A PagKNAT5a gene and its applications

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 PagKNAT5a gene in poplar trees is highly expressed; improving the traits of poplar trees includes: increasing the plant height of poplar trees; increasing the ground diameter of poplar tree plants; increasing the internode length of poplar trees; increasing the cell length of cortical cells in the xylem of poplar trees; increasing the fiber cell length of poplar tree plants; increasing the vessel length of poplar tree plants; increasing the width of the vascular xylem of poplar tree plants; increasing the cell wall thickness of xylem fiber cells in poplar tree plants; increasing the secondary cell wall thickness of the xylem of poplar tree plants; increasing the lignin content in poplar tree stem segments.

Description

Technical Field

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

[0002] As one of the world's most abundant biomass resources, wood is a 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 of woody plants, and is the most important renewable resource on earth. Cultivating new varieties of high-yield and high-quality forest trees for building a large number of artificial forests helps to alleviate the contradiction between supply and demand. However, forest trees have the characteristics of slow growth, difficult and long cycle of new variety cultivation, high genomic heterozygosity, and conservative structure, and it is relatively difficult to integrate distant genetic resources through traditional breeding methods for directional hybridization improvement of specific shapes. With the development of molecular biology, using modern biotechnology means to reveal the tree growth mechanism and cultivate new varieties of high-quality and high-yield forest trees is an important way to improve the wood yield and quality.

[0003] In recent years, with the development of genomics and molecular biology and the successive completion of the whole-genome sequencing of some forest tree species, certain progress has been made in the research on the molecular mechanism of wood formation. Analyzing the molecular regulation mechanism of xylem development and using it as a theoretical basis to support modern molecular breeding technology for improving the wood yield and quality of forest tree varieties is of great significance. Summary of the Invention

[0004] To solve the problems existing in the prior art, a first aspect of the present invention provides a method for improving the traits of poplar, and the method is: by means of genetic engineering, making the PagKNAT5a gene highly expressed in poplar;

[0005] The improvement of poplar traits includes:

[0006] Increasing the plant height of poplar;

[0007] Increasing the ground diameter of poplar plants;

[0008] Increasing the internode length of poplar;

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

[0010] Increasing the fiber cell length of poplar plants;

[0011] Increasing the vessel length of poplar plants;

[0012] Increasing the width of vascular xylem of poplar plants;

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

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

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

[0016] In some embodiments, the poplar variety is Populus alba×Populus glandulosa '84K'.

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

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

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

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

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

[0022] S3: Infect wounded poplar leaves with the recombinant Agrobacterium containing the KNAT5a gene to obtain infected poplar leaves;

[0023] S4: Place 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 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, pH 5.5 - 6.5;

[0025] S5: Place 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-(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, pH 5.5 - 6.5;

[0027] S6: Insert the differentiated poplar buds on a rooting and screening medium to obtain rooted poplar plants;

[0028] The rooting and screening medium is based on WPM, and the rooting and screening medium also contains 8 - 12 g / L of sucrose, 6 - 10 g / L of agar, 0.4 - 0.6 g / L of 2 - morpholinoethanesulfonic acid, 150 - 250 mg / L of cefamycin, 150 - 250 mg / L of ticarcillin, 1.5 - 2.5 mg / L of hygromycin, and the pH is 5.5 - 6.5.

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

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

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

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

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

[0034] C4: In S3, the poplar leaf is the leaf of a poplar tissue culture seedling;

[0035] C5: In S3, the poplar leaf is pre - cultured on the co - culture medium for 1.5 - 2.5 days before use;

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

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

[0038] C8: In S6, the culture condition is: the culture temperature is 20 - 28 °C, the light intensity is 40 - 60 μmol·m -2 ·s -1 , and the photoperiod is 14 - 18 h of light / 6 - 10 h of darkness per day.

