Application of eucalyptus grandis EgrDof5 gene in poplar

Abstract

The invention relates to the technical field of plant biology, in particular to application of eucalyptus grandis EgrDof5 gene in poplar, and the application comprises at least one of the following items: a, improving poplar biomass; b, improving photosynthetic performance of the poplar; c, regulating and controlling secondary growth of the poplar; the nucleotide sequence of the eucalyptus grandis EgrDof5 gene is as shown in SEQ ID No. 1. By transferring the EgrDof5 gene into the populus genome, the poplar biomass can be increased, the photosynthetic performance of the poplar can be improved, the secondary growth of the poplar can be regulated and controlled, a new method is provided for genetic improvement of the poplar, the gene bank of the poplar can be enriched, and more choices are provided for forest breeding in the future.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, and particularly relates to application of Eucalyptus grandis EgrDof5 gene in poplar. BACKGROUND

[0002] Trees are perennial woody plants, which have unique secondary growth characteristics. In the process of secondary growth for many years, trees are affected by internal genetic factors and external environmental conditions. Therefore, it is of great significance to deeply analyze the genetic mechanism of regulating tree secondary growth, to purposefully change the expression of related genes, to improve the wood quality, wood properties and adaptive traits of trees, to improve the level of molecular breeding of Chinese forest trees, and to promote the technological innovation of forest tree seed industry.

[0003] Dof transcription factors play a core regulatory role in multiple processes of plant growth and development, including seed germination, photosynthesis, carbon and nitrogen metabolism, organogenesis and stress response, and are key factors in the molecular genetic regulation network of plants. Eucalyptus grandis, as a globally important fast-growing commercial forest tree species, has the advantages of fast growth, high biomass accumulation efficiency, and strong adaptability, and is an ideal material for studying the growth and development mechanism of woody plants. However, the current systematic study of Eucalyptus grandis Dof gene family is still blank, and the sequence characteristics, evolutionary rules, expression patterns and functional regulation mechanisms of its members are not well understood, which limits the application of this gene family in genetic improvement of woody plants.

[0004] Poplar (such as 84k poplar) is an important industrial timber forest and ecological protection forest tree species in northern China, and its biomass yield directly determines its economic value and ecological benefit. The existing biomass improvement technology of poplar mainly relies on traditional hybrid breeding, which has the problems of long breeding cycle and poor directionality. SUMMARY

[0005] The purpose of the present application is to provide an application of Eucalyptus grandis EgrDof5 gene in poplar, which can at least solve some defects in the prior art.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is an application of Eucalyptus grandis EgrDof5 gene in poplar, which includes at least one of the following: a. increasing the biomass of poplar; b. improving the photosynthetic performance of poplar; c. regulating the secondary growth of poplar; The nucleotide sequence of the Eucalyptus grandis EgrDof5 gene is shown in SEQ ID No. 1.

[0007] Further, the improving the biomass of the poplar comprises at least one of improving the plant height, increasing the leaf area, improving the height of stem node, improving the root length, and increasing the root surface area.

[0008] Further, the improving the photosynthetic performance of the poplar comprises at least one of improving the net photosynthetic rate, improving the transpiration rate, improving the stomatal conductance, and reducing the intercellular CO2 concentration.

[0009] Further, the regulating the secondary growth of the poplar comprises at least one of widening the xylem, increasing the fiber cell area, and increasing the cell diameter.

[0010] Further, the application comprises the following steps: The overexpression vector pK2GW7-EgrDof5 of the EgrDof5 gene is constructed, and the overexpression vector is transformed into the poplar genome by using the agrobacterium-mediated genetic transformation method.

[0011] Further, the method for constructing the overexpression vector pK2GW7-EgrDof5 is as follows: specific primers are designed, then the CDS of the EgrDof5 gene is transferred to the plant expression vector pK2GW7 by the LR recombination reaction, and the overexpression vector pK2GW7-EgrDof5 is obtained through Spe resistance screening and colony PCR verification.

[0012] Further, the nucleotide sequence of the specific primer is shown in SEQ ID No. 2 and SEQ ID No. 3.

