Application of eucalyptus grandis egrdof5 gene in poplar

Abstract

The present application relates to the field of plant biotechnology, and particularly relates to application of Eucalyptus grandis EgrDof5 gene in poplar, the application comprises at least one of the following: a, improving 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 as SEQ ID No. 1. The present application can improve the biomass of poplar, improve the photosynthetic performance of poplar and regulate the secondary growth of poplar by transferring the Eucalyptus grandis EgrDof5 gene into the genome of poplar, which provides a new method for genetic improvement of poplar, helps to enrich the gene library of poplar and provides more choices for future forest breeding.

Description

TECHNICAL FIELD

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

[0002] Trees are perennial woody plants with unique secondary growth characteristics. During 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 regulatory 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 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 of the 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:

[0007] a. improving the biomass of poplar;

[0008] b. improving the photosynthetic performance of poplar;

[0009] c. regulating the secondary growth of poplar;

[0010] The nucleotide sequence of the Eucalyptus grandis EgrDof5 gene is shown in SEQ ID No. 1.

[0011] 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 the stem node, improving the root length, and increasing the root surface area.

[0012] 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.

[0013] 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.

[0014] Further, the application comprises the following steps:

[0015] 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.

[0016] 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 by performing Spe resistance screening and colony PCR verification.

[0017] Further, the nucleotide sequence of the specific primer is shown in SEQ ID No. 2 and SEQ ID No. 3.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] 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

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0021] Figure 1 The phylogenetic tree of the Dof family of different species;

[0022] Figure 2 The expression heat map of the EgrDof gene family in different tissues and development stages;

[0023] Figure 3 Heatmap of EgrDof gene expression under abiotic stress and phytohormone treatment;

[0024] Figure 4 Transgenic poplar positive identification analysis, wherein A is transgenic plant semi-quantitative PCR identification, B is poplar Actin PCR identification, and C is transgenic expression level analysis;

[0025] Figure 5 Transgenic poplar growth and development analysis, wherein A is 80 d growth poplar height, B is 60 d growth transgenic poplar crown width, C is 30 d, 60 d, 80 d transgenic poplar height change, and D is 30 d, 60 d, 80 d transgenic poplar ground diameter change;

[0026] Figure 6 Transgenic poplar leaf analysis, wherein A is different parts of poplar leaf morphology, B is different parts of poplar leaf area, and C is different parts of poplar leaf diameter;

[0027] Figure 7 Transgenic poplar stem node analysis, wherein A is transgenic poplar 7th and 14th stem node length phenotype diagram, B is transgenic poplar stem node length, and C is transgenic poplar diameter;

[0028] Figure 8 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, and D is transgenic poplar unit volume root system length;

[0029] Figure 9 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, and D is transgenic poplar intercellular CO2 concentration

[0030] Figure 10 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, and F is transgenic poplar longitudinal section cell diameter;

[0031] Figure 11 Poplar sample infrared spectrum. DETAILED DESCRIPTION

[0032] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of the present application.

[0033] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents and the like used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0034] I. Screening and analysis of EgrDof5 gene

[0035] 1. Phylogenetic analysis of Eucalyptus grandis Dof gene family

[0036] Eucalyptus grandis has 26 EgrDof genes, which are named EgrDof1-EgrDof26 according to their chromosomal location order. To clarify the functional relevance of EgrDof genes, the MEGA software was used to construct a phylogenetic tree of Eucalyptus grandis, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa Dof proteins using the neighbor-joining (NJ) method. Figure 1 The results showed that all Dof proteins were divided into five subfamilies (I-V), with the largest number of members in subfamily III (accounting for 34.6%), suggesting that the functional module corresponding to this subfamily may be strengthened in Eucalyptus grandis; and subfamily V was completely missing in Eucalyptus grandis, which was presumably eliminated by natural selection due to functional redundancy. The phylogenetic relationship showed that EgrDof5 had the closest genetic relationship with the functionally known AtDof2.3 and AtDof1.5 in Arabidopsis thaliana.

