Application of PdeMYB73 gene in improving growth performance and / or waterlogging stress resistance in poplars

Abstract

The present invention belongs to the field of molecular biology breeding technology, and in particular relates to the application of the PdeMYB73 gene in improving the growth performance and / or waterlogging stress resistance of poplars. The nucleotide sequence of the PdeMYB73 gene is shown in SEQ ID NO.1. The present invention cloned the poplar PdeMYB73 gene and explored the role and response mechanism of the gene in poplar growth regulation and waterlogging stress resistance. It was found that the gene has a positive regulatory effect on the growth and waterlogging stress resistance of poplars, providing an excellent gene resource for improving the growth performance and waterlogging stress resistance of poplars, and providing a new solution for cultivating new fast-growing and waterlogging-resistant poplar varieties. It has broad application prospects and good social benefits in forestry production such as the improvement of excellent forest tree stress-resistant varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological breeding, and in particular to application of the PdeMYB73 gene in improving the growth performance and / or waterlogging stress resistance of poplars. Background Art

[0002] Waterlogging stress can occur in various ecosystems such as river floodplains, swamps, and wetlands. Excessive accumulation of water in these areas will cause a sharp decrease in the absorption of most mineral nutrients in the soil by plants, weakening the growth and development of new roots and shoots. Moreover, under waterlogging stress, the oxygen content in the rhizosphere of plants will decrease rapidly, causing the plant root system and / or leaf tissue to present a low oxygen or anoxic state, thereby limiting the plant's aerobic respiration, inhibiting the plant's photosynthesis and nutrient uptake, and hindering its normal life activities. In addition, the low oxygen or anoxic state caused by accumulated water will cause the lipid peroxidation of plant tissues to increase, the protective enzyme system in the body to be damaged, and then lead to the accumulation of toxic substances. These factors will cause harm to the growth and development of plants and pose a serious threat to the survival of plants. Poplar (Populus) has the characteristics of fast growth, easy reproduction and wide application, and has become an important afforestation tree species worldwide with important economic and ecological value. In production practice, waterlogging remains the primary stress facing poplar trees and has become one of the main factors contributing to their poor growth. The resulting losses in timber production and ecological function may exceed those caused by all other factors. Furthermore, tree height and growth rate are key factors influencing poplar timber production. Therefore, enhancing poplar growth rates under normal conditions and their resilience to waterlogging is crucial for the sustainable development of the poplar industry.

[0003] There is an urgent need to develop new fast-growing, flood-resistant poplar varieties. However, traditional forest breeding relies on morphological selection, which presents numerous challenges, including long breeding cycles and difficulty in selection. With the advancement of molecular biology, the use of genetic engineering techniques to cultivate new fast-growing, flood-resistant forest germplasm has broad prospects. Understanding poplar adaptation mechanisms to flooding stress and identifying flood-resistance genes are crucial prerequisites for developing highly resilient and flood-tolerant poplar varieties. Summary of the Invention

[0004] To address the above-mentioned technical problems in the prior art, the present invention provides a PdeMYB73 gene isolated from poplar trees. This gene has a positive regulatory effect on the growth and waterlogging stress resistance of poplar trees, providing an excellent genetic resource for improving poplar growth performance and waterlogging stress resistance, and a new solution for breeding new fast-growing and waterlogging-resistant poplar varieties. It has broad application prospects and good social benefits in forestry production, such as improving high-quality, stress-resistant varieties of forest trees. The present invention is specifically implemented through the following technical solutions:

[0005] In a first aspect, the present invention provides an application of the PdeMYB73 gene in regulating the growth performance of poplar trees. The nucleotide sequence of the PdeMYB73 gene is shown in SEQ ID NO.1.

[0006] A second aspect of the present invention provides the use of the PdeMYB73 gene in improving the resistance of poplars to waterlogging stress. The nucleotide sequence of the PdeMYB73 gene is shown in SEQ ID NO.1.

[0007] The third aspect of the present invention provides a breeding method for fast-growing and / or flood-resistant poplars, comprising the following steps: constructing a PdeMYB73 gene overexpression vector, transforming wild-type poplars, and cultivating transgenic poplars with enhanced PdeMYB73 gene expression.

[0008] The advantages and positive effects of the present invention are:

[0009] The present invention cloned the poplar PdeMYB73 gene and explored the role and response mechanism of the gene in poplar growth regulation and resistance to waterlogging stress. The results showed that after the expression level of the PdeMYB73 gene in poplar was increased, its growth rate was significantly accelerated, and in the waterlogging stress experiment, the overexpression transgenic strain was significantly enhanced compared to the wild type and PdeMYB73 gene suppression expression type plants, which can improve the growth and / or survival ability of plants under waterlogging stress conditions and alleviate the damage of waterlogging stress to plants. It was confirmed that the PdeMYB73 gene plays a key role in regulating poplar growth and improving its waterlogging resistance. The PdeMYB73 gene provided by the present invention can be applied to the breeding of new varieties of fast-growing and waterlogging-resistant poplars and variety improvement, and has broad application prospects and good social benefits in forestry production such as the improvement of high-quality forest tree stress-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0011] Figure 1 This is a diagram showing the difference in seedling height between wild-type and transgenic poplars at 30 days after tissue culture subculture in an embodiment of the present invention;

[0012] Figure 2 This is a diagram showing the difference in seedling height between wild-type and transgenic poplars at 60 days after transplantation of tissue culture seedlings according to an embodiment of the present invention;

[0013] Figure 3 Graphs showing the height phenotype observation and growth measurement results of wild-type and transgenic poplar seedlings at 60-80 days after transplantation of tissue-cultured seedlings according to an embodiment of the present invention, wherein graphs AD represent seedling height, ground diameter, seedling height growth, and ground diameter growth, respectively;

[0014] Figure 4 The following are pictures of leaves of wild-type and transgenic poplars and bar graphs of relative chlorophyll content according to the embodiments of the present invention;

[0015] Figure 5 Figure 4 is a graph showing the leaf number and internode growth curves of wild-type and transgenic poplars at different cultivation times according to an embodiment of the present invention, wherein Figure A shows the leaf number, Figure B shows the average internode length, and Figures C and E show the internode lengths of different internodes at 60, 70, and 80 days, respectively;

[0016] Figure 6 The bar graphs are of the net photosynthetic rates of wild-type and transgenic poplars at different cultivation times according to the embodiment of the present invention, wherein Figures A to C are for 60 days, 70 days, and 80 days, respectively;

[0017] Figure 7 This is a bar graph showing the lignin, cellulose, and hemicellulose contents of wild-type and transgenic poplars according to an embodiment of the present invention;

[0018] Figure 8 This is a bar graph showing the indoleacetic acid content in roots, stems, and leaves of wild-type and transgenic poplars according to an embodiment of the present invention;

[0019] Figure 9 This is a bar graph showing the gibberellin content in roots, stems, and leaves of wild-type and transgenic poplars according to an embodiment of the present invention;

[0020] Figure 10 This is a bar graph showing the cytokinin content in roots, stems, and leaves of wild-type and transgenic poplars according to an embodiment of the present invention;

[0021] Figure 11 The microscopic images of the longitudinal sections of the stem tips of wild-type and transgenic poplars in the examples of the present invention are shown;

[0022] Figure 12 This application example is a statistical graph of stem tip cell length of wild-type and transgenic poplars according to an embodiment of the present invention;

[0023] Figure 13 The phenotypes of wild-type and transgenic poplars after waterlogging stress treatment in the examples of the present invention are shown in FIG.

[0024] Figure 14 This is a bar graph showing the growth of seedling height and ground diameter of wild-type and transgenic poplars after waterlogging stress treatment in an embodiment of the present invention;

[0025] Figure 15 This is a graph showing the change in net photosynthetic rate of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0026] Figure 16 This is a graph showing the changing patterns of transpiration rates of wild-type and transgenic poplars under waterlogging stress treatment for different periods of time according to an embodiment of the present invention;

[0027] Figure 17 This is a graph showing the stomatal conductance changes of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0028] Figure 18 This is a graph showing the changing pattern of the ratio of intercellular CO2 concentration to ambient CO2 concentration of wild-type and transgenic poplars under waterlogging stress treatment at different times according to an embodiment of the present invention;

[0029] Figure 19 This is a graph showing the changing regularity of the instantaneous water use efficiency of wild-type and transgenic poplars under waterlogging stress treatment at different times according to an embodiment of the present invention;

[0030] Figure 20 This is a graph showing the change in maximum photochemical efficiency of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0031] Figure 21 This is a graph showing the change pattern of PSⅡ potential activity of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0032] Figure 22 This is a graph showing the change in effective photochemical quantum yield of PSⅡ of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0033] Figure 23 This is a graph showing the change in actual photochemical efficiency of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0034] Figure 24 This is a graph showing the change in photochemical quenching of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0035] Figure 25 This is a graph showing the changes in non-photochemical quenching of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0036] Figure 26 This is a graph showing the change in apparent electron transfer rate of wild-type and transgenic poplars under waterlogging stress treatment for different time periods according to an embodiment of the present invention;

[0037] Figure 27 This is a graph showing the change in relative chlorophyll content of wild-type and transgenic poplars under waterlogging stress treatment at different times according to an embodiment of the present invention;

[0038] Figure 28 This is a bar graph showing the malondialdehyde content in leaves and fine roots of wild-type and transgenic poplars under waterlogging stress treatment at different times according to an embodiment of the present invention;

[0039] Figure 29This is a bar graph showing root activity of wild-type and transgenic poplars under waterlogging stress treatment at different times according to an embodiment of the present invention;

[0040] Figure 30 This is a cluster analysis result of waterlogging resistance of wild-type and transgenic poplars under waterlogging stress treatment at different times according to an embodiment of the present invention. DETAILED DESCRIPTION

[0041] To make the objects, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. The examples described herein are intended only to illustrate the present invention and are not intended to limit the present invention. All numbers used in the present invention to express amounts, percentages, and other numerical values ​​should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values ​​and may vary depending on the desired properties to be achieved. Each numerical parameter should at least be considered to be based on the reported significant figures and obtained by conventional rounding methods. The meaning of "comprising," "including," "containing," "having," and similar words is non-restrictive, and other steps and other ingredients that do not affect the results can be added. "And / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0042] In order to make the purpose and features of the present invention more obvious and easy to understand, the technical principles and specific implementation methods of the present invention are described in detail below.