[0013] Compared with the prior art, the present application has the following beneficial effects: The present application can improve the biomass of the poplar, improve the photosynthetic performance of the poplar, and regulate the secondary growth of the poplar by transforming the EgrDof5 gene of Eucalyptus grandis into the poplar genome, thereby providing a new method for genetic improvement of the poplar. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0015] Figure 1 The phylogenetic tree of the Dof family of different species; Figure 2 The expression heat map of the EgrDof gene family in different tissues and development stages; Figure 3 The heat map of the expression of the EgrDof gene under abiotic stress and plant hormone treatment; Figure 4 For transgenic poplar positive identification analysis, wherein A is transgenic plant semi-quantitative PCR identification, B is poplar Actin PCR identification, C is transgenic expression level analysis; Figure 5 For transgenic poplar growth and development analysis, wherein A is 80 d poplar height, B is 60 d transgenic poplar crown width, C is 30 d, 60 d, 80 d transgenic poplar height change, D is 30 d, 60 d, 80 d transgenic poplar ground diameter change; Figure 6 For transgenic poplar leaf analysis, wherein A is different parts of poplar leaf shape, B is different parts of poplar leaf area, C is different parts of poplar leaf diameter; Figure 7 For transgenic poplar stem node analysis, wherein A is transgenic poplar 7th, 14th stem node length phenotype diagram, B is transgenic poplar stem node length, C is transgenic poplar diameter; Figure 8 For transgenic poplar root system analysis, wherein A is transgenic poplar root system phenotype diagram, B is transgenic poplar root system length, C is transgenic poplar root surface area, D is transgenic poplar unit volume root system length; Figure 9 For transgenic poplar photosynthetic parameter analysis, wherein A is transgenic poplar net photosynthetic rate, B is transgenic poplar transpiration rate, C is transgenic poplar stomatal conductance, D is transgenic poplar intercellular CO2 concentration Figure 10 For transgenic poplar stem section staining analysis, wherein A-C are transgenic poplar 14th stem node tissue section toluidine blue staining observation, D is transgenic poplar xylem width, E is transgenic poplar fiber cell area, F is transgenic poplar longitudinal section cell diameter; Figure 11 For poplar sample infrared spectrum. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0017] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0018] I. Screening and Analysis of the EgrDof5 Gene 1. Phylogenetic analysis of the Eucalyptus macrocarpa Dof gene family Eucalyptus macrocarpa has 26 EgrDof genes, which are named EgrDof1-EgrDof26 according to their chromosomal position. To clarify the functional relationships of EgrDof genes, a phylogenetic tree of Dof proteins from Eucalyptus macrocarpa, Arabidopsis thaliana, rice, and Populus tomentosa was constructed using MEGA software with the neighbor-joining (NJ) method. Figure 1 The results showed that all Dof proteins were divided into five subfamilies (IV), with subfamily III having the largest number of members (34.6%), suggesting that the functional modules corresponding to this subfamily may be enhanced in *Eucalyptus macrocarpa*. Subfamily V was completely absent in *Eucalyptus macrocarpa*, presumably due to functional redundancy and elimination by natural selection. Phylogenetic analysis showed that EgrDof5 is most closely related to the known functional AtDof2.3 and AtDof1.5 in *Arabidopsis thaliana*.

[0019] 2. Screening and molecular characterization of EgrDof5 candidate genes The CDS length of the EgrDOF5 gene is 579 bp, as shown in SEQ ID No. 1, encoding 193 amino acids. The N-terminal region contains a conserved Dof domain, and the gene has no introns, suggesting that it can participate in rapid physiological regulation through efficient transcription. WoLF PSORT prediction and transient expression in tobacco confirmed that EgrDOF5 is located in the cell nucleus, consistent with the functional characteristics of nuclear regulation of transcription factors. The EgrDOF5 promoter region contains auxin / jasmonic acid response elements, light-responsive elements (G-box), and stress-responsive elements (LTR / ARE), which can integrate hormones and environmental signals to regulate growth.

