Application of pagmyb057 protein in regulating poplar adaptation to nitrogen deficiency

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of a PagMYB057 protein in regulating nitrogen deficiency adaptation of poplar. The amino acid sequence of the PagMYB057 protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The application proves through experiments that under low nitrogen conditions, PagMYB057 Gene overexpression can promote xylan biosynthesis, increase fiber cell wall thickness, and thus improve the adaptability of silver gland poplar to a low nitrogen environment.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of PagMYB057 protein in regulating the adaptation of poplar trees to nitrogen deficiency. Background Technology

[0002] Nitrogen (N) is one of the essential macronutrients for plant growth and development, and as a key element in plant growth and development, it plays a vital role in the process of wood formation. Poplar ( Populus Poplar (spp.) is a woody model plant with significant applications in papermaking, biocarbon sequestration, and biofuel production due to its strong reproductive capacity, rapid growth, and adaptability. However, most poplar plantations in my country are located on nitrogen-poor land, while the rapid growth of poplar trees results in a high nitrogen demand, making nitrogen supply a key factor limiting timber quality and yield. Transcription factors can alter the chemical composition and anatomical characteristics of wood by regulating the expression of key enzyme genes in cell wall biosynthesis pathways. MYB transcription factor is one such transcription factor that regulates wood formation in plants.

[0003] In poplar trees, overexpression PtrMYB2 / 3 / 20 / 21 This leads to ectopic deposition of the secondary xylem wall, and its functional inhibition results in reduced secondary wall thickening in transgenic poplar wood. Furthermore, white spruce (… Picea glauca )of PtMYB1 and PtMYB8 and eucalyptus EgMYB2 With poplar PtrMYB2 / 3 / 20 / 21 These genes are homologous, and functional studies have shown that they can also activate the entire process of secondary wall biosynthesis. Furthermore, PtrMYB18 / 74 / 75 / 128 have also been shown to activate the expression of secondary wall biosynthesis genes. On the other hand, secondary wall biosynthesis can also be regulated by repressive mechanisms. For example, PtrMYB156, PdMYB221, and PtrMYB189 negatively regulate secondary wall thickening by inhibiting the expression of secondary wall biosynthesis genes.

[0004] However, there are no reports on the role of PagMYB057 protein in regulating xylan biosynthesis to adapt to nitrogen deficiency. Summary of the Invention

[0005] To fill the gap in the existing technology, this invention provides the application of PagMYB057 protein in regulating the adaptation of poplar trees to nitrogen deficiency.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of PagMYB057 protein in regulating the biosynthesis of secondary xylem cell walls in poplar trees to adapt to nitrogen deficiency, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the PagMYB057 protein enhances the adaptability of poplar to low nitrogen by positively regulating the biosynthesis of xylan in the poplar cell wall.

[0009] Furthermore, the nucleotide sequence of the gene encoding the PagMYB057 protein is shown in SEQ ID NO.2.

[0010] Furthermore, the amplification primer sequences for the encoded gene are shown in SEQ ID NO.3-4.

[0011] Based on the same inventive concept, this invention also provides a method for constructing a transgenic poplar tree, comprising:

[0012] The step of overexpressing the PagMYB057 protein.

[0013] Furthermore, the step of overexpressing the PagMYB057 protein includes: introducing the PagMYB057 protein encoding gene or biological material containing the encoding gene into poplar trees, and further cultivating transgenic poplar trees with improved adaptability to low nitrogen.

[0014] Furthermore, the biological material includes an overexpression vector or genetically engineered bacteria containing the encoding gene.

[0015] Based on the same inventive concept, this invention also provides a method for identifying the ability of poplar trees to adapt to low nitrogen, comprising:

[0016] The expression level of the gene encoding the PagMYB057 protein in the test poplar was detected. If the expression level of the PagMYB057 protein in the test poplar was significantly higher than the average expression level of this type of poplar, then it was a poplar with high and low nitrogen adaptability.

[0017] Furthermore, the method for detecting the expression level of the encoding gene in the tested poplar includes quantitative PCR. Even further, the sequences of the primers used for quantitative PCR identification are shown in SEQ ID NO. 7-8.

