Application of PdeMYB73 gene in improving growth performance and / or flooding stress resistance of poplar

Abstract

The invention belongs to the technical field of molecular biology breeding, and particularly relates to application of a PdeMYB73 gene to improvement of poplar growth performance and / or flooding stress resistance. The nucleotide sequence of the PdeMYB73 gene is as shown in SEQ ID NO. 1 (sequence identifier number 1). The poplar PdeMYB73 gene is cloned, the action and response mechanism of the poplar PdeMYB73 gene in poplar growth regulation and control and flooding stress resistance are explored, it is found that the poplar PdeMYB73 gene has the positive regulation and control effect on poplar growth and flooding stress resistance improvement, and an excellent gene resource is provided for improving poplar growth performance and flooding stress resistance; a new solution is provided for cultivating a new fast-growing and waterlogging-resistant poplar variety, and the method has wide application prospects and good social benefits in forestry production aspects such as improvement of excellent forest stress-resistant varieties and the like.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biological breeding, and in particular to application of a PdeMYB73 gene in improving growth performance and / or flood 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 reduction in the absorption of most mineral nutrients in the soil by plants, weakening the growth and development of new roots and new shoots. Moreover, under waterlogging stress, the oxygen content in the rhizosphere of plants will decrease rapidly, causing hypoxia or anoxic conditions in the root system and / or leaf tissues of the plants, thereby limiting the aerobic respiration of the plants, inhibiting photosynthesis and nutrient uptake of the plants, and hindering their normal life activities. In addition, the hypoxia or anoxic conditions caused by stagnant water will cause lipid peroxidation in plant tissues to intensify, 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 values. In production practice, flooding stress is still the main stress faced by poplars and has become one of the main factors leading to poor growth of poplars. The loss of wood production and ecological functions caused by it may exceed the loss caused by all other reasons. In addition, tree height and growth rate are the key factors affecting poplar wood production. Therefore, enhancing the growth rate of poplars under normal conditions and their resistance to flooding stress is an important factor in improving the sustainable development of the poplar industry.

[0003] At present, there is an urgent need to carry out the breeding of new varieties of fast-growing and flood-resistant poplar trees, but traditional forest breeding is based on morphological screening, which has many difficulties such as long cycle and difficult breeding. With the development of molecular biology, the use of genetic engineering technology to cultivate new germplasm of fast-growing and flood-resistant forest trees has broad development prospects. Analyzing the adaptation mechanism of poplars to flooding stress and exploring flood-resistant genes are important prerequisites for breeding poplar varieties with strong stress resistance and flooding tolerance. Summary of the invention

[0004] In view of the above technical problems in the prior art, the present invention provides a PdeMYB73 gene isolated from poplar, which has a positive regulatory effect on the growth and flooding stress resistance of poplar, provides excellent gene resources for improving the growth performance and flooding stress resistance of poplar, and provides a new solution for breeding new fast-growing and flood-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. The present invention is specifically implemented through the following technical solutions:

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

[0006] The second aspect of the present invention provides the use of the PdeMYB73 gene in improving the resistance of poplar to flooding 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 flooding 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 flooding stress experiment, the waterlogging resistance of the overexpression transgenic strain was significantly enhanced compared with the wild type and the PdeMYB73 gene inhibition expression type plants, which can improve the growth and / or survival ability of plants under flooding stress conditions and alleviate the damage of flooding stress to plants. It was confirmed that the PdeMYB73 gene plays a key role in regulating poplar growth and improving its flooding resistance. The PdeMYB73 gene provided by the present invention can be applied to the breeding of new varieties of fast-growing and flood-resistant poplars and variety improvement, and has a wide range of application prospects and good social benefits in forestry production such as the improvement of excellent forest tree stress resistance 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 the wild-type and transgenic poplars at 60 days after transplanting tissue culture seedlings of an embodiment of the present invention;

[0013] Figure 3 The diagrams are the results of the phenotypic observation and growth measurement of wild-type and transgenic poplar seedling height at 60-80 days after transplanting tissue culture seedlings of the embodiment of the present invention, wherein Figures AD are the seedling height, ground diameter, seedling height growth and ground diameter growth, respectively;

[0014] Figure 4 The actual picture of leaves of wild-type and transgenic poplars and the bar graph of relative chlorophyll content in the embodiment of the present invention;

