Method for evaluating plant property of transgenic poplar at seedling stage and application of overexpressed pagEXPA1 gene in promoting xylem development of poplar

Abstract

The invention provides a method for evaluating plant property of a transgenic poplar at a seedling stage and application of an overexpressed pagEXPA1 gene in promoting xylem development of the poplar, and relates to the technical field of forest wood property determination. The method for evaluating the plant property of the transgenic poplar at the seedling stage comprises the following steps: S1, detecting whether an exogenous gene is integrated into a poplar genome or not through PCR (Polymerase Chain Reaction); s2, detecting the expression level of the exogenous gene in the poplar; s3, carrying out microstructure analysis on the stem vascular tissue of the transgenic poplar; and S4, separating fibers, conduits and sieve tube cells through fiber separation, dyeing the separated fibers, and observing the forms and structures of the fibers, conduits and sieve tube cells. According to the method, the wood property can be comprehensively evaluated in the early growth stage of the poplar. The invention further provides application of the overexpressed pagEXPA1 gene in promoting development of xylem of poplar, and a foundation is laid for improvement of poplar planting resources.

Description

Technical Field

[0001] The invention relates to the technical field of forest wood property determination, and in particular to a method for evaluating the wood property of transgenic poplar seedlings and the application of overexpressing pagEXPA1 gene in promoting the development of poplar xylem. Background Art

[0002] Transgenic poplar plants refer to poplar individuals that have been grown and developed in the seedling stage by introducing exogenous genes or vectors into the poplar plants through genetic engineering technology. Transgenic poplar plants show some characteristics that are different from non-transgenic poplars in terms of growth rate, morphological characteristics, physiological and biochemical characteristics, stress resistance or disease and pest resistance in the early stages of growth. However, direct measurement of wood properties (such as density, strength, etc.) usually needs to be carried out after the trees have grown to a certain age. High-quality, fast-growing transgenic poplars need to be identified and analyzed for wood properties in the early stages of growth in order to ensure that their fast-growing and high-quality characteristics are reflected and meet subsequent application needs, and to achieve the screening of early excellent strains. Therefore, it is necessary to develop a method for identifying wood properties in the early stages of poplar growth.

[0003] Expansin is the main factor inducing cell wall extension. As a plant cell wall protein, it can loosen the components of plant cell walls and increase the flexibility of cell walls. Studies have shown that the Expansin family plays an important role in plant root extension, fruit ripening, petiole shedding, plant development and stress resistance. At present, research on poplars is mostly focused on root growth and elongation, plant morphological development and stress resistance. Fiber elongation and xylem development are important biological processes in the formation and development of forest wood. Therefore, whether expansins can affect the development and changes of fiber cells and xylem cells by extending the cell wall requires further research and improvement. Summary of the invention

[0004] The purpose of the present invention is to provide a method for evaluating the wood properties of transgenic poplar seedlings and the application of overexpressing the pagEXPA1 gene in promoting the development of poplar xylem, so as to solve the problem that the existing technology cannot identify and analyze wood properties in the early growth stage of poplars.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for evaluating the wood properties of transgenic poplar seedlings, comprising the following steps:

[0007] S1. Detect whether the exogenous gene is integrated into the poplar genome by PCR;

[0008] S2. Detect the expression level of exogenous genes in poplar;

[0009] S3. Microstructural analysis of the stem vascular tissue of transgenic poplars;

[0010] S4. Separate the fibers, ducts, and sieve tube cells through fiber separation, dye the separated fibers, and observe the morphology and structure of the fibers, ducts, and sieve tube cells.

[0011] Preferably, the microstructural analysis comprises the following steps:

[0012] (1) cutting a poplar stem segment and then slicing the segment, wherein the thickness of the slice is 55 to 65 μm;

[0013] (2) mixing the slices with a dye to perform staining;

[0014] (3) removing the stained sections from the dye and observing the vascular structure and tissue characteristics of the poplar stem under a microscope;

[0015] (4) The cross-sectional diameter, xylem width, and number of xylem cell layers of transgenic and non-transgenic poplar plants were measured and counted to identify the effects of exogenous genes on the development of the xylem region of poplar plants.