[0043] Water conditions are one of the key environmental factors for plant growth and are also one of the main abiotic stresses to which plants are subjected. Waterlogging stress refers to the phenomenon in which excessive soil moisture or prolonged exposure to waterlogging during plant growth, resulting in excessive water and insufficient oxygen supply, limits the plant's aerobic respiration and energy production required to sustain life activities, leading to physiological dysfunction and stunted growth and development. Excessive water in the soil generally occurs in two states: one is when the soil moisture exceeds its maximum water holding capacity and is saturated, with the gas phase of the soil completely replaced by the liquid phase. This is known as "waterlogging," also known as waterlogging damage. The other is when the water not only fills the soil but also fills the ground, submerging parts of the plant or the entire plant, commonly referred to as "waterlogging damage."

[0044] Under long-term waterlogging stress, plants have evolved various mechanisms to cope with it. Transcription factors are protein molecules in eukaryotes that can bind to cis-acting elements in gene promoters. By enhancing or inhibiting the expression of target genes at specific intensities and in specific time and space, they regulate the expression of downstream genes. When plants are subjected to adverse stress, transcription factors rapidly participate in the regulation of many important processes, such as activating related enzyme systems within the cell, participating in hormone secretion, affecting cellular metabolic activity and signal transduction. At the same time, they can also act as special messenger molecules to participate in various physiological and biochemical reactions, thereby regulating plant growth and development and coping with abiotic stress.

[0045] The present invention isolated and cloned a PdeMYB73 gene involved in regulating poplar growth rate and waterlogging stress adaptability from wild Nanlin 895 poplar. The CDS sequence of the PdeMYB73 gene is shown in SEQ ID NO. 1, with a total length of 924 bp.

[0046] The PdeMYB73 gene was the target of research. Overexpression and suppression expression vectors were constructed and transformed into wild-type poplars, resulting in PdeMYB73 functional disruption mutants and overexpression lines. The growth performance of the transgenic and wild-type poplars was evaluated, revealing differences in growth among the different lines. The PdeMYB73 overexpression lines showed significantly higher growth indicators than the wild-type plants, including seedling height, stem elongation, root, stem, and leaf biomass, and total biomass. Photosynthetic parameters such as chlorophyll content and net photosynthetic rate, as well as levels of hormones related to cell elongation (auxin, cytokinin, and gibberellin), were also significantly higher in the wild-type plants than in the PdeMYB73 suppression lines. This suggests that the PdeMYB73 overexpression lines exhibited faster growth and improved photosynthetic performance, which is beneficial for their rapid growth and biomass accumulation. The above results fully highlight the importance of the PdeMYB73 gene in regulating poplar growth performance, and provide a theoretical basis for analyzing how the PdeMYB73 gene regulates poplar growth rate.

[0047] The waterlogging resistance of transgenic and wild-type poplar plants was further evaluated. They were treated with waterlogging using the hydroponic method. The effects of waterlogging stress on different strains were compared by measuring their phenotypic indicators combined with physiological indicators. It was found that under waterlogging stress, multiple growth indicators such as root activity, seedling height, ground diameter, leaf growth phenotype, internode length, and plant biomass accumulation were inhibited. In addition, the photosynthesis of the plants was significantly inhibited, and their chlorophyll content, leaf gas exchange parameters (including net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), intercellular CO2 concentration / ambient CO2 concentration (Ci / Ca), instantaneous water use efficiency (R) and so on) were significantly inhibited. WUEi)) and chlorophyll fluorescence parameters (including maximum photochemical efficiency (Fv / Fm), potential PSⅡ activity (Fv / Fo), effective PSⅡ photochemical quantum yield (Fv' / Fm'), actual photochemical efficiency (ФPSⅡ), photochemical quenching (qP), non-photochemical quenching (NPQ), and apparent electron transport rate (ETR)) all showed a downward trend under waterlogging stress. Each parameter was negatively correlated with stress duration. Increased levels of lipid peroxidation products, such as malondialdehyde, indicated that the plants were damaged by hypoxia caused by waterlogging. However, overall, the inhibitory effect on the growth of the PdeMYB73-overexpressing lines was significantly less than that of wild-type plants and PdeMYB73-repressed lines. The PdeMYB73-overexpressing lines exhibited higher growth, biomass accumulation, stronger photosynthetic adaptability, and photochemical conversion efficiency under waterlogging, indicating that the growth inhibition was least affected by waterlogging. Furthermore, during drainage, the aforementioned parameters recovered more quickly, indicating a good ability to recover from waterlogging. It can be seen that overexpressing the PdeMYB73 gene is beneficial for enhancing and alleviating plant damage symptoms under flooding stress conditions, reducing leaf wilting, death, and shedding, maintaining a high photosynthetic rate and normal photosynthetic characteristics, and improving the ability to resist oxidative damage under flooding stress, thereby increasing plant growth rate and survival under flooding stress, and enhancing plant resistance to flooding stress. Therefore, the present invention also confirms the key role of the PdeMYB73 gene in coping with plant response to flooding stress, providing a theoretical basis for analyzing the PdeMYB73 gene's regulation of poplar waterlogging resistance.

[0048] In summary, this study uncovers the positive regulatory role of the PdeMYB73 gene in regulating height growth and waterlogging resistance in poplars, laying a foundation for further elucidating the molecular mechanisms underlying these traits. Furthermore, it provides excellent genetic resources for improving poplar growth performance and waterlogging stress resistance, and offers a new approach for breeding new fast-growing, waterlogging-resistant poplar varieties. Furthermore, as a woody model plant, the discovery and functional characterization of poplar's excellent genetic resources will also provide a reference for related research in other plant species.

[0049] Based on this, one embodiment of the present invention provides the use of the PdeMYB73 gene in regulating the growth performance of poplar.

[0050] The PdeMYB73 gene has a positive regulatory effect on improving the growth performance of poplar trees. By enhancing the expression level of the PdeMYB73 gene through genetic engineering breeding, the growth and development capabilities of poplar trees can be enhanced by increasing their growth rate and photosynthetic capacity, providing a new breeding strategy for improving poplar growth performance and increasing their growth rate. By inhibiting the expression level of the PdeMYB73 gene, the growth and development of plants can be inhibited, providing a target and direction for cultivating dwarf plants. The PdeMYB73 gene provided by the present invention has broad application prospects and good social benefits in improving high-quality forest tree varieties.

[0051] Optionally, the poplar growth performance includes at least one of the following (1)-(6): (1) seedling height or ground diameter growth; (2) root, stem or leaf biomass; (3) stem tip length or internode length; (4) net photosynthetic rate; (5) lignin, cellulose or hemicellulose content; (6) indoleacetic acid, gibberellin or cytokinin content.

[0052] Optionally, the regulatory effect described above includes positive regulation or negative regulation. By increasing the expression level of the PdeMYB73 gene, positive regulation of plant growth performance can be achieved; by reducing the expression level of the PdeMYB73 gene, negative regulation of plant growth performance can be achieved. It should be noted that the expression level of the PdeMYB73 gene includes the expression level at the gene level (mRNA) and / or the expression level at the protein level. The gene level can be achieved by RNA interference technology, CRISPR-Cas9 technology or overexpression vectors, and the protein level can be achieved by protein activators or inhibitors. This is a conventional technical means in the art and will not be described in detail here.

[0053] Specifically, the positive regulation of poplar growth performance includes the following steps: constructing a PdeMYB73 gene overexpression vector, transforming wild-type poplars, and cultivating transgenic poplars with enhanced PdeMYB73 gene expression. More specifically, the construction of the PdeMYB73 gene overexpression vector includes the following steps: using poplar cDNA as a template, using the PdeMYB73-OE-F and PdeMYB73-OE-R primer pairs for PCR amplification to obtain the CDS sequence of the PdeMYB73 gene, and inserting the PdeMYB73 gene sequence into the pKGW-RR-MGW expression vector using gateway gene cloning technology to construct the PdeMYB73 gene overexpression vector; wherein the nucleotide sequences of PdeMYB73-OE-F and PdeMYB73-OE-R are shown in SEQ ID NOs. 2-3, respectively.

[0054] Specifically, negatively regulating poplar growth performance includes the following steps: constructing a PdeMYB73 gene inhibition expression vector, transforming wild-type poplars, and cultivating transgenic poplars with reduced PdeMYB73 gene expression. More specifically, constructing the PdeMYB73 gene inhibition expression vector includes the following steps: using poplar cDNA as a template, using the PdeMYB73-RE-F and PdeMYB73-RE-R primer pairs for PCR amplification to obtain a PdeMYB73 gene RNAi sequence, and inserting the RNAi sequence into the pK7GWIWG2(II) expression vector using gateway gene cloning technology to construct the PdeMYB73 gene inhibition expression vector; wherein the nucleotide sequences of PdeMYB73-RE-F and PdeMYB73-RE-R are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequence of RNAi is shown in SEQ ID NO.6.