[0015] Figure 5 The graphs are the leaf number and internode growth curves of wild-type and transgenic poplars at different cultivation times in the embodiment of the present invention, wherein Figure A is the leaf number, Figure B is the average internode length, and Figures CE are the internode lengths of different internodes at 60, 70 and 80 days, respectively;

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

[0017] Figure 7 The bar graphs are the lignin, cellulose and hemicellulose contents of wild-type and transgenic poplars in the embodiments of the present invention;

[0018] Figure 8 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] Fig. 9 This is a bar graph of gibberellin content in roots, stems and leaves of wild-type and transgenic poplars according to an embodiment of the present invention;

[0020] Fig.10 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] Fig.11 The microscopic examination images of the stem tip longitudinal sections of the wild-type and transgenic poplars in the embodiments of the present invention;

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

[0023] Fig.13 The phenotype diagram of wild-type and transgenic poplars after waterlogging stress treatment in the embodiment of the present invention;

[0024] Fig.14 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] Fig.15 This is a graph showing the change in net photosynthetic rate of wild-type and transgenic poplars under flooding stress treatment at different times according to an embodiment of the present invention;

[0026] Fig.16 This is a graph showing the changing rules of transpiration rates of wild-type and transgenic poplars under flooding stress treatment at different times according to an embodiment of the present invention;

[0027] Fig.17 This is a graph showing the stomatal conductance variation of wild-type and transgenic poplars under flooding stress treatment at different times in an embodiment of the present invention;

[0028] Fig.18 This is a graph showing the change regularity of the ratio of intercellular CO2 concentration to ambient CO2 concentration of wild-type and transgenic poplars under flooding stress treatment at different times in an embodiment of the present invention;

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

[0030] Fig. 20 This is a graph showing the variation of the maximum photochemical efficiency of wild-type and transgenic poplars under flooding stress treatment at different times according to an embodiment of the present invention;

[0031] Fig.21 This is a graph showing the change pattern of the potential activity of PSⅡ of wild-type and transgenic poplars under flooding stress treatment at different times in an embodiment of the present invention;

[0032] Fig. 22 This is a graph showing the change regularity of the effective photochemical quantum yield of PSⅡ of wild-type and transgenic poplars under different flooding stress treatment times in an embodiment of the present invention;

[0033] Fig.23 This is a graph showing the variation of the actual photochemical efficiency of wild-type and transgenic poplars under flooding stress treatment at different times according to an embodiment of the present invention;

[0034] Fig.24 This is a graph showing the change rules of photochemical quenching of wild-type and transgenic poplars under flooding stress treatment at different times according to an embodiment of the present invention;

[0035] Fig.25 This is a graph showing the variation of non-photochemical quenching of wild-type and transgenic poplars under flooding stress treatment at different times according to an embodiment of the present invention;

[0036] Fig.26 This is a graph showing the change in apparent electron transfer rate of wild-type and transgenic poplars under flooding stress treatment at different times according to an embodiment of the present invention;

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

[0038] Fig.28 A bar graph showing the malondialdehyde content in leaves and fine roots of wild-type and transgenic poplars under flooding stress treatment at different times in an embodiment of the present invention;

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

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

[0041] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. The examples described herein are only used to explain the present invention and are not used to limit the present invention. All the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the attached claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. The meaning of similar words such as "include", "comprise", "contain", "have" is non-restrictive, that is, 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 principle and specific implementation methods of the present invention are described in detail below.

[0043] Water condition is one of the important environmental factors for plant growth, and it is also one of the main abiotic stresses suffered by plants. Waterlogging stress refers to the phenomenon that excessive soil moisture or long-term flooding in a flooded environment during the growth process of plants limits the aerobic respiration of plants and the energy production required to maintain life activities, leading to disorders in plant physiological functions and stunted growth and development. There are generally two states of excessive water in the soil. One is that the soil moisture exceeds the maximum water holding capacity and is in a saturated state. The gas phase of the soil is completely replaced by the liquid phase, which is called "waterlogging", also known as waterlogging damage; the other is that the water not only fills the soil but also the ground is flooded, submerging parts of the plant or the entire plant, which is usually called "waterlogging damage".

