SEQ ID NO.1): 5’-ATGGTGAAGGCTGTAGCTGTTCTTAATAGCAGTGAAGGTGTGAGTGG CACCATCTTCTTTACCCAAGAAGGAGATGGCCCAACTACTGTAACTGGAAACCTTTCTGGTCTTAAGCCAGGCCTTCATGGCTTCCACGTCCATGCCCTTGGAGACACCACAAATGGCTGCATGTCAACTGGGCCGCATTTTAATCCTGTAGGCAAGGAGCATGGTGCCCCTGAGGATGAGAATCGTCATGCTGGTGATCTGGGAAATGTCACTGTTGGTGATGATGGCACTGCTACTTTCACAATCATTGACAAACAGATTCCTCTTACTGGACCACATTCCATTATTGGAAGGGCTGTTGTTGTTCATGGAGATCCTGATGATCTTGGCAAGGGAGGACATGAACTCAGCAAAACCACCGGTAATGCTGGTGGCAGAGTAGCATGCGGTATTATTGGTCTGCAAGGTTGA-3’

[0097] The protein sequence expressed by the female parent PagSOD2 gene is as follows (SEQ ID NO.2):

[0098] MVKAVAVLNSSEGVSGTIFFTQEGDGPTTVTGNLSGLKPGLHGFHVHALGDTTNGCMSTGPHFNPVGKEHGAPEDENRHAGDLGNVTVGDDGTATFTIIDKQIPLTGPHSIIGRAVVVHGDPDDLGKGGHELSKTTGNAGGRVACGIIGLQG

[0099] The α-expansin gene is highly expressed in the cambium and expanding xylem cells. The accession number of the homologous gene of the α-expansin gene in Populus trichocarpa in the aforementioned Populus trichocarpa reference genome (cultivar name selected as Populus trichocarpa v3.1) is Potri.001G240900. The α-expansin gene of Populus alba×Populus glandulosa is called the Populus EXPA1 gene, abbreviated as PagEXPA1 gene, and its promoter is called proPagEXPA1.

[0100] The coding sequence of the paternal PagEXPA1 gene is as follows (SEQ ID NO.3): 5’-ATGGCAATGAGCAGTTTAATTTGCATTGCCACTAGTTTACTAATAATA GTGTCATCGTTGTGGATGGCTAAAGCTAGAATTCCTGGTGTTTACTCCGGGGGTGCTTGGGAAAATGCTCATGCAACCTTCTATGGCGGTTCTGATGCCTCTGGCACAATGGGAGGAGCTTGTGGATATGGAAATCTGTACAGCCAAGGGTATGGAGTGAGCACTGCAGCCCTAAGCACAGCACTGTTCAACAACGGGTTAAGTTGCGGTTCTTGCTTCGAGATAAAATGTGCAAGTGACCCGAGATGGTGCCACTCAGGCAGCCCGTCTATTTTCATCACTGCAACCAACTTTTGCCCTCCAAATTATGCACTTCCTAGTGACAATGGAGGCTGGTGCAACCCTCCTCGCCCCCACTTTGACCTTGCCATGCCCATGTTCCTTAAGATCGCCGAGTATCGTGCCGGTATCGTCCCTGTTGCCTACCGCCGAGTGCCATGCCGCAAGAGGGGAGGTATAAGGTTCACTATAAACGGATTCCGTTACTTCAACTTGGTATTGATCAGCAACGTGGCGGGTGCAGGGGATATAGTGCAGGTGAGCGTGAAGGGTTCAAAGACTGGTTGGATGAGCATGAGCCGTAACTGGGGCCAGAACTGGCAGTCAAACGCTGTTCTGGTTGGCCAGACACTCTCCTTCAGGGTTAGGGCCAGTGACAGACGCTCCTCCACTTCATGGAACATTGTCCCAGCCCACTGGCAGTTTGGTCAAACTTTTACCGGCAAGAATTTCAGGGTTTAA-3’

[0101] In the present invention, the transgenic material proPagEXPA1 sequence was cloned from the paternal parent.

[0102]

[0103] (3) Testing on the gene expression characteristics of PagSOD2

[0104] Taking the UBQ gene of Populus alba × Populus glandulosa as the internal reference gene, the quantitative primer sequences are UBQF and UBQR.

