Application of PagKIN10 gene in regulation and control of growth and development of poplar

Abstract

The invention discloses application of a PagKIN10 gene in regulation and control of growth and development of poplars, belongs to the technical field of genetic engineering, and provides application of the PagKIN10 gene in regulation and control of growth and development of the poplars, and the nucleotide sequence of the PagKIN10 gene is shown as SEQ ID NO.1. The invention further discloses application of the PagKIN10 gene in regulation and control of growth and development of the poplars. According to the application of the PagKIN10 gene in improving the saccharification efficiency of the poplar, the saccharification efficiency of the poplar is improved by overexpressing the PagKIN10 gene. Through overexpression of the PagKIN10 gene, rapid growth of the poplar can be promoted, the cell wall structure of the poplar can be optimized, and the saccharification efficiency is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of PagKIN10 gene in regulating the growth and development of poplar. Background Art

[0002] Poplar is one of the most widely distributed and fastest growing deciduous trees in the world, and is widely used in wood production, pulp manufacturing, and biomass energy development. Its rapid growth, high wood yield, and strong adaptability make it an important economic tree. As the global demand for renewable energy increases, poplar has attracted much attention as a potential bioenergy crop due to its efficient photosynthesis ability and high biomass accumulation.

[0003] Biomass saccharification refers to the process of converting lignocellulosic biomass into fermentable sugars by enzymatic or chemical methods. This process plays a key role in biofuel production and bio-based chemical synthesis. However, the saccharification efficiency of lignocellulosic materials such as poplar is limited by their complex cell wall structure, especially the tight cross-linking between cellulose, hemicellulose and lignin, which makes the enzymatic efficiency low, thereby increasing the cost of biomass conversion.

[0004] In recent years, genetic engineering technology has provided new solutions for improving the growth and development of poplars and the efficiency of biomass utilization. Studies have shown that certain key genes can significantly affect the growth and development characteristics of poplars, such as plant height, stem thickness, and branch angle. For example, regulating genes related to the cytokinin signaling pathway can promote apical dominance and reduce the number of branches, so that more energy is concentrated on trunk growth and wood yield is increased. In addition, by regulating genes related to secondary metabolism, the xylem composition of poplars can also be changed to optimize their quality as biomass raw materials. Gene regulation can not only affect the growth and development of poplars, but also change its cell wall composition at the molecular level, thereby improving saccharification efficiency. Specifically, lignin is one of the main factors that hinder enzymatic hydrolysis. By inhibiting genes related to lignin synthesis (such as C4H, COMT, etc.), the deposition of lignin can be reduced and the accessibility of cellulose and hemicellulose can be enhanced. The expression of certain genes can affect the crystalline structure of cellulose, making it easier to be enzymatically hydrolyzed. By regulating genes related to hemicellulose synthesis (such as XTH, GT43, etc.), the molecular structure of hemicellulose can be changed to improve its compatibility with enzymes.

[0005] Although gene regulation technology has made significant progress in poplar improvement, there are still some urgent problems to be solved. The growth, development and saccharification efficiency of poplar are regulated by multiple genes. How to ensure that the quality of wood and other economic values ​​are not affected while improving the saccharification efficiency is also a problem that needs to be weighed. Summary of the invention

[0006] To solve the above technical problems, the present invention proposes the application of the PagKIN10 gene in regulating the growth and development of poplar. Overexpression of the PagKIN10 gene can enhance the growth and development rate of poplar, achieving a higher plant height and thicker stem in a shorter time. It can not only promote the rapid growth of poplar, but also optimize its cell wall structure and significantly improve the saccharification efficiency.

[0007] To achieve the above object, the present invention provides the application of the PagKIN10 gene in regulating the growth and development of poplar, and the nucleotide sequence of the PagKIN10 gene is shown as SEQ ID NO.1.

[0008] Preferably, overexpression of the PagKIN10 gene promotes the growth and development of poplar.

[0009] The present invention also provides a vector containing the PagKIN10 gene.

