PagKNAT5a gene and application thereof

Abstract

The invention discloses a method for improving properties of poplar, which comprises the following steps of: performing high expression on PagKNAT5a gene in the poplar by a genetic engineering means; poplar character improvement includes: increasing poplar plant height; increasing the ground diameter of the poplar plant; the internode length of the poplar is increased; the cortical cell length of the aspen xylem is increased; the poplar plant fiber cell length is increased; the length of poplar plant conduits is increased; the vascular xylem width of the poplar plant is increased; the cell wall thickness of xylem fiber cells of poplar plants is increased; the thickness of xylem secondary cell walls of poplar plants is increased; the lignin content of the poplar stems is increased.

Description

Technical Field

[0001] The invention belongs to the field of plant breeding and relates to a PagKNAT5a gene and an application thereof. Background Art

[0002] As one of the most abundant biomass resources in the world, wood is the raw material for many industries such as construction, papermaking and biofuels, and has important economic value. Wood refers to the secondary xylem produced by the proliferation and differentiation of vascular cambium cells in woody plants, and is the most important renewable resource on earth. Cultivating new high-yield and high-quality forest varieties for the construction of a large number of artificial forests will help alleviate the contradiction between supply and demand. However, trees have the characteristics of slow growth, difficulty in cultivating new species, long cycles, high genome heterozygosity, and conservative structure. It is difficult to integrate distant genetic resources through traditional breeding methods for targeted hybridization improvement of specific shapes. With the development of molecular biology, the use of modern biotechnology to reveal the growth mechanism of trees and cultivate new high-quality and high-yield forest varieties is an important way to improve wood production and quality.

[0003] In recent years, with the development of genomics and molecular biology and the completion of whole genome sequencing of some afforestation tree species, research on the molecular mechanism of wood formation has also made some progress. It is of great significance to analyze the molecular regulatory mechanism of xylem development and use it as a theoretical basis to support modern molecular breeding technology to improve the wood yield and quality of forest tree species. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a method for improving the traits of poplars, the method comprising: using genetic engineering means to highly express the PagKNAT5a gene in poplars;

[0005] The improved poplar traits include:

[0006] Increase the height of poplar plants;

[0007] Increase the ground diameter of poplar plants;

[0008] Increase internode length of poplar trees;

[0009] Increase the length of cortical cells in poplar xylem;

[0010] Increase the length of fiber cells in poplar plants;

[0011] Increase the length of the poplar plant's duct;

[0012] Increase the width of vascular xylem in poplar plants;

[0013] Increase the cell wall thickness of xylem fiber cells in poplar plants;

[0014] Increase the thickness of xylem secondary cell walls in poplar plants;

[0015] Increase the lignin content of poplar stem segments.

[0016] In some embodiments, the poplar tree is of the 84K variety.

[0017] In some embodiments, the KNAT5a gene is a poplar KNAT5a gene.

[0018] In some embodiments, the protein sequence encoded by the KNAT5a gene is shown as SEQ ID NO.12 or SEQ ID NO.14.

[0019] In some embodiments, the method comprises the steps of:

[0020] S1: transferring the coding sequence of the KNAT5a gene into an expression vector to obtain a recombinant vector containing the KNAT5a gene;

[0021] S2: transforming Agrobacterium with the recombinant vector containing the KNAT5a gene to obtain recombinant Agrobacterium containing the KNAT5a gene;

[0022] S3: infecting a poplar leaf with a wound with the recombinant Agrobacterium containing the KNAT5a gene to obtain an infected poplar leaf;

[0023] S4: placing the infected poplar leaves on a co-culture medium to obtain co-cultured leaves;

[0024] 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;

[0025] S5: placing the co-cultured leaves on a differentiation medium to obtain differentiated poplar buds;

[0026] 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 pH 5.5-6.5;

[0027] S6: inserting the differentiated poplar buds into a rooting screening medium to obtain rooted poplar plants;

[0028] 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.

[0029] In some embodiments, the method is selected from any one of the following situations C1, C2, C3, C4, C5, C6, C7 and C8 or a combination thereof;

[0030] C1: In S1, the backbone of the expression vector is pMDC32 vector;

[0031] C2: In S2, the Agrobacterium is Agrobacterium GV3101;

[0032] C3: In S2, the Agrobacterium is activated in the activation medium for 1.5-2.5 days before use;

[0033] The activation culture medium is based on liquid MS, and the activation culture medium also contains 40-60 mg / L kanamycin, 40-60 mg / L gentamicin and 40-60 mg / L rifamycin;

[0034] C4: In S3, the poplar leaves are leaves of poplar tissue culture seedlings;

[0035] C5: In S3, the poplar leaves are placed on the co-culture medium for pre-culture for 1.5-2.5 days before use;

[0036] C6: In S4, the culture conditions are: dark culture for 1.5-2.5 days;

[0037] C7: In S5, the culture conditions are: culture temperature 20-28°C, light intensity 40-60 μmol·m -2 ·s -1 , the photoperiod was 14-18 h light / 6-10 h dark per day;

[0038] C8: 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 every day.