[0105] UBQF (SEQ ID NO.5):

[0106] 5’-GACTTTGACCGGAAAGACCA-3’

[0107] UBQR (SEQ ID NO.6):

[0108] 5’-GGAGACGAAGGACAAGGTGA-3’

[0109] Design upstream quantitative primer PagSOD2F and downstream quantitative primer PagSOD2R for the coding sequence of PagSOD2 gene, and the specific sequences are as follows.

[0110] PagSOD2F (SEQ ID NO.7):

[0111] 5’-TGGCACCATCTTCTTTACCC-3’

[0112] PagSOD2R (SEQ ID NO.8):

[0113] 5’-TGACATTTCCCAGATCACCA-3’

[0114] Using the conventional tissue culture seedlings of Populus alba × Populus glandulosa transplanted for 30 days, obtaining the bark, developing xylem and mature xylem of the stem segments from the 7th to the 15th internodes, and mixing the same tissues from different internodes for use as test samples.

[0115] Using Plant RNAKit (ER301, TransGen) to extract total RNA, and using PrimeScript TMcDNA synthesis was performed using the RT reagent Kit (RR047A, Takara). The reaction system was prepared using ChamQ SYBR qPCR Master Mix (Q311, Vazyme biotech), and qRT-PCR was performed using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Wilmington, DE, USA). The primers used were the above-mentioned reference gene quantitative primer pair and the PagSOD2 gene quantitative primer pair. There were 3 biological replicates.

[0116] The statistical results of the expression level of the PagSOD2 gene relative to the reference gene are shown in Figure 1 .

[0117] According to Figure 1 It can be seen that the PagSOD2 gene is expressed in the developing xylem, mature xylem, and bark of the plant stem segments. It is thus speculated that the PagSOD2 gene may regulate the development of the xylem of Populus alba × Populus glandulosa '84K'.

[0118] Example 2: Preparation of PagSOD2 transgenic plants

[0119] (I) Preparation of recombinant vectors

[0120] The specific primers proPagEXPA1F and proPagEXPA1R for amplifying the PagEXPA1 promoter of the female parent of Populus alba × Populus glandulosa '84K' and the primers PagSOD2CDSF and PagSOD2CDSR for amplifying the PagSOD2 gene of the female parent of Populus alba × Populus glandulosa '84K' were designed using Snapgene software.

[0121] The genomic DNA of Populus alba × Populus glandulosa '84K' was extracted using the CTAB method. Using the genomic DNA of Populus alba × Populus glandulosa '84K' as a template, the promoter sequence of the PagEXPA1 gene was amplified by polymerase chain reaction (PCR) using 2×Phanta Max Master Mix high-fidelity polymerase to obtain amplification fragment 1.

[0122] Using Extract RNA from the stems of Populus alba×Populus glandulosa using the Plant RNAKit (ER301, TransGen), and then reverse transcribe it into cDNA for use as a template for gene cloning. Using the cDNA of Populus alba×Populus glandulosa as a template, the PagSOD2 CDS sequence was amplified by polymerase chain reaction (PCR) using the 2×Phanta Max Master Mix high-fidelity polymerase to obtain amplified fragment 2.

[0123] The above two PCR reaction systems are the same, as shown in Table 1. The PCR reaction program is as follows:

[0124] Pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 60 s, denaturation to extension cycle 35 times, and final extension at 72°C for 5 min.

[0125] Homologous recombination technology was used for vector construction. The pMDC32 empty vector was digested with the restriction endonucleases HindIII / SacI and reacted at 37°C for 3 h. The digestion system is shown in Table 2. Use MultiS One Step Cloning Kit (C11, Novizan) to ligate the amplified fragment 1, amplified fragment 2 of the target fragment with the homologous recombination-specific linker and the linearized backbone of the pMDC32 vector, and react at 37°C for 30 min. By performing PCR detection and sequencing verification on the recombinant vector, it was confirmed that the vector construction was successful and named proPagEXPA1::PagSOD2.

[0126] proPagEXPA1F (SEQ ID NO.9)

[0127] 5’-acgacggccagtgccaagctATGTCCGATTACCCAAATGC-3’

[0128] proPagEXPA1 R (SEQ ID NO.10)

[0129] 5’-taagaacagctacagccttcaccatTTTCTCTCCGCAATACTACT-3’

[0130] PagSOD2CDSF (SEQ ID NO.11)

[0131] 5’-ATGGTGAAGGCTGTAGCTGTTCTTA-3’

[0132] PagSOD2CDSR (SEQ ID NO.12)

[0133] 5’-cgatcggggaaattcgagctTCAACCTTGCAGACCAATAATAC-3’

[0134] Table 1. PCR reaction system

[0135]

[0136] Table 2. Restriction enzyme digestion system

[0137]

[0138] (2) Preparation of transgenic plants

[0139] Genetic transformation of PagSOD2 is carried out as follows.