[0010] The present invention also provides the application of the vector in regulating the growth and development of poplar. The vector promotes the growth and development of poplar by overexpressing the PagKIN10 gene.

[0011] The present invention also provides the application of the PagKIN10 gene in regulating the plant height of poplar. Overexpression of the PagKIN10 gene increases the growth rate of poplar plant height.

[0012] The present invention also provides the application of the PagKIN10 gene in regulating xylem differentiation of poplar. Overexpression of the PagKIN10 gene promotes the widening of the xylem region of poplar, thereby promoting xylem differentiation of poplar.

[0013] The present invention also provides the application of the PagKIN10 gene in regulating the activity of the cambium of poplar. Overexpression of the PagKIN10 gene promotes an increase in the number of cambium cell layers of poplar and improves the activity of the cambium of poplar.

[0014] The present invention also provides the application of the PagKIN10 gene in regulating the cellulose content of poplar. Overexpression of the PagKIN10 gene increases the cellulose content in poplar.

[0015] The present invention also provides the application of the PagKIN10 gene in enhancing the saccharification efficiency of poplar. Overexpression of the PagKIN10 gene enhances the saccharification efficiency of poplar.

[0016] The present invention also provides a method for enhancing the saccharification efficiency of poplar stems. Overexpression of the PagKIN10 gene enhances the saccharification efficiency of poplar stems.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] The present invention has found that overexpression of the PagKIN10 gene can promote the growth and development of poplar, help poplar better adapt to various environments, and can make poplar reach a higher plant height in a shorter time by overexpressing the PagKIN10 gene.

[0019] The present invention promotes the widening of the xylem region, increases the number of cambium cell layers, and affects the thickness and arrangement of the xylem cell wall by overexpressing the PagKIN10 gene.

[0020] The present invention promotes the growth and development of poplar and improves the saccharification efficiency by overexpressing the PagKIN10 gene. Through precise regulation of the gene, it can not only promote the rapid growth of poplar, but also optimize its cell wall structure and significantly improve the saccharification efficiency. This will provide technical support for the large-scale application of poplar as a bioenergy crop and promote the development of sustainable forestry and clean energy industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 It is the positive identification result of the 3HA tag primer fragment. Among them, A is the first part of the identification result, B is the second part of the identification result, C is the third part of the identification result, and the labels 1 to 48 in the figure represent 48 positive seedlings;

[0023] Figure 2 It is the result of fluorescence quantitative PCR analysis. In the figure, WT represents the wild-type plant, and OE represents the transgenic plant;

[0024] Figure 3 It is the phenotypic comparison diagram of wild-type plants and transgenic plants at the same growth stage. In the figure, WT represents the wild-type plant, and KIN10-OE-24 and KIN10-OE-7 represent the transgenic plants;

[0025] Figure 4 It is the statistical analysis diagram of the plant height of wild-type plants and transgenic plants at the same growth stage. In the figure, WT represents the wild-type plant, and KIN10-OE-24 and KIN10-OE-7 represent the transgenic plants, and "*" represents significant difference;

[0026] Figure 5Cross-section diagrams of wild-type plants and transgenic plants at the same growth stage. Among them, A shows the xylem width of wild-type plants, where the red xy in the figure represents the xylem width; B shows the xylem width of transgenic plant KIN10-OE-24, where the red xy in the figure represents the xylem width; C shows the xylem width of transgenic plant KIN10-OE-7, where the red xy in the figure represents the xylem width; D shows the number of cambium cell layers of wild-type plants; E shows the number of cambium cell layers of transgenic plant KIN10-OE-24; F shows the number of cambium cell layers of transgenic plant KIN10-OE-7;

[0027] Figure 6 Statistical analysis diagrams of the xylem region width and the number of cambium cell layers of wild-type plants and transgenic plants at the same growth stage. Among them, A shows the xylem region width, B shows the number of cambium cell layers. In the figure, WT represents wild-type plants, KIN10-OE-24 and KIN10-OE-7 represent transgenic plants, and "*" represents significant differences;