[0020] 3. Tissue-specific expression pattern analysis of the Dof gene in Eucalyptus macrocarpa The expression patterns of the EgrDof gene in different tissues and developmental stages of Eucalyptus macrocarpa were analyzed using RNA-seq technology. Figure 2 The results showed that the EgrDof gene expression was generally low in leaves and high in stems and phloem. Twenty EgrDof genes were highly expressed in the stems, with significantly increased expression levels in the phloem of 6-month-old and 3-year-old Eucalyptus trees. Furthermore, the expression levels of most genes were higher in the 9th stem node than in other nodes.

[0021] Expression characterization of EgrDof5 showed high expression in the phloem and shoot tip of 6-month-old Eucalyptus macrocarpa and the xylem of 3-year-old Eucalyptus macrocarpa. Figure 2 During stem node development, as the stem nodes mature (3-11 nodes), the expression level of EgrDof5 shows a gradient increasing trend. Figure 2), which is highly consistent with the strengthening trend of secondary growth during wood development. This expression pattern indicates that EgrDof5 is directly involved in the process of stem development and xylem formation in E. grandis and is a key candidate gene for regulating biomass accumulation in E. grandis.

[0022] 4. Response analysis of E. grandis Dof gene to abiotic stress and hormone treatment Online RNA-Seq analysis was used to analyze 2-month-old E. grandis under boron deficiency, phosphorus deficiency, salt stress, and spraying of salicylic acid (SA) and jasmonic acid (JA) treatment. As shown in Figure 3 , the results show that: (1) Hormone response: After SA or JA treatment, the expression level of EgrDof5 at different time points showed significant differences, and the expression level increased by 2.3 times at 6h after JA treatment, and decreased to 0.4 times of the control at 24h after SA treatment, indicating that it can accurately respond to hormone signals, which is consistent with the gene expression characteristics of growth and development regulation; (2) Nutrient stress response: Under phosphorus deficiency treatment, the expression level of EgrDof5 decreased to the lowest (0.3 times of the control) at 24h, but recovered to 1.8 times of the control at 96h, reflecting its regulatory role in nutrient stress adaptation; (3) Salt stress response: The expression level of EgrDof5 significantly increased (3.1 times of the control) at 168h under salt stress, while its expression level decreased under SA treatment at the same period. This dual regulation characteristic indicates that EgrDof5 can coordinate hormone signals and stress signals, and plays a core role in maintaining the balance between plant growth and stress adaptation.

[0023] II. Introduction of EgrDof5 gene into poplar 1. Construction of overexpression vector pK2GW7-EgrDof5 Specific primers were designed based on the annotation information from Phytozome, and the nucleotide sequences of the specific primers are shown in SEQ ID No. 2 and SEQ ID No. 3, wherein SEQ ID No. 2 is the forward primer and SEQ ID No. 3 is the reverse primer. Then, the CDS of EgrDof5 was efficiently transferred to the plant expression vector pK2GW7 through LR recombination reaction, and the recombinant vector pK2GW7-EgrDof5 was successfully constructed through Spe resistance screening and colony PCR verification.

[0024] 2. Plant genetic transformation and detection of positive seedlings of transgenic poplar plants The 84K poplar tissue culture seedlings with 30 d growth were infected with Agrobacterium, and the overexpression vector pK2GW7-EgrDof5 was introduced into the poplar genome by Agrobacterium-mediated genetic transformation method. The infected explants were transferred to co-culture medium for dark culture for 2 d, and then to the differentiation screening medium for photoperiod culture to obtain resistant buds Figure 4 ). The buds with a length of about 2 cm were transferred to rooting medium, and finally the transformed plants with well-developed root systems were subjected to molecular identification. The initial screening was completed by PCR amplification with NPTII gene-specific primers. For the positive strains in the initial screening, total RNA was further extracted and reverse transcribed to synthesize cDNA, and the gene expression level was detected by qRT-PCR. Based on the ΔΔCT method, EgrDof5 gene overexpression strains (named OE-3, OE-17, OE-29) with significantly high expression were screened for further study.