[0016] Preferably, the dye comprises toluidine blue solution or phloroglucinol dye.

[0017] Preferably, the concentration of the toluidine blue solution is 0.8-1.2% w / v, and the dyeing time of the toluidine blue solution is 8-12 seconds.

[0018] Preferably, the phloroglucinol dye comprises a phloroglucinol solution and a hydrochloric acid solution, the concentration of the phloroglucinol solution is 2-4%, the concentration of the hydrochloric acid is 30-40%; the volume ratio of the phloroglucinol solution to the hydrochloric acid solution is 1.5-2.5:1.

[0019] Preferably, the dyeing time of the phloroglucinol dye is 28 to 32 seconds.

[0020] Preferably, the fiber separation step is as follows:

[0021] (1) cutting the poplar xylem tissue into strip samples with a thickness of 0.4 to 0.6 mm, and then mixing the strip samples with the separation liquid to obtain a mixture;

[0022] (2) reacting the mixture under heating conditions for 72 to 96 hours until white fibrous tissue appears in thin strips.

[0023] Preferably, the separation liquid comprises hydrogen peroxide and glacial acetic acid; the concentration of the hydrogen peroxide is 25-35%; and the volume ratio of the hydrogen peroxide to the glacial acetic acid is 1:2-3.

[0024] Preferably, the heating method is metal bath heating, and the heating temperature is 60-70°C.

[0025] The present invention also provides the use of overexpressing the pagEXPA1 gene in promoting the development of poplar fiber cells and xylem cells.

[0026] By adopting the above technical scheme, the present invention has the following beneficial effects: the method of the present invention for evaluating the wood properties of transgenic poplar seedlings comprises the following steps: S1. detecting whether the exogenous gene is integrated into the poplar genome by PCR; S2. detecting the expression level of the exogenous gene in the poplar; S3. performing microstructural analysis on the stem vascular tissue of the transgenic poplar; S4. separating the fibers, vessels and sieve tube cells by fiber separation, staining the separated fibers, and observing the morphology and structure of the fibers, vessels and sieve tube cells. The method of the present invention can comprehensively evaluate the wood properties of the poplar in the early growth stage. The present invention also provides the application of overexpressing the pagEXPA1 gene in promoting the development of poplar xylem, laying a foundation for the improvement of poplar planting resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the electrophoresis diagram for the identification of PagEXPA1 overexpressing transgenic plants; wherein, M: Marker; P: positive control, PagEXPA1 gene overexpression vector plasmid; CK-: negative control, non-transgenic silver gland poplar plants; 1-7: detected OE: PagEXPA1 transgenic positive plants.

[0028] Figure 2 The graph shows the expression level detection results of the target gene in the PagEXPA1 overexpressing transgenic plants; among them, CK: non-transgenic control plants; OE-1~OE-3: OE:EXPA1-1~OE:EXPA1-3, positive PagEXPA1 overexpressing transgenic plants.

[0029] Figure 3 The vascular structure of the 15th internode of PagEXPA1-overexpressing transgenic and non-transgenic 84K poplars; A is the vascular tissue of the 15th internode of PagEXPA1-overexpressing transgenic and non-transgenic 84K poplars; B is the cross-sectional diameter of the 15th internode of PagEXPA1-overexpressing transgenic and non-transgenic 84K poplars; C is the xylem width (μm); D: statistics of the number of xylem cell layers.

[0030] **P-value<0.01.

[0031] Figure 4The vascular structure of the 15th internode of PagEXPA1-overexpressing transgenic and non-transgenic 84K poplars was stained with phloroglucinol. A and B are the vascular tissues of the 15th internode of non-transgenic 84K poplars, A: objective magnification 10 times, B: objective magnification 20 times; CH are the vascular tissues of the 15th internode of PagEXPA1-overexpressing transgenic poplars, B, E, G: objective magnification 10 times, C, F, H: objective magnification 20 times. C and D: OE:EXPA1-1 (OE-1); E and F: OE:EXPA1-2 (OE-2); G and H: OE:EXPA1-3 (OE-3).