[0028] Figure 7 Statistical analysis diagram of the cellulose content of wild-type plants and transgenic plants at the same growth stage. In the figure, WT represents wild-type plants, KIN10-OE-24 and KIN10-OE-7 represent transgenic plants, and "*" represents significant differences;

[0029] Figure 8 Statistical analysis diagram of the lignin content of wild-type plants and transgenic plants at the same growth stage. In the figure, WT represents wild-type plants, KIN10-OE-24 and KIN10-OE-7 represent transgenic plants, and "*" represents significant differences;

[0030] Figure 9 Standard curve obtained from the determination of glucose standard solution;

[0031] Figure 10 Statistical graph of the saccharification efficiency of wild-type plants and transgenic plants. In the figure, WT represents wild-type plants, KIN10-OE-24 and KIN10-OE-7 represent transgenic plants, and "*" represents significant differences. Detailed implementation manners

[0032] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0033] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0037] Example 1

[0038] I. Construction of a poplar genetic transformation system.

[0039] 1. Construction of the expression vector.

[0040] (1) Take the stems of 3-month-old '84k' poplar for gene cloning and isolation;

[0041] (2) Extract poplar stem RNA using an RNA extraction kit (Shandong Sikejie Biotechnology Co., Ltd.);

[0042] (3) Reverse transcribe the extracted poplar stem RNA into cDNA using a reverse transcription kit (Cuisheng Biotechnology (Shanghai) Co., Ltd.);

[0043] (4) The PagKIN10 gene (SEQ ID NO.1) was amplified by the reagent 2x Taq Master Mix (Nanjing Novoprotein Scientific Inc.) using the PCR method. The DNA sequence of the PagKIN10 gene was retrieved from the phytozome website with the gene ID Potri.004G115900;

[0044] (5) The pCAMBIA1300 vector was digested with the "BamHⅠ" restriction endonuclease (New England Biolabs (Beijing) Co., Ltd.);

[0045] (6) By the homologous recombination method, using the One Step Cloning Kit (Nanjing Novoprotein Scientific Inc.) to religate the amplified PagKIN10 fragment to the BamHⅠ digestion site of the pCAMBIA1300 vector;

[0046] (7) It was transformed into Agrobacterium rhizogenes EHA105 (Beijing Tsingke Biotechnology Co., Ltd.) by experimental methods such as ice bath and heat shock. After correct identification and sequencing of the bacterial solution, the Agrobacterium was mixed with 50% glycerol and stored at -80°C for subsequent poplar infection.

[0047]

[0048] The specific primers for the PagKIN10 gene fragment are: forward primer: 5’-TAACATGTCGACACGTGGATCCATGGATGGGTCATCTT-3’ (SEQ ID NO.2);

[0049] reverse primer: 5’-AGCCTGCGGCCGCGCCGGATCCCAGGACCCGGAGCTG-3’ (SEQ ID NO.3).

[0050] 2. Genetic transformation of poplar leaves.

[0051] (1) Take out the preserved bacterial liquid from the -80°C refrigerator and incubate it in 1 mL of liquid LB medium containing "kanamycin and rifampicin" resistance at 28°C and 220 rpm for 24 h to activate the strain. Then, take a certain amount of the bacterial liquid and add it to 50 mL of liquid LB medium containing the corresponding resistance and culture it until the OD 600 is 0.6 - 0.8;

[0052] (2) Centrifuge at 5000 rpm for 10 min to collect the bacterial cells, and culture the bacterial cells in the co-culture liquid medium containing 100 μM / L acetosyringone (AS) at 28°C and 220 rpm in an incubator until the OD 600 is 0.3 - 0.4 to obtain the infection solution;

[0053] (3) Take the 2nd - 5th young leaves at the upper part of the sterile seedlings, make several cuts on the main vein with a sterile scalpel, put them into the infection solution and soak for 7 min. Place the leaves on sterile filter paper to absorb the moisture, and lay them flat on the co-culture solid medium (WPM (purchased from PhytoTech Labs) 2.3 g, MES 0.5 g, sucrose 20 g, agar 8 g, AS (concentration 1 g / 50 mL) 1 mL, water 1 L), and conduct dark treatment for 2 d;