[0055] Based on the same inventive concept, an embodiment of the present invention also provides the use of the PdeMYB73 gene in improving the resistance of poplar to flooding stress.

[0056] The PdeMYB73 gene has a positive regulatory effect on improving the resistance of poplar to flooding stress. By enhancing the expression level of the PdeMYB73 gene through genetic engineering breeding, the damage to plants caused by adversity can be reduced, and leaf wilting, death and shedding, as well as oxidative damage and photoinhibition in plants under flooding stress conditions can be alleviated. It significantly improves the waterlogging tolerance of plants and enhances the growth and / or survival ability of plants under flooding stress conditions. This provides a new method for breeding waterlogging-resistant poplar varieties using genetic engineering technology and has important application potential.

[0057] Optionally, the resistance of the poplar to flooding stress includes at least one of the following (1)-(5): (1) seedling height or ground diameter growth under flooding stress; (2) leaf gas exchange parameters under flooding stress, the leaf gas exchange parameters including at least one of net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration / ambient CO2 concentration; (3) chlorophyll fluorescence parameters under flooding stress, the chlorophyll fluorescence parameters including at least one of maximum photochemical efficiency, PSⅡ potential activity, PSⅡ effective photochemical quantum yield, actual photochemical efficiency, photochemical quenching coefficient, non-photochemical quenching coefficient and apparent electron transfer rate; (4) leaf chlorophyll content or malondialdehyde content under flooding stress; (5) root activity under flooding stress.

[0058] The method for improving the resistance of poplar trees to flooding stress is basically the same as the method for positively regulating plant growth performance, and will not be described in detail here.

[0059] Optionally, the poplar tree mentioned above is Nanlin 895 poplar.

[0060] The embodiment of the present invention also provides a breeding method for fast-growing and / or flood-resistant poplars, comprising the following steps: constructing a PdeMYB73 gene overexpression vector, transforming wild-type poplars, and cultivating transgenic poplars with enhanced PdeMYB73 gene expression.

[0061] In a typical embodiment, constructing a PdeMYB73 gene overexpression vector includes the following steps: using poplar cDNA as a template, PCR amplification is performed using PdeMYB73-OE-F and PdeMYB73-OE-R primer pairs to obtain the CDS sequence of the PdeMYB73 gene, and inserting the PdeMYB73 gene sequence into the pKGW-RR-MGW expression vector through gateway gene cloning technology to construct the PdeMYB73 gene overexpression vector; wherein the nucleotide sequences of PdeMYB73-OE-F and PdeMYB73-OE-R are shown in SEQ ID NO.2-3, respectively.

[0062] Transformation of host cells with expression vectors can be performed using conventional techniques familiar to those skilled in the art. Vectors carrying the PdeMYB73 gene can be introduced into poplar cells or tissues using DNA transfection methods such as calcium phosphate co-precipitation, Ti plasmids, Ri plasmids, viral vectors, gene guns, microinjection, electroporation, or Agrobacterium-mediated transfection.

[0063] The present invention will be further described below with reference to specific examples. The experimental methods in the following examples, where specific conditions are not specified, generally follow conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or the conditions recommended by the manufacturer. In addition, the materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0064] Main biological materials: The plant genetic transformation material is the wild-type Populus × euramericana 'Nanlin 895'. The competent Escherichia coli is DH5α, purchased from Beijing Qingke Biological Co., Ltd. The competent Agrobacterium tumefaciens is the nopaline type EHA105, purchased from Shanghai Weidi Biotechnology Co., Ltd. The plant overexpression vector is pKGW-RR-MGW, and the relevant information is disclosed in the literature "Evgenia Ovchinnikova, Etienne-Pascal Journet, Mireille Chabaud, Viviane Cosson, Pascal Ratet, Gerard Duc, Elena Fedorova, Wei Liu, Rik Op den Camp, Vladimir Zhukov, Igor Tikhonovich, Alexey Borisov, Ton Bisseling, Erik Limpens. IPD3 Controls the Formation of Nitrogen-Fixing Symbiosomes in Pea and Medicago Spp. Molecular Plant-Microbe Interactions, 2011, 24(11): 1333-1344. doi: 10.1094 / MPMI-01-11-0013”, the plant inhibitory expression vector is pK7GWIWG2(II) containing the dsRed selection marker, purchased from Kelei Biotechnology Co., Ltd., product access link: http: / / www.kelei-biology.com / m / view.php?aid=983.

[0065] Genes and Primers: The PdeMYB73 gene was cloned from Nanlin 895 Poplar (a member of the Populus deltoides family) by designing primers with reference to the genome sequence of Populus deltoides. Since this gene has not been reported in Populus deltoides, a BLAST-P analysis was performed in Arabidopsis thaliana, and the most homologous gene was found to be AtMYB73. The newly cloned gene in this application was named PdeMYB73. The PdeMYB73 gene and related primer sequences involved in the embodiments of the present invention are shown in Table 1.

[0066] Table 1 DNA sequence of PdeMYB73 gene and related primer sequences in the embodiment of the present invention

[0067]

[0068]

[0069] Example

[0070] 1. Construction of transgenic poplars transformed with the PdeMYB73 gene

[0071] 1.1. Construction of PdeMYB73 gene overexpression and suppression vectors

[0072] The primer pairs PdeMYB73-OE-F / PdeMYB73-OE-R and PdeMYB73-RE-F / PdeERF53-RE-R were designed with reference to the genome sequence of Populus deltoides (access link: https: / / phytozome-next.jgi.doe.gov / info / PdeltoidesWV94_v2_1) and were used to amplify the full-length CDS sequence (924 bp, see SEQ ID NO. 1) and RNA interference (RNAi) sequence (243 bp, see SEQ ID NO. 6) of the PdeMYB73 gene, respectively, for construction of the PdeMYB73 overexpression vector (OE) and repression expression vector (RE).

[0073] Nanlin 895 poplar leaves were used as materials to extract mRNA, and the mRNA was reverse transcribed into cDNA using the reverse transcription kit HifairШ1st StrandcDNA Synthesis Super Mix (purchased from Shanghai Yisheng Biological, cat. no. 11137ES). Using cDNA as a template, PCR amplification was performed using the above primer pairs to obtain the target gene fragments PdeMYB73-CDS and PdeMYB73-RNAi. By gateway gene cloning technology, the target gene fragments PdeMYB73-CDS and PdeMYB73-RNAi were obtained, respectively. BP Clonase TM II Enzyme Mix (purchased from Hangzhou Bailibo, product number 11789020) and LR Clonase TM Under the catalysis of II Enzyme Mix (purchased from Hangzhou Bailibo, product number 11791020), the full-length PdeMYB73 gene was cloned into the overexpression vector pKGW-RR-MGW through BP and LR recombination reaction; the RNAi interference fragment of the PdeMYB73 gene was cloned into the inhibition expression vector pK7GWIWG2 (II).

[0074] 1.2. Agrobacterium-mediated PdeMYB73 gene overexpression and suppression vector transformation in poplar

[0075] The overexpression vector and the suppression expression vector carrying the PdeMYB73 gene were transformed into Agrobacterium tumefaciens EHA105 respectively by electroporation, and the young leaves of Nanlin 895 poplar that were about 30 days old and strong were selected as explants for Agrobacterium infection. The specific experimental steps are as follows: 1) Activation and culture of bacterial strains: Take the EHA105 strain stored at -80°C, melt it on ice, dip a small amount of bacterial liquid on the LB medium containing the corresponding antibiotics (spectinomycin (Spe) + rifampicin (Rif)), and culture it upside down in the dark at 28°C for 2-3 days; pick a single colony and inoculate it into SOB liquid medium containing Spe and Rif resistance, and culture it in the dark at 200r / min and 28°C until the OD 600 =0.5-0.8; 2) Material collection and infection: Select tissue culture seedlings that have been rooted for 30 days, take the 3rd to 5th dark green, flat leaves from top to bottom, the size, shape and color of the leaves are basically the same, cut off the leaf edges, and cut into 2-3 cm 2 The obtained bacterial solution was centrifuged at room temperature for 10 min at 5000 r / min, the supernatant was discarded, and the precipitate was resuspended in 50-75 mL of resuspension solution (WPM without agar) to an OD of 600 =0.5-0.8, and then infect for 10-15 min; 3) Co-cultivation: Remove the leaves from the infection solution, place them on sterile filter paper to absorb the infection solution remaining on the surface, and spread the leaves with the back facing down on the culture medium (WPM + 100 μM AS), and culture them in the dark at 24°C for 2 days; 4) Induction of callus: Transfer the co-cultivated leaves to the culture medium for inducing callus differentiation (WPM + 1 mg / L 2,4-D + 0.5 mg / L KT + 300 mg / L Cef + 50 mg / L Kan + 300 mg / L TMT), and culture them in the dark at 24°C. The culture medium was changed every 15 days to obtain leaves with callus tissue; 5) Induction of buds: Place callus tissue (about 1 cm in size) on the stalk of the leaf. 2 ) were excised and transferred to a bud differentiation induction medium (WPM + 0.02 mg / L TDZ + 300 mg / L Cef + 50 mg / L Kan + 300 mg / L TMT), cultured at 24°C under light, and the medium was replaced every 15 days until adventitious buds grew on the surface of the callus tissue; 6) Bud elongation induction: when the buds differentiated from the leaves grew to 1 cm in length, the adventitious buds were cut and transferred to a bud elongation medium (WPM + 300 mg / L Cef + 50 mg / L Kan) for culture, cultured at 24°C under light, and the medium was replaced every 15 days; 7) Root induction: resistant seedlings with stem segments longer than 3 cm in the bud elongation medium were excised and placed in a rooting medium (WPM + 250 mg / L Cef + 50 mg / L Kan), cultured at 24°C under light, and the medium was replaced every 30 days.