[0018] Based on the same inventive concept, this invention also provides a method for improving the adaptability of poplar trees to low-nitrogen environments, comprising:

[0019] Transgenic poplar trees were constructed using the method described above, and the transgenic poplar trees were planted in low-nitrogen soil.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention has been proven through experiments. PagMYB057 Gene overexpression can significantly promote the biosynthesis of xylan in poplar cell walls, thereby enhancing their adaptation to low nitrogen. This study created a poplar variety with high adaptability to low nitrogen, providing new materials for breeding new tree varieties with strong adaptability to barren site conditions. Attached Figure Description

[0022] Figure 1 for PagMYB057 Full-length cDNA identification results, M: DL2000 marker; lane 4 is a clone using a mixed sample as a template.

[0023] Figure 2 for PagMYB057 DNA and RNA identification results of gene overexpression and silencing lines. (a) PagMYB057 DNA identification results of gene overexpression lines, (b) PagMYB057 DNA identification results of gene-silenced strains; (c) PagMYB057 Results of RNA expression level identification in gene overexpression lines; (d) PagMYB057 Results of RNA expression level identification in gene silencing lines.

[0024] Figure 3 for PagMYB057 Phenotypic and biomass of overexpressing, silenced and wild-type *Populus amurensis* plants under low nitrogen (0.4 mM KNO3) / normal nitrogen (4 mM KNO3) treatments: (a) phenotype; (b) shoot height; (c) shoot diameter; (d) shoot biomass.

[0025] Figure 4 for PagMYB057 Anatomical features and quantitative analysis results of the tenth internode of overexpressing, silencing and wild-type *Populus silvereris* under low nitrogen (0.4 mM KNO3) / normal nitrogen (4 mM KNO3) treatments: (a) typical image; (b) xylem width; (c) fiber cell diameter; (d) vessel diameter; (e) cambium cell layer number.

[0026] Figure 5 for PagMYB057 Cell wall thickness and xylose content of overexpressing, silenced and wild-type *Populus aegyptium* plants under low nitrogen (0.4 mM KNO3) / normal nitrogen (4 mM KNO3) treatments. (a) Typical image; (b) Cell wall thickness; (c) Xylose content. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0028] The following examples involve experimental materials:

[0029] 1) Plant materials

[0030] The silver-leaved poplar used in this experiment ( P. alba × P. glandulosa It is preserved by the State Key Laboratory of Forest Tree Genetics and Breeding, Chinese Academy of Forestry.

[0031] 2) Strains and vectors

[0032] 21) Carrier

[0033] Gateway technology entry vector pDNOR207, plant overexpression vector pCAMBIA2306, and plant silencing expression vector pK7GWIWG2D are all commercially available.

[0034] 3) Reagent preparation:

[0035] Poplar subculture medium (1L): MS powder 2.2 g + sucrose 30 g + agar powder 7 g, pH 5.8, autoclaved at 121 ℃ for 20 min.

[0036] Poplar co-culture medium (1L): MS powder 2.2 g + sucrose 30 g + agar powder 7 g, pH 5.8, autoclaved at 121 ℃ for 20 min, then add 0.5 mg 6-BA / L + 0.05 mg NAA / L.

[0037] Poplar selective medium (1L): MS powder 2.2 g + sucrose 20 g + agar powder 7 g, pH 5.8, autoclaved at 121 ℃ for 20 min, then add 0.5 mg 6-BA / L + 0.01 mg NAA / L, 200 mg / L termethin and 30 mg / L kanamycin.

[0038] Poplar rooting medium (1L): MS powder 2.2 g + sucrose 30 g + agar powder 7 g, pH 5.8, autoclaved at 121 ℃ for 20 min, then add 0.05 mg IBA / L + 0.01 mg NAA / L and 30 mg / L kanamycin.

[0039] The following specific examples demonstrate the function of the PagMYB057 protein in regulating the biosynthesis of the secondary xylem cell wall in poplar trees to adapt to nitrogen deficiency:

[0040] Example 1: Silver Gland Poplar PagMYB057 Gene cloning and construction of overexpression plasmids and recombinant Agrobacterium.

[0041] 1. Silver gland poplar PagMYB057 Cloning of genes

[0042] RNA was extracted from *Populus alba* tissue culture seedlings using the CTAB method, and then Primescript was used. ™ The 1st strand cDNA synthesis kit was used to reverse transcribe DNase-digested RNA to obtain cDNA, which was then used as a template. Specific primers PagMYB057-F (SEQ ID NO.3) and PagMYB057-R (SEQ ID NO.4) were designed for gene cloning. The amplified band of approximately 1000 bp was then... Figure 1 The recombinant plasmid was recovered from the gel, ligated into the pMD19-T vector, and then sequenced. The sequenced sequence was compared with the genome sequence, and the recombinant plasmid was named pM- PagMYB057 Obtained through cloning PagMYB057 The coding region of the gene is 957 bp in length (SEQ ID NO.2), encoding 318 amino acids (SEQ ID NO.1), with a molecular weight of 35.55 kDa. The sequencing-positive plasmid was named pM- PagMYB057 .