[0037] 2. Screening and molecular characteristics of EgrDof5 candidate gene

[0038] The CDS length of 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 intron, suggesting that it can be involved in rapid physiological regulation through efficient transcription; through WoLF PSORT prediction and tobacco transient expression verification, EgrDof5 is located in the nucleus, which is consistent with the functional characteristics of nuclear regulation of transcription factors; the promoter region of EgrDOF5 contains auxin / jasmonic acid response elements, light response elements (G-box) and stress response elements (LTR / ARE), which can integrate hormone and environmental signal regulation of growth.

[0039] 3. Analysis of tissue-specific expression pattern of Eucalyptus grandis Dof gene

[0040] The expression patterns of EgrDof genes in different tissues and developmental stages of E. grandis were analyzed by RNA-seq technology Figure 2 ), and the results showed that the overall expression of EgrDof genes was characterized by "low expression in leaves and high expression in stems and phloem". The expression of 20 EgrDof genes was higher in stems, and the expression of EgrDof genes in the phloem of 6-month-old and 3-year-old E. grandis was significantly increased, and the expression of most genes in the 9th stem segment was higher than that in other segments.

[0041] The analysis of the expression characteristics of EgrDof5 showed that it was highly expressed in the phloem of 6-month-old E. grandis, the stem tip, and the xylem of 3-year-old E. grandis Figure 2 During the development of stem segments, the expression of EgrDof5 showed a gradient increase trend from young to mature (3-11 segments) Figure 2 , which was highly consistent with the strengthening trend of secondary growth during wood development. This expression pattern indicated that EgrDof5 was directly involved in the stem development and xylem formation of E. grandis, and was a key candidate gene for regulating biomass accumulation in E. grandis.

[0042] 4. Response analysis of EgrDof genes in E. grandis to abiotic stress and hormone treatment

[0043] The 2-month-old E. grandis was subjected to boron deficiency, phosphorus deficiency, salt stress, and spraying of salicylic acid (SA) and jasmonic acid (JA) using online RNA-Seq analysis. As shown in Figure 3 , the results showed that:

[0044] (1) Hormone response: After SA or JA treatment, the expression of EgrDof5 showed significant differences at different time points, with a 2.3-fold increase at 6h after JA treatment and a 0.4-fold decrease at 24h after SA treatment, indicating that it could accurately respond to hormone signals, which was consistent with the expression characteristics of growth and development regulation genes;

[0045] (2) Nutrient stress response: Under phosphorus deficiency, the expression 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;

[0046] (3) Salt stress response: The expression of EgrDof5 significantly increased (3.1 times of the control) at 168h under salt stress, while its expression decreased under SA treatment at the same time, indicating that EgrDof5 could coordinate hormone signals and stress signals, and played a core role in maintaining the balance between plant growth and stress adaptation.

[0047] II. Introduction of EgrDof5 gene into poplar

[0048] 1. Construction of overexpression vector pK2GW7-EgrDof5

[0049] Specific primers were designed according to the annotation information of 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 a forward primer and SEQ ID No. 3 is a reverse primer. Then, the CDS of EgrDof5 was efficiently transferred to the plant expression vector pK2GW7 by LR recombination reaction. After Spe resistance screening and colony PCR verification, the recombinant vector pK2GW7-EgrDof5 was successfully constructed.

[0050] 2. Plant genetic transformation and detection of positive seedlings of transgenic poplar plants

[0051] The 84K poplar tissue culture seedlings grown for 30 d 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 subjected to photoperiod culture in differentiation screening medium to obtain resistant buds (B). 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, and OE-29) with significantly high expression were screened and used for subsequent research.