[0032] Figure 5 The figures show the statistics of xylem fiber cells and length in the 20th internode of PagEXPA1-overexpressing transgenic and non-transgenic 84K poplars; A: xylem fiber cells in the 20th internode of non-transgenic 84K poplar; B: xylem fiber cells in the 20th internode of different PagEXPA1-overexpressing transgenic poplar lines OE:EXPA1-1 (OE-1), OE:EXPA1-2 (OE-2), and OE:EXPA1-3 (OE-3); E: statistics of fiber length of non-transgenic 84K poplar and OE-1~OE-3 transgenic poplar plants. DETAILED DESCRIPTION

[0033] The present invention provides a method for evaluating the wood properties of transgenic poplar seedlings, comprising the following steps:

[0034] S1. Detect whether the exogenous gene is integrated into the poplar genome by PCR;

[0035] S2. Detect the expression level of exogenous genes in poplar;

[0036] S3. Microstructural analysis of the stem vascular tissue of transgenic poplars;

[0037] S4. Separate the fibers, tracheae, and sieve tube cells through fiber separation, and observe the morphology and structure of the fibers, tracheae, and sieve tube cells.

[0038] The microstructural analysis comprises the following steps:

[0039] (1) cutting a poplar stem segment and then slicing the segment, wherein the thickness of the slice is 55 to 65 μm;

[0040] (2) mixing the slices with a dye to perform staining;

[0041] (3) taking out the stained sections from the toluidine blue solution and observing the vascular structure and tissue characteristics of the poplar stem under a microscope;

[0042] (4) The cross-sectional diameter, xylem width, and number of xylem cell layers of transgenic and non-transgenic poplar plants were measured and counted to identify the effects of exogenous genes on the development of the xylem region of poplar plants.

[0043] In the present invention, a stem segment of a poplar is first cut, and the stem segment is preferably a stem segment of the 14th to 16th internode, and more preferably a stem segment of the 15th internode; the internode is calculated as follows: the stem segment below the first unfolded leaf at the top of the poplar plant is the first internode, and the internode is calculated from top to bottom; the length of the stem segment is 1.5 to 2.5 cm, more preferably 1.8 to 2.3 cm, and more preferably 2 cm. Then the poplar stem segment is vibrated and sliced, preferably, the vibrated slices are performed after being fixed with FAA fixative; the FAA fixative includes glacial acetic acid and anhydrous ethanol, and the volume ratio of glacial acetic acid to anhydrous ethanol is 1:2 to 4, more preferably 1:2.5 to 3.5, and more preferably 1:3. After the stem segment is fixed with FAA fixative, it is washed 2 to 3 times before slicing, and the water for washing is preferably sterilized deionized water. The oscillating slices are preferably performed on an oscillating slicer. Before slicing, the stem segments are cut into segments of about 1 cm in length, and after the two ends of the stem segments are flattened, they are pasted on the sample stage of the oscillating slicer, and the position between the blade and the sample is adjusted. After the stem segment sample is fixed, the oscillating slices are performed. The thickness of the slices is 55 to 65 μm, more preferably 58 to 62 μm, and more preferably 60 μm. The cut stem segment slices are then transferred to sterilized deionized water with tweezers for storage or directly stained for observation.