[0039] 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;

[0040] The improvement of poplar traits includes:

[0041] Increase the plant height of poplar;

[0042] Increase the ground diameter of poplar plants;

[0043] Increase the internode length of poplar;

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

[0045] Increase the fiber cell length of poplar plants;

[0046] Increase the vessel length of poplar plants;

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

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

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

[0050] Increase the lignin content in 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 the 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: Gene engineering vector

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

[0064] P7: Cell

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

[0066] The coding proteins in the gene expression cassette of the gene engineering vector are constitutively expressed, tissue-specifically expressed or artificially induced to express; 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 gene 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 gene engineering vector described in P6 or the cell described in P7.

[0071] In some embodiments, the KNAT5a gene is the Populus alba×Populus glandulosa KNAT5a gene.

[0072] In some embodiments, the amino acid sequence of the KNAT5a protein is as shown in 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 cases D1, D2, D3 and D4;

[0074] D1: The variety of the Populus alba×Populus glandulosa is 84K Poplar;

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

[0076] D3: The cell is Agrobacterium tumefaciens GV3101 cell;

[0077] D4: The functional protein fragment is a tag peptide and / or a signal peptide for protein isolation and purification. Description of the Drawings

[0078] Figure 1 It is a comparison diagram of the relative expression levels of PagKNAT5a in different tissue parts of 84K Poplar.

[0079] Figure 2 It is a photo of the GUS staining result of the ProPagKNAT5a::GUS transgenic material.

[0080] Figure 3Results of the identification of the expression level of the positive plants overexpressing the PagKNAT5a gene.

[0081] Figure 4 Phenotype of the PagKNAT5a overexpressing plants grown in soil for 2 months.

[0082] Figure 5 Longitudinal section of the vascular tissue morphology and stem segment cell length statistics of the PagKNAT5a overexpressing plants grown in soil for 2 months. Among them, the blue arrow represents the vessel, and the yellow arrow represents the fiber cell.

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

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

[0085] Figure 8 Results of the staining and content determination of the cell wall components of the PagKNAT5a overexpressing plants. Detailed implementation manners

[0086] To make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0087] 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.

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

[0089] (1) Analysis of the expression of the PagKNAT5a gene

[0090] To analyze the expression of the PagKNAT5a gene (KNAT5a is the gene name, and Pag represents that the gene comes from Populus alba×P. glandulosa ‘84K’) in different tissue parts of Populus alba×P. glandulosa ‘84K’ (hereinafter referred to as ‘84K poplar’), in this invention, a polysaccharide / polyphenol plant RNA rapid extraction kit (HLINGENE, NG3021S) was used to extract RNA from different tissue parts of 84K poplar, such as bark, developmental xylem (DX), mature xylem (MX), stem segments (N1-9), leaves (Leaf, L1-3), roots, and root tips. The Evo M-MLV reverse transcription kit (Accurate, AG11705) was used to reverse transcribe the extracted RNA into cDNA. Quantitative primers for the PagKNAT5a gene (F1 and R1) were designed, and qRT-PCR was performed using cDNA as a template to analyze the relative expression levels of the PagKNAT5a gene ( Figure 1 ) in different tissue parts. The PagACTIN gene was used as an internal reference, and the primers for the internal reference were F2 and R2, with the expression level of the internal reference being 1. The results showed that this gene was widely expressed in different tissue parts.

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

[0092] R1 (SEQ ID NO.2): 5’-GCGTGGCAATCCTTAAACAA-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 the PagKNAT5a gene promoter

[0096] 1. Vector construction

[0097] (1) Amplification of the target fragment

[0098] In this invention, a recombinant pMDC164 vector was constructed by double digestion combined with homologous recombination. First, primers (F3, R3) were designed according to the promoter region sequence of the obtained target gene PagKNAT5a from the male parent of 84K poplar, and PCR amplification was performed using the genomic DNA sample of 84K poplar as a template.

[0099] The promoter region sequence of the PagKNAT5a gene in the male parent of Populus alba×Populus glandulosa 84K 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 of the leaves of Populus alba×P. glandulosa clone 84K tissue culture seedlings was extracted by the CTAB method.