[0052] III. Effect of EgrDof5 gene on poplar

[0053] 1. Effect of EgrDof5 gene on growth and development of poplar

[0054] The growth of transgenic poplar was compared, and the growth data of poplar at different time periods were recorded, as shown in Table 1. Figure 5 By comparing the morphological characteristics of wild-type poplar (WT) and EgrDof5 gene overexpression strains (OE-3, OE-17, and OE-29), it was found that the OE-3 strain had the most significant increase in plant height (A, C) among the poplars planted for 80 d. Figure 5 The continuously measured growth index records showed that the crown expansion of the EgrDof5 overexpression poplar strain was better than that of the wild-type poplar (B), and the plant height was significantly higher than that of the wild-type (C). Figure 5 Figure 5 ​C). Meanwhile, the ground diameter of the OE-29 line after 30 days of growth was significantly larger than that of the wild type, while the ground diameter of transgenic poplars at other time points showed varying degrees of increase compared to the wild type. Figure 5 D).

[0055] 2. Effects of the EgrDof5 gene on poplar leaves

[0056] The leaf area and diameter of the transgenic lines showed a synergistic growth trend. When the plants stabilized after 80 days of planting, the leaves were photographed for data recording. Figure 6 As shown, the leaf area and leaf diameter of the three poplar lines overexpressing the EgrDof5 gene were generally higher than those of the wild-type poplar, with the OE-3 line showing the most significant increase in leaf area.

[0057] 3. Effects of EgrDof5 on poplar stem nodes

[0058] Measurements of the 7th and 14th stem nodes of poplars grown for 80 days revealed that the stem node length of the overexpression line was greater than that of the wild type. Figure 7 A). 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.

[0059] 4. Effects of EgrDof5 on poplar root system

[0060] 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).

[0061] IV. Effects of the EgrDof5 gene on the photosynthetic performance of poplar

[0062] from Figure 9It can be seen that the net photosynthetic rate of transgenic plants OE-3 and OE-17 is significantly higher than that of wild type, which is increased by 14% and 13% respectively, while OE-29 has no obvious change Figure 9 A). In terms of transpiration rate, the transpiration rate of OE-3, OE-17 and OE-29 transgenic poplar lines is significantly higher than that of wild type poplar Figure 9 B), which is increased by 36%, 28% and 25% respectively, among which OE-3 transgenic line is the most obvious. The change of stomatal conductance is consistent with the trend of transpiration rate Figure 9 C), the stomatal conductance of OE-3, OE-17 and OE-29 transgenic poplar lines is increased by 25%, 18% and 15% respectively. The intercellular CO2 concentration of transgenic poplar is lower than that of wild type poplar, among which the decrease of OE-29 line is the most obvious, which is decreased by 19%, and the decrease of OE-3 and OE-17 is 16% and 14% respectively Figure 9 D).

[0063] The correlation analysis of four photosynthetic parameters of poplar is carried out (Table 1). The results show that Pn is positively correlated with Gs and Tr, and the correlation coefficients are 0.759 and 0.714 respectively, and is negatively correlated with Ci. The correlation between Gs and Tr is very high, and the correlation coefficient is 0.996, and is significantly correlated at 0.01 significance level. In addition, Gs and Tr are negatively correlated with Ci, and the correlation coefficients are-0.874 and-0.913 respectively.

[0064] Table 1 Correlation analysis of photosynthetic parameters of transgenic poplar EgrDof5

[0065] P n ]]> G s ]]> [TECHNICAL FIELD] r ]] [C i ]]> P n ]]> 1 Gs 0.759 1 [CAT r ]]> 0.714 0.996** 1 [C i ]]> -0.392 -0.874 -0.913 1

[0066] Note: ** represents 0.01 significant level.