[0025] III. Effects of EgrDof5 gene on poplar 1. Effects of EgrDof5 gene on the growth and development of poplar The growth of transgenic poplar was compared, and the growth data of poplar at different time periods were recorded. As shown in Figure 5 , by comparing the morphological characteristics of wild-type poplar (WT) and EgrDof5 gene overexpression strains (OE-3, OE-17, OE-29), it was found that in the poplar planted for 80 d, the OE-3 strain had the most significant increase in plant height Figure 5 A, C), and the continuous determination of growth indicators showed that the crown expansion of EgrDof5 overexpression poplar strains was better than that of wild-type poplar Figure 5 B), and the plant height was significantly higher than that of wild-type Figure 5 C). At the same time, the diameter of the OE-29 strain planted for 30 d was significantly thicker than that of the wild type, while the diameter of the transgenic poplar at other time periods was differentially thickened compared with the wild type Figure 5 D).

[0026] 2. Effects of EgrDof5 gene on poplar leaves The leaf area and diameter of transgenic strains showed a synergistic growth trend, and the leaves were photographed and recorded when the plants were planted for 80 d and grew stably, as shown in Figure 6 , the leaf area and diameter of the three EgrDof5 gene overexpression poplar strains were higher than those of the wild-type poplar, and the leaf area of the OE-3 strain increased most significantly.

[0027] 3. Effects of EgrDof5 on poplar stem nodes The 7th and 14th stem node heights of the 80 d poplar were measured, and it was found that the stem node length of the overexpression strains was greater than that of the wild type Figure 7A). Except for the OE-29 line, which showed no significant increase in the length of the 14th stem segment compared to the wild-type poplar, the other two overexpression lines had significantly longer 7th and 14th stem segments than the wild-type (p<0.05), indicating that overexpression of the EgrDof5 gene promoted poplar stem segment elongation. Figure 7 B). Regarding internode diameter, the diameter of the 14th internode in the OE-17 and OE-29 lines was significantly larger than that in the wild type (B). Figure 7 C) indicates that some overexpression lines also promote radial growth of stem nodes.

[0028] 4. Effects of EgrDof5 on poplar root system During plant growth, it was observed that the roots of transgenic plants were more likely to break through the culture medium and become visible than those of wild-type plants. Therefore, the root systems of both wild-type and transgenic lines were observed and measured. The results showed that the transgenic poplar root system was more dense (…). Figure 8 A). In terms of root length and root surface area, the OE-3 line had significantly higher root length and root surface area than the wild type ( Figure 8 B and C), while the OE-17 and OE-29 lines showed some increase compared to the wild type, but the difference was not significant. In terms of root length per unit volume, transgenic plants showed a trend of increasing compared to the wild type. Figure 8 D).

[0029] IV. Effects of the EgrDof5 gene on the photosynthetic performance of poplar from Figure 9 It can be seen that, compared with the wild type, the net photosynthetic rates of transgenic plants OE-3 and OE-17 were significantly increased, by 14% and 13% respectively, while the change in OE-29 was not significant. Figure 9 A). Regarding transpiration rate, the transpiration rates of OE-3, OE-17, and OE-29 transgenic poplar lines were all significantly increased compared to wild-type poplar. Figure 9 B), increasing by 36%, 28%, and 25% respectively, with the OE-3 transgenic line showing the most significant improvement. The change in stomatal conductance followed a trend consistent with the transpiration rate. Figure 9 (C) The stomatal conductance of transgenic poplar lines OE-3, OE-17, and OE-29 increased by 25%, 18%, and 15%, respectively. However, the intercellular CO2 concentration of transgenic poplars decreased compared to wild-type poplars, with the OE-29 line showing the most significant decrease (19%), followed by OE-3 and OE-17 (16% and 14%, respectively). Figure 9 D).

[0030] Correlation analysis of four photosynthetic parameters of poplar was conducted (Table 1). The results showed that Pn was positively correlated with Gs and Tr, with correlation coefficients of 0.759 and 0.714, respectively, and was negatively correlated with Ci. Gs was highly correlated with Tr, with a correlation coefficient of 0.996, and was significantly correlated at the 0.01 significance level. In addition, Gs and Tr were both negatively correlated with Ci, with correlation coefficients of -0.874 and -0.913, respectively.