[0106] The enzyme used for amplification was 2×Phanta Flash Master Mix (Vazyme, P510). The PCR system was 1 μL of the DNA template extracted by the CTAB method, 1 μL of the F primer, 1 μL of the R primer, 10 μL of 2×Phanta Flash Master Mix, and ddH 2 O was used to make up the system to 20 μL. The PCR program was: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 60 s. After repeating the denaturation, annealing, and extension steps 34 times, full extension was carried out at 72 °C for 10 min, and the product was stored at 4 °C.

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

[0108] The PCR products and the digested products were ligated by homologous recombination using the ClonExpress MultiS One Step Cloning Kit (Vazyme, C113). The reaction system was: 2 μL of 5×CE MultiS Buffer, 200 ng of the linearized vector, 200 ng of the PCR products, 1 μL of Exnase MultiS, and ddH 2 O was used to make up the system to 10 μL. The ligation reaction was carried out at 37 °C for 30 min, and the ligation products could be directly used for the transformation of Escherichia 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 placed on ice for 5 min; heat shock reaction was carried out at 42 °C for 1 min and then placed on ice for 15 min; 200 μL of antibiotic-free LB liquid medium was added and cultured on a shaker at 37 °C and 200 rpm for 1 h; 100 μL of the cultured bacterial solution was taken and spread on the LB solid medium supplemented with 50 mg / L Kan (kanamycin) antibiotic. After sealing, it was inverted and cultured overnight in a 37 °C incubator, and the colony situation was observed the next day.

[0111] (3) Plasmid extraction

[0112] After screening by the resistant medium, verifying by colony PCR, and confirming that the recombinant vector was correct by Sanger sequencing, the transformed Escherichia coli was cultured in a corresponding resistant LB liquid medium for expansion, and then the plasmid was extracted using a high-purity plasmid mini-prep midiprep kit (Huiling, NG211S) for standby.

[0113] 2. Preparation of transgenic plants

[0114] (1) Transformation of Agrobacterium tumefaciens GV3101

[0115] After obtaining the plasmid, the Agrobacterium tumefaciens GV3101 was transformed by the chemical transformation method. The experimental steps were as follows: The competent Agrobacterium tumefaciens GV3101 was taken out from the -80 °C refrigerator and thawed on ice. After thawing, 100 ng of the above-extracted plasmid was added; it was placed on ice for 5 min, frozen in liquid nitrogen for 5 min, water-bathed at 37 °C for 5 min, and then placed on ice for 5 min; 200 μL of antibiotic-free LB liquid medium was added and cultured on a shaker at 28 °C and 200 rpm for 2 h to obtain an Agrobacterium suspension. 100 μL of the culture solution was taken and spread on the LB solid medium supplemented with 50 mg / L Kan, 50 mg / L Gent (gentamicin), and 50 mg / L Rif (rifampicin) antibiotics. After sealing, it was inverted and cultured in a 37 °C incubator for two days, and the colony growth situation was observed.

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

[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 laminar flow hood. First, select the leaves of 84K tissue culture seedlings with good growth status, remove the leaf tips and petioles using a sterile blade, scratch the leaves on the main veins, and place them in the co-culture medium (the co-culture medium is based on WPM medium, and also contains 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES (2-morpholinoethanesulfonic acid), 100 μM acetosyringone, pH 5.9) for pre-culture for 2 days; pick the above-mentioned Agrobacterium tumefaciens GV3101 carrying the recombinant pMDC164 vector and activate it in 1 mL of LB liquid medium added with 50 mg / L Kan, 50 mg / L Gent, and 50 mg / L Rif antibiotics for 2 days; take 100 μL of the activated bacterial liquid and add it to 50 mL of LB liquid medium added with the corresponding antibiotics (50 mg / L Kan, 50 mg / L Gent, and 50 mg / L Rif), and culture it on a shaker at 28 °C and 200 rpm for 12 h; centrifuge at 4000 rpm for 10 min to remove the supernatant, and 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 liquid, gently shake for 10 - 15 min, then clamp out the leaves and blot the remaining bacterial liquid on autoclaved filter paper, place the leaves with the back side up in the co-culture medium and culture in the dark for 2 days.