[0043] SEQ ID NO.1:MGRSPCCEKEHTNKGAWTKEEDERLINYIKSHGEGCWRSLPKAAGLLRCGKSCRLRWINYLRPDLKRGNFSDEEDELIINLHSLLGNKWSLIAARLPGRTDNEIKNYWNTHIKRKLFSRGIDPQTHRPLNSSTTSSTTSTTTNSTCNKNSNMGTKRI TNFKLEEQNYSFVQAQPEFMMSNIIKKASDSSIIKVGGSSSDSAEDSNSSSGVTAELEVYPNHKLNLELSIGLPCQSQLSSINDLNDSNQANQQHQEQAVTHQLFGTSATPTSGAPAVVHRTACLCSYNRGFKNREACSCCNAVEKFVTADSLYRFYRPLDA.

[0044] SEQ ID NO.2:。

[0045] SEQ ID NO. 3: ATGGGCAGATCTCCTTGTTG.

[0046] SEQ ID NO. 4: TCAAGCATCCAAAGGTCTGT.

[0047] 2. PagMYB057 Construction of gene overexpression plasmids and transformation of Agrobacterium

[0048] (1) with pM- PagMYB057 Using the plasmid as a template, PCR was performed using primers PagMYB057-OE-F / R (SEQ ID NO.5 / SEQ ID NO.6). The target fragment was recovered after detection by agarose gel electrophoresis.

[0049] SEQ ID NO. 5: GGGGACGAGCTCGGTACATGGGCAGATCTCCTTGTTG.

[0050] SEQ ID NO. 6: ATTGGTCGACTCTAGAGGATCCAGCATCCAAAGGTCTGT.

[0051] (2) Use Xba The pCAMBIA2306 vector was digested with an enzyme I endonuclease at a temperature of 37°C for 1 h, with a reaction volume of 10 μL.

[0052] (3) The DNA fragment obtained in step (1) was ligated to the linearized vector obtained in step (2) using homologous recombination. The ligation product was then transformed into DH5α *E. coli* using a heat shock method, and single colonies with normal growth were selected for sequencing. The positive plasmid was transformed into *Agrobacterium* to obtain... PagMYB057 Overexpression of recombinant Agrobacterium.

[0053] 3. PagMYB057 Construction and transformation of gene silencing plasmids into Agrobacterium

[0054] With pM- PagMYB057 Using plasmids as templates, primers were used. PagMYB057 PCR was performed using -207-F / R (SEQ ID NO.9 / SEQ ID NO.10), and the 151 bp fragment was recovered after detection by agarose gel electrophoresis. PagMYB057 Excerpt.

[0055] SEQ ID NO. 9: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGATTCCAATAGTAGCAGTGGC.

[0056] SEQ ID NO. 10: GGGGACCACTTTGTACAAGAAAGCTGGGTCATGCTGTTGGTTTTGCTTGG.

[0057] The recovered fragment and pDONR207 were reacted via a BP reaction to obtain pDONR207- PagMYB057 plasmid, pDONR207- PagMYB057 The plasmid and the plant expression vector pK7GWIWG2D were ligated via an LR reaction to obtain... PagMYB057- pK7GWIWG2D, PagGT43B After transforming DH5α competent E. coli cells with -pK7GWIWG2D, single colonies with normal growth were selected for sequencing. The positive plasmid was then transformed into Agrobacterium to obtain recombinant Agrobacterium.

[0058] Example 2: PagMYB057 Functional analysis of genes in *Populus alba*

[0059] 1. Experimental Methods

[0060] 1.1 PagMYB057 Genetic transformation steps for overexpression of *Populus alba*

[0061] (1) The sample obtained in Example 1 PagMYB057 Recombinant Agrobacterium was overexpressed and activated. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and cultured on a shaker at 28°C and 180 rpm until OD. 600 =0.6-0.8. Transfer 1% of the bacterial culture into freshly prepared LB liquid culture medium. Incubate under the same conditions for approximately 6 hours, OD... 600 When the concentration is 0.2-0.5, the bacterial cells are suspended in 20 mL of MS liquid medium for transformation experiments.