[0044] The staining includes toluidine blue staining and phloroglucinol staining. Toluidine blue (TBO for short) can distinguish different vascular tissue cells, and the acidic substances in the cells can be combined with the cations in the TBO dye to be stained. In plant cells, suberized tissue and lignified cell walls are stained blue-green; non-lignified (such as polysaccharide properties) cell walls are reddish purple; proteins and protein-containing mixtures are stained blue or blue-purple. The steps of toluidine blue staining are: soaking the cut tissue in a toluidine blue solution, the concentration of the toluidine blue solution is 0.8-1.2% w / v, more preferably 0.9-1.1% w / v, more preferably 1% w / v, the staining time is 8-12s, more preferably 9-11s, more preferably 10s, after staining, the toluidine blue solution is sucked away with a pipette, and then rinsed 1-2 times, the washing is preferably washed with sterilized deionized water, and then the vascular structure and tissue characteristics of the poplar stem are observed under a microscope. Next, imageJ software (https: / / github.com / imagej) was used to measure and count the cross-sectional diameter, xylem region width, and number of xylem cell layers of transgenic and non-transgenic poplar plants overexpressing PagEXPA1 to identify the effect of the target gene on the development of the xylem region of the plants.

[0045] Phloroglucinol is a lignin dye, which can specifically bind to lignin in the cell wall in an acidic environment to produce a cherry red or purple red reaction, and can be used to determine the lignified cell wall. The phloroglucinol dye includes a phloroglucinol solution and a hydrochloric acid solution, wherein the concentration of the phloroglucinol solution is 2-4%, more preferably 2.5-3.5%, and more preferably 3%; the concentration of the hydrochloric acid is 30-40%, more preferably 32-38%, and more preferably 37%; the volume ratio of the phloroglucinol to the hydrochloric acid is 1.5-2.5:1, more preferably 1.8-2.2:1, and more preferably 2:1. The cut tissue is immersed in the phloroglucinol dye for staining, and the staining time is 28-32s, more preferably 29-31s, and more preferably 30s, and then washed 2-3 times and the lignin content of the cell wall in the xylem area of ​​the poplar stem segment is observed under a microscope.

[0046] In the present invention, it also includes the step of separating fibers, vessels, and sieve tube cells by fiber separation, and observing the morphology and structure of fibers, vessels, and sieve tube cells. The specific operation of the fiber separation is as follows: cut the stem segment of poplar, the stem segment is preferably the stem segment between the 19th and 21st nodes, and more preferably the stem segment between the 20th node; cut the poplar wood xylem tissue into a strip sample with a thickness of 0.4 to 0.6 mm, and the thickness of the strip sample is further preferably 0.45 to 0.55 mm, and more preferably 0.5 mm; then mix the strip sample with the separation liquid, and react under heating conditions for 72 to 96 hours, more preferably 80 to 90 hours, and more preferably 85 hours; until white fiber tissue appears in the thin strip. The separation liquid includes hydrogen peroxide and glacial acetic acid; the concentration of hydrogen peroxide is 25 to 35%; more preferably 27 to 32%, and more preferably 30%; the volume ratio of hydrogen peroxide to glacial acetic acid is 1:2 to 3, more preferably 1:2.2 to 2.8, and more preferably 1:2.5. The heating method is metal bath heating, and the heating temperature is 60-70°C, more preferably 62-68°C, and more preferably 65°C.

[0047] The separated fibers are then mixed with a toluidine blue solution for dyeing, wherein the concentration of the toluidine blue solution is 0.4-0.6%, more preferably 0.5%, and the dyeing time is 28-32 seconds, further preferably 29-31 seconds, and more preferably 30 seconds; after dyeing, the fibers are washed 2-3 times, preferably with sterilized deionized water, and then the dyed fibers are placed under a microscope for observation.

[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1 Obtaining transgenic plants and detecting expression levels

[0050] 1. Transformation of PagEXPA1 overexpressing transgenic plants

[0051] Gateway cloning primers were designed according to the sequence of Populus trichocarpa Expansin (Potri.001G240900), as shown in Table 1.

[0052] Table 1 Gateway cloning primer sequences

[0053]

[0054] The transcript sequence of PagEXPA1 was obtained by PCR cloning using the cDNA of Populs alba × P. glandulosa 84K as template and then cloned using Gateway TM BP Clonase TM II Enzyme mix kit (#11789020, Invitrogen) was used to connect the cloned product to the intermediate vector pDNOR207 according to the instructions. The vector plasmid was then sequenced to confirm that the cloned product had been connected to the intermediate vector. TM LR Clonase TM IIEnzyme mix kit (#11791100, Invitrogen) used the multiple cloning site to connect the intermediate vector to the overexpression vector pMDC32 to construct the PagEXPA1 overexpression vector. The constructed PagEXPA1 overexpression vector was introduced into Populus argentea 84K by the Agrobacterium-mediated leaf disc method, and resistant poplar tissue culture seedlings were obtained by resistance screening (hygromycin resistance).