[0118] (3) Differentiation and rooting culture

[0119] After the co-culture, transfer the leaves to the screening induction differentiation medium added with antibiotics (based on WPM medium, and also contains 20 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 0.05 mg / L NAA, 0.5 mg / L 6-BA, 200 mg / L cefotaxime, 200 mg / L ticarcillin, 2 mg / L hygromycin, pH 5.9) for culture, and change the medium in time according to the Agrobacterium contamination situation. The culture conditions are a temperature of 25 °C, a light intensity of 50 μmol·m -2 ·s -1 , and a photoperiod of 16 h light / 8 h dark. Culture until adventitious buds differentiate and grow to about 2 cm, take out the buds and place them in the screening rooting medium (based on WPM medium, and also contains 10 g / L sucrose, 7.8 g / L agar, 0.5 g / L MES, 200 mg / L cefotaxime, 200 mg / L ticarcillin, 2 mg / L hygromycin, pH 5.9) to continue the screening rooting culture and make serial number marks. The culture conditions are a temperature of 25 °C, a light intensity of 50 μmol·m -2 ·s -1 , and a photoperiod of 16 h light / 8 h dark, and culture until rooting.

[0120] 3. Identification of transgenic positive plants

[0121] Take the leaves of the material with buds and rooted plants, extract the leaf DNA by the CTAB method. Using this DNA as a template, detect the inserted fragment by using the universal primer R4 (SEQ ID NO.8) of the pMDC164 vector and the primer F3 for the GUS reporter gene expression vector construction. Use the genomic DNA of Populus alba×Populus glandulosa 84K as a negative control and the recombinant plasmid as a positive control. After the PCR products are electrophoresed at 135V for 15 min in a 1% concentration agarose gel added with nucleic acid dye, image under ultraviolet light, and identify whether the material is a transgenic positive plant according to whether the band is consistent with the positive control.

[0122] Sequence of R4: 5’-TTGCCCGGCTTTCTTGTAAC-3’

[0123] 4. Detection of GUS reporter gene

[0124] Take the identified positive transgenic plants (named: ProPagKNAT5a::GUS), after routine tissue culture subculture for four weeks, soak the whole plant in 90% acetone and fix it at 4°C for 2 h; after fixation, wash it 3 times with GUS staining buffer; add GUS staining solution, evacuate for 30 min to make the staining solution fully contact the plant tissue; stain at 37°C for 15 h; place it in 75% ethanol to remove pigments such as chlorophyll to observe the distribution of GUS signals.

[0125] After staining, take pictures of the whole plant material, and take samples of different tissue parts for vibratome sectioning, and further observe under an optical microscope.

[0126] The results are as Figure 2 shown. Among them, A: longitudinal section of the apical bud (bar = 100 μm); B: transverse section of the stem segment of the third internode (bar = 100 μm); C: transverse section of the stem segment of the fifth internode (bar = 100 μm); D: leaf section (bar = 100 μm); E: transverse section of the leaf vein (bar = 100 μm); F: transverse section of the root (bar = 100 μm); G: ProPagKNAT5a::GUS transgenic plant (bar = 1 cm). Thus, relatively significant GUS signals are detected in the apical bud, leaf and root ( Figure 2 A, D, G). Further histological section studies reveal that there are strong GUS signals 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 process of xylem development.

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

[0128] (1) Vector construction

[0129] In this example, the vector used was the pMDC32 vector. Using the Populus alba×Populus glandulosa 84K cDNA sample as a template, primers F5 (SEQ ID NO.9) and R5 (SEQ ID NO.10) were designed to amplify the full-length coding sequence of the parental gene of the PagKNAT5a gene. The pMDC32 vector was linearized using the restriction enzymes XbaⅠ and SacⅠ, and the recombinant expression vector (named: 35S::PagKNAT5a) was constructed using the homologous recombination method. Each operation other than the vector, restriction enzyme, and inserted sequence was the same as the "vector construction" method in Example 1.