[0039] The second aspect of the present invention provides a use of a biomaterial in preparing a formulation for poplar breeding for improving poplar traits;

[0040] The improved poplar traits include:

[0041] Increase the height of poplar plants;

[0042] Increase the ground diameter of poplar plants;

[0043] Increase internode length of poplar trees;

[0044] Increase the length of cortical cells in poplar xylem;

[0045] Increase the length of fiber cells in poplar plants;

[0046] Increase the length of the poplar plant's duct;

[0047] Increase the width of vascular xylem in poplar plants;

[0048] Increase the cell wall thickness of xylem fiber cells in poplar plants;

[0049] Increase the thickness of xylem secondary cell walls in poplar plants;

[0050] Increase the lignin content of poplar stem segments;

[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 KNAT5a protein;

[0054] P2: Fusion protein

[0055] The amino acid sequence of the fusion protein contains the amino acid sequence of the KNAT5a 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 KNAT5a protein described in P1 or the fusion protein described in P2;

[0058] P4: Gene

[0059] The coding sequence of the gene can encode the KNAT5a 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: Cells

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

[0066] The encoded protein in the gene expression cassette of the genetic engineering vector is expressed constitutively, tissue-specifically, or artificially induced; and

[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: Test 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, the KNAT5a gene is a poplar KNAT5a gene.

[0072] In some embodiments, the amino acid sequence of the KNAT5a protein is shown as SEQ ID NO.12 or SEQ ID NO.14.

[0073] In some embodiments, the method is selected from any one or a combination of the following situations D1, D2, D3 and D4;

[0074] D1: The variety of the poplar is 84K poplar;

[0075] D2: The backbone of the genetic engineering vector is pMDC32 vector;

[0076] D3: The cells are Agrobacterium GV3101 cells;

[0077] D4: The functional protein fragment is a tag peptide and / or signal peptide used for separating and purifying proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is a comparison of the relative expression levels of PagKNAT5a in different tissues of 84K poplar.

[0079] Figure 2 This is a photograph of the GUS staining results of ProPagKNAT5a::GUS transgenic material.

[0080] Figure 3This is the result of identifying the expression levels of PagKNAT5a gene overexpression positive plants.

[0081] Figure 4 This is the phenotype of PagKNAT5a overexpressing plants cultured in soil for 2 months.

[0082] Figure 5 The longitudinal section of the vascular tissue morphology and stem segment cell length statistics of PagKNAT5a overexpressing plants cultured in soil for 2 months, where the blue arrows represent vessels and the yellow arrows represent fiber cells.

[0083] Figure 6 The cross-section vascular tissue morphology and xylem width statistics of PagKNAT5a-overexpressing plants cultured in soil for 2 months. The red arrow indicates the xylem.

[0084] Figure 7 Observation of the cell wall morphology and thickness statistics of xylem fiber cells in PagKNAT5a overexpressing plants under electron microscopy.

[0085] Figure 8 The results of staining and content determination of cell wall components in PagKNAT5a overexpressing plants. DETAILED DESCRIPTION

[0086] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0087] Materials and instruments not described in the present invention are conventional materials and instruments in the art, and operation details not described in the present invention are conventional operations in the art. Unless otherwise specified, the nucleic acid sequences shown in the present invention are written from left to right in the direction of 5' to 3'.

[0088] Example 1: Analysis of PagKNAT5a gene expression specificity

[0089] (I) Analysis of PagKNAT5a gene expression

[0090] In order to analyze the expression of PagKNAT5a gene (KNAT5a is the gene name, Pag represents the gene from 84K poplar) in different tissues of Populus alba×P.glandulosa'84K' (referred to as "84K poplar"), the present invention used a polysaccharide / polyphenol plant RNA rapid extraction kit (HLINGENE, NG3021S) to extract RNA from different tissues of Populus alba×P.glandulosa'84K', including bark, developmental xylem (DX), mature xylem (MX), stem segment (N1-9), leaf (Leaf, L1-3), root and root tip, and used Evo The extracted RNA was reverse transcribed into cDNA using the M-MLV reverse transcription kit (Accurate, AG11705). The quantitative primers (F1 and R1) of the PagKNAT5a gene were designed. The cDNA was used as a template for qRT-PCR to analyze the relative expression of the PagKNAT5a gene in different tissues ( Figure 1 ), with PagACTIN gene as internal reference, primers of internal reference were F2 and R2, and the internal reference expression level was 1. The results showed that the gene was widely expressed in different tissues.

[0091] F1 (SEQ ID NO.1): 5'-CGACGACGTTTTAACCCAAA-3'

[0092] R1 (SEQ ID NO.2): 5'-GCGTGGCAATCCCTTAAACAA-3'

[0093] F2 (SEQ ID NO.3): 5'-AAACTGTAATGGTCCTCCCTCCG-3'

[0094] R2 (SEQ ID NO.4): 5'-GCATCATCACAATCACTCTCCGA-3'

[0095] (II) GUS reporter gene detection of PagKNAT5a gene promoter

[0096] 1. Vector Construction

[0097] (1) Target fragment amplification

[0098] The invention adopts double enzyme digestion combined with homologous recombination method to construct recombinant pMDC164 vector. Firstly, primers (F3, R3) are designed according to the promoter region sequence of the obtained target gene PagKNAT5a silver gland poplar 84K poplar father, and PCR amplification is performed with silver gland poplar 84K genomic DNA sample as template.