[0054] (4) Transfer the leaves to the induction callus medium (WPM 2.3 g, MES 0.5 g, sucrose 20 g, agar 8 g, kinetin KT (concentration 1 mg / mL) 0.5 mL, 2,4-D (concentration 1 mg / mL) 1 mL, water 1 L) and conduct dark culture;

[0055] (5) When the callus grows to the size of rice grains, cut it off with a sterile scalpel and transfer it to the screening and differentiation medium for inducing buds (WPM 2.3 g, MES 0.5 g, sucrose 20 g, agar 8 g, 2,4-dichlorobenzoic acid TDZ (concentration 1 mg / mL) 20 μL, water 1 L) for culture;

[0056] (6) After the callus grows buds, cut the buds and insert them into the rooting medium (1 / 2MS (purchased from Sangon Biotech Co., Ltd.) 2.37 g, MES 0.5 g, sucrose 20 g, agar 8 g, water 1 L) for cultivation.

[0057] II. Identification of positive plants.

[0058] (1) Take the genetically transformed seedlings that have grown in the rooting medium for 1 month, and extract the DNA of poplar leaves using the CTAB method;

[0059] (2) Conduct positive identification on poplar leaves through the 3HA tag primer fragment, and the obtained results are as Figure 1 shown in A, Figure 1 shown in B, and Figure 1 shown in C, and 48 positive seedlings are obtained.

[0060] 3HA tag primer sequence: Forward primer: 5’-AACGAATCTCAAGCAATCAAG-3’ (SEQ ID NO.4); Reverse primer: 5’-AGACAAGTTGGTAATGGTAGC-3’ (SEQ ID NO.5).

[0061] III. Gene transcription identification.

[0062] (1) Extract RNA from the leaves of wild-type and positive seedlings using an RNA extraction kit (Shandong Sikejie Biotechnology Co., Ltd.);

[0063] (2) Reverse transcribe it into cDNA using a reverse transcription kit (Cuisheng Biotechnology (Shanghai) Co., Ltd.);

[0064] (3) Use fluorescence quantitative PCR analysis, repeat each gene three times, use the PagUBQ gene as an internal reference, and the obtained results are as Figure 2 shown.

[0065] (4) Through fluorescence quantitative PCR analysis, select the two lines OE-24 and OE-7 with the highest expression levels.

[0066] Specific primers for PagUBQ fluorescence quantitative PCR analysis are: Forward primer: 5’-AGACCTACACCAAGCCCAAGAAGAT-3’ (SEQ ID NO.6); Reverse primer: 5’-CCAGCACCGCACTCAGCATTAG-3’ (SEQ ID NO.7).

[0067] The specific primers for PagKIN10 fluorescence quantitative PCR analysis are: forward primer: 5’-CCATCAAGATACTTAACCGCCGT-3’ (SEQ ID NO.8); reverse primer: 5’-CATAAAGTCGTATAATGTGAGGATGC-3’ (SEQ ID NO.9).

[0068] IV. Poplar phenotypic identification.

[0069] Transfer the wild-type ‘84k’ poplar and PagKIN10-OE transgenic positive seedlings from the rooting medium to the greenhouse (photoperiod 16h / 8h, light intensity 80 μmol / m 2 / s, temperature 24 - 26 °C, humidity 70%) and culture for 90 days. Measure the plant height of the poplars in the greenhouse every two weeks.

[0070] The results are as Figure 3 and Figure 4 shown. Statistical analysis found that the plant height of the transgenic line OE-24 was slightly increased compared to the wild type, and the plant height of the transgenic line OE-7 was significantly increased compared to the wild type.

[0071] V. Cambium and xylem development identification.