[0076] 1.3. Identification of PdeMYB73 transgenic positive plants

[0077] The PdeMYB73 transgenic positive seedlings were screened by detecting the expression of red fluorescent protein in the plants and combining PCR to detect whether the genomic DNA of the transgenic resistant plants contained the target fragment. The PCR screening steps were as follows: DNA of the overexpression and suppression expression transgenic resistant plants was amplified by specific primers, with PdeMYB73 gene overexpression and suppression expression vector plasmids as positive controls, wild-type Nanlin 895 poplar leaf DNA as negative controls, and ddH2O as blank controls. The PCR reaction system was calculated as 10 μL and contained: 0.5 μL each of forward and reverse primers, 2×Hieff TM PCR Master Mix (purchased from Shanghai Yisheng Biotechnology, Cat. No. 10102ES08) (5 μL), template (3 μL), and ddH2O (balance). The PCR protocol for the DsRed-F1 / AtUBQ10-R1 primer pair was as follows: 94°C denaturation for 5 min; 30 cycles of 94°C denaturation for 30 s, 59°C annealing for 30 s, and 72°C extension for 90 s; 72°C extension for 5 min, followed by storage at 4°C. The PCR protocol for the DsRed-R2 / DsRed-F3 primer pair was as follows: 94°C denaturation for 5 min; 30 cycles of 94°C denaturation for 30 s, 60°C annealing for 30 s, and 72°C extension for 30 s; 72°C extension for 5 min, followed by storage at 4°C. The PCR program for the Attb1 / Attb2 primer pair amplification was as follows: 94°C denaturation for 5 min, 32 cycles of 94°C denaturation for 30 s, 65°C annealing for 30 s, and 72°C extension for 30 s (for expression suppression) or 90 s (for overexpression), followed by 72°C extension for 5 min and storage at 4°C. The PCR program for the PdeMYB73-F5 / AtUBQ10-R5 primer pair amplification was as follows: 94°C denaturation for 5 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 90 s, followed by 32 cycles of 72°C extension for 5 min and storage at 4°C.

[0078] 2. Detection of the expression level of the target gene PdeMYB73 in transgenic poplars

[0079] Based on the sequence information of the PdeMYB73 gene, the primer pair PdeMYB73-F1 / PdeMYB73-R1 was designed for real-time quantitative PCR (qRT-PCR) to detect the relative expression of PdeMYB73 in the leaves and roots of transgenic plants. Actin7 (gene ID: Podel.01G470900 in the Populus deltoides genome) was used as an internal reference gene, and the internal reference primer pair was Actin-qF / Actin-qR.

[0080] Using Hieff TM qPCR Green Master Mix (purchased from Shanghai Yisheng Biological, product number 11201ES) was added to the qRT-PCR reactions were performed on a 96-well plate (Roche, Switzerland); the qRT-PCR system consisted of 10 μL of polymerase, 5 μL of forward and reverse primers, 0.5 μL each of template cDNA, and 3 μL of ddH2O. A standard three-step PCR amplification protocol was used, including: 95°C for 600 s; 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s; and 95°C for 10 s, 65°C for 60 s, and 97°C for 1 s. The specificity of the PCR reaction was analyzed using melting curves of the amplified products. Three biological replicates and two technical replicates were performed. The relative expression levels of genes were expressed as −2 △△CT Method calculation, by Light 96SW1.1 software analysis is completed.

[0081] Expression levels of the PdeMYB73 gene were measured in 48 transgenic lines, revealing significant differences in PdeMYB73 expression levels in leaves and fine roots. Three overexpression (OE) lines (OE5, OE32, and OE33) with the highest expression levels and fastest growth in leaves and fine roots, and three repressed expression (RE) lines with the best inhibitory effects among the repressed expression lines (RE40, RE45, and RE72), were selected for subsequent high-growth function evaluation. Furthermore, three overexpression (OE) lines with significant expression changes in leaves and fine roots (OE4, OE24, and OE30) and three repressed expression (RE) lines with the best inhibitory effects among the repressed expression lines (RE40, RE45, and RE72) were selected for subsequent waterlogging resistance evaluation. Among them, the expression levels of OE strains OE5, OE32, OE33, OE4, OE24, and OE30 in leaves were 11.43 times, 10.20 times, 13.00 times, 17.92 times, 21.28 times, and 24.85 times that of wild-type Nanlin 895 poplar (WT), and their expression levels in fine roots were 10.00 times, 28.74 times, 48.84 times, 33.98 times, 19.54 times, and 16.51 times that of WT, respectively; the expression levels of RE strains RE40, RE45, and RE72 in leaves were 0.33 times, 0.55 times, and 0.18 times that of WT, respectively, and their expression levels in fine roots were 0.47 times, 0.65 times, and 0.54 times that of WT, respectively.

[0082] 3. Growth performance evaluation of PdeMYB73 transgenic poplars

[0083] Four-week-old, robust, uniformly growing, and contaminant-free tissue culture seedlings of wild-type Nanlin 895 poplar (WT), OE5, OE32, OE33, RE40, RE45, and RE72 were selected. Terminal buds approximately 1.5 cm long were excised in a laminar flow hood and inoculated into WPM medium of equal size and thickness (i.e., identical volume of medium). Rooting time was observed, and seedling height was measured 28 days later. Four-week-old, robust, uniformly growing, and contaminant-free rooting tissue culture seedlings were selected, the rooting medium washed with clean water, and transplanted into pots (16 cm × 14 cm × 12 cm) using nutrient soil (pH 6.0) containing 2-5% N, P₂O₅, and K₂O, with an organic matter content exceeding 20% ​​(dry weight). Four weeks after transplanting, all plants were watered weekly with a modified 1 / 2 Hoagland nutrient solution and tap water, and maintained as per standard procedures. All transplanted seedlings were cultured in a culture room with a temperature of (25 ± 2) °C, a photoperiod of 16 h light and 8 h dark (16 h / d), and a light intensity of 400 μmol·m -2 ·s -1 The light source is a cold white light, and the relative humidity in the room is 70-80%. Observe the seedling height and ground diameter growth throughout the growth process. Seedling height is measured from the base of the seedling to the base of the terminal bud, accurate to 0.1 cm. Ground diameter is measured 1 cm above the root of the seedling, accurate to 0.01 cm.

[0084] When all plants reached their 60th day of growth, robust, uniformly growing plants were randomly divided into two groups: 9 plants per group, 3 plants per replicate, and randomly arranged. One group was used to measure net photosynthetic rate, growth, stem tip length, leaf number, and internode length, measured every 10 days for a total of three measurements. The other group was used to collect roots, stems, and leaves for determination of lignin, cellulose, hemicellulose, and related hormone contents.

[0085] 3.1 Phenotypic observation and growth measurement

[0086] Figure 1 and Figure 2 The differences in seedling height between wild-type poplars and transgenic lines are shown 30 days after tissue culture and 60 days after transplanting. Significant differences in seedling height were observed between OE, RE, and WT lines. The OE line, which overexpresses PdMYB73, grew significantly taller than the WT line, while the RE line, which inhibits PdMYB73 expression, grew significantly shorter than the WT line. This suggests that PdeMYB73 positively regulates poplar growth.

[0087] Figure 3Shown are the results of seedling height (Panel A) and ground diameter (Panel B), as well as the growth rate of seedling height (Panel C) and ground diameter (Panel D) for wild-type poplar and transgenic lines at 60, 70, and 80 days after transplantation. It can be seen that at all three time points, the seedling height of the OE line was greater than that of the WT plants, while the seedling height of the WT plants was greater than that of the RE line. The ground diameter of both the OE and WT lines was greater than that of the RE line (except RE72). During the experimental period (60-80 days), the growth rate of seedling height of the OE line was significantly greater than that of the WT plants, while the growth rate of seedling height of the WT plants was significantly greater than that of the RE line. There were differences in the growth rate of ground diameter among the OE, WT, and RE lines, but these differences were not significant.

[0088] 3.2 Biomass determination

[0089] After the experiment ended (80 days), wild-type and transgenic poplar plants were harvested, the soil carefully rinsed with tap water, and then wiped clean. The plants were then separated into roots, stems, and leaves. The plants were then oven-dried at 105°C for 2 hours and then dried at 60°C to constant weight. The dry weights of the roots, stems, and leaves were weighed, and the biomass differences between the different strains were compared. The results are shown in Table 2. The root-to-shoot ratio was calculated as: root-to-shoot ratio = root dry weight / (stem dry weight + leaf dry weight). Total biomass was calculated as: total biomass = root dry weight + stem dry weight + leaf dry weight.

[0090] Table 2 Biomass determination results of wild-type and transgenic poplars in the present invention

[0091]

[0092] As can be seen from the table, the root, stem, leaf biomass and total biomass accumulation of OE plants were higher than those of RE and WT, indicating that they had a faster nutrient accumulation rate, which was manifested as faster and more robust plant growth, which was consistent with the Figure 1-Figure 2 The conclusions are consistent.

[0093] 3.3 Determination of relative chlorophyll content

[0094] The fifth fully expanded leaf of each plant after the experiment was selected as the measurement leaf, and the relative chlorophyll content (SPAD value) was determined using a chlorophyll meter SPAD-502Plus (Minolta Co, Japan). Five points were measured on each leaf, and the average value was taken as the relative chlorophyll content of the leaf, with three repetitions per treatment.