[0140] (1) Agrobacterium transformation: Take out the Agrobacterium tumefaciens GV3101 chemically competent cells stored at -80°C, pipette 1 μL of the expression vector plasmid proPagEXPA1::PagSOD2 into 50 μL of Agrobacterium tumefaciens GV3101 competent cells, and gently pipette to mix. First, let it stand on ice for 5 min, then put it into liquid nitrogen for 5 min, then transfer it to a 37°C water bath for 5 min, and finally on ice for 5 min. Add 800 μL of liquid LB medium without antibiotics in a laminar flow hood, mix well, and recover in a shaker at 28°C and 200 rpm for 2 - 3 h. Centrifuge at 5000 rpm for 1 min, collect the bacteria at the bottom of the centrifuge tube, leave about 200 μL of the supernatant, gently pipette to suspend the bacteria in the laminar flow hood, and evenly spread it on a solid LB medium supplemented with Kan (kanamycin), Gent (gentamicin), and Rif (rifampicin) antibiotics. Invert the petri dish and culture it in an incubator at 28°C for 60 - 72 h. Pick a single colony in the laminar flow hood, add about 600 μL of liquid LB medium supplemented with Kan, Gent, and Rif antibiotics, mix well, and culture it in a shaker at 28°C and 200 rpm. Take a part of the bacterial solution for PCR amplification test. The amplification result shows that the aforementioned bacterial solution is the Agrobacterium tumefaciens GV3101 bacterial solution that has been successfully transformed with proPagEXPA1::PagSOD. Add 50% glycerol aqueous solution to it and store it at -80°C for genetic transformation.

[0141] (2) Agrobacterium infection and co-cultivation: Take out the bacterial solution stored at -80℃, and use a sterile pipette tip to isolate single colonies by plate streaking method on solid LB medium supplemented with Kan, Gent and Rif antibiotics. In the clean bench, pick a single clone for colony test, inoculate the positive single colony into 1mL of liquid LB medium supplemented with Kan, Gent and Rif antibiotics, and place it in a shaker at 28℃ and 200rpm for small shaking. After overnight culture, take 50μL of the small shaking bacterial solution and add it to 50mL of liquid LB medium supplemented with the same antibiotics, and place it in a shaker at 28℃ and 200rpm for large shaking. Culture overnight until OD 600 =0.4-0.6. The bacterial solution was centrifuged at 3500rpm for 15min, the bacterial cells were collected, and then the resuspension solution (the resuspension solution was based on WPM culture medium and also contained 20g / L sucrose, 0.5g / L MES (2-morpholineethanesulfonic acid), 100μM acetosyringone, 1mg / L 2,4-D, 0.1mg / L KT, pH 5.4) was added to resuspend the bacterial cells until OD 600 =0.3-0.4.

[0142] Select the leaves of 84K tissue culture seedlings with good growth status, scratch the leaves at the main veins with a sterile scalpel blade, and soak them in the above-mentioned Agrobacterium resuspension for 15-20 minutes, shaking the bacterial solution during the period. After the infection, use tweezers to pick up the leaves and place them in a sterile filter to absorb the bacterial solution attached to the surface. Place the back of the leaves facing up on the co-culture medium and culture them in the dark for 3 days. The co-culture medium is based on WPM, and also contains 20g / L sucrose, 7.8g / L agar, 0.5g / LMES, 100μM acetosyringone, pH 5.9.

[0143] (3) Differentiation culture: After the co-culture, the leaves were transferred to the screening differentiation medium supplemented with the corresponding antibiotics and cultured under normal light with a light intensity of 50 μmol·m -2 ·s -1 , the culture temperature is 25℃, and the photoperiod is 16h light / 8h dark. Induce differentiation of adventitious buds. The differentiation medium is changed every 20d, and the leaves are observed for contamination. This stage is about 40 days. 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, 2mg / L hygromycin, pH 5.9.

[0144] (4) Rooting culture: When the adventitious buds differentiated from the leaves 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 cefamycin, 200 mg / L ticarcillin, 2 mg / L hygromycin, and the pH was 5.9.