[0072] 1. Vibration section observation:

[0073] (1) Take the stem segment of the 10th internode of poplar (counting from the first leaf towards the root except for the apical bud) as the experimental material, fix it on the placement table of the vibration slicer, and perform vibration slicing;

[0074] (2) Carefully pick out the cut sections (section thickness is 30 μm) with forceps, and then they can be stained;

[0075] (3) Stain the section material with 0.03% concentration of toluidine blue (TBO). After 10 minutes, rinse with water, and then it can be observed and photographed with a ZEISS microscope.

[0076] The results are as Figure 5 and Figure 6 shown. As Figure 5 shown, in the PagKIN10-OE transgenic plants (OE-24 and OE-7), the xylem width thickens, and the number of cambium cell layers in the 10th internode stem increases. After further statistical analysis as Figure 6 shown, Figure 6 A in it shows that the xylem width of the PagKIN10-OE transgenic plants (OE-24 and OE-7) increases significantly, and the xylem of OE-24 and OE-7 thickens by 26% and 28% respectively; Figure 6In B, it shows that the number of cambial cell layers in PagKIN10-OE transgenic plants (OE-24 and OE-7) increases by about 1 to 1.5 layers.

[0077] In summary, the PagKIN10 gene enhances the cambial activity of poplar and promotes the differentiation of xylem.

[0078] VI. Identification of cell wall components.

[0079] The changes in the cell wall components of the stems of PagKIN10-OE plants and wild-type plants were measured.

[0080] 1. AIR extraction.

[0081] (1) Select wild-type '84k' poplar and PagKIN10-OE transgenic positive seedlings grown in the greenhouse for 90 days, and take the basal stems as experimental materials;

[0082] (2) Put the taken materials into liquid nitrogen for quick freezing, grind them into powder using a tissue grinder, and put them into centrifuge tubes for AIR extraction;

[0083] (3) Place 10 mL of the powder sample in a 50 mL centrifuge tube, add 80% ethanol to 10 mL, centrifuge at 12000 rpm for 10 min, and gently suck out the upper liquid with a pipette after centrifugation, avoiding sucking the precipitate;

[0084] (4) Add 10 mL of 80% ethanol and 10 mL of absolute ethanol to the above precipitate twice, and repeat the above operation;

[0085] (5) Add 10 mL of chloroform:methanol = 1:1 mixture to the above precipitate, heat in a water bath at 37°C for 40 min, centrifuge at 12000 rpm for 10 min, and gently shake the liquid up and down, repeat once;

[0086] (6) Place it in a fume hood to dry the precipitate;

[0087] (7) After drying in a ventilated place, a pure AIR sample can be obtained.

[0088] 2. Determination of cellulose content.

[0089] The cellulose content of PagKIN10-OE and wild-type plants was determined using a cellulose (CLL) content kit (Suzhou Keming Biotechnology Co., Ltd.), and the operation steps are as follows:

[0090] (1) Weigh 10 mg of the above self-made AIR sample into an EP tube, add 1 mL of reagent one to the tube, mix well, and heat in a water bath at 90°C for 30 min;

[0091] (2) Take out 8000 g after cooling, centrifuge at 25 °C for 10 min, and discard the supernatant;

[0092] (3) Wash the precipitate three times with distilled water (add 1 mL of distilled water, mix well, vortex for 2 min, centrifuge at 8000 g and 25 °C for 10 min, and discard the supernatant);

[0093] (4) Add 1 mL of acetone to the precipitate, mix well, centrifuge at 8000 g and 25 °C for 10 min, discard the supernatant, and dry the precipitate for later use;

[0094] (5) Add 0.5 mL of distilled water to the dried precipitate above, place it in an ice-water bath, slowly add 0.75 mL of concentrated sulfuric acid, mix well, let it stand in the ice-water bath for 30 min, centrifuge at 8000 g and 4 °C for 10 min, take the supernatant, dilute it 20 times with distilled water, and wait for determination;

[0095] (6) Preparation of working solution: Add 4 mL of reagent three to reagent two and dissolve it completely;