[0095] Figure 4Shown are actual leaves of wild-type poplars and transgenic lines (left) and SPAD values ​​for chlorophyll content (right). The results show that the chlorophyll content of OE lines (OE5, OE32, and OE33) was significantly lower than that of WT plants, while that of RE lines (RE40, RE45, and RE72) was not significantly different from WT. Based on the differences in chlorophyll content among the lines, it is speculated that the reduction in chlorophyll content is one of the factors that causes the changes in leaf color in OE plants.

[0096] 3.4 Measurement of stem tip length, leaf number, and internode length

[0097] On the 60th, 70th, and 80th day of transplanted seedling growth, internode lengths of OE5, OE32, OE33, WT, RE40, RE45, and RE72 plants were measured using a vernier caliper, and the number of fully expanded leaves was counted. The stem segment between two adjacent leaves was considered an internode. Internode length was measured from the first fully expanded leaf at the top of the plant to the last leaf at the bottom, and the average internode length was calculated. The lengths of the first to fifth internodes at the top at the three time points were plotted to investigate whether differences in seedling height were caused by an increase in internode number or internode length.

[0098] Figure 5 The leaf number (Figure A) and average internode length (Figure B) of wild-type poplar and transgenic lines at 60, 70, and 80 days after transplantation are shown. The results show that the leaf number and average internode length of the OE line are significantly higher than those of the WT, which are higher than those of the RE line. Figure 5 Also shown are internode growth curves for wild-type poplars and transgenic lines at 60 days (C), 70 days (D), and 80 days (E) after transplantation. The results show that the slope of the OE line is higher than that of the WT, which in turn is higher than that of the RE line, indicating that the difference in seedling height between the OE overexpressing line, the WT, and the RE-suppressed expression line is primarily due to increased internode length.

[0099] 3.5 Net photosynthetic rate

[0100] The net photosynthetic rate (Pn) of wild-type poplar and transgenic lines was measured starting from the 60th day of plant growth and every 10 days for a total of 3 times.

[0101] Figure 6 The net photosynthetic rate (Pn) of wild-type poplar and transgenic lines at 60 days (A), 70 days (B), and 80 days (C) after transplantation is shown. Pn values ​​differ among the OE overexpressing lines, WT, and RE-suppressed expression lines. The Pn values ​​of the OE lines are significantly higher than those of the WT lines, and the Pn values ​​of the WT lines are significantly higher than those of the RE lines, indicating that the OE lines have better photosynthetic performance, which is conducive to their rapid growth and biomass accumulation.

[0102] 3.6 Determination of lignin, cellulose and hemicellulose content

[0103] The stem segment between the 4th and 6th fully expanded leaves at the top of the soil-cultured seedlings 80 days after transplantation was taken, and the lignin (product number G0708F), cellulose (product number G0715F), and hemicellulose (product number G0716W) contents were determined using kits from Suzhou Gres Biotechnology Co., Ltd.

[0104] Figure 7 The results of lignin, cellulose, and hemicellulose content measurements in wild-type poplars and transgenic lines are shown. Lignin content in OE plants was greater than that in WT plants and greater than that in RE plants. Lignin content in OE plants was 56.97% higher than that in WT plants, while that in RE plants was 32.02% lower. Cellulose content in OE plants was greater than that in WT plants and greater than that in RE plants. Cellulose content in OE plants was 28.45% higher than that in WT plants, while that in RE plants was 30.01% lower than that in WT plants. Hemicellulose content in OE plants was significantly higher than that in WT plants, increasing by 43.19%, while no significant difference in hemicellulose content was found between WT and RE lines.

[0105] 3.7 Hormone content determination

[0106] The roots (young fine roots), stem tips (stem segments between the 1st and 3rd fully expanded leaves at the top of the plant) and leaves (the 3rd fully expanded leaf at the top of the plant) of OE5, OE32, OE33, WT, RE40, RE45 and RE72 plants transplanted 80 days ago were collected and the contents of auxin indoleacetic acid (IAA, Product No. YX-E21770), gibberellin (GA3, Product No. YX-E21766) and cytokinin (CTK, Product No. YX-22136P) were determined by enzyme-linked immunosorbent assay (ELISA kit, Shanghai Youxuan Biotechnology Co., Ltd.). The results are shown in Table 2. Figures 8-10 .

[0107] Figure 8 The results showed that in the root system, IAA content in OE plants was lower than that in WT plants and significantly lower than that in RE plants. IAA content in RE plants was higher than that in WT plants, but the difference was not significant. In the shoot apex and leaves, IAA content in OE plants was significantly higher than that in both WT and RE plants, while IAA content in RE plants was not significantly different from that in WT plants. Among all OE and RE plants, IAA content in OE overexpression plants was significantly increased in the aboveground parts of the plants and decreased in the belowground parts. IAA content in RE suppression plants was increased in the belowground parts of the plants and decreased to varying degrees in the aboveground parts, but the changes were not significant.

[0108] Figure 9The results showed that in the root system, the GA3 content of OE plants was lower than that of WT, but the difference was not significant; the GA3 content of RE lines was significantly higher than that of WT. In the shoot apex, the GA3 content of OE lines was higher than that of WT, while the GA3 content of RE lines was lower than that of WT. In leaves, the GA3 content of OE lines was significantly higher than that of WT and RE lines, while the GA3 content of RE lines was not significantly different from that of WT. GA3 content showed roughly the same trend in all OE and RE lines. GA3 content in OE overexpression lines increased to varying degrees in the aboveground parts of the entire plant, while no significant changes were observed in the underground parts. GA3 content in RE suppression expression lines increased significantly in the underground parts of the entire plant, while no significant changes were observed in the aboveground parts.

[0109] Figure 10 The results showed that in the roots, CTK content in OE plants was lower than that in WT plants, while it was higher in RE plants, although the differences were not significant. In the shoot apex, CTK content in OE plants was higher than that in WT plants, while CTK content in RE plants remained unchanged. In leaves, CTK content in OE plants was significantly higher than that in both WT and RE plants, while CTK content in RE plants did not differ significantly from that in WT plants. CTK content showed similar trends in all OE and RE plants. CTK content in the OE overexpression lines increased to varying degrees in the shoot apex and leaves, but the change was not significant in the shoot apex, while it was significantly increased in the leaves. CTK content in the roots of the OE overexpression lines was lower, but the difference was not significant. CTK content in the RE suppression lines increased throughout the underground plant, but the difference was not significant, and remained largely unchanged in the shoot apex and leaves.

[0110] Auxin, a growth-promoting substance produced primarily at the stem tip and in the expanding young leaves, is a hormone. Cytokinin, a hormone that promotes cytoplasmic division, promotes the differentiation and growth of various tissues. Gibberellic acid, a hormone that regulates elongation, also promotes cell division. Elevated levels of these hormones play a key role in the rapid elongation growth of poplars.

[0111] 3.8. Anatomy of the stem apex

[0112] Anatomy of the stem apex: Stem segments between the first and second fully expanded leaves at the apex of OE5 and RE72 plants, 80 days after transplantation, were cut into approximately 0.5 cm long segments using a scalpel blade. Semi-thin sections were prepared to observe differences in stem apical cellular structure. Semi-thin sections were prepared and observed according to the instructions for Technovit 7100 resin semi-thin sectioning.

[0113] Figure 11The longitudinal sections of the stem tips of wild-type and transgenic poplars are shown in the figure. It can be seen that the stem tips of each strain are composed of epidermis, phloem, cambium and pith. Based on the microscopic examination results, the lengths of various cell types in the stem tips were counted, and the results are shown in Figure 12 . It can be seen that there are significant differences in the lengths of epidermal cells, phloem cells, and pith cells among WT, OE5, and RE72. The epidermal cell length of OE5 plants increased by 97.3% compared with WT, while the epidermal cell length of RE72 decreased by 21.79% compared with WT; the phloem cell length of OE5 plants increased by 133.25% compared with WT, while the phloem cell length of RE decreased by 13.2% compared with WT; the pith cell length of OE5 plants increased by 96.69% compared with WT, while the pith cell length of RE decreased by 15.11% compared with WT. It can be seen that there are significant differences in the size of the shoot tip cells of different strains, which may be an important reason for the differences in growth rates among different strains.

[0114] 4. Evaluation of waterlogging resistance of PdeMYB73 transgenic poplars

[0115] The tissue culture seedlings of PdeMYB73 gene overexpression lines (OE4, OE24, OE30), RNAi-inhibited expression lines (RE40, RE45, RE72), and wild-type Nanlin 895 poplar (WT) were used as experimental materials. The tissue culture seedlings were cultured in a light-sensitive culture room at a temperature of (25±2)°C and a light intensity of 400 μmol·m -2 ·s -1 After six weeks of growth, the root culture medium was washed with clean water and the plants were transplanted into nutrient pots. Four weeks after transplanting, all plants were watered once a week with modified 1 / 2 Hoagland nutrient solution and once with tap water, and routine management was implemented.

[0116] When the average height of all plants reached 40-50 cm, the flooding stress test was started. Plant materials with strong growth and uniform growth were selected and randomly assigned to two treatments for 35 days: (1) Control treatment (CK): a small amount of water was applied to the nutrient pots every day to maintain the soil moisture content at 70-75% of the maximum field water capacity; (2) Flooding treatment (PS): a thin layer of fine sand was covered on the surface of the nutrient pots (to prevent the nutrient soil from diffusing in the water), and the pots were placed in a water tank with the water level kept 5 cm above the soil surface. The water was flooded for 28 days and then drained for 7 days. Each treatment was divided into two groups. One group was sampled and measured for physiological indicators at 0, 7, 28, and 35 days; the other group was observed daily for plant phenotype, and chlorophyll content (SPAD value), leaf gas exchange, and chlorophyll fluorescence parameters were measured at 0, 7, 14, 21, 28, and 35 days.