[0145] (III) Identification of transgenic plants

[0146] Forty-eight transgenic plants to be tested were obtained through the above steps. Leaves of each plant were cut and genomic DNA was extracted. Using the universal primers F1 and R1 of the hygromycin resistance gene on the vector as primers, 24 positive plants were identified by PCR.

[0147] Through the NBT histochemical staining method, some identified overexpression positive plants were screened for detecting the superoxide anion level in stem segments. Using the identified positive plants as materials, the 7th internode of the seedlings 30 days after transplanting of tissue culture seedlings was taken and incubated for 8 h under the dark condition of 1 mg / mL NBT (IN0110, Solarbio) at 25 °C, and the staining was terminated with 75% ethanol. The NBT-stained stem segments were sectioned with a vibratome (VT1200S, Leica), and the section thickness was 50 μm. Observation was carried out using an upright fluorescence microscope (DM6B, Leica). Two transgenic lines with low superoxide anion levels (OE#2, OE#9) were selected for transcriptional level analysis. Using the aforementioned PagSOD2F and PagSOD2R as primers, RNA was extracted from the 7th internode stem segments of the soil-grown seedlings grown for 30 days as materials, reverse transcribed into cDNA and subjected to qRT-PCR detection.

[0148] The results are as Figure 2 shown. A shows the overall morphology of PagSOD2 overexpression transgenic plants (OE#2, OE#9) and the parental 84K poplar (Control). B is the analysis of the SOD2 expression level of PagSOD2 overexpression transgenic lines (OE#2, OE#9) and the parental 84K poplar.

[0149] F1 (SEQ ID NO.13):

[0150] 5’-ACGGTGTCGTCCATCACAGTTTGCC-3’

[0151] R1 (SEQ ID NO.14):

[0152] 5’-TTCCGGAAGTGCTTGACATTGGGGA-3’

[0153] Example 3. Characterization of traits of genetically engineered poplar plants

[0154] Select the overexpressing PagSOD2 transgenic lines OE#2 and OE#9 and the wild type 84K that have been in conventional soil culture for two months, compare their vascular tissue morphological structures. The PagSOD2 overexpressing plants showed an increase in the number of vessels and the ratio of vessel area, an increase in the cross-sectional area of fiber cells and an increase in length. Analysis of cell wall components found that the lignin content decreased in PagSOD2 overexpressing plants. Scanning electron microscopy results showed that the cell walls of fiber cells in PagSOD2 overexpressing plants were thinner than those of wild type plants. The specific measurement indicators are as follows.

[0155] For two-month-old seedlings (two months after transplantation of conventional tissue culture) of three poplar plants, namely the parental 84K poplar (Control), PagSOD2 transgenic lines OE#2 and OE#9 (referred to as three poplar germplasms), the following parallel test experiments were carried out respectively.

[0156] (I) Determination of the ratio of vessel area, the number of vessels per unit area and the cross-sectional area of fiber cells in the xylem region of the stem cross-section

[0157] For the above three poplar germplasms, conventional tissue culture seedlings were prepared in parallel respectively. Two months after transplantation, the stem segments of the 7th internode were taken and cut into slices with a thickness of 50 μm using a vibrating microtome (VT1200S, Leica). Subsequently, the slices were laid flat on a glass slide, 0.1% Toluidine Blue O (TBO) staining solution was added, and after staining for 30 - 60 s, the staining solution was washed off. A upright fluorescence microscope (DM6B, Leica) was used to observe and photograph and record. (The results are shown in Figure 3 A, scale bar = 50 μm) and the number of vessels per unit area, the ratio of vessel area, and the cross-sectional area of fiber cells in the 7th internode were statistically measured (6 plants were used for each germplasm for statistics, and the results are shown in Figure 3 B). The results showed that the PagSOD2 overexpressing plants showed an increase in the number of vessels per unit area and the ratio of vessel area, and an increase in the cross-sectional area of fiber cells. Thus, it can be seen that PagSOD2 overexpression promotes the enlargement of the xylem cell lumen.