[0062] (2) Take leaves from healthy, rooted, sterile seedlings of *Populus alba* that are 4 weeks old, with similar size, shape, and color. Remove the leaf tip and leaf edge from each leaf, make 2-3 cuts with a scalpel, and then immerse the leaf in the bacterial solution in (1) for 15 minutes, gently shaking it during this time so that each leaf can come into contact with the bacterial solution.

[0063] (3) Transfer the explants to poplar co-culture medium and co-culture in the dark for 3 days.

[0064] (4) After the co-culture stage, the explants were transferred to a culture dish of poplar selective medium and cultured in the dark for about 6-8 weeks.

[0065] (5) After two months of cultivation, when the transformed buds have grown to about 2 cm, cut them off and inoculate them into the rooting medium for continued cultivation.

[0066] 1.2 PagMYB057 Identification and screening of overexpressing *Populus silverae* strains

[0067] Extraction using CTAB method PagMYB057 Overexpression of total DNA and RNA in leaves of wild-type Populus aurea.

[0068] PCR was performed using pCAMBIA2306-F (SEQ ID NO.11) and pCAMBIA2306-R (SEQ ID NO.12) as primers to identify... PagMYB057 Overexpressing transgenic plants. Positive control: PagMYB057 The recombinant vector plasmid was overexpressed, and the negative control was a wild-type *Populus simonii* plant.

[0069] SEQ ID NO. 11: CCATCATTGCGATAAAGGAAAG.

[0070] SEQ ID NO. 12: TGTTTCCAAGTAAGCTGTGTAG.

[0071] by PagMYB057 Using cDNA overexpressing both *Populus aegyptium* and wild-type *Populus aegyptium* as templates, PagMYB057 Quantitative PCR was performed using primers RT-F / R (SEQ ID NO.7 / SEQ ID NO.8). Identification PagMYB057 Different transgenic lines of *Populus alba* overexpressing *Philippines argyrophylla* PagMYB057 The relative expression level. The control group consisted of wild-type plants. PagActin This is an internal reference gene, and the primer sequences are shown in SEQ ID NO.13-14.

[0072] SEQ ID NO.7: GTTCAAGCCCAGCCAGAGT.

[0073] SEQ ID NO.8: CTGTCACGCCACTGCTACTAT.

[0074] SEQ ID NO. 13: CCATTGAGCACGGTATTGT.

[0075] SEQ ID NO. 14: TACGACCACTGGCATACAGG.

[0076] 1.3 PagMYB057 Silent Silver Gland Poplar Genetic Transformation Steps

[0077] (1) The sample obtained in Example 1 PagMYB057The silenced recombinant Agrobacterium was activated, and single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 mg / L kanamycin solution and 25 mg / L rifampin. The cultures were incubated at 28°C and 180 rpm on a shaker until OD (October Expiratory Time). 600 =0.6-0.8. Transfer 1% of the bacterial culture into freshly prepared LB liquid culture medium. Incubate under the same conditions for approximately 6 hours, OD... 600 When the concentration is 0.2-0.5, the bacterial cells are suspended in 20 mL of MS liquid medium for transformation experiments.

[0078] (2) Take leaves from healthy, rooted, sterile seedlings of *Populus alba* that are 4 weeks old, with similar size, shape, and color. Remove the leaf tip and leaf edge from each leaf, make 2-3 cuts with a scalpel, and then immerse the leaf in the bacterial solution in (1) for 15 minutes, gently shaking it during this time so that each leaf can come into contact with the bacterial solution.

[0079] (3) Transfer the explants to poplar co-culture medium and co-culture in the dark for 3 days.

[0080] (4) After the co-culture stage, the explants were transferred to a culture dish of poplar selective medium and cultured in the dark for about 6-8 weeks.

[0081] (5) After two months of cultivation, when the transformed buds have grown to about 2 cm, cut them off and inoculate them into the rooting medium for continued cultivation.

[0082] 1.4 PagMYB057 Identification and screening of silent silver gland poplar strains

[0083] Extraction using CTAB method PagMYB057 Total DNA and RNA from leaves of silent and wild-type *Populus silverae*.