[0055] The CDS sequence of the PagEXPA1 gene is shown in SEQ ID NO.1:

[0056]

[0057] 2. PCR detection of whether the exogenous gene has been integrated into the poplar genome

[0058] The leaf tissues of hygromycin-resistant 84K poplar and non-transgenic 84K poplar tissue culture seedlings were used as materials, and their genomic DNA was extracted for PCR molecular detection. Because the overexpression vector contains the resistance gene hygromycin phosphotransferase gene HptII, the upstream primer HYP-F: 5'-CGTCTCCGACCTGATGCAGCTCT-3' (SEQ ID NO.4) and the downstream primer HYP-R: 5'-ATCCTGCAAGCTCCGGATGCCTC-3' (SEQ ID NO.5) were designed according to the sequence of the gene for PCR amplification. If the result has an amplified band with a size of about 700 bp, it can be used as a sign of successful transgenic. The non-transgenic 84K tissue culture seedlings were used as negative controls, and the overexpression vector plasmid was used as a positive control, and the obtained resistant tissue culture seedlings were tested by PCR.

[0059] The 50 μL PCR reaction system is as follows: 0.25 μL Taq DNA polymerase (5 U / μL), 5 μL 10× PCR buffer (containing Mg 2+ ), 4μL dNTP Mixture (2.5mM each), 2μL Primer (10μM), 500ng plant DNA, and make up to 50μL with sterile deionized water. Place the reaction tube in the PCR instrument and set the PCR amplification program according to the following parameters: 95℃5min, 95℃20s, 58℃30s, 72℃30s, 35 cycles, 72℃7min, and store at 4℃. Each tissue culture seedling that can detect a band of the same size as the positive control (about 700bp) is used as a positive transgenic strain for propagation; and the tissue culture seedlings that do not detect the target fragment are false positive or non-transgenic strains. The results showed that there were 7 positive transgenic strains, from which 3 strains (OE:EXPA1-1, OE:EXPA1-2, OE:EXPA1-3) were randomly selected for subsequent functional studies.

[0060] 3. Detection of PagEXPA1 expression in transgenic plants

[0061] Total RNA was extracted from leaf tissues of three transgenic 84K poplar lines (OE:EXPA1-1, OE:EXPA1-2, OE:EXPA1-3) and non-transgenic 84K poplars using RNAprep Pure Plant Plus Kit (#DP441, TianGen, China). The integrity of total RNA was assessed by 1.5% agarose gel electrophoresis and analyzed by NanoDrop TM RNA concentration and purity were determined by 8000 spectrophotometer. Only RNA samples with A260 / A280 ratios between 1.9 and 2.1 and A260 / A230 greater than 1.80 were used for cDNA synthesis. TM RT reagent Kit (#RR037, TaKaRa, Japan) was used to reverse transcribe and synthesize cDNA from 1 μg of qualified RNA samples. The quantitative amplification primers qEXP-F: 5'-GAGCAGTTTAATTTGCATTGCCACT-3' (SEQ ID NO.6) and qEXP-R: 5'-AGAGTAAACACCAGGAATTCTAGCTT-3' (SEQ ID NO.7) of the PagEXPA1 gene were used to amplify the cDNA. The relative expression of the target PagEXPA1 gene in transgenic plants was detected in a 480System quantitative PCR instrument (Roche, Germany). The 20μL PCR reaction system was as follows: 10μL KAPA SYBR FAST qPCR MasterMix (#K4601, KAPABiosystems, USA), 2μL 20-fold diluted cDNA, 0.4μM forward primer (qEXP-F) and 0.4μM reverse primer (qEXP-R), and sterilized deionized water was used to make up to 20μL. Quantitative PCR amplification was performed according to the following procedure: 95℃ 3s, 95℃ 10s, 60℃ 30s, and 72℃ 3s for 40 cycles. The reaction without template was used as a negative control, and PP2A-2 was used as an internal reference gene. Each sample was evaluated with four technical replicates, and the experiment was repeated 3 times. 2 -ΔΔCT Methods The relative expression of the data was calculated. By comparing the expression levels of the target gene PagEXPA1 in transgenic and non-transgenic plants, the effect of overexpression of PagEXPA1 on the development of poplar fiber cells and xylem cells could be evaluated.