[0099] The promoter region sequence of the PagKNAT5a gene in Populus argentea 84K male parent is as follows (SEQ ID NO.5):

[0100]

[0101] The sequence of F3 (SEQ ID NO.6) is as follows:

[0102] 5'-agtgccaagctctagttaatATGACATGACATTTATGCTGGA-3'

[0103] The sequence of R3 (SEQ ID NO.7) is as follows:

[0104] 5'-cccggggatcgatcctctagTAGAAGTGGGTTTTCATTTC-3'

[0105] The genomic DNA from the leaves of Populus argentatus 84K tissue culture seedlings was extracted using the CTAB method.

[0106] The enzyme used for amplification was 2×Phanta Flash Master Mix (Vazyme, P510), and the PCR system was 1 μL of DNA template extracted by the CTAB method, 1 μL of F primer, 1 μL of R primer, 10 μL of 2×Phanta Flash Master Mix, and ddH 2 O was used to fill the system to 20 μL, and the PCR program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s. The denaturation, annealing and extension steps were repeated 34 times, and then fully extended at 72°C for 10 min. The products were stored at 4°C.

[0107] The PCR product was mixed with loading buffer and spotted in the 1.5% agarose gel spotting wells. After electrophoresis at 100 V for 15 min, the correct size band was cut under ultraviolet light to recover the PCR product. At the same time, the pMDC164 vector was linearized using restriction endonucleases XbaⅠ and PacⅠ, then purified and recovered using a kit (HLINGENE, NG208S), and stored at -20°C.

[0108] The PCR product and the enzyme digestion product were homologously recombined using ClonExpress MultiS One Step Cloning Kit (Vazyme, C113). The reaction system was: 5×CE MultiS Buffer 2 μL, linearized vector 200 ng, PCR product 200 ng, Exnase MultiS 1 μL, ddH 2 0 to complete the system to 10 μL. Incubate the ligation reaction at 37°C for 30 min. The ligation product can be directly used to transform E. coli.

[0109] (2) Transformation of Escherichia coli DH5α

[0110] After the homologous recombination reaction, the ligation product was transformed into Escherichia coli DH5α. The experimental steps were as follows: the competent Escherichia coli DH5α was taken out from the -80°C refrigerator and thawed on ice, the above homologous recombination product was added, and it was placed on ice for 5 minutes; a heat shock reaction was carried out at 42°C for 1 minute and it was placed on ice for 15 minutes; 200 μL of resistance-free LB liquid culture medium was added and cultured at 37°C 200rpm shaking for 1 hour; 100 μL of the cultured bacterial liquid was aspirated and spread on the LB solid culture medium added with 50 mg / L Kan (kanamycin) antibiotic, and after sealing, it was inverted and cultured in a 37°C incubator overnight, and the colony was observed the next day.

[0111] (3) Plasmid extraction

[0112] After resistance culture medium screening, colony PCR verification, and Sanger sequencing to confirm that the recombinant vector is correct, the transformed Escherichia coli is expanded and cultured in the corresponding resistance LB liquid culture medium, and the plasmid is extracted using a high-purity plasmid mini-extraction kit (Welling, NG211S) for use.

[0113] 2. Preparation of transgenic plants

[0114] (1) Transformation of Agrobacterium GV3101

[0115] After obtaining the plasmid, Agrobacterium GV3101 was transformed by chemical transformation. The experimental steps are as follows: take out the competent Agrobacterium GV3101 from the -80℃ refrigerator and thaw it on ice. After thawing, add 100ng of the above-mentioned extracted plasmid; place it on ice for 5min, quick freeze it with liquid nitrogen for 5min, water bath it at 37℃ for 5min, and place it on ice for 5min; add 200μL of non-resistant LB liquid culture medium, and culture it on a shaking table at 28℃ and 200rpm for 2h; obtain Agrobacterium suspension. 100μL of culture solution was applied to LB solid culture medium with 50mg / L Kan, 50mg / LGent (gentamicin) and 50mg / L Rif (rifamycin) antibiotics added, sealed, and inverted in a 37℃ incubator for two days to observe the growth of the colony.