[0067] V. Effect of EgrDof5 gene on secondary growth of poplar

[0068] The 7th and 14th stem nodes were selected for toluidine blue staining analysis. The results show that the xylem of transgenic plants has been connected into a ring at the 7th stem node, while WT has not been xylemized into a ring. Quantitative analysis of the staining images of the 14th internode sections by ImageJ software shows that the xylem of poplar overexpressing EgrDof5 gene is wider than that of WT Figure 10 A, D). At the same time, it is found that the fiber cell wall thickness of transgenic plants is thinner, and the fiber cell area is significantly larger Figure 10 B, E). The observation of the staining of the longitudinal section of the fourteenth stem node of poplar shows that the cell diameter of the transgenic poplar is wider and the cell area is larger than that of the wild type Figure 10 ​(C, F). This demonstrates that the EgrDof5 gene plays a regulatory role in the secondary growth of poplar stems.

[0069] To further verify the effect of the EgrDof5 gene on the biosynthesis of the secondary cell wall in 84K poplar, the lignin content and monosaccharide content of transgenic poplar were measured, and the results are shown in Table 2. Plants overexpressing the EgrDof5 gene showed increased levels of both acid-insoluble and acid-soluble lignin in their stems, with total lignin content increasing by 3.30%, 4.07%, and 5.2% respectively compared to wild-type plants. Furthermore, the cellulose content of plants overexpressing the EgrDof5 gene was significantly increased, increasing by 15.02%, 15.5%, and 17.18% compared to wild-type, while xylan and arabinose showed no significant difference compared to wild-type.

[0070] Table 2. Detection of secondary cell wall components in transgenic and wild-type plants.

[0071] 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

[0072] 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.

[0073] VI. Infrared Spectroscopic Analysis of EgrDof5 Transgenic Poplar

[0074] Fourier transform infrared spectroscopy was used to conduct an in-depth analysis of the structure of EgrDof5 transgenic poplar.

[0075] 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 -1The absorption peaks at 1050 cm -1 The absorption peaks at 1506 cm -1 The absorption peaks at 1740 cm -1 The absorption peaks at 1740 cm

[0076] Seven, Summary

[0077] EgrDof5 gene can improve the biomass of poplar. The plant height, ground diameter, leaf area and crown width of poplar overexpressing EgrDof5 gene are higher than those of wild type poplar, among which the OE-3 strain changes most obviously; and the length of the 7th stem node is significantly higher than that of the wild type, and the diameter of the 14th stem node of OE-17 and OE-29 strains is also significantly improved; the root system data statistics show that the root length and surface area of OE-3 strain are significantly increased.

[0078] EgrDof5 gene can improve the photosynthetic performance of poplar. Overexpression of EgrDof5 gene can improve the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of poplar, and reduce the intercellular CO2 concentration (Ci). Correlation analysis shows that the Pn of transgenic poplar is positively correlated with Gs and Tr, and negatively correlated with Ci, and Gs is extremely significantly positively correlated with Tr.

[0079] EgrDof5 gene can regulate the secondary growth of poplar. EgrDof5 gene plays an important role in the regulation of cell wall components of poplar. The lignification process of overexpression of EgrDof5 plants is advanced, and the secondary xylem of the 14th stem node of transgenic plants is widened, the fiber cell area is significantly increased, and the cell wall thickness is thinned. The total lignin content of transgenic strains is increased, and the cellulose content is increased. Joint Fourier transform infrared spectroscopy analysis shows that the S-type lignin is increased and the cellulose structure is changed. These changes will enhance the mechanical strength and anti-degradation ability of cell wall, thereby affecting the growth and development of poplar.

[0080] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and 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, including at least one of the following: increase plant height, increase leaf area, increase stem internode length, and increase root length; b. Improve the photosynthetic performance of poplar trees, including at least one of the following: increase transpiration rate, increase stomatal conductance, and reduce intercellular CO2 concentration; c. Regulate the secondary growth of poplar trees, including at least one of increasing the fiber cell area and increasing the longitudinal section cell diameter; The nucleotide sequence of the Eucalyptus macrocarpa EgrDof5 gene is shown in SEQ ID No. 1; The application 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.

2. The application of the Eucalyptus macrocarpa EgrDof5 gene in poplar as described in claim 1, 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.

3. The application of the Eucalyptus giantis EgrDof5 gene in poplar as described in claim 2, 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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