[0084] Using leaf DNA as a template, PCR was performed using pK7GWIWG2D-F (SEQ ID NO.15) and pK7GWIWG2D-R (SEQ ID NO.16) as primers to identify... PagMYB057 Silent transgenic plants. Positive control: PagMYB057 The recombinant vector plasmid was silenced, and the negative control was a wild-type Populus spp.

[0085] SEQ ID NO. 15: TGAGACTTTTCAACAAAGGGTA.

[0086] SEQ ID NO. 16: ATGCTGTTGGTTTGCTTGGTTT.

[0087] by PagMYB057 Using cDNA from silent and wild-type *Populus simonii* as templates, PagMYB057 Quantitative PCR was performed using primers -RT-F / R (SEQ ID NO.7 / SEQ ID NO.8). Identification PagMYB057 Silent Silver Gland Poplar Different Transgenic Lines PagMYB057 The relative expression level. The control group consisted of wild-type plants. PagActin This is an internal reference gene, and the primer sequences are shown in SEQ ID NO.13-14.

[0088] 1.5 Treatment with Salicylic Acid

[0089] The next generation of wild-type silver gland poplar, PagMYB057 overexpression and PagMYB057 Silent transgenic *Populus silverae* was cultured in a culture room for 4-6 weeks. Plants with similar growth were selected and cultured in sand in nitrogen-deficient rice-based nutrient solution (supplemented with 4 mM KNO3). After two months of sand culture, wild-type *Populus silverae* and three... PagMYB057 Overexpression of *Populus alba* strains and three PagMYB057 The silent silver-glanded poplar strains were divided into two groups, each containing nine silver-glanded plants of each genotype. The silver-glanded poplars in each group were treated with 0.4 or 4 mM KNO3 for eight weeks.

[0090] 1.6 Sample Harvesting

[0091] Before nitrogen treatment, the plant tips were marked with a marker. After treatment, young leaves (the part formed after nitrogen treatment), mature leaves, bark, wood, and root tissue were collected. The harvested samples were immediately frozen in liquid nitrogen. The frozen samples were ground into a fine powder using a ball mill and stored at -80°C for further analysis.

[0092] 1.7 Histochemical staining

[0093] Wild-type silver poplar, PagMYB057 overexpression and PagMYB057 The tenth internode of the silent transgenic *Populus simonii* was cut into 1 cm long stem segments. The stem segments were immediately immersed in 4% paraformaldehyde solution for fixation, and vacuumed for 20 min, repeated twice until the material settled to the bottom of the tube. One end of the fixed stem segment was fixed to the sample holder of a vibratory microtome (LEICA VT1000) with 502 glue. The section thickness was set to 50 μm, and cross-sections of the stem segments were prepared. The sections were stained with 0.1% toluidine blue solution for 30 s. The sections were washed 2-3 times with sterile water to remove excess staining solution. The stained sections were placed on glass slides and observed and photographed using an Olympus BX51 microscope for preservation.

[0094] 1.8 Analysis of Wood Anatomical Characteristics

[0095] The anatomical features of the microscopic images were analyzed using the image analysis software ImageJ. The main anatomical parameters of the wood measured included: xylem width, the diameter of the lumen of vessel units and fiber cells in the direction perpendicular to the wood rays, and the thickness of the double-layered fiber cell wall.

[0096] 1.9 Scanning electron microscopy observation

[0097] Wild-type Populus spp. with different nitrogen concentrations were selected. PagMYB057 overexpression and PagMYB057 Stem sections from the tenth internode of the silent transgenic *Populus simonii* were prepared to a thickness of approximately 2 mm. The sections were fixed in 4% paraformaldehyde solution and subjected to vacuum degassing (30 min / time, repeated 2-3 times) until the material completely settled. Subsequently, the fixed samples were rinsed three times with 0.1 mol / L phosphate buffer (pH = 7.2), followed by gradient ethanol dehydration. The dehydration process used ethanol concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100%, with each stage lasting 20 min. After dehydration, the samples were dried using an automated critical point desiccator. The dried samples were then sputter-coated with gold to enhance conductivity. Finally, the cell walls of the samples were observed and images were acquired using a Regulus 8230 scanning electron microscope.