[0116] (2) Agrobacterium infection and co-cultivation

[0117] The transgenic materials used in the present invention are all obtained by Agrobacterium-mediated leaf disc transformation, and the genetic transformation steps are all carried out under sterile conditions in a clean bench. First, select the leaves of 84K tissue culture seedlings with good growth status, use a sterile blade to remove the leaf tip and petiole, and scratch the leaves on the main veins, and place them in a co-culture medium (the co-culture medium is based on WPM as the basic medium, and also contains 20g / L sucrose, 7.8g / L agar, 0.5g / L MES (2-morpholineethanesulfonic acid), 100μM acetosyringone, pH5.9) for pre-culture for 2 days; pick the above-mentioned Agrobacterium GV3101 carrying the recombinant pMDC164 vector and activate it in 1mL LB liquid medium to which 50mg / L Kan, 50mg / L Gent and 50mg / L Rif antibiotics have been added for two days; take 100μL of the activated bacterial solution and add 50mL to which the corresponding antibiotics (50mg / L Kan, 50mg / L Gent and 50mg / L Rif) LB liquid medium, 28 ° C, 200 rpm shaker culture for 12 hours; 4000 rpm centrifugation for 10 minutes to remove the supernatant, add an appropriate amount of autoclaved suspension to dilute to OD 600 =0.3-0.4; place the pre-cultured leaves in the resuspended bacterial solution, shake gently for 10-15 minutes, then remove the leaves and absorb the remaining bacterial solution on autoclaved filter paper, place the leaves with the back side facing up in the co-culture medium and culture in the dark for 2 days.

[0118] (3) Differentiation and rooting culture

[0119] After the co-cultivation, the leaves were transferred to the screening and induction differentiation medium (WPM as the basic medium, containing 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 cephalosporin, 200 mg / L timentin, 2 mg / L hygromycin, pH 5.9) with antibiotics added. The medium was replaced in time according to the contamination of Agrobacterium. The culture conditions were 25 °C and the light intensity was 50 μmol·m -2 ·s -1 , the photoperiod was 16h light / 8h dark. Cultivate until adventitious buds differentiate and grow to about 2cm, take out the buds and place them in screening rooting medium (based on WPM, containing 10g / L sucrose, 7.8g / L agar, 0.5g / L MES, 200mg / L cephalosporin, 200mg / L timentin, 2mg / L hygromycin, pH 5.9) to continue screening rooting culture and mark the serial number. The culture conditions are temperature 25℃, light intensity 50μmol·m -2 ·s -1 The photoperiod was 16h light / 8h dark, and the plants were cultured until roots took shape.

[0120] 3. Identification of transgenic positive plants

[0121] The leaves of the budding and rooting materials were taken, and the leaf DNA was extracted by the CTAB method. The DNA was used as a template, and the universal primer R4 (SEQ ID NO.8) of the pMDC164 vector and the primer F3 constructed by the GUS reporter gene expression vector were used to detect the inserted fragment. The silver gland poplar 84K genomic DNA template was used as a negative control, and the recombinant plasmid was used as a positive control. The PCR product was electrophoresed at 135V for 15min in a 1% concentration agarose gel with a nucleic acid dye added, and then imaged under an ultraviolet lamp. Whether the material was a transgenic positive plant was identified based on whether the band was consistent with the positive control.

[0122] R4 sequence: 5'-TTGCCCGGCTTTCTTGTAAC-3'

[0123] 4. GUS reporter gene detection

[0124] Take the identified positive transgenic plants (named: ProPagKNAT5a::GUS), and after four weeks of conventional tissue culture subculture, take the whole plant and immerse it in 90% acetone at 4°C for 2 hours; after fixation, wash it three times with GUS staining buffer; add GUS staining solution and vacuum for 30 minutes to allow the staining solution to fully contact the plant tissue; stain at 37°C for 15 hours; place it in 75% ethanol to remove chlorophyll and other pigments in order to observe the distribution of GUS signals.

[0125] After staining, the whole material was photographed, and samples from different tissue parts were taken for vibration sectioning and further observation under an optical microscope.

[0126] The results are as follows Figure 2 As shown, A: longitudinal section of the apical bud (bar = 100 μm); B: cross section of the third internode stem segment (bar = 100 μm); C: cross section of the fifth internode stem segment (bar = 100 μm); D: leaf section (bar = 100 μm); E: leaf vein cross section (bar = 100 μm); F: root cross section (bar = 100 μm); G: ProPagKNAT5a::GUS transgenic plants (bar = 1 cm). It can be seen that relatively significant GUS signals were detected in the apical buds, leaves and roots ( Figure 2 A, D, G). Further tissue section studies revealed that there was a strong GUS signal in the xylem of the stem segment ( Figure 2 B, C). The above results are consistent with the results of qRT-PCR analysis. It is speculated that this gene may be involved in the development of xylem.

[0127] Example 2: Construction of transgenic plants overexpressing PagKNAT5a

[0128] (I) Vector construction

[0129] In this example, the vector used was pMDC32 vector, and the silver gland poplar 84K cDNA sample was used as a template. Primers F5 (SEQ ID NO.9) and R5 (SEQ ID NO.10) were designed to amplify the full length of the coding sequence of the paternal gene of the PagKNAT5a gene. The pMDC32 vector was linearized using restriction endonucleases XbaⅠ and SacⅠ, and the homologous recombination method was still used to construct a recombinant expression vector (named: 35S::PagKNAT5a). The operations other than the vector, endonuclease, and insertion sequence were the same as the "vector construction" method in Example 1.