[0031] Table 1 Correlation analysis of photosynthetic parameters of transgenic poplar EgrDof5 P n ]]> G s ]]> [TECHNICAL FIELD] r ]] [C i ]]> P n ]]> 1 Gs 0.759 1 [TECHNICAL FIELD] r ]] 0.714 0.996** 1 [C i ]]> -0.392 -0.874 -0.913 1 Note: ** represents the 0.01 significant level.

[0032] V. Effect of EgrDof5 gene on secondary growth of poplar The seventh and fourteenth stem nodes were selected for toluidine blue staining analysis. The results showed that the xylem of the transgenic plants had already connected into a ring at the seventh stem node, while the WT had not yet lignified into a ring. Quantitative analysis of the staining images of the fourteenth internode sections by ImageJ software showed that the xylem of the transgenic plants was wider than that of the WT Figure 10 A, D). At the same time, observation of the secondary xylem region of poplar showed that the fiber cell wall thickness of the transgenic plants was thinner, and the fiber cell area was significantly larger Figure 10 B, E). Observation of the staining of the fourteenth stem node longitudinal section of poplar showed that the cell diameter of the transgenic poplar was wider and the cell area was larger than that of the wild type Figure 10 C, F). Therefore, EgrDof5 gene has a regulatory effect on the secondary growth of poplar stems.

[0033] To further verify the effect of EgrDof5 gene on the biosynthesis of secondary wall of 84K poplar, the lignin content and monosaccharide of the transgenic poplar were determined, and the results are shown in Table 2. The acid-insoluble lignin and acid-soluble lignin in the stems of the plants overexpressing EgrDof5 gene were increased, and the total lignin content was increased by 3.30%, 4.07%, and 5.2% compared with the wild type. In addition, the cellulose content of the plants overexpressing EgrDof5 gene was significantly increased, which was significantly increased by 15.02%, 15.5%, and 17.18% compared with the wild type, while the xylan and arabinose had no obvious difference compared with the wild type.

[0034] Table 2 Detection of secondary wall components of transgenic plants and wild type Sample WT OE-3 OE-17 OE-29 Lignin Acid-insoluble lignin 18.62±0.22 19.19±0.18* 19.17±0.28* 19.41±0.11** Acid-soluble lignin 5.93±0.12 6.17±0.21 6.37±0.16 6.41±0.17* Total lignin 24.54±0.27 25.35±0.37* 25.54±0.43* 25.82±0.15* Polysaccharides Glucose 31.94±0.24 36.74±0.07** 36.79±0.11** 37.43±0.06** Xylose 17.31±0.2 17.06±0.09 17.19±0.09 17.12±0.04 Arabinose 4.6±0.04 4.71±0.04 4.66±0.11 4.91±0.11 Note: Data are expressed as mean ± standard deviation (n = 3), * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, according to Duncan's multiple range test.​

[0035] VI. Infrared Spectroscopic Analysis of EgrDof5 Transgenic Poplar Fourier transform infrared spectroscopy was used to conduct an in-depth analysis of the structure of EgrDof5 transgenic poplar.

[0036] like Figure 11 As shown, at 834 cm -1 An absorption peak of S-type lignin was observed at 898 cm⁻¹. Compared with the OE-3, OE-17, and OE-29 lines, the absorption intensity of the WT sample was the weakest, suggesting that the content of S-type lignin in WT may be low. -1 The absorption peak corresponds to the vibrational peaks of the β-1,4 glycosidic bond and the CH bond. It is located at 1056 cm⁻¹. -1 The absorption peaks indicate the CO and C-C stretching vibrations or C-OH bending vibrations of hemicellulose in xylan, suggesting that the poplar sample hemicellulose contains xylan-arabinose components. The deformation absorption peak of lignin (CH) appears at 1245 cm⁻¹. -1 , and 1328 cm -1 The absorption peak at 1424 cm⁻¹ is a characteristic manifestation of the CH bending vibration. Furthermore, the absorption peak at 1424 cm⁻¹... -1 and 1460 cm -1 The absorption peaks represent the stretching vibrations of -CH2 and CH in cellulose, respectively, and the amplitudes of these vibrations in the transgenic plants are larger than those in the wild type, consistent with previous experiments showing that EgrDof5 enhances the cellulose content of poplar. (1506 cm⁻¹) -1 The absorption peak is related to the vibration of the C=C bond on the aromatic ring of lignin. (1740 cm⁻¹) -1 The absorption peaks revealed the vibration of the acetyl CO bond in hemicellulose, indicating that the side chains of the poplar sample hemicellulose may have acetyl modification.