[0117] 4.1 Phenotypic observation and growth measurement

[0118] During the flooding treatment, plant morphological characteristics, including leaf yellowing, wilting, and shedding, were observed and recorded daily. Surviving plants were counted at the end of the experiment (PS35). At the end of the experiment, all plants survived, with a survival rate of 100%. Figure 13 The phenotypes of wild-type poplars and transgenic lines at the end of waterlogging stress (PS28) are shown. WT and RE lines exhibit significant phenotypic changes after waterlogging, including slowed leaf expansion and growth, yellowing, wilting, and leaf shedding, lenticel enlargement, and the induction of adventitious root formation. However, the OE line is less affected by waterlogging. At 28 days (PS28) of waterlogging, WT and RE lines (RE40, RE45, and RE72) experience severe leaf shedding, while OE lines (OE4, OE24, and OE30) experience only a small number of lower leaves yellowing and shedding. As can be seen, OE overexpression lines exhibit mild symptoms and are more resistant to waterlogging; RE repression lines exhibit severe symptoms and poorer waterlogging resistance, while WT lines fall somewhere in between. This suggests that increasing PdeMYB73 expression can enhance plant tolerance to waterlogging.

[0119] On the 0th and 35th day of the experiment, the seedling height and ground diameter of the plants were measured, and the growth of the seedlings during the experiment was calculated. Figure 14 Shown are the results of seedling height (left) and ground diameter (right) growth measurements for wild-type poplars and transgenic lines. The impact of waterlogging stress on various plant indicators was assessed using the waterlogging tolerance coefficient (WC) = WC / Control × 100%. As can be seen from the figure, waterlogging stress inhibited seedling height and ground diameter growth for all plants, but the extent of inhibition varied among the different lines. At the end of the experiment (PS35), the seedling height and ground diameter growth of the OE strain were significantly higher than those of the WT and RE strains; at this time, the waterlogging resistance coefficients of the seedling height growth of each strain were as follows: OE4 (74.62%) > OE24 (68.3%) > OE30 (68.15%) > WT (65.02%) > RE45 (62.2%) > RE40 (61.19%) > RE72 (57.83%); the waterlogging resistance coefficients of the ground diameter growth were as follows: OE30 (74.69%) > OE24 (65.20%) > OE4 (60.67%) > WT (51.05%) > RE45 (48.25%) > RE40 (45.49%) > RE72 (37.90%). The results showed that the waterlogging resistance of OE strain was stronger than that of WT strain, and the waterlogging resistance of WT strain was stronger than that of RE strain.

[0120] 4.2 Leaf gas exchange parameters

[0121] Leaf gas exchange parameters refer to the parameters of gas exchange between the leaf surface and the external environment, reflecting physiological processes such as photosynthesis, respiration, and transpiration in plant leaves. Leaf gas exchange parameters, including net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO₂ concentration / ambient CO₂ concentration (Ci / Ca), were measured in plants from the control (CK) and flooded (PS) treatments on days 0, 7, 14, 21, 28, and 35 of the experiment using an LI-6400 photosynthetic meter (LI-COR Inc., Lincoln, NE, USA). These parameters, along with environmental conditions such as saturated water vapor pressure and leaf surface temperature, were also measured. Measurements were taken between 9:00 AM and 11:00 AM, with three replicates per treatment. Before the experiment began, the fifth fully expanded leaf at the top of the plant was marked and used as the leaf for photosynthetic measurement. In the event of severe leaf loss later in the experiment, the upper, fully expanded, healthy leaf was used as the leaf for measurement. During the measurement, a standard LI-COR leaf chamber and a red and blue light source (6400-02LED light source) were used, and the light intensity was set to 1500 μmol·m -2 ·s -1 , air flow rate is 500 μmol·s -1 .

[0122] 4.2.1 Net photosynthetic rate

[0123] Net photosynthetic rate (Pn) is a direct expression of the instantaneous photosynthetic capacity of leaves. Figure 15 The results of net photosynthetic rate measurements for WT, OE, and RE plants under waterlogging stress are shown. While the net photosynthetic rate (Pn) values ​​of the control plants remained unchanged during the experiment, the Pn values ​​of the waterlogged plants decreased significantly with prolonged stress. However, the Pn value of the OE strain remained consistently higher than that of the WT and RE strains. During rewatering, the Pn values ​​of all strains increased to varying degrees, but the increase in the RE strain was much smaller than that of the WT and OE strains. At the end of waterlogging (28 days), the Pn resistance coefficient of each strain was ranked as follows: OE30 (73.17%) > OE24 (67.32%) > OE4 (67.06%) > WT (64.24%) > RE40 (55.38%) > RE45 (53.97%) > RE72 (52.83%). The results showed that the effect of waterlogging stress on the photosynthetic capacity of the OE line was significantly smaller than that of the WT and RE lines, indicating that increasing the expression of the PdeMYB73 gene is beneficial to enhancing the photosynthetic performance of plants under waterlogging stress.

[0124] 4.2.2 Transpiration rate

[0125] Figure 16The results of transpiration rate (Tr) measurements for WT, OE, and RE plants under waterlogging stress are shown. Tr values ​​for the leaves of flooded plants decreased significantly with increasing waterlogging stress. By day 14 of waterlogging, Tr values ​​for all strains were significantly lower than those in the control. By day 28, Tr values ​​for all strains had reached their lowest values. The magnitude of the decrease in Tr values ​​for the RE strain was significantly greater than for the WT and OE strains during the experimental period. During rewatering, Tr values ​​for all strains rebounded, but the OE strain saw a greater increase than the WT and RE strains. At the end of waterlogging (day 28), the Tr resistance coefficients for the strains were ranked as follows: OE30 (63.7%) > OE24 (56.96%) > OE4 (56.80%) > WT (49.22%) > RE45 (34.15%) > RE40 (31.87%) > RE72 (30.30%).

[0126] 4.2.3 Stomatal conductance

[0127] Figure 17 The results of stomatal conductance (Gs) measurements of WT, OE, and RE plants under waterlogging stress are shown. It can be seen that the Gs of the leaves of the flooded plants showed a continuous downward trend as the degree of waterlogging stress intensified. Reduced stomatal conductance will hinder the entry of CO2 into the leaves, resulting in a decrease in photosynthetic rate. After one day of waterlogging, the Gs values ​​of all strains were significantly lower than those of the control treatment; by the 28th day, the Gs values ​​of all strains had dropped to the lowest value. During the experimental period, the Gs value of the OE strain was significantly higher than that of the WT and RE strains. During the rewatering period, the Gs values ​​of all strains rebounded, with the OE strain's Gs value increasing more significantly than the WT and RE strains. At the end of flooding (28 days), the waterlogging resistance coefficients of the Gs strains were ranked as follows: OE4 (55.45%) > OE30 (52.85%) > OE24 (52.56%) > WT (41.49%) > RE40 (32.06%) > RE72 (31.65%) > RE45 (30.76%).

[0128] 4.2.4 Intercellular CO2 concentration / ambient CO2 concentration

[0129] Figure 18The results of intercellular CO₂ concentration / ambient CO₂ concentration (Ci / Ca) measurements of WT, OE, and RE plants under waterlogging stress are shown. As can be seen, the Ci / Ca ratio of the leaves of the flooded plants continued to decrease as the level of waterlogging increased. By day 14 of waterlogging, the Ci / Ca values ​​of all strains were significantly lower than those of the control. By day 28, the Ci / Ca values ​​of all strains had reached their lowest values. The Ci / Ca value of the OE strain was significantly higher than that of the WT and RE strains during the experimental period. During the rewatering period, the Ci / Ca values ​​of all strains rebounded, but the increase in the OE strain was greater than that of the WT and RE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of Ci / Ca of each strain was ranked as follows: OE30 (95.11%) > OE4 (93.09%) > OE24 (92.66%) > WT (87.58%) > RE72 (80.14%) > RE45 (74.34%) > RE40 (69.89%).

[0130] 4.2.5 Instantaneous water utilization rate

[0131] Instantaneous water use efficiency (R WUEi ) is calculated as follows: WUEi =Pn / Gs. Figure 19 The results of calculation of instantaneous water use efficiency of WT, OE and RE plants under flooding stress are shown in Figure 2. WUEi The values ​​did not change significantly, while the R WUEi The value showed a continuous upward trend. WUEi The values ​​were significantly higher than those of WT and OE strains. WUEi There was no significant change in the value. At the end of flooding (28 days), the R WUEi The order of flood resistance coefficient is: RE45 (175.60%) > RE72 (167.00%) > RE40 (161.11%) > WT (150.59%) > OE30 (135.36%) > OE24 (128.57%) > OE4 (118.35%)

[0132] 4.3 Chlorophyll fluorescence parameters

[0133] Chlorophyll fluorescence parameters are a set of variables or constant values ​​used to describe the photosynthetic mechanism and physiological status of plants. They reflect the photosynthetic mechanism and photosynthetic physiological status of plants and are regarded as intrinsic probes for studying the relationship between plant photosynthesis and the environment. On days 0, 7, 14, 21, 28, and 35 of the experiment, chlorophyll fluorescence parameters including initial fluorescence (Fo), variable fluorescence (Fv), maximum fluorescence (Fm), potential activity of PSⅡ (Fv / Fo), maximum photochemical quantum yield (Fv / Fm), effective photochemical quantum yield of PSⅡ (Fv' / Fm'), actual photochemical efficiency of PSⅡ (ФPSⅡ), photosynthetic electron transfer rate (ETR) of PSⅡ, photochemical quenching coefficient (qP), and non-photochemical quenching coefficient (NPQ) of leaves of control (CK) and flooded (PS) plants of each line were measured and calculated using an LI-6400 photosynthetic meter (LI-COR Inc., Lincoln, NE, USA). Where qP = (Fm'-Fs) / (Fm'-Fo'), and NPQ = Fm / Fm'-1. Before the experiment began, the fifth fully expanded leaf at the top of the plant was marked as the leaf for fluorescence measurement. If leaf shedding became severe, a healthy, fully expanded leaf at the top was used as the measurement leaf. Before measurement, the plant leaves were dark-adapted for 30 minutes. The same leaves were then photoactivated with activating light for 30 minutes before testing. Three replicates per treatment were used.