[0044] In the environment of long-term flooding stress, plants have evolved various mechanisms to cope with flooding stress. Transcription factors are protein molecules in eukaryotes that can bind to cis-acting elements of gene promoters. They ensure the expression of target genes at a specific intensity and in a specific time and space by enhancing or inhibiting them, and play a switch regulating role in the expression of downstream genes. When plants are subjected to adverse stress, transcription factors will quickly participate in the regulation of many important processes, such as activating related enzyme systems in cells, participating in hormone secretion, affecting cell metabolic activities and signal transduction, etc. At the same time, it can also act as a special messenger molecule to participate in a variety of physiological and biochemical reactions, thereby regulating the growth and development of plants and coping with abiotic stress.

[0045] The present invention isolates and clones a PdeMYB73 gene involved in regulating poplar growth rate and flooding 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 used as the research object, and its overexpression and inhibition expression vectors were constructed. The wild-type poplar was transformed to obtain the PdeMYB73 gene function interference mutant strain and overexpression strain. The growth performance of transgenic strains and wild-type poplar plants was evaluated, and it was found that there were differences in the growth status of plants of different strains. The PdeMYB73 gene overexpression strain had significantly higher growth indicators such as seedling height growth, stem elongation growth, root, stem and leaf biomass and total biomass, photosynthetic performance parameters such as chlorophyll content and net photosynthetic rate, and levels of hormones related to cell elongation growth (auxin, cytokinin and gibberellin) than the wild-type plants. At the same time, the wild-type plants were significantly higher than the PdeMYB73 gene inhibition expression strain, showing that the PdeMYB73 gene overexpression strain had a faster growth rate and better photosynthetic performance, which was also conducive to its rapid growth and accumulation of biomass. The above results fully highlight the importance of the PdeMYB73 gene in regulating the growth performance of poplar, and provide a theoretical basis for analyzing the regulation of poplar growth rate by the PdeMYB73 gene.

[0047] The flooding resistance of transgenic strains and wild-type poplar plants was further evaluated. They were flooded using hydroponics. The effects of flooding stress on different strains were compared by measuring their phenotypic indicators combined with physiological indicators. It was found that under flooding stress, multiple growth indicators such as plant root activity, seedling height, ground diameter, leaf growth phenotype, internode length, and plant biomass accumulation were inhibited, and the photosynthesis of the plants was significantly inhibited. The 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 WUEi)) and chlorophyll fluorescence parameters (including maximum photochemical efficiency (Fv / Fm), PSⅡ potential activity (Fv / Fo), PSⅡ effective photochemical quantum yield (Fv' / Fm'), actual photochemical efficiency (ФPSⅡ), photochemical quenching (qP), non-photochemical quenching (NPQ), apparent electron transfer rate (ETR)) all showed a downward trend under flooding stress, and all indicators were negatively correlated with the stress time. The content of lipid peroxidation products such as malondialdehyde increased, indicating that the plants were damaged by hypoxia caused by flooding stress. However, in general, the inhibition of the strains overexpressing the PdeMYB73 gene was significantly lower than that of the wild-type plants and the strains inhibited by PdeMYB73 expression. The strains overexpressing the PdeMYB73 gene had higher growth, biomass accumulation, stronger photosynthetic adaptability and photochemical conversion efficiency under flooding stress, showing that the growth inhibition was the weakest under flooding stress; in addition, during the drainage period, the above indicators had a faster recovery speed, indicating that they had a good recovery ability after flooding. It can be seen that overexpression of the PdeMYB73 gene is beneficial to enhance and alleviate the symptoms of plant damage under flooding stress conditions, reduce leaf wilting, death and shedding, maintain a high photosynthetic rate and normal photosynthetic characteristics, and improve the ability to resist oxidative damage under flooding stress, thereby increasing the growth rate and survival of plants under flooding stress, and enhancing the resistance of plants to flooding stress. Therefore, the present invention also confirms the key role of the PdeMYB73 gene in responding to plant response to flooding stress, and provides a theoretical basis for analyzing the regulation of poplar waterlogging resistance by the PdeMYB73 gene.

[0048] In summary, the present invention has explored the positive regulatory role of the PdeMYB73 gene in regulating the height growth and waterlogging resistance of poplars, laid a research foundation for further elucidating the molecular mechanism of the formation of the height growth and waterlogging resistance of poplars, and provided excellent gene resources for improving the growth performance and resistance to flooding stress of poplars, and provided a new solution for breeding new varieties of fast-growing and waterlogging-resistant poplars. At the same time, as a woody model plant, the excavation and functional identification of excellent gene resources of poplars will also provide reference and reference for related research on other plant species.