[0130] F5:5'-ggagaggacctcgactctagATGGCTTTCAAGACCACCA-3'

[0131] R5:5'-cgatcggggaaattcgagctTCACTTCTTTCGTTTGCTCT-3'

[0132] PagKNAT5a gene paternal coding sequence (SEQ ID NO.11):

[0133]

[0134] PagKNAT5a gene paternal protein sequence (SEQ ID NO.12):

[0135] MAFQDHHHTPQEMAFQLPQHHHISASPSTGPTWLSNAVLRRHDDVLTQTRIEKPENNTNNGSEEELIDSVSDNWERAKCKAEILGHPLYEQLLAAHVACLRIATPVDQLARIDTQLAQSQDVVAKYSGVGRSHVVDEKELDQFMTHYVLLLCSFKDQLQQHVRVHAMEAVMACWELEQSLQSLTGVSPGEGTGATMSDDDDDQADSDANLYDGNLDGMDTMGFGPLVPTETERSLMERVRQELKHELKQDYKEKIVDIREEILRKRRAGKLPGDTTSLLKAWWQTHSKWPYPTEEDKARLVQETGLHLKQINNWFINQRKRNWHSSPSGSTSKSKRKK.

[0136] PagKNAT5a gene maternal coding sequence (SEQ ID NO.13):

[0137]

[0138] PagKNAT5a gene maternal protein sequence (SEQ ID NO.14):

[0139] MAFQDDHHTSQEMAFQLPQHHHISASPSTGPTWLSNAVLRRHDDVLSQTRIEKPENNTNNGSEEELIDSVSDNWERAKCKAEILGHPLYEQLLAAHVACLRIATPVDQLARIDTQLAQSQDVVAKYSGVGRSHVVDEKELDQFMTHYVLLLCSFKDQLQQHVRVHAMEA VMACWELEQSLQGLTGVSPGEGTGATMSDDDDDQADSDANLYDGNLDGLDTMGFGPLVPTETERSLMERVRQELKHELKQDYKEKIVDIREEILRKRRAGKLPGDTTSLLKAWWQTHSKWPYPTEEDKARLVQETGLHLKQINNWFINQRKRNWHSSPSGSTSKSKRKK.

[0140] (II) Genetic transformation

[0141] The 35S::PagKNAT5a expression vector constructed and verified to be correct was transformed into Agrobacterium and then used to mediate genetic transformation of poplar. Except for the different recombinant vectors used, the experimental steps were the same as those of "transgenic plant preparation" in Example 1.

[0142] (III) Identification of transgenic positive plants

[0143] The leaves of the genetically transformed rooted plants were taken, and the leaf DNA was extracted by CTAB method. The insertion fragment was detected by using the universal primer R6 (SEQ ID NO. 15) of the pMDC32 vector and the primer F5 constructed by the PagKNAT5a overexpression vector. The other experimental steps were the same as those for the identification of transgenic positive plants in Example 1. A total of 8 positive lines were identified.

[0144] R6:5'-GATAATCATCGCAAGACCGG-3'

[0145] (IV) Analysis of relative expression levels in PagKNAT5a overexpressing transgenic plants

[0146] The 8 transgenic positive plants identified above were further propagated by tissue culture. The wild type 84K poplar was used as a control sample. The fifth internode leaf of the tissue culture seedlings with consistent growth was taken to extract RNA and reverse transcribed into cDNA. The cDNA was used as a template for qRT-PCR analysis using quantitative primers (F1, R1). Except for the strain material and specific tissues, the experimental steps were the same as those for the PagKNAT5a gene expression analysis in Example 1.

[0147] The relative expression levels of PagKNAT5a gene in 8 positive strains were obtained by qRT-PCR analysis. Among them, the expression levels of PagKNAT5a in strains OE#9 and OE#11 were the highest, which were 13 times and 24 times that of the control plant (parent 84K poplar), respectively. The results are shown in Figure 3 Therefore, strains OE#9 and OE#11 were selected as experimental materials for tissue culture and propagation for subsequent research.

[0148] Example 3: Characterization of the traits of PagKNAT5a overexpressing transgenic plants

[0149] (I) Determination of growth indicators of PagKNAT5a overexpressing transgenic plants

[0150] The control (control, parent 84K poplar) and transgenic plants of OE#9 and OE#11 that were cultured for 1 month and grew in the same manner were selected and transplanted into nutrient soil. The 1st to 9th internode stem segments of the control and transgenic plants that were cultured in soil for 1 month were mixed and RNA was extracted and PagKNAT5a expression was detected. Except for the strain material and specific tissues, the operation steps were the same as the PagKNAT5a gene expression analysis in Example 1. The growth indicators such as plant height, ground diameter, and length of each stem segment were measured for the control plants and transgenic materials that were cultured in soil for 2 months.

[0151] Results Figure 4 , where A: phenotypes of control and PagKNAT5a transgenic plants; B: PagKNAT5a gene expression levels in control and transgenic plants; C: plant height statistics of control and PagKNAT5a overexpressing transgenic plants; D: ground diameter statistics of control and PagKNAT5a overexpressing transgenic plants; E: internode number statistics of control and PagKNAT5a overexpressing transgenic plants; F: internode length statistics of control and PagKNAT5a transgenic plants (where OE#9 and OE#11 are represented by bars, and the control is represented by a line graph, and the intersection of the line graph and the two bars is the length of the corresponding internode) (*p<0.05; **p<0.01).