[0062] Example 2 Method for measuring wood property indexes of transgenic plants

[0063] 1. Cultivation and Growth of Transgenic Plants

[0064] The transgenic seedlings with overexpression of PagEXPA1 and the non-transgenic poplar seedlings were subcultured at the same time. The rooting of the transgenic poplar seedlings was taken as the starting point. After the seedlings grew for 40 days, three transgenic lines with overexpression of PagEXPA1 and non-transgenic poplars were selected. Four seedlings from each line were transplanted to the soil at the same time and cultured in the culture room at a temperature of 25-28°C and a light duration of 16 hours. After 80 days of culture in the soil, the transgenic and non-transgenic poplar plants with overexpression of PagEXPA1 were subjected to microscopic observation and statistical analysis such as tissue sectioning and fiber separation.

[0065] 2. Microstructural analysis of stem vascular tissue

[0066] Objective: By comparing the microstructure of the stems of one-year-old transgenic and non-transgenic poplar plants using an optical microscope or an electron microscope, we can visually observe how exogenous gene modification affects the cell morphology, tissue structure and arrangement of poplars, which will help us gain a deeper understanding of how exogenous genes affect the growth, development, phenotype and wood properties of poplars.

[0067] Method: Generally speaking, one-year-old poplars have almost no lateral branches, and each leaf or lateral bud represents a node. Therefore, the stem segment below the first unfolded leaf at the top of the poplar plant cultured for 80 days was taken as the first internode, and the internodes were counted from top to bottom. The 15th internode was selected for tissue sectioning and microscopic observation.

[0068] (1) Slicing

[0069] Use a single-sided blade to cut a stem segment of about 2 cm in the middle of the 15th internode, fix it with FAA fixative (glacial acetic acid: anhydrous ethanol = 1:3) or directly oscillate it. If the stem segment is fixed with FAA fixative, it needs to be washed three times with sterilized deionized water before slicing. The cross-section of the stem segment is performed on a Leica VT1000S oscillating slicer. Before slicing, use a single-sided blade to cut a stem segment of about 1 cm in the middle of the sample, and flatten the two ends of the stem segment and stick it to the sample stage of the oscillating slicer with Loctite 495 glue. Adjust the position between the blade and the sample, and oscillate the stem segment sample after it is fixed. The slice thickness is 60 μm. The cut stem segment slices are stained and observed.

[0070] (2) Dyeing

[0071] ① Toluidine blue staining: Toluidine blue (TBO for short) can distinguish different vascular tissue cells. The acidic substances in the cells can be combined with the cations in the TBO dye to be stained. In plant cells, suberized tissue and lignified cell walls are stained blue-green; non-lignified (such as polysaccharide) cell walls are stained red-purple; proteins and protein-containing mixtures are stained blue or blue-purple.