[0158] (II) Statistics of fiber cells and fiber cell length

[0159] For the above-mentioned 3 poplar germplasms, conventional tissue culture seedlings were prepared in parallel. Two months after transplantation, the stem segments of the 7th internode were taken and the bark was removed. They were placed in a mixed solution of hydrogen peroxide (aqueous solution with a volume ratio of 30%) and glacial acetic acid (analytical pure) with a volume ratio of 1:1 and heated in a water bath at 65 °C for several hours. During this period, the solution was shaken appropriately until the samples were separated into single fibers and fully dissociated. After dissociation, centrifugation was carried out at 5000 rpm / min for 10 min, the supernatant dissociation solution was poured off, and it was washed several times with ddH2O. An appropriate amount of the dissociated fiber sample was taken, stained with 0.1% toluidine blue O (solvent is water), and made into a temporary slide. It was observed and photographed with an upright fluorescence microscope (DM6B, Leica) (the results are shown in Figure 4 A, scale bar = 100 μm), and the fiber cell length was statistically analyzed (6 plants were used for each germplasm for statistics, and the results are shown in Figure 4 B). The results showed that compared with the wild type, the length of xylem fiber cells in transgenic plants increased. Thus, overexpression of PagSOD2 promoted the elongation of xylem fiber cells, which was beneficial to improving the tearing degree, breaking length, and bursting strength of paper.

[0160] (III) Analysis of xylem cell wall components

[0161] For the above-mentioned 3 poplar germplasms, conventional tissue culture seedlings were prepared in parallel. Two months after transplantation, the stem segments of the 7th internode were taken and cut into slices with a thickness of 50 μm using a vibrating microtome (VT1200S, Leica). They were stained with 1% phloroglucinol staining solution (solvent is 12% hydrochloric acid solution), and the distribution and content changes of lignin were observed using an upright fluorescence microscope (DM6B, Leica) and photographed; to observe the distribution and content changes of cellulose, Calcofluor White (CW) staining was used, and the stained slices were observed and photographed using an upright fluorescence microscope (DM6B, Leica) (the results are shown in Figure 5 A, scale bar = 50 μm). The results showed that the transverse section staining of the stem segments of transgenic plants was lighter than that of the wild type. In addition, the acetyl bromide method (M1711A, Suzhou Mixi Biotechnology) was used for quantitative determination of lignin content, and the anthrone method (M1718A, Suzhou Mixi Biotechnology) was used for quantitative determination of cellulose content (9 plants were used for each germplasm for statistics, and the results are shown in Figure 5 B). The results showed that the lignin content and cellulose content in overexpressing plants were both decreased compared with the wild type. Thus, overexpression of PagSOD2 could reduce the lignin content in the xylem cell wall, which was beneficial to improving the pulping and papermaking performance.

[0162] (IV) Analysis of the thickness of fiber cell walls

[0163] For the above-mentioned 3 poplar germplasms, conventional tissue culture seedlings were prepared in parallel. Two months after transplantation, the stem segments of the 7th internode were taken and cut into slices with a thickness of 50 μm using a vibratome (VT1200S, Leica), and observed with a scanning electron microscope (TM4000, Hitachi) (see the results in Figure 6 A, scale bar = 25 μm), and the cell wall thickness was statistically analyzed using Image J software (6 plants were used for each germplasm for statistics, see the results in Figure 6 B). The results showed that compared with the wild type, the cell walls of the stem segments of PagSOD2 overexpressing transgenic poplars (OE#2, OE#9) became thinner. Combining the characteristics of the larger lumen of the fiber cells in the overexpressing transgenic poplars, thin walls and large lumens are beneficial to improving the tensile strength and burst index of the paper prepared from the transgenic germplasm materials.

[0164] In this invention, Populus alba × Populus glandulosa 84K was used as the material to clone the PagSOD2 gene; at the same time, an overexpression vector proPagEXPA1::PagSOD2 was constructed. Driven by the PagEXPA1 promoter, PagSOD2 can be specifically overexpressed in the vascular cambium and developing xylem of poplar. Compared with the wild type 84K, the number of vessels per unit area and the ratio of vessel area in PagSOD2 overexpressing poplars increased, the cross-sectional area of fiber cells increased, the length increased, and the cell walls became thinner, and the lignin content in the xylem decreased. It shows that PagSOD2 is involved in regulating the development of poplar xylem, improving the wood properties, and enhancing the pulp and paper-making performance of poplar wood, which has application significance in cultivating new poplar varieties for pulp and paper-making.