[0152] The statistical results showed that compared with the control plants, the plant height and ground diameter of the PagKNAT5a overexpressing transgenic lines increased significantly ( Figure 4A, C, D), the internode number statistics showed that there was no significant difference in the internode number between the transgenic lines overexpressing PagKNAT5a and the control plants ( Figure 4 D). Further analysis of internode length showed that, starting from the ninth internode, the internode length of the two transgenic lines overexpressing PagKNAT5a was significantly longer than that of the control plants ( Figure 4 E), indicating that the differences in plant height are mainly caused by changes in internode length.

[0153] (II) Observation of vascular tissue in transgenic plants overexpressing PagKNAT5a

[0154] (1) Optical microscope observation

[0155] Toluidine Blue O (TBO) staining: Take the 10th internode stem segment of the control plants and transgenic materials that have been cultivated in soil for 2 months, trim the samples with a blade and glue them to the stage with glue, slice them with an oscillating slicer, and slice them with a thickness of 50 μm. Make transverse and longitudinal slices respectively, and collect the sliced ​​samples. Use tweezers or a dropper to gently take out the complete slice, spread it on a glass slide, add 0.1% toluidine blue dye solution, and stain for 40 seconds. Then, add a small amount of deionized water to wash away the dye solution. The temporary mount prepared in this way is placed under an optical microscope, observed with a 20x objective lens, and photographed and recorded for observation of the transverse vascular tissue morphology of the stem segment and statistics of the longitudinal cell length.

[0156] Fiber separation: Take the 10th internode stem segment of the control plant and the transgenic material that have been cultivated in soil for 2 months, and peel off the bark. Place it in a volume ratio of 1:1 hydrogen peroxide (30%) and glacial acetic acid (analytical grade) and heat it in a 65℃ water bath for several hours. Shake the solution appropriately during the period until the sample is separated into single fibers and fully dissociated. After the dissociation is completed, centrifuge at 5000rpm / min for 10min, pour off the supernatant dissociation solution, and use ddH 2 O was washed several times, and an appropriate amount of the dissociated fiber sample was taken out and stained with safranin to make a temporary glass slide, which was placed under an optical microscope for observation to count the length of xylem fibers and vessels.

[0157] Results Figure 5 , 6, where, 5A: TBO staining results of longitudinal sections of the 10th internode of the control and PagKNAT5a transgenic plants; 5B: Observation of xylem fiber cells and vessels in the 10th internode of the control and transgenic plants (yellow double arrows indicate fiber cells; blue double arrows indicate vessels); 5C: Statistics of the length of cortical cells near the phloem, xylem fiber cells and vessels in the control and PagKNAT5a transgenic plants (*p<0.05; **p<0.01); 6A: TBO staining results of transverse sections of the 10th internode of the control and PagKNAT5a transgenic plants (red double arrows indicate xylem); 6B: Statistics of the width of xylem in the 10th internode of the control and PagKNAT5a transgenic plants.

[0158] Since PagKNAT5a promotes the growth of poplar stem segments and the increase of ground diameter, the 10th internode stem segment was taken for longitudinal sectioning and toluidine blue staining analysis. The results showed that the length of cortical cells near the xylem increased with overexpression ( Figure 5 A, C). In addition, after isolating xylem fibers, it was found that the fiber cell length and vessel length in plants overexpressing the PagKNAT5a gene were increased ( Figure 5 B, C). This indicates that PagKNAT5a promotes stem segment elongation by promoting vascular cell elongation, ultimately leading to plant height growth. In addition, transverse sectioning and toluidine blue staining of the 10th internode stem segment showed that the width of the vascular xylem in the overexpressing plant increased ( Figure 6 A, B), indicating that PagKNAT5a promotes the thickening of ground diameter by promoting the development of vascular xylem.

[0159] (2) Electron microscope observation

[0160] Transmission electron microscopy observation:

[0161] (a) Fixation: The 10th internode stem segment in the soil culture material that has been cultivated for two months was cut into a length of about 3 mm, and then the cut sample was placed in a 2.5% glutaraldehyde solution for vacuum extraction for 15 minutes. The sample was then placed in a 2.5% glutaraldehyde solution for overnight fixation. After fixation, the fixative was removed and the sample was rinsed three times with 0.1M phosphate buffer (pH7.0), each time for 15 minutes. Subsequently, the sample was fixed for a second time with 1% osmic acid solution for 1-2 hours, and the sample was rinsed three times with 0.1M phosphate buffer (pH7.0) again, each time for 15 minutes.

[0162] (b) Dehydration: Dehydrate the samples using a series of ethanol solutions with increasing concentrations (30%, 50%, 70%, 80%, 90% and 95%), with each concentration treated for 15 min. Finally, the samples were placed in 100% ethanol for 20 min. The samples were replaced with new 100% ethanol and treated overnight.