[0072] The cut tissue was completely immersed in 1% (w / v) TBO solution. After 10 seconds, the slices were taken out, the excess TBO dye was removed with a pipette, and then washed twice with sterile deionized water. The vascular structure and tissue characteristics of the stems of transgenic poplars and non-transgenic recipient poplars were observed under the bright field of an Olympus BX51 microscope. The cross-sectional diameter, xylem area width, and number of xylem cell layers of transgenic and non-transgenic poplar plants overexpressing PagEXPA1 were measured and counted using imageJ software (https: / / github.com / imagej) to identify the effect of the target gene on the development of the xylem area of ​​the plant. The results are shown in Figure 2. Figure 3 As shown in the figure, compared with the non-transgenic 84K plants, the cross-sectional diameter and xylem tissue of the plants with different PagEXPA1 overexpression lines increased significantly ( Figure 3 The cross-sectional diameters of OE:PagEXPA1-1 to OE:PagEXPA1-3 plants increased by 9.4%, 7.2%, and 7.9%, respectively ( Figure 3 B), the xylem width increased by 24.8%, 19.2% and 22.3% respectively ( Figure 3 C), the number of cell layers in the xylem region increased by 11.6%, 9.5% and 18.3% respectively ( Figure 3 D). This indicates that the thickening of the xylem region in PagEXPA1-overexpressing plants may be caused by an increase in the number of xylem cell layers. This also suggests that changes in PagEXPA1 expression alter the radial growth and xylem development rate of poplar plants.

[0073] ②Phloroglucinol staining: Phloroglucinol is a lignin dye, which can specifically bind to lignin in the cell wall in an acidic environment to produce a cherry red or purple-red reaction, and can be used to determine lignified cell walls. Phloroglucinol dye is a 3% phloroglucinol solution mixed with 37% concentrated hydrochloric acid in a volume ratio of 2:1 to form a phloroglucinol-HCl solution. Place the tissue section in the phloroglucinol-HCl solution for staining, aspirate the solution after about 30 seconds, and wash it three times with sterile deionized water. Then observe the lignin content of the cell wall in the xylem area of ​​the stem of the transgenic poplar and non-transgenic 84K poplar under the bright field of an Olympus BX51 microscope. The results are as follows: Figure 4 shown.

[0074] from Figure 4 It can be seen that after phloroglucinol staining, the three strains overexpressing the PagEXPA1 gene ( Figure 4 The color of the xylem cell wall in CH) was similar to that in non-transgenic 84K plants ( Figure 4A and B in the figure are not much different, but the specific content changes still require chemical determination of lignin content. It can be seen from this that the application of this method can predict the lignin content of plants in advance in the early stage of plant growth.

[0075] Example 3 Fiber Isolation and Cell Observation of Stem Xylem Cells

[0076] Purpose: Fiber separation can be used to separate fibers, vessels and other cells from the cell wall, so as to observe the morphology and structure of fibers, vessels, sieve tubes and other cells, and to gain an in-depth understanding of basic information such as the microstructure and chemical composition of wood, and to help understand the physical and mechanical properties of wood, providing basic data for further analysis of wood properties.

[0077] Methods: Fiber separation was performed by acid treatment, and the separation solution was a mixture of 30% hydrogen peroxide and glacial acetic acid in a volume ratio of 3:7. The 20th stem segment of PagEXPA1 overexpressing transgenic and non-transgenic poplar plants cultured in soil for 80 days was used as the material for fiber separation.

[0078] (1) Fiber separation method

[0079] Use a single-edged blade to cut off a stem segment of about 2 cm in the middle of the 20th node, remove the outer bark of the stem segment and keep only the tissue sample within the woody part, cut it into thin strips of about 0.5 mm, and then immerse it in a 2 mL centrifuge tube containing separation solution. The sample in the centrifuge tube is incubated in a metal bath at 65°C for 80 hours until white fibrous tissue appears in the thin strips. After a short centrifugation in a desktop microcentrifuge, use a pipette to suck away all the separation solution as much as possible, then wash it with sterile deionized water three times (i.e., add sterile water to mix, centrifuge it slightly, and then suck away the sterile water) and store it in sterile water.

[0080] (2) Staining method and observation statistics

[0081] The separated fibers were transferred to a 2.0 mL centrifuge tube containing 0.5% (w / v) toluidine blue (TBO) solution and stained for 30 seconds, and then washed three times with sterile deionized water. The stained fibers can be imaged and measured using an Olympus BX51 microscope. The fiber length of transgenic and non-transgenic poplar plants overexpressing PagEXPA1 was measured using imageJ software (https: / / github.com / imagej), and 200 fibers were randomly measured for each plant. The measurement results were statistically analyzed using SPSS software to calculate the average fiber length and variance, as well as significant difference analysis. The results are shown in the figure. Figure 5 shown.