[0130] F5: 5’-ggagaggacctcgactctagATGGCTTTTCAAGACCACCA-3’

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

[0132] Parental coding sequence of the PagKNAT5a gene (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] Maternal protein sequence of PagKNAT5a gene (SEQ ID NO.14):

[0139] MAFQDDHHTSQEMAFQLPQHHHISASPSTGPTWLSNAVLRRHDDVLSQTRIEKPENNTNNGSEEELIDSVSDNWERAKCKAEILGHPLYEQLLAAHVACLRIATPVDQLARIDTQLAQSQDVVAKYSGVGRSHVVDEKELDQFMTHYVLLLCSFKDQLQQHVRVHAMEAVMACWELEQSLQGLTGVSPGEGTGATMSDDDDDQADSDANLYDGNLDGLDTMGFGPLVPTETERSLMERVRQELKHELKQDYKEKIVDIREEILRKRRAGKLPGDTTSLLKAWWQTHSKWPYPTEEDKARLVQETGLHLKQINNWFINQRKRNWHSSPSGSTSKSKRKK.

[0140] (II) Genetic transformation

[0141] The constructed and verified 35S::PagKNAT5a expression vector was transformed into Agrobacterium tumefaciens, and Agrobacterium-mediated genetic transformation of poplar was carried out. Except for the different recombinant vectors used, the experimental procedures were the same as those in "Preparation of transgenic plants" in Example 1.

[0142] (III) Identification of transgenic positive plants

[0143] Leaves of the rooted plants obtained from genetic transformation were taken, and leaf DNA was extracted by the CTAB method. The inserted fragment was detected using the universal primer R6 (SEQ ID NO.15) of the pMDC32 vector and the primer F5 for the PagKNAT5a overexpression vector construction. Other experimental procedures 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 of PagKNAT5a overexpressing transgenic plants

[0146] The 8 transgenic positive plants obtained from the above identification were further propagated by tissue culture. Using the wild-type 84K poplar as a control sample, the leaves of the 5th internode of the tissue-cultured seedlings with consistent growth were taken to extract RNA and reverse-transcribed into cDNA. Using the cDNA as a template, qRT-PCR analysis was performed using quantitative primers (F1, R1). Except for the line materials and specific tissues, the experimental procedures were the same as those for the PagKNAT5a gene expression analysis in Example 1.

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

[0148] Example 3: Characterization of PagKNAT5a Overexpressing Transgenic Plants

[0149] (I) Determination of Growth Indexes of PagKNAT5a Overexpressing Transgenic Plants

[0150] The control (Control, parent 84K poplar), OE#9 and OE#11 transgenic plants that had been tissue-cultured for 1 month and had consistent growth were selected and transplanted into nutrient soil. The stem segments of the 1st to 9th internodes of the control and transgenic plants after 1 month of soil culture were mixed to extract RNA and detect the expression level of PagKNAT5a. Except for the line materials and specific tissues, the operation steps were the same as those for the PagKNAT5a gene expression analysis in Example 1. The growth indexes such as plant height, ground diameter, and the length of each stem segment of the control plants and transgenic materials after 2 months of soil culture were measured.

[0151] The results are shown in Figure 4 , where A: phenotypes of the control and PagKNAT5a transgenic plants; B: expression levels of the PagKNAT5a gene in the control and transgenic plants; C: plant height statistics of the control and PagKNAT5a overexpressing transgenic plants; D: ground diameter statistics of the control and PagKNAT5a overexpressing transgenic plants; E: internode number statistics of the control and PagKNAT5a overexpressing transgenic plants; F: length statistics of each internode of the control and PagKNAT5a transgenic plants (where OE#9 and OE#11 are represented by bars, and the control is represented by a line graph. The intersection point 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 were significantly increased ( Figure 4(A, C, D). The statistical results of internode numbers showed that there was no significant difference in internode numbers between the transgenic lines overexpressing PagKNAT5a and the control plants. Figure 4 (D). Further analyzing the internode lengths, we observed that starting from the ninth internode, the internode lengths of the two transgenic lines overexpressing PagKNAT5a were significantly longer than those of the control plants. Figure 4 (E), indicating that the difference in plant height was mainly caused by the change in internode length.