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

[0050] The PdeMYB73 gene has a positive regulatory effect on the improvement of poplar growth performance. By genetic engineering breeding, the expression level of the PdeMYB73 gene can be enhanced, and the growth and development ability of the poplar can be enhanced by increasing the growth rate and photosynthetic capacity of the poplar, providing a new breeding idea for improving the growth performance of the poplar and increasing the growth rate of the poplar; and by inhibiting the expression level of the PdeMYB73 gene, the growth and development of the plant can be inhibited, providing a goal and direction for cultivating dwarf plants. The PdeMYB73 gene provided by the present invention has broad application prospects and good social benefits in the improvement of fine forest varieties and other aspects.

[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 of the PdeMYB73 gene, positive regulation of plant growth performance can be achieved; by reducing the expression of the PdeMYB73 gene, negative regulation of plant growth performance can be achieved. It should be noted that the expression of the PdeMYB73 gene includes the expression of the gene level (mRNA) and / or the expression of the protein level. The gene level can be achieved by RNA interference technology, CRISPR-Cas9 technology or overexpression vector, 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, positively regulating the growth performance of poplars includes the following steps: constructing a PdeMYB73 gene overexpression vector, transforming wild-type poplars, and cultivating transgenic poplars with enhanced expression of the PdeMYB73 gene. More specifically, constructing a PdeMYB73 gene overexpression vector includes the following steps: using poplar cDNA as a template, using PdeMYB73-OE-F and PdeMYB73-OE-R primer pairs for PCR amplification to obtain the CDS sequence of the PdeMYB73 gene, inserting the PdeMYB73 gene sequence into the pKGW-RR-MGW expression vector through gateway gene cloning technology, and constructing 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.

[0054] Specifically, negatively regulating the growth performance of poplars includes the following steps: constructing a PdeMYB73 gene inhibition expression vector, transforming wild-type poplars, and cultivating transgenic poplars with reduced expression of the PdeMYB73 gene. More specifically, constructing a PdeMYB73 gene inhibition expression vector includes the following steps: using poplar cDNA as a template, using PdeMYB73-RE-F and PdeMYB73-RE-R primer pairs for PCR amplification to obtain a PdeMYB73 gene RNAi sequence, inserting the RNAi sequence into the pK7GWIWG2 (II) expression vector through gateway gene cloning technology, and constructing 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, oxidative damage, photoinhibition, etc. under flooding stress conditions can be alleviated, thereby significantly improving the waterlogging resistance of plants and improving the growth and / or survival ability of plants under flooding stress conditions. This provides a new method for breeding poplar flood-resistant varieties using genetic engineering technology and has important application potential.

[0057] Optionally, the poplar's resistance 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 poplars 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 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, using PdeMYB73-OE-F and PdeMYB73-OE-R primer pairs to perform PCR amplification to obtain the CDS sequence of the PdeMYB73 gene, inserting the PdeMYB73 gene sequence into the pKGW-RR-MGW expression vector through gateway gene cloning technology, and constructing 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] The expression vector can be used to transform host cells using conventional techniques known to those skilled in the art. The vector carrying the PdeMYB73 gene can be introduced into poplar cells or tissues by using the following DNA transfection methods: calcium phosphate coprecipitation method, Ti plasmid method, Ri plasmid method, viral vector method, gene gun method, microinjection method, electroporation method or Agrobacterium-mediated method, etc.

[0063] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or the conditions recommended by the manufacturer. In addition, the materials, reagents, etc. 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 a dsRed selection marker, purchased from Kelei Biotechnology Co., Ltd., product access link: http: / / www.kelei-biology.com / m / view.php?aid=983.

[0065] Gene and primer: PdeMYB73 gene was cloned from Nanlin 895 poplar (belonging to the black poplar school poplar) by designing primers with reference to the genome sequence of Populus deltoides. Since this gene has not been reported in Populus deltoides, BLAST-P was performed in Arabidopsis thaliana, and the most homologous gene was found to be AtMYB73, and the newly cloned gene of this application was named PdeMYB73. The PdeMYB73 gene and related primer sequences involved in the embodiment 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 poplar transformed with PdeMYB73 gene

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

[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), which 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 constructing the PdeMYB73 overexpression vector (OE) and suppression expression vector (RE).