[0165] As is known by common technical knowledge, the present invention can be implemented by other embodiments that do not depart from its spiritual essence or essential features. Therefore, the above-disclosed embodiments are illustrative in all aspects and are not the only ones. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. A method for improving poplar traits, the method comprising: transferring a gene expression cassette into the poplar genome by genetic engineering means, so that the superoxide dismutase gene in the poplar tissue is highly expressed; The variety of the poplar is 84K poplar; The gene expression cassette contains a superoxide dismutase gene coding sequence; The protein sequence encoded by the superoxide dismutase gene is shown in SEQ ID NO.2; The promoter in the gene expression cassette is poplar EXPA1 The promoter of a gene; The poplar EXPA1 The nucleotide sequence of the promoter of the gene is shown in SEQ ID NO.4; The improved poplar traits include: Increase the xylem vessel area of ​​poplar stems; Increase the number of vessels per unit area in the xylem of poplar stems; Increase the cross-sectional area of ​​xylem fiber cells in poplar stems; Increase the xylem cell cavity of poplar stems; Increase the length of xylem fiber cells in poplar stems; Reducing the lignin content of poplar stems; and Reduce the cell wall thickness of poplar fiber cells.

2. The method according to claim 1, characterized in that The poplar EXPA1 The coding sequence of the gene is shown in SEQ ID NO.

3.

3. The method according to claim 1, characterized in that The method comprises the following steps: S1: transferring the gene expression cassette into an expression vector to obtain a recombinant vector containing the superoxide dismutase gene; S2: transforming Agrobacterium with the recombinant vector containing the superoxide dismutase gene to obtain recombinant Agrobacterium containing the superoxide dismutase gene; S3: infecting a poplar leaf with a wound with the recombinant Agrobacterium containing the superoxide dismutase 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 differentiation medium to obtain differentiated poplar adventitious 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, 1.5-2.5 mg / L hygromycin, and a pH of 5.5-6.5; S6: inserting the differentiated poplar adventitious buds into a rooting screening medium to obtain rooted poplar plants; The rooting screening medium is based on WPM as a basic 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-morpholineethanesulfonic acid, 150-250 mg / L cephalosporin, 150-250 mg / L timentin, 1.5-2.5 mg / L hygromycin, and a pH of 5.5-6.

5.

4. The method according to claim 3, characterized in that Selected from any one or a combination of the following situations A1, A2 and A3; A1: In S4, the culture conditions are: dark culture for 2.5-3.5 days; A2: In S5, 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 30-50 days; A3: In S6, the culture conditions are: culture temperature is 20-28℃, 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.

5. The method according to claim 3, characterized in that Selected from any one or a combination of the following situations B1, B2 and B3; B1: In S3, the poplar leaves are leaves of poplar tissue culture seedlings; The backbone of the expression vector is pMDC32 vector; B2: In S2, the Agrobacterium is Agrobacterium GV3101; B3: The resuspension of Agrobacterium is based on WPM as the basic culture medium, and the resuspension also contains 15-25 g / L sucrose, 0.4-0.6 g / L 2-morpholineethanesulfonic acid, 80-120 μΜ acetosyringone, 0.8-1.2 mg / L 2,4-D, 0.08-0.12 mg / L KT, pH 5.0-6.

0.

6. 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 include: Increase the xylem vessel area of ​​poplar stems; Increase the number of vessels per unit area in the xylem of poplar stems; Increase the cross-sectional area of ​​xylem fiber cells in poplar stems; Increase the xylem cell cavity of poplar stems; Increase the length of xylem fiber cells in poplar stems; Reducing the lignin content of poplar stems; and Reduce the thickness of poplar fiber cell walls; The biological material is selected from any one of the following P4, P5, P6, P7, P8 and P9: P4: Gene The coding sequence of the gene can encode the superoxide dismutase protein as shown in SEQ ID NO.2; the nucleotide sequence of the promoter of the gene is shown in SEQ ID NO.4; P5: Gene expression cassette The gene expression product in the gene expression cassette can be translated to obtain a superoxide dismutase protein with a sequence as shown in SEQ ID NO.2; the promoter in the gene expression cassette is poplar EXPA1 Gene promoter; The poplar EXPA1 The nucleotide sequence of the promoter of the gene is shown in SEQ ID NO.4; 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; P8: Composition The composition contains the genetic engineering vector described in P6 or the cell described in P7; P9: Test kit The kit contains the genetic engineering vector described in P6 or the cells described in P7.

7. The use according to claim 6, characterized in that: The skeleton of the genetic engineering vector is a pMDC32 vector; and / or The cells are Agrobacterium GV3101 cells.

8. The use according to claim 6, characterized in that The poplar EXPA1 The coding sequence of the gene is shown in SEQ ID NO.3.

Images