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

[0154] (1) Observation under optical microscope

[0155] Toluidine Blue O (TBO) staining: Take the stem segments of the 10th internode of the control plants and transgenic materials grown in soil for 2 months. After trimming the samples with a blade, glue them to the stage. Use a vibratome to section, with a section thickness of 50 μm. Make transverse and longitudinal sections respectively, and collect the sections. Gently take out the complete sections with forceps or a dropper, lay them flat on the glass slide, and add 0.1% toluidine blue staining solution, and stain for 40 s. Subsequently, add a small amount of deionized water to wash away the staining solution. The prepared temporary mounts are placed under an optical microscope, observed with a 20× objective lens, and photographed and recorded for observing the transverse vascular tissue morphology of the stem segments and counting the longitudinal cell lengths.

[0156] Fiber isolation: Take the stem segments of the 10th internode of the control plants and transgenic materials grown in soil for 2 months, and peel off the bark. Place them in a 1:1 volume ratio of hydrogen peroxide (30%) and glacial acetic acid (analytical grade) and heat in a 65 °C water bath for several hours. Shake the solution appropriately during this period until the samples are separated into single fibers and fully dissociated. After dissociation, centrifuge at 5000 rpm / min for 10 min, pour off the supernatant dissociation solution, and wash several times with ddH 2 O. Appropriate amounts of the dissociated fiber samples are taken, stained with safranin, made into temporary slides, and placed under an optical microscope for observation to count the lengths of xylem fibers and vessels.

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

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

[0159] (2) Electron microscopy observation

[0160] Transmission electron microscopy observation:

[0161] (a) Fixation: Cut the 10th internode stem segments of the soil-cultured materials cultured for two months into lengths of about 3 mm, and then place the cut samples in a 2.5% glutaraldehyde solution for 15 min of vacuum extraction. Subsequently, the samples were fixed overnight in a 2.5% glutaraldehyde solution. After fixation, remove the fixative and rinse the samples three times with 0.1 M phosphate buffer (pH 7.0), each time for 15 min. Subsequently, the samples were secondarily fixed with a 1% osmium tetroxide solution for 1-2 h and rinsed three times again with 0.1 M phosphate buffer (pH 7.0), each time for 15 min.

[0162] (b) Dehydration: Dehydrate the samples with a series of ethanol solutions with increasing concentration gradients (30%, 50%, 70%, 80%, 90%, and 95%), each concentration for 15 min. Finally, place the samples in 100% ethanol for 20 min. Replace them into fresh 100% ethanol and treat them overnight.

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

[0164] Observation with scanning electron microscope:

[0165] The steps of fixation, dehydration, and drying were the same as those in the sample preparation process for transmission electron microscopy. The sample holder was placed in the vacuum chamber of the microscope for vacuum pumping. Subsequently, the cross-section of the stem segment was gradually scanned, and the structural characteristics of xylem cells were observed.

[0166] The scanning electron microscope measures the width of the cell wall between two cells, and the transmission electron microscope measures the thickness of the cell wall of a single cell. The results are as Figure 7 shown, where A: Observation results of xylem cells with scanning electron microscope; B: Statistical results of the cell wall thickness between two xylem fiber cells at 100 μm, 200 μm, and 300 μm away from the cambium cells based on the scanning electron microscope observation results (*p < 0.05; **p < 0.01); C: Observation results of xylem cells with transmission electron microscope (ve represents vessel, xf represents xylem fiber cell); D: Statistical results of the cell wall thickness between xylem fiber cells based on the scanning electron microscope observation results (*p < 0.05; **p < 0.01). The results show that the xylem cell walls of PagKNAT5a overexpressing transgenic plants are significantly thickened at 100 μm, 200 μm, and 300 μm away from the cambium cells compared with the control plants ( Figure 7 A, B). The observation results with transmission electron microscope were used to further confirm that the secondary cell wall thickness of the xylem in overexpressing plants increased compared with the control plants, indicating that PagKNAT5a promotes the thickening of the secondary cell wall of xylem fiber cells ( Figure 7 C, D).