[0134] 4.3.1 Maximum photochemical efficiency

[0135] The maximum photochemical efficiency (Fv / Fm) reflects the maximum photochemical efficiency of the photosystem II (PSⅡ) reaction center under completely dark-adapted conditions and is usually used to evaluate the photosynthetic capacity and light energy conversion efficiency of plants. Figure 20The results of the maximum photochemical efficiency (Fv / Fm) measurements of WT, OE, and RE plants under flooding stress are shown. As shown in the figure, under the control treatment, the Fv / Fm values ​​of all plants did not change significantly. The Fv / Fm values ​​of the flooded strains showed a continuous downward trend, and the Fv / Fm decrease of the RE strain was significantly greater than that of the WT and OE strains. By 28 days, the Fv / Fm values ​​of all strains had dropped to the lowest value. At this time, the Fv / Fm value of the OE strain was higher than that of the WT and RE strains. During the drainage recovery period, the Fv / Fm values ​​of the WT, RE, and OE strains all rebounded to a certain extent, and the recovery degree of the RE strain was significantly lower than that of the OE strain, indicating that the leaves of the OE plants have a higher photosynthetic response capacity. At the end of flooding (28 days), the Fv / Fm resistance coefficients of each strain were ranked as follows: OE4 (84.36%) > OE24 (83.04%) > OE30 (82.50%) > WT (78.09%) > RE40 (68.91%) > RE45 (68.33%) > RE72 (65.22%).

[0136] 4.3.2. Potential PSⅡ Activity

[0137] Figure 21 The results of the potential PSII activity (Fv / Fo) assay in WT, OE, and RE plants under waterlogging stress are shown. Based on the degree of waterlogging, there was no significant difference in Fv / Fo values ​​between the control and the 7-day waterlogging treatment. By 14 days after waterlogging, the Fv / Fo values ​​of all strains decreased, but the decrease in the RE strain was much greater than that of the WT and OE strains, indicating that waterlogging had a lesser inhibitory effect on the Fv / Fo values ​​of the WT and OE strains. By 21 days after waterlogging, the Fv / Fo values ​​of all strains decreased significantly, but the decrease in the RE strain was more pronounced. By 28 days after waterlogging, the Fv / Fo values ​​of all strains had reached their lowest level, with the RE strain's Fv / Fo value significantly lower than those of the WT and OE strains. During rewatering, the Fv / Fo values ​​of all strains recovered, but the recovery in the RE strain was less than that of the WT and OE strains. At the end of flooding (28 days), the Fv / Fo resistance coefficient of each strain was ranked as follows: OE30 (77.03%) > OE4 (75.52%) > OE24 (70.08%) > WT (60.77%) > RE72 (54.67%) > RE45 (53.99%) > RE40 (43.36%).

[0138] 4.3.3 PSⅡ Effective Photochemical Quantum Yield

[0139] The effective photochemical quantum yield of PSⅡ (Fv' / Fm') reflects the maximum photochemical efficiency of the PSⅡ reaction center under light adaptation and is used to characterize the photochemical efficiency when the PSⅡ reaction center is fully open in the presence of heat dissipation. Figure 22Shown are the results of Fv' / Fm' measurements of WT, OE, and RE plants under waterlogging stress. Under the control treatment, the Fv' / Fm' values ​​of all plants remained unchanged. During waterlogging, the Fv' / Fm' values ​​of all strains decreased, with the decrease in the RE strain being more significant compared to the OE strain. During drainage recovery, the Fv' / Fm' values ​​of all strains recovered to some extent, but the recovery in the RE strain was less than that of the WT and OE strains. At the end of flooding (28 days), the Fv' / Fm' resistance coefficients of each strain were ranked as follows: OE4 (78.79%) > OE24 (76.63%) > OE30 (76.13%) > WT (64.80%) > RE45 (54.68%) > RE72 (52.20%) > RE40 (48.05%).

[0140] 4.3.4 Actual Photochemical Efficiency

[0141] The actual photochemical efficiency (ΦPSⅡ) indicates the quantum yield of the PSⅡ reaction center actually used for photochemical electron transfer under light conditions, reflecting the conversion efficiency of light energy in plant leaves during photosynthesis. The higher the ΦPSII value, the stronger the leaf's ability to absorb and convert light. Figure 23 Shown are the results of ΦPSⅡ measurements for WT, OE, and RE plants under waterlogging stress. Under the control treatment, the actual photochemical efficiency (ΦPSⅡ) of all plants remained unchanged. During waterlogging, the ΦPSⅡ values ​​of all strains showed a downward trend, but the OE strain had a higher ΦPSⅡ value than the WT, which in turn was higher than the RE strain. During drainage recovery, the ΦPSⅡ values ​​of all strains recovered to some extent, but the recovery in the RE strain was less than that of the WT and OE strains, indicating that the light energy conversion efficiency of the RE strain was more significantly suppressed. At the end of flooding (28 days), the waterlogging resistance coefficient of each strain ФPSⅡ was ranked as follows: OE24 (62.35%) > OE30 (61.63%) = OE4 (61.63%) > WT (51.16%) > RE72 (41.86%) > RE45 (41.57%) > RE40 (40.70%).

[0142] 4.3.5 Fluorescence Quenching

[0143] There are two types of chlorophyll fluorescence quenching: photochemical quenching, represented by qP, and non-photochemical quenching, expressed in two ways: NPQ or qN. qP reflects fluorescence quenching caused by the capture of excitation energy by open PSII reaction centers and its conversion into chemical energy. It indicates the capacity of PSII to undergo photochemical reactions under light-acclimated conditions, specifically the proportion of open PSII reaction centers. Non-photochemical quenching (NPQ) is the process by which leaves eliminate excess energy under intense light exposure through heat transfer and photoprotection mechanisms. Higher NPQ values ​​indicate greater tolerance to light damage. Figure 24 and Figure 25 The results of qP and NPQ measurements for WT, OE, and RE plants under waterlogging stress are shown. Waterlogging significantly affected the qP and NPQ processes of each strain. Under waterlogging, qP values ​​of all strains decreased significantly. Compared with the WT and RE strains, the qP value of the OE strain was relatively less affected, indicating higher photosynthetic efficiency. During drainage recovery, qP values ​​of all strains recovered to some extent, but the recovery of qP values ​​in the RE strain was less than that of the WT and OE strains. At the end of waterlogging (28 days), the qP resistance coefficient of each strain was ranked as follows: OE4 (46.72%) > OE30 (40.15%) > OE24 (39.86%) > WT (38.33%) > RE72 (30.19%) > RE45 (27.69%) > RE40 (23.65%). Under flooding stress, the NPQ of each strain increased significantly. During drainage recovery, the NPQ of each strain decreased rapidly, with the RE strain decreasing more than the OE strain. At the end of flooding (28 days), the NPQ resistance coefficient of each strain was ranked as follows: RE45 (584.16%) > RE40 (568.77%) > RE72 (565.54%) > WT (410.15%) > OE24 (350.89%) > OE4 (348.73%) > OE30 (341.30%).

[0144] 4.3.6 Apparent Electron Transfer Rate

[0145] The apparent electron transfer rate (ETR) refers to the rate at which electrons in leaves are transferred in the photosynthetic system under light conditions, and can be used to evaluate the efficiency of leaves in utilizing light. Figure 26The ETR measurements of WT, OE, and RE plants under waterlogging stress are shown. Waterlogging stress significantly affected the ETR values ​​of each strain. Under waterlogging, the ETR values ​​of each strain decreased significantly. By day 28, all strains had reached their lowest values, with highly significant differences compared to the control. At this point, the ETR value of the OE strain was higher than that of the WT and higher than that of the RE strain. During drainage recovery, the ETR values ​​of all strains rebounded to some extent, but the recovery of the RE strain was less than that of the WT and OE strains. At the end of waterlogging (day 28), the ETR resistance coefficient of each strain was ranked as follows: OE24 (67.62%) > OE4 (66.62%) > OE30 (65.61%) > WT (46.02%) > RE72 (37.56%) > RE40 (35.86%) > RE45 (32.88%).

[0146] 4.4 Relative chlorophyll content

[0147] Chlorophyll is a light energy harvester during photosynthesis, and its content directly affects a plant's light energy utilization efficiency and photosynthesis efficiency. On days 0, 7, 14, 21, 28, and 35 of the experiment, the fifth fully expanded leaf of each control (CK) and waterlogged (PS) plant was selected as the measurement leaf. The relative chlorophyll content (SPAD value) was determined using a chlorophyll meter, SPAD-502 Plus (Minolta Co., Japan). Five measurements were taken on each leaf, and the average value was used as the relative chlorophyll content for that leaf. Three replicates were performed per treatment.