[0073] Nanlin 895 poplar leaves were used as materials to extract mRNA, and the reverse transcription kit Hifair Ш1st Strand cDNA Synthesis Super Mix (purchased from Shanghai Yisheng Biological, catalog number 11137ES) was used to reverse transcribe the mRNA into cDNA. Using cDNA as a template, the above primer pairs were used for PCR amplification to obtain the target gene fragments PdeMYB73-CDS and PdeMYB73-RNAi. The gateway gene cloning technology was used to clone the target gene fragments PdeMYB73-CDS and PdeMYB73-RNAi in BP Clonase TM II Enzyme Mix (purchased from Hangzhou Bailibo, catalog 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 of poplar

[0075] The overexpression vector and the inhibition expression vector carrying the PdeMYB73 gene were transformed into Agrobacterium tumefaciens EHA105 by electroporation, and the young leaves of Nanlin 895 poplar, which 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 and streak it 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 culture medium containing Spe and Rif resistance, and culture it in the dark at 200r / min and 28°C until 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 leaves that are dark green and flat 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 at 5000r / min for 10min, the supernatant was discarded, and the precipitate was resuspended in 50-75mL resuspension solution (WPM without agar) to OD 600 =0.5-0.8, and then infect for 10-15min; 3) Co-cultivation: remove the leaves from the infection solution, place them on sterile filter paper to absorb the residual infection solution on the surface, and lay the leaves with the back facing down on the culture medium (WPM+100μM AS), and culture them in the dark at 24℃ for 2d; 4) Induce callus: transfer the co-cultivated leaves to the culture medium for inducing callus differentiation (WPM+1mg / L2,4-D+0.5mg / LKT+300mg / L Cef+50mg / LKan+300mg / L TMT), and culture them in the dark at 24℃. The culture medium was changed every 15d to obtain leaves with callus tissue; 5) Induce budding: Place callus tissue (about 1cm in size) on the 2 ) were cut and transferred to a medium for inducing bud differentiation (WPM+0.02mg / L TDZ+300mg / L Cef+50mg / L Kan+300mg / 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) Inducing bud elongation: 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+300mg / L Cef+50mg / L Kan) for culture, cultured at 24°C under light, and the medium was replaced every 15 days; 7) Inducing rooting: the resistant seedlings with stem segments longer than 3 cm in the bud elongation medium were cut and placed in a rooting medium (WPM+250mg / L Cef+50mg / LKan) for culture, 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: the DNA of the overexpressed and suppressed transgenic resistant plants was amplified by specific primers, the PdeMYB73 gene overexpression and suppression expression vector plasmid was used as the positive control, the wild-type Nanlin 895 poplar leaf DNA was used as the negative control, and ddH2O was used as the blank control. The PCR reaction system was calculated as 10 μL, including: 0.5 μL of each forward and reverse primer, 2×Hieff TM PCR Master Mix enzyme (purchased from Shanghai Yisheng Biology, catalog number 10102ES08) 5μL, template 3μL, balance ddH2O. Among them, the PCR program for the amplification of the DsRed-F1 / AtUBQ10-R1 primer pair: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 59℃ annealing for 30s, 72℃ extension for 90s, 30 cycles; 72℃ extension for 5min, 4℃ storage. The PCR program for the amplification of the DsRed-R2 / DsRed-F3 primer pair: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, 30 cycles; 72℃ extension for 5min, 4℃ storage. The PCR program for the amplification of the Attb1 / Attb2 primer pair was as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 30 s (inhibition of expression) or 90 s (overexpression), 32 cycles; 72°C extension for 5 min, and storage at 4°C. The PCR program for the amplification of the PdeMYB73-F5 / AtUBQ10-R5 primer pair was as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 90 s, 32 cycles, 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] According to the sequence information of PdeMYB73 gene, primer pair PdeMYB73-F1 / PdeMYB73-R1 was designed for real-time fluorescence quantitative PCR (qRT-PCR) experiment to detect the relative expression of PdeMYB73 in leaves and roots of transgenic positive plants. Actin7 (gene id number in the genome of Populus deltoides: Podel.01G470900) was used as the 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, catalog number 11201ES) was added to the qRT-PCR reaction was completed on 96 (Roche, Switzerland); the qRT-PCR system was 10 μL, including 5 μL of polymerase, 0.5 μL of forward primer and reverse primer, 1 μL of template cDNA, and 3 μL of ddH2O. The three-step PCR amplification standard procedure was used, including: 95℃600s; 95℃10s, 60℃20s, 72℃30s, 40 cycles; 95℃10s, 65℃60s, 97℃1s. The melting curve of the amplified product was used to analyze the specificity of the PCR reaction, with 3 biological replicates and 2 technical replicates. The relative expression level of the gene was -2 △△CT Method calculation, by Light 96SW1.1 software analysis is complete.