[0096] (7) Add 300 μL of distilled water, 70 μL of working solution, and 630 μL of concentrated sulfuric acid to the blank tube in sequence, add 300 μL of sample, 70 μL of working solution, and 630 μL of concentrated sulfuric acid to the measurement tube in sequence, mix well, place it in a 95 °C water bath for 10 min, cool to room temperature, and read the absorbance values of the blank tube and the measurement tube at 620 nm respectively. ΔA = A measurement tube - A blank tube;

[0097] (8) Calculate according to the sample mass: Cellulose (mg / g dry weight) = [(ΔA + 0.0043) ÷ 7.875 × V1] ÷ (W × V1 ÷ V2) × 20 = 3.17 × (ΔA + 0.0043) ÷ W, where V1 is the volume of the added sample, 0.3 mL; V2 is the volume of the added extraction solution, 1.25 mL; W is the sample dry weight, 0.01 g; the sample dilution factor is 20.

[0098] The results are as Figure 7 shown. Statistical analysis found that the cellulose content of the transgenic line OE-24 and the cellulose content of the transgenic line OE-7 were significantly increased compared with the wild type WT.

[0099] 3. Determination of lignin content.

[0100] Determine the lignin content of the wild type and PagKIN10-OE plants using a lignin content detection kit (BIXBIO). The operation steps are as follows:

[0101] (1) Weigh 3 mg of the above AIR sample into a 10 mL quartz tube (one extra empty tube is used as a control);

[0102] (2) Slowly add 250 μL of reagent one and 10 μL of perchloric acid along the tube wall;

[0103] (3) After sealing, mix well thoroughly, incubate in a water bath at 80 °C for 40 min for acetylation, and mix gently every 10 min. After the reaction is completed, cool naturally to room temperature;

[0104] (4) Add 250 μL of Reagent II to the cooled reaction sample, mix well thoroughly, let stand at room temperature for a while, and then take 10 μL of the supernatant;

[0105] (5) Transfer the supernatant and 490 μL of glacial acetic acid to a 2 mL centrifuge tube and mix well;

[0106] (6) Pipette 200 μL of the reaction solution into a micro quartz enzyme-linked immunosorbent assay (ELISA) plate and measure the absorbance at 280 nm;

[0107] (7) Calculation formula: Lignin content (mg / g) = ΔA × (V supernatant + V glacial acetic acid) × V acetylation / ε × d2 × V supernatant × W = 1.092W × ΔA / W; Percentage of lignin content (%) = Lignin content × 100% / 1000 = 0.1092 × ΔA / W, where ΔA = A measurement tube - A blank tube; V supernatant: Volume of the supernatant, 0.01 mL; V glacial acetic acid: Volume of glacial acetic acid added to the reaction system, 0.49 mL; V acetylation: Volume of the acetylation reaction, 0.51 mL; ε: Lignin extinction coefficient, 23.35 mL / mg / cm; d2: Optical path of the 96-well UV plate, 1 cm; W: Sample mass, g.

[0108] The results are as Figure 8 shown. Statistical analysis found that the lignin content of the transgenic line OE-24 increased compared to the wild type WT.

[0109] 4. Alkaline treatment saccharification efficiency reaction.

[0110] (1) Weigh 20 mg of the prepared AIR sample and transfer it to a 15 mL glass tube;

[0111] (2) Add 1.8 mL of 1% NaOH, incubate in a water bath at 30 °C for 30 min, and sterilize at 121 °C for 1 h;

[0112] (3) Centrifuge the glass tube at 5000 g for 15 min, take the supernatant and put it into a new 15 mL test tube, add 2 mL of 2.5 N HCl to neutralize the precipitate, and centrifuge to take the supernatant and combine it into the 15 mL test tube;

[0113] (4) Centrifuge the above test tube at 5000 g, and take the supernatant, which is the sugar solution pretreated with alkali and regarded as alkali one, and store it in a -20 °C refrigerator;

[0114] (5) Resuspend the precipitate with ultrapure water, transfer it to a new 15 mL test tube, continue to add water to the old glass tube until all the precipitate is transferred to the new 15 mL test tube, centrifuge to remove the supernatant, and then wash the residue with citrate solution three times until the pH is 4.8;