[0037] VII. Conclusion The EgrDof5 gene can increase poplar biomass. Poplars overexpressing the EgrDof5 gene have higher height, diameter at ground level, leaf area, and crown width than wild-type poplars, with the OE-3 line showing the most significant changes. Furthermore, the length of the 7th stem node is significantly greater in OE-3 lines than in wild-type poplars, and the diameter of the 14th stem node is also significantly increased in OE-17 and OE-29 lines. Root data analysis shows that the root length and surface area of ​​the OE-3 line are significantly increased.

[0038] The EgrDof5 gene can improve the photosynthetic performance of poplar. Overexpression of the EgrDof5 gene can increase the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) of poplar, and decrease the intercellular CO2 concentration (Ci). Correlation analysis showed that Pn in transgenic poplar was positively correlated with Gs and Tr, and negatively correlated with Ci, while Gs was significantly positively correlated with Tr.

[0039] EgrDof5 gene can regulate the secondary growth of poplar. EgrDof5 gene plays an important role in the regulation of poplar cell wall components. Overexpression of EgrDof5 plants lignification process in advance, and the transgenic plants 14 stem node secondary xylem widened, fiber cell area significantly larger, and the cell wall thickness thin. The total lignin content of transgenic lines increased, and the cellulose content increased. Fourier transform infrared spectroscopy analysis found that S-type lignin increased and the cellulose structure changed. These changes will enhance the mechanical strength and anti-degradation ability of cell wall, thereby affecting the growth and development of poplar.

[0040] The above merely describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An application of the EgrDof5 gene from Eucalyptus macrocarpa in poplar, characterized in that, The application includes at least one of the following: a. Increase poplar biomass; b. Improve the photosynthetic performance of poplar trees; c. Regulate the secondary growth of poplar trees; The nucleotide sequence of the Eucalyptus EgrDof5 gene is shown in SEQ ID No.

1.

2. The application of the Eucalyptus macrocarpa EgrDof5 gene in poplar as described in claim 1, characterized in that: The improvement of poplar biomass includes at least one of the following: increasing plant height, increasing leaf area, increasing stem node height, increasing root length, and increasing root surface area.

3. The application of the Eucalyptus macrocarpa EgrDof5 gene in poplar as described in claim 1, characterized in that: The improvement of poplar photosynthetic performance includes at least one of increasing net photosynthetic rate, increasing transpiration rate, increasing stomatal conductance, and reducing intercellular CO2 concentration.

4. The application of the Eucalyptus macrocarpa EgrDof5 gene in poplar as described in claim 1, characterized in that: The regulation of poplar secondary growth includes at least one of widening the xylem, increasing the area of ​​fiber cells, and increasing the cell diameter.

5. The application of the Eucalyptus macrocarpa EgrDof5 gene in poplar as described in claim 1, characterized in that, Includes the following steps: An overexpression vector pK2GW7-EgrDof5 for the EgrDof5 gene was constructed and transferred into the poplar genome using Agrobacterium-mediated genetic transformation.

6. The application of the Eucalyptus macrocarpa EgrDof5 gene in poplar as described in claim 5, characterized in that, The method for constructing the overexpression vector pK2GW7-EgrDof5 is as follows: First, specific primers are designed, and then the CDS of the EgrDof5 gene is transferred into the plant expression vector pK2GW7 through LR recombination reaction. After Spe resistance screening and colony PCR verification, the overexpression vector pK2GW7-EgrDof5 is obtained.

7. The application of the Eucalyptus giantis EgrDof5 gene in poplar as described in claim 6, characterized in that: The nucleotide sequences of the specific primers are shown in SEQ ID No. 2 and SEQ ID No. 3.

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