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

[0168] (1) Sampling

[0169] Take the stem segments of the 10th internode of the control plants and transgenic materials grown in soil for 2 months, and make transverse sections with a thickness of 50 μm for phloroglucinol and Calcofluor White staining. Select the stem segments of the 7th - 15th internodes of the soil-grown plant materials cultured for 2 months, carefully remove the leaves and peel off the bark. Put the remaining stem materials into the oven and dry them to a constant weight at a temperature of 80 °C. Subsequently, grind the dried samples into powder and sieve them through a 30 - 50 mesh sieve to obtain a fine powder sample as the test sample for subsequent cell wall component analysis.

[0170] (2) Phloroglucinol staining

[0171] Mix HCl and 3% phloroglucinol solution at 1:2 (v / v) (prepare before use), place the transverse tissue sections in the mixed solution for staining for 5 min, then make temporary mounts, observe under a 20× optical microscope, and take pictures for preliminary analysis of the lignin content in the vascular tissue.

[0172] (3) Calcofluor White staining

[0173] Place the transverse tissue sections on a glass slide, add Calcofluor White staining solution, stain for 5 min, suck out the staining solution with deionized water, then observe under a 20× optical microscope and take pictures for preliminary analysis of the cellulose content in the vascular tissue.

[0174] (4) Determination of lignin content

[0175] In this experiment, a lignin content test kit (Mengxi Bio, M1711A) was used. The experiment was carried out according to the instructions of the kit. The absorbance of the sample was measured at a wavelength of 340 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the lignin content of the sample was calculated according to the standard curve formula.

[0176] (5) Determination of cellulose content

[0177] In this experiment, a cellulose content test kit (Mengxi Bio, M1718A) was used. The experiment was carried out according to the instructions of the kit. The absorbance of the sample was measured at a wavelength of 540 nm using an ELISA reader, and the cellulose content of the sample was calculated according to the standard curve formula.

[0178] (6) Determination of hemicellulose content

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

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

[0181] As is known by technical common sense, 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 not exclusive. 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 the characteristics of poplars, the method comprising: using genetic engineering to KNAT5a High gene expression; The variety of the poplar is 84K poplar; Said KNAT5a The protein sequence encoded by the gene is shown in SEQ ID NO.12; 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 cell 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 method comprises the following steps: S1: Put the KNAT5a The coding sequence of the gene is transferred into the expression vector to obtain the KNAT5a Recombinant vectors of genes; S2: containing the KNAT5a The recombinant vector of the gene is transformed into Agrobacterium to obtain KNAT5a Genetic recombinant Agrobacterium; S3: containing the KNAT5a 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 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 a pH of 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.

3. The method according to claim 2, 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 medium is based on liquid MS, and the activation 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 was 14-18 h light / 6-10 h dark per day.

4. Use of a biomaterial in the preparation of a formulation for poplar breeding for improving poplar traits; The variety of the poplar is 84K poplar; The improved poplar traits include: Increase the height of poplar plants; Increase the ground diameter of poplar plants; Increase internode length of poplar trees; Increase the cell 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 P3, P4, P5, P6, P7, P8 and P9: P3: RNA The RNA can be translated into KNAT5a Protein; KNAT5a The amino acid sequence of the protein is shown in SEQ ID NO.12; P4: Gene The coding sequence of the gene can encode KNAT5a Protein; KNAT5a The amino acid sequence of the protein is shown in SEQ ID NO.12; 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; and P9: Test kit The kit contains the RNA described in P3, the genetic engineering vector described in P6 or the cells described in P7.

5. The use according to claim 4, characterized in that The skeleton of the genetic engineering vector is a pMDC32 vector.

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