[0098] 1.10 Determination of the content of major components in wood

[0099] First, the alcohol-insoluble matter (AIR) was prepared: Transgenic lines and wild-type materials were ground into powder under liquid nitrogen conditions. A suitable amount of sample was freeze-dried, and then 3-4 times its volume of 80% ethanol solution was added. The mixture was then treated in a water bath at 80°C for 1 h, followed by centrifugation at 4000 g for 5 min to collect the precipitate. The precipitate was then sequentially treated with a chloroform-methanol mixture (2:1, v / v) at 37°C for 2 h (repeated twice), and then with 65%, 80%, and 100% ethanol solutions at 37°C for 30 min each (repeated twice). After each treatment, the precipitate was collected by centrifugation at 4000 g for 5 min. Finally, the precipitate was treated with 100% acetone at 37°C for 2 h (repeated twice). The centrifuged precipitate was then vacuum-dried at 70°C to obtain the final AIR sample.

[0100] The monosaccharide composition of hemicellulose was analyzed using acid hydrolysis-ion chromatography. 2 mg of AIR sample was accurately weighed and added to 0.5 mL of 2M TFA solution. Hydrolysis was carried out at 120℃ for 120 min, followed by cooling and centrifugation at 12,000 rpm for 5 min. An equal volume of 2M TFA and anhydrous ethanol was added to the supernatant, and the mixture was dried under vacuum at 50-100℃. After drying, 2 mL of anhydrous ethanol was added, and the mixture was dried again under vacuum. Finally, the sample was dissolved in 5 mL of distilled water, filtered through a 0.22 μM filter membrane, and analyzed by ion chromatography to determine the content of each monosaccharide component in hemicellulose.

[0101] 2. Experimental Results

[0102] 2.1 PagMYB057 Identification of overexpression and silencing strains of *Populus alba*

[0103] like Figure 2 As shown, a total of 12 were identified. PagMYB057 Overexpression positive lines ( Figure 2 a) and 8 PagMYB057 Silent positive strains ( Figure 2 b). Through expression level identification, three genes with high expression levels were identified. PagMYB057 Overexpression lines, namely PagMYB057 -OE9、 PagMYB057 -OE11 and PagMYB057 -OE12 ( Figure 2 c) and 3 with lower expression levels PagMYB057 Silent strains, namely PagMYB057 -RNAi2, PagMYB057 -RNAi5 and PagMYB057 -RNAi8 ( Figure 2 d). The selected strains were subcultured into antibiotic-free MS solid medium for propagation.

[0104] Select 3 samples with high expression levels after screening. PagMYB057 Overexpression lines ( PagMYB057 -OE9、 PagMYB057 -OE11 and PagMYB057 -OE12), 3 PagMYB057 Silent strains ( PagMYB057 -RNAi2, PagMYB057 -RNAi5 and PagMYB057 After hardening off, seedlings of Populus alba var. mongolica (-RNAi8) and wild-type Populus alba var. mongolica (WT) were transferred to a nitrogen-deficient rice nutrient solution supplemented with 4 mM KNO3 for sand culture.

[0105] 2.2 Low nitrogen pair PagMYB057 Effects of overexpression and silencing on poplar phenotype and biomass

[0106] Depend on Figure 3It can be seen that under normal nitrogen conditions, PagMYB057 The height of newly formed plants in the overexpression lines was 19.4%-23.1% lower than that of the wild type; however, under low nitrogen conditions, PagMYB057 The height of newly formed plants in the overexpression lines was significantly increased by 25.8%-32.5% compared to the wild type. Figure 3 a, Figure 3 b). Further research PagMYB057 Statistical analysis was performed on the stem diameters of transgenic lines at marked locations during nitrogen treatment. Under normal nitrogen conditions, PagMYB057 The stem diameter of the overexpression lines was reduced by 13.2%-16.0% compared to the wild type; however, under low nitrogen conditions, PagMYB057 The stem diameter of the overexpression lines was significantly increased by 12.7%-15.0% compared to the wild type. Figure 3 c). Furthermore, statistical analysis of the dry weight of the newly formed portion after nitrogen treatment showed that, under normal nitrogen conditions, PagMYB057 The dry weight of the overexpression lines was reduced by 9.9%-17.5% compared to the wild type; under low nitrogen conditions, PagMYB057 The aboveground dry weight of the overexpressing strains increased by 47.8%-58.9% compared to the wild type. Figure 3 d). Regardless of whether the nitrogen level is normal or low, PagMYB057 The plant height, stem diameter, and dry weight of new shoots of the silent strains were not significantly different from those of the wild type. Figure 3 b- Figure 3 d).