[0163] (c) Drying and observation: The samples were dried by critical point drying method using Hitachi HCP-2 critical point dryer. After drying, they were coated and placed in Hitachi H-7650 transmission electron microscope for observation of xylem cell subcellular structure.

[0164] Scanning electron microscope observation:

[0165] The steps of fixation, dehydration and drying are the same as those of transmission electron microscope sample preparation. The sample holder is placed in the vacuum chamber of the microscope for vacuum treatment. The cross section of the stem segment is then scanned step by step to observe the structural characteristics of the xylem cells.

[0166] The scanning electron microscope counts the width of the cell wall between two cells, while the transmission electron microscope counts the thickness of the cell wall of a single cell. Figure 7 As shown, A: Scanning electron microscopy observation of xylem cells; B: The thickness of the cell wall between two xylem fiber cells at 100μm, 200μm, and 300μm from the cambium cells was calculated based on the results of scanning electron microscopy observation (*p<0.05; **p<0.01); C: Transmission electron microscopy observation of xylem cells (ve represents vessels, xf represents xylem fiber cells); D: The thickness of the cell wall between xylem fiber cells was calculated based on the results of scanning electron microscopy observation (*p<0.05; **p<0.01). The results showed that the xylem cell walls of PagKNAT5a overexpressing transgenic plants were significantly thicker than those of the control plants at 100μm, 200μm, and 300μm from the cambium cells ( Figure 7 A, B). Transmission electron microscopy observations further confirmed that the thickness of the xylem secondary cell wall in the overexpression plants was greater than that in the control plants, indicating that PagKNAT5a promotes the thickening of the secondary wall of xylem fiber cells ( Figure 7 C, D).

[0167] (III) Determination of the contents of major cell wall components in PagKNAT5a overexpressing transgenic plants

[0168] (1) Sampling

[0169] The 10th internode stem segment of the control plants and transgenic materials cultured in soil for 2 months was cut into 50 μm thick transverse sections for phloroglucinol and Calcofluor White staining. The 7th to 15th internode stem segments of the soil-cultured plant materials cultured for 2 months were selected, and the leaves and bark were carefully removed. The remaining stem material was placed in an oven and dried at 80°C to constant weight. Subsequently, the dried sample was ground into powder and sieved through a 30 to 50 mesh sieve to obtain a fine powder sample as the sample to be tested for subsequent cell wall component analysis.

[0170] (2) Phloroglucinol staining

[0171] HCl and 3% phloroglucinol solution were mixed at a ratio of 1:2 (v / v) (prepared and used immediately), and transverse tissue sections were placed in the mixed solution for staining for 5 minutes, and then temporary slides were made. The slides were observed under a 20x optical microscope and photographed for preliminary analysis of the lignin content in vascular tissues.

[0172] (3) Calcofluor White staining

[0173] Place the transverse tissue sections on a glass slide, add Calcofluor White stain, stain for 5 minutes, absorb the stain with deionized water, observe under a 20x optical microscope, and take photos for preliminary analysis of the cellulose content in the vascular tissue.

[0174] (4) Determination of lignin content

[0175] This test uses a lignin content test kit (Mengxi Biotechnology, M1711A) and follows the instructions to carry out the test. An ELISA reader is used to measure the sample absorbance at a wavelength of 340 nm, and the sample lignin content is calculated according to the standard curve.

[0176] (5) Determination of cellulose content

[0177] This test uses a cellulose content test kit (Mengxi Biotechnology, M1718A) and follows the instructions to carry out the test. An ELISA reader is used to measure the sample absorbance at a wavelength of 540 nm, and the sample cellulose content is calculated according to the standard curve.

[0178] (6) Determination of hemicellulose content

[0179] This experiment used a hemicellulose content test kit (Mengxi Biotechnology, M1719A), carried out the test according to the instructions, and used an ELISA reader to measure the absorbance of the sample at a wavelength of 620nm. Finally, the hemicellulose content in the sample was calculated based on the hemicellulose standard curve and its calculation formula.

[0180] Results Figure 8 , where A: Phloroglucinol staining of transverse sections of the 10th internode of control and PagKNAT5a overexpressing transgenic plants; B: Calcofluor White staining of transverse sections of the 10th internode of control and PagKNAT5a overexpressing transgenic plants; C: Determination of lignin, cellulose, and hemicellulose contents (*p<0.05; **, p<0.01). The results showed that the phloroglucinol staining of the vascular xylem of the overexpressing plants was deeper than that of the control plants ( Figure 8A), indicating that the lignin content of the vascular xylem of the overexpression plants was higher than that of the control plants. Calcofluor White staining results showed that there was no significant difference in the fluorescence signal intensity between the vascular xylem of the overexpression plants and the control plants ( Figure 8 B). At the same time, we quantitatively determined the main chemical components of plant secondary cell walls, lignin, cellulose, and hemicellulose (Figure C). The results showed that the lignin content of transgenic plants was significantly higher than that of control plants, while the content of cellulose and hemicellulose was similar to that of control plants. This suggests that PagKNAT5a may promote the thickening of the secondary wall of xylem cells by promoting lignin synthesis, thereby promoting the lignification process.