[0082] from Figure 5It can be seen that compared with the non-transgenic 84K plants, the fiber length of the plants with different PagEXPA1 overexpression lines increased significantly ( Figure 5 The fiber lengths of OE:PagEXPA1-1 to OE:PagEXPA1-3 plants increased by 12.7%, 8.2%, and 8.6%, respectively ( Figure 5 Middle E), indicating that overexpression of PagEXPA1 promoted the growth of fibers in the cell wall of plants.

[0083] As can be seen from the above embodiments, the present invention provides a method for evaluating the wood properties of transgenic poplar seedlings and the application of overexpressing the pagEXPA1 gene in promoting the development of poplar xylem. The method for identifying the wood properties of transgenic poplar seedlings of the present invention includes multiple aspects such as molecular biological detection and measurement of basic physiological indicators. Molecular biological detection can detect the stability of the exogenous genes of transgenic plants and the influence of exogenous genes on the wood properties of plants at the molecular level. The microstructural analysis of the stem vascular tissue and the fiber separation and cell observation of the stem xylem cells can comprehensively evaluate the wood properties in the early growth stage of poplars.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for evaluating the wood properties of transgenic poplar seedlings, characterized in that: The following steps are involved: S1. Detect whether the exogenous gene is integrated into the poplar genome by PCR; S2. Detect the expression level of exogenous genes in poplar; S3. Microstructural analysis of the stem vascular tissue of transgenic poplars; S4. Separate the fibers, ducts, and sieve tube cells through fiber separation, dye the separated fibers, and observe the morphology and structure of the fibers, ducts, and sieve tube cells.

2. The method according to claim 1, characterized in that The microstructural analysis comprises the following steps: (1) cutting a poplar stem segment and then slicing it, with the thickness of the slice being 55 to 65 μm; (2) mixing the slices with dyes for staining; (3) removing the stained sections from the dye and observing the vascular structure and tissue characteristics of the poplar stem under a microscope; (4) The cross-sectional diameter, xylem width, and number of xylem cell layers of transgenic and non-transgenic poplar plants were measured and counted to identify the effects of exogenous genes on the development of the xylem region of poplar plants.

3. The method according to claim 2, characterized in that The dye includes toluidine blue solution or phloroglucinol dye.

4. The method according to claim 3, characterized in that The concentration of the toluidine blue solution is 0.8-1.2% w / v, and the dyeing time of the toluidine blue solution is 8-12 seconds.

5. The method according to claim 3, characterized in that: The phloroglucinol dye comprises a phloroglucinol solution and a hydrochloric acid solution. The concentration of the phloroglucinol solution is 2-4%, and the concentration of the hydrochloric acid is 30-40%. The volume ratio of the phloroglucinol solution to the hydrochloric acid solution is 1.5-2.5:

1.

6. The method according to claim 5, characterized in that The dyeing time of the phloroglucinol dye is 28 to 32 seconds.

7. The method according to claim 1, characterized in that The steps of fiber separation are as follows: (1) cutting the poplar xylem tissue into strip samples with a thickness of 0.4 to 0.6 mm, and then mixing the strip samples with the separation liquid to obtain a mixture; (2) reacting the mixture under heating conditions for 72 to 96 hours until white fibrous tissue appears in thin strips.

8. The method according to claim 7, characterized in that The separation liquid comprises hydrogen peroxide and glacial acetic acid; the concentration of the hydrogen peroxide is 25-35%; and the volume ratio of the hydrogen peroxide to the glacial acetic acid is 1:2-3.

9. The method according to claim 7, characterized in that The heating method is metal bath heating, and the heating temperature is 60-70°C.

10. Application of overexpression of pagEXPA1 gene in promoting xylem development of poplar.

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