[0148] Figure 27 The results of relative chlorophyll content measurements of WT, OE, and RE plants under waterlogging stress are shown. Under waterlogging, the relative chlorophyll content of each strain showed a downward trend, but the decrease was greater in the WT and RE strains. By the 14th day, the SPAD values ​​of each strain had not decreased significantly. By the 28th day, the SPAD values ​​of OE and WT had not decreased significantly, while the SPAD value of RE had decreased significantly. After drainage was restored, the SPAD values ​​of each strain gradually increased to the control level. At the end of waterlogging (28 days), the waterlogging resistance coefficient of the relative chlorophyll content of each strain was ranked as follows: OE4 (95.13%) > OE30 (90.73%) > OE24 (86.55%) > WT (81.67%) > RE72 (78.24%) > RE40 (75.37%) > RE45 (72.75%).

[0149] 4.5 Malondialdehyde content

[0150] Under waterlogging stress, plants experience an increase in harmful free radicals due to low oxygen or anoxic environments, disrupting the stability of their cell membranes. Malondialdehyde (MDA) content reflects the degree of oxidative damage to cell membranes, with greater damage associated with higher values. Therefore, MDA content and its waterlogging resistance coefficient are negatively correlated with plant waterlogging resistance, with higher values ​​indicating poorer resistance. Leaves and fine roots of control (CK) and waterlogged (PS) plants from each strain were collected on days 0, 7, 28, and 35 of the experiment, and MDA content was measured using the thiobarbituric acid (TBA) method, with three replicates per treatment.

[0151] Figure 28 The results of malondialdehyde (MDA) content measurements in leaves and roots of WT, OE, and RE plants under waterlogging stress are shown. As shown, waterlogging stress significantly increased MDA content in leaves and fine roots of all strains. On the 7th day, leaf MDA content increased slightly in all strains, while root MDA content remained unchanged. On the 28th day, MDA content in leaves and roots of all strains increased significantly, with the RE strain experiencing a significantly greater increase than the OE-expressing strain, and the WT falling in between. Seven days after drainage (PS 35 days), MDA content in both leaves and roots decreased, with the RE strain experiencing a smaller decrease than the WT and OE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of leaf MDA content of each strain was ranked as follows: OE30 (212.54%) > RE45 (206.38%) > RE72 (203.47%) > RE40 (188.67%) > WT (178.64%) > OE24 (160.75%) > OE4 (152.41%); the waterlogging resistance coefficient of root MDA content was ranked as follows: RE45 (401.00%) > OE4 (389.24%) > WT (383.18%) > OE24 (381.08%) > RE40 (342.91%) > RE72 (329.39%) > OE30 (326.59%).

[0152] 4.6 Root activity

[0153] On the 0th, 7th, 28th and 35th days of the experiment, fine roots of the control treatment (CK) and flooding treatment (PS) plants of each strain were collected, and the root activity was measured using the Plant Root Activity Test Kit (TTC method, Shanghai Yuanye Biotechnology Co., Ltd., product number R30350), with 3 replicates per treatment.

[0154] Figure 29Figure 2 shows the results of root activity measurements for WT, OE, and RE plants under waterlogging stress. Root activity values ​​for each plant decreased with increasing waterlogging duration, indicating that waterlogging stress can severely reduce root activity and affect normal plant growth. On the 7th day of waterlogging, root activity decreased for all plant types, but the differences from the control were not significant. On the 28th day, root activity for all plant types dropped to its lowest level, significantly lower than that of the control plants, but the decline in the OE plant was smaller than that of the WT and RE plants. During drainage recovery, root activity values ​​for all plant types recovered to varying degrees. At the end of flooding (28 days), the root activity and waterlogging resistance coefficients of the various strains were ranked as follows: OE4 (63.58%) > OE24 (62.87%) > OE30 (59.43%) > WT (48.86%) > RE72 (46.02%) > RE40 (44.69%) > RE45 (44.45%). This indicates that the OE strain was less affected by waterlogging stress, indicating that it possesses greater waterlogging tolerance.

[0155] 5. Comprehensive evaluation of flood resistance

[0156] Cluster analysis: Cluster analysis (minimum distance method) was performed using leaf gas exchange parameters, chlorophyll fluorescence parameters, chlorophyll content, malondialdehyde content, and waterlogging resistance coefficient of root activity of OE, RE, and WT plants on the 28th day, as well as seedling height growth and ground diameter growth at the end of the experiment (35 days). The results are shown in Figure 30 , showing that OE4, OE24, and OE30 are clustered into one class; RE40, RE45, and RE72 are clustered into one class; and the WT strain is a single class.

[0157] Membership function analysis: Membership function analysis was performed using the gas exchange parameters, chlorophyll fluorescence parameters, chlorophyll content, malondialdehyde content and waterlogging resistance coefficient of root activity of OE, RE and WT at the end of flooding on the 28th day, as well as the waterlogging resistance coefficient of seedling height growth and ground diameter growth at the end of the experiment (35 days). The results are shown in Table 3, showing that the waterlogging resistance of each strain was ranked as follows: OE4>OE30>OE24>WT>RE72>RE40>RE45, indicating that the waterlogging resistance of the OE strain was stronger than that of the RE strain, and the waterlogging resistance of WT was between OE and RE. The results were consistent with the cluster analysis.

[0158] Table 3 Membership function analysis results of wild-type and transgenic poplars in the examples of the present invention

[0159]

[0160] The above results indicate that overexpression of the PdeMYB73 gene improves the tolerance of poplar to waterlogging stress, while inhibition of the expression of the PdeMYB73 gene reduces the waterlogging resistance of poplar, confirming that the PdeMYB73 gene has the function of regulating the waterlogging resistance of poplar.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of the PdeMYB73 gene in regulating the growth performance of poplars, characterized in that: The application includes: overexpressing the PdeMYB73 gene to positively regulate the growth performance of poplar trees, wherein the nucleotide sequence of the PdeMYB73 gene is shown in SEQ ID NO.1; wherein the growth performance of the poplar trees includes at least one of the following (1)-(6): (1) Seedling height; (2) stem or leaf biomass; (3) stem tip length or internode length; (4) Net photosynthetic rate; (5) lignin, cellulose or hemicellulose content; (6) Contents of indoleacetic acid, gibberellins or cytokinins in stem tips and leaves.

2. The use of the PdeMYB73 gene in regulating the growth performance of poplar according to claim 1, characterized in that: The positive regulation of poplar growth performance includes the following steps: constructing a PdeMYB73 gene overexpression vector, transforming wild-type poplars, and cultivating transgenic poplars with enhanced PdeMYB73 gene expression.

3. The use of the PdeMYB73 gene in regulating poplar growth performance according to claim 2, characterized in that: The construction of the PdeMYB73 gene overexpression vector includes the following steps: Using poplar cDNA as a template, PCR amplification was performed using the PdeMYB73-OE-F and PdeMYB73-OE-R primer pairs to obtain the CDS sequence of the PdeMYB73 gene. The PdeMYB73 gene sequence was inserted into the pKGW-RR-MGW expression vector using gateway gene cloning technology to construct the PdeMYB73 gene overexpression vector. Among them, the nucleotide sequences of PdeMYB73-OE-F and PdeMYB73-OE-R are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

4. The use of the PdeMYB73 gene in regulating poplar growth performance according to claim 1, characterized in that: The poplar tree is Nanlin 895 poplar.

5. Application of the PdeMYB73 gene in improving the resistance of poplars to flooding stress, characterized in that: The application includes: overexpressing the PdeMYB73 gene to improve the resistance of poplar to flooding stress, and the nucleotide sequence of the PdeMYB73 gene is shown in SEQ ID NO.

1.

6. The use of the PdeMYB73 gene according to claim 5 in improving the resistance of poplar to flooding stress, characterized in that: The poplar's resistance to flooding stress includes at least one of the following (1)-(5): (1) Seedling height or ground diameter growth under waterlogging stress; (2) leaf gas exchange parameters under waterlogging stress, wherein the leaf gas exchange parameters include at least one of net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration / ambient CO2 concentration; (3) Chlorophyll fluorescence parameters under waterlogging stress, wherein the chlorophyll fluorescence parameters include at least one of maximum photochemical efficiency, PS II potential activity, PS II effective photochemical quantum yield, actual photochemical efficiency, photochemical quenching coefficient, non-photochemical quenching coefficient, and apparent electron transfer rate; (4) Leaf chlorophyll content under waterlogging stress; (5) Root activity under waterlogging stress.

7. A method for breeding fast-growing and / or flood-resistant poplars, characterized in that: The following steps are involved: A PdeMYB73 gene overexpression vector was constructed and transformed into wild-type poplars to obtain transgenic poplars with enhanced PdeMYB73 gene expression.

8. The method for breeding fast-growing and / or flood-resistant poplars according to claim 7, characterized in that: The construction of the PdeMYB73 gene overexpression vector includes the following steps: Using poplar cDNA as a template, PCR amplification was performed using the PdeMYB73-OE-F and PdeMYB73-OE-R primer pairs to obtain the CDS sequence of the PdeMYB73 gene. The PdeMYB73 gene sequence was inserted into the pKGW-RR-MGW expression vector using gateway gene cloning technology to construct the PdeMYB73 gene overexpression vector. Among them, the nucleotide sequences of PdeMYB73-OE-F and PdeMYB73-OE-R are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

Images