[0115] (6) Centrifuge the test tube to remove the supernatant. Add 2 mL of enzyme digestion solution to the 15 mL test tube and mix well (enzyme digestion solution formula: 1.76 mL of 0.1 M sodium citrate buffer with pH 4.8 + 0.04 mL of 2% NaN3 + 0.2 mL of cellulase);

[0116] (7) After mixing well, incubate at 50 °C and 100 rpm for 72 h, then centrifuge at 5000 g for 10 min. Take the supernatant and the obtained alkali-pretreated enzyme hydrolysis released sugar solution as alkali two, and store it in a -20 °C refrigerator.

[0117] 5. Determine the saccharification efficiency by the phenol-sulfuric acid method.

[0118] (1) Prepare a glucose standard curve: Weigh 10 mg of glucose standard sample and dissolve it in 10 mL of ultrapure water to prepare a stock solution. Dilute it into solutions of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1 mg / mL respectively. Add 150 μL of phenol (5%) and mix well, then add 750 μL of sulfuric acid (98%) and mix well. Then incubate the mixed solution in a water bath at 30 °C for 30 min. Measure the absorbance values of the above solutions with an enzyme-labeled instrument at 490 nm, and prepare a glucose standard curve according to the diluted solution concentrations and the corresponding absorbance values;

[0119] (2) Take 100 μL of the alkali-pretreated sugar solution sample alkali one and the alkali-pretreated enzyme hydrolysis released sugar solution sample alkali two respectively, add 150 μL of 5% phenol and 750 μL of concentrated sulfuric acid to them for mixing, then incubate the mixed solution in a water bath at 30 °C for 30 min, and measure the absorbance values of the above solutions with an enzyme-labeled instrument at 490 nm;

[0120] (3) Calculate the saccharification efficiency.

[0121] The results are as Figure 9 and Figure 10 shown. Figure 9 is the standard curve determined for the glucose standard solution. According to Figure 9 Calculate the saccharification efficiencies of the transgenic lines OE-24 and OE-7 and the wild-type WT plants. The saccharification efficiency of the transgenic line OE-24 is slightly higher than that of the wild-type WT, and the saccharification efficiency of the transgenic line OE-7 is significantly higher than that of the wild-type WT.

[0122] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of PagKIN10 gene in regulating poplar growth and development, characterized in that, The nucleotide sequence of the PagKIN10 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, Overexpressing the PagKIN10 gene promotes the growth and development of poplar.

3. A vector containing the PagKIN10 gene described in claim 1.

4. Use of the carrier according to claim 3 in regulating the growth and development of poplar, characterized in that, The vector promotes the growth and development of poplar by overexpressing the PagKIN10 gene.

5. Use of the PagKIN10 gene as described in claim 1 in regulating the plant height of poplar, characterized in that, Overexpressing the PagKIN10 gene increases the growth rate of poplar plant height.

6. Use of the PagKIN10 gene as described in claim 1 in regulating poplar wood differentiation, characterized in that, Overexpressing the PagKIN10 gene promotes the widening of the xylem region of poplar, thereby promoting the differentiation of poplar wood.

7. Use of the PagKIN10 gene as described in claim 1 in regulating the activity of the vascular cambium of poplar, characterized in that, Overexpressing the PagKIN10 gene promotes an increase in the number of cambium cell layers in poplar and improves the cambium activity of poplar.

8. Use of the PagKIN10 gene as described in claim 1 in regulating the cellulose content of poplar, characterized in that, Overexpressing the PagKIN10 gene increases the cellulose content in poplar.

9. Use of the PagKIN10 gene as described in claim 1 in enhancing the saccharification efficiency of poplar, characterized in that, Overexpressing the PagKIN10 gene improves the saccharification efficiency of poplar.

10. A method for improving the saccharification efficiency of poplar stems, characterized in that Overexpressing the PagKIN10 gene described in claim 1 improves the saccharification efficiency of poplar stems.

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