[0181] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A method for improving poplar traits, the method comprising: using genetic engineering to highly express the KNAT5a gene in poplars; The improved poplar traits include: Increase the height of poplar plants; Increase the ground diameter of poplar plants; Increase internode length of poplar trees; Increase the length of cortical cells in poplar xylem; Increase the length of fiber cells in poplar plants; Increase the length of the poplar plant's duct; Increase the width of vascular xylem in poplar plants; Increase the cell wall thickness of xylem fiber cells in poplar plants; Increase the thickness of xylem secondary cell walls in poplar plants; Increase the lignin content of poplar stem segments.

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

3. The method according to claim 1, characterized in that The KNAT5a gene is a poplar KNAT5a gene.

4. The method according to any one of claims 1 to 3, characterized in that The protein sequence encoded by the KNAT5a gene is shown as SEQ ID NO.12 or SEQ ID NO.

14.

5. The method according to any one of claims 1 to 4, characterized in that The method comprises the following steps: S1: transferring the coding sequence of the KNAT5a gene into an expression vector to obtain a recombinant vector containing the KNAT5a gene; S2: transforming Agrobacterium with the recombinant vector containing the KNAT5a gene to obtain recombinant Agrobacterium containing the KNAT5a gene; S3: infecting a poplar leaf with a wound with the recombinant Agrobacterium containing the KNAT5a 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-cultured leaves 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, 1.5-2.5 mg / L hygromycin, and pH 5.5-6.5; S6: inserting the differentiated poplar buds into a rooting screening medium to obtain rooted poplar plants; The rooting screening medium is based on WPM as a basic medium, and 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.

6. The method according to claim 5, characterized in that The method is selected from any one or a combination of the following situations C1, C2, C3, C4, C5, C6, C7 and C8; C1: In S1, the backbone of the expression vector is pMDC32 vector; C2: In S2, the Agrobacterium is Agrobacterium GV3101; C3: In S2, the Agrobacterium is activated in the activation medium for 1.5-2.5 days before use; The activation culture medium is based on liquid MS, and the activation culture medium also contains 40-60 mg / L kanamycin, 40-60 mg / L gentamicin and 40-60 mg / L rifamycin; C4: In S3, the poplar leaves are leaves of poplar tissue culture seedlings; C5: In S3, the poplar leaves are placed on the co-culture medium for pre-culture for 1.5-2.5 days before use; C6: In S4, the culture conditions are: dark culture for 1.5-2.5 days; C7: In S5, the culture conditions are: culture temperature 20-28°C, light intensity 40-60 μmol·m -2 ·s -1 , the photoperiod was 14-18 h light / 6-10 h dark per day; C8: 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 every day.

7. Use of a biomaterial in the preparation of a formulation for poplar breeding for improving poplar traits; The improved poplar traits include: Increase the height of poplar plants; Increase the ground diameter of poplar plants; Increase internode length of poplar trees; Increase the length of cortical cells in poplar xylem; Increase the length of fiber cells in poplar plants; Increase the length of the poplar plant's duct; Increase the width of vascular xylem in poplar plants; Increase the cell wall thickness of xylem fiber cells in poplar plants; Increase the thickness of xylem secondary cell walls in poplar plants; Increase the lignin content of poplar stem segments; The biological material is selected from any one of the following P1, P2, P3, P4, P5, P6, P7, P8 and P9: P1: Protein The protein is KNAT5a protein; P2: Fusion protein The amino acid sequence of the fusion protein contains the amino acid sequence of the KNAT5a protein described in P1 and the amino acid sequence of a functional protein fragment or an inert protein fragment; P3: RNA The RNA can be translated to obtain the KNAT5a protein described in P1 or the fusion protein described in P2; P4: Gene The coding sequence of the gene can encode the KNAT5a protein described in P1 or the fusion protein described in P2; P5: Gene expression cassette The gene expression product in the gene expression cassette is the RNA described in P3; P6: Genetic Engineering Vector The genetic engineering vector contains the gene expression cassette described in P5; P7: Cells The cell contains the genetic engineering vector described in P6; The encoded protein in the gene expression cassette of the genetic engineering vector is expressed constitutively, tissue-specifically, or artificially induced; and P8: Composition The composition contains the 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 P9: Test kit 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.

8. The use according to claim 7, characterized in that The KNAT5a gene is a poplar KNAT5a gene.

9. The use according to claim 7 or 8, characterized in that The amino acid sequence of the KNAT5a protein is shown in SEQ ID NO.12 or SEQ ID NO.

14.

10. The use according to any one of claims 7 to 9, characterized in that The method is selected from any one or a combination of the following situations D1, D2, D3 and D4; D1: The variety of the poplar is 84K poplar; D2: The backbone of the genetic engineering vector is pMDC32 vector; D3: The cells are Agrobacterium GV3101 cells; D4: The functional protein fragment is a tag peptide and / or signal peptide used for separating and purifying proteins.

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