[0107] 2.3 Low nitrogen pair PagMYB057 Effects of overexpression and silencing of poplar anatomical features

[0108] Depend on Figure 4 It can be seen that, under normal nitrogen conditions, compared with the wild type, PagMYB057 The xylem width of the overexpressing lines was significantly reduced by 16.9%-17.2%, while PagMYB057 The xylem width of the silent lines increased by 13.9%-15.8%; however, under low nitrogen conditions, the xylem width of each transgenic line was not significantly different from that of the wild type. Figure 4 a, Figure 4 b). Under normal nitrogen conditions, compared to the wild type, PagMYB057 The overexpression lines showed an increase in fibroblast diameter of 4.6%-5.0% and an increase in vascular unit diameter of 8.0%-1.2%; while PagMYB057 The diameter of fibroblasts in the silent strains decreased by 7.1%-7.8%, while the diameter of vessel units showed no significant change. Figure 4 c, Figure 4 d). Under low nitrogen conditions, compared to the wild type, PagMYB057 The overexpression lines showed a 4.2%-7.1% decrease in fibroblast diameter and a 9.5%-11.3% increase in vascular unit diameter, while... PagMYB057 The diameters of fiber and vascular units in the silent strains were not significantly different from those in the wild type. Figure 4 c, Figure 4 d). Under low nitrogen conditions, compared to the wild type, PagMYB057 Overexpression lines and PagMYB057 There was no significant difference in the number of cambium cell layers among the silent strains. Figure 4 e).

[0109] 2.4 Low nitrogen pair PagMYB057 Effects of overexpression and silencing on cell wall thickness and xylose content in poplar fibers

[0110] like Figure 5 As shown, under normal nitrogen conditions, PagMYB057 The overexpression lines exhibited a 11.2%-14.0% reduction in bilayer fibrous cell wall thickness compared to the wild type; however, under low nitrogen conditions, the bilayer fibrous cell wall thickness increased by 7.8%-8.2% compared to the wild type. In contrast, PagMYB057 The thickness of fibroblast cells in the silent strains showed no significant difference compared to the wild type under different nitrogen levels. Figure 5 a, Figure 5 b).

[0111] Under normal nitrogen conditions PagMYB057 The xylose content of the overexpression lines was significantly increased by 18.7%-26.1% compared to the wild type, while PagMYB057 Silent lines showed no significant changes; under low nitrogen conditions, PagMYB057 The xylose content of the overexpression lines increased by 14.9%-18.8% compared to the wild type, while PagMYB057 The xylose content of the silent strains was significantly lower than that of the wild type by 7.5%-11.1%. Figure 5 c).

[0112] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0113] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of PagMYB057 protein in regulating the biosynthesis of secondary xylem cell walls in poplar trees to adapt to nitrogen deficiency, characterized in that... The amino acid sequence of the PagMYB057 protein is shown in SEQ ID NO.

1. The PagMYB057 protein improves the adaptability of poplar to low nitrogen by positively regulating the biosynthesis of poplar xylan.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the PagMYB057 protein is shown in SEQ ID NO.

2.

3. A method for constructing a transgenic poplar tree, characterized in that, include: The step of overexpressing the PagMYB057 protein as described in claim 1.

4. The method according to claim 3, characterized in that, The steps for overexpressing the PagMYB057 protein include: The gene encoding the PagMYB057 protein or biological material containing the gene is introduced into poplar trees to further cultivate transgenic poplar trees with improved adaptability to low nitrogen.

5. The method according to claim 4, characterized in that, The biological material includes an overexpression vector or genetically engineered bacteria containing the encoding gene.

6. A method for identifying the ability of poplar trees to adapt to low nitrogen, characterized in that, include: The expression level of the gene encoding the PagMYB057 protein described in claim 1 is detected in the test poplar. If the expression level of the gene encoding the PagMYB057 protein in the test poplar is significantly higher than the average expression value of this type of poplar, then it is a poplar with strong adaptability to low nitrogen. If the expression level of the gene encoding the PagMYB057 protein in the tested poplar is significantly lower than the average expression level of this type of poplar, then it is a poplar with weak adaptability to low nitrogen.

7. The method according to claim 6, characterized in that, The method for detecting the expression level of the coding gene in the test poplar is quantitative PCR, and the sequences of the primers used for identification by the quantitative PCR method are shown in SEQ ID NO.7-8.

8. A method for improving the adaptability of poplar trees to low-nitrogen environments, characterized in that, include: Transgenic poplar trees were constructed using the method described in claim 3, and the transgenic poplar trees were planted in low-nitrogen soil.

Images