[0081] The expression levels of 48 transgenic lines of the PdeMYB73 gene were measured, and it was found that the expression levels of the PdeMYB73 gene in different lines were significantly different in leaves and fine roots. The three overexpression (OE) lines (OE5, OE32, OE33) with the highest expression levels and the fastest growth in leaves and fine roots and the three repressed expression (RE) lines with the best inhibition effect among the repressed expression lines (RE40, RE45, RE72) were selected for the subsequent identification test of high growth function. At the same time, the three overexpression (OE) lines with significant expression changes in leaves and fine roots (OE4, OE24, OE30) and the three repressed expression (RE) lines with the best inhibition effect among the repressed expression lines (RE40, RE45, RE72) were selected for the subsequent identification of waterlogging resistance function. 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 the 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 the expression levels in fine roots were 0.47 times, 0.65 times, and 0.54 times that of WT, respectively.

[0082] 3. Evaluation of growth performance of PdeMYB73 transgenic poplar

[0083] Select 4-week-old wild-type Nanlin 895 poplar (WT), OE5, OE32, OE33, RE40, RE45 and RE72 tissue culture seedlings with robust growth, uniform growth and no pollution, cut out the terminal buds of about 1.5 cm in length in the clean bench, inoculate them in WPM medium of the same size and thickness (i.e., the volume of the medium is the same), observe their rooting time, and measure their seedling height after 28 days. Then select 4-week-old rooting tissue culture seedlings with robust growth, uniform growth and no pollution, wash the root culture medium with clean water, and transplant them into culture pots (16cm×14cm×12cm). The culture medium is nutrient soil with pH 6.0, containing 2-5% N, P2O5 and K2O, and the organic matter (dry weight) exceeds 20%. After 4 weeks of transplanting, all plants are watered with modified 1 / 2 Hoagland nutrient solution and tap water once a week, and routine management is implemented. 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 lamp, and the relative humidity of the indoor air is 70-80%. The seedling height and ground diameter growth are observed throughout the growth process. The seedling height is measured from the base of the seedling to the base of the terminal bud, accurate to 0.1cm. The ground diameter is measured at 1cm above the root of the seedling, accurate to 0.01cm.

[0084] When all plants grew to the 60th day, the plant materials with strong growth and uniform growth were selected and randomly divided into 2 groups: 9 plants / group, 3 plants / replicate, and randomly arranged. One group was used to measure its net photosynthetic rate, growth, stem tip length, leaf number and internode length, which were measured every 10 days for a total of 3 times. The other group was used to collect roots, stems and leaves for the 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 poplar and transgenic lines at 30 days after tissue culture and 60 days after transplanting tissue culture seedlings are shown respectively. It can be seen that there are obvious differences in seedling height between OE, RE and WT. The seedling height growth of the OE line overexpressing the PdMYB73 gene is significantly higher than that of the WT line, while the seedling height growth of the RE line inhibiting the expression of the PdMYB73 gene is significantly lower than that of the WT line; indicating that the PdeMYB73 gene has a positive effect on regulating the growth performance of poplar.

[0087] Figure 3The results of the determination of seedling height (Figure A) and ground diameter (Figure B) of wild-type poplar and transgenic lines at 60d, 70d, and 80d after transplantation of tissue culture seedlings, as well as the growth of seedling height (Figure C) and ground diameter (Figure D) are shown. It can be seen that at the three time points, the seedling height of the OE line was greater than that of the WT plant, and the seedling height of the WT plant was greater than that of the RE line; the ground diameter of the OE line and WT was greater than that of the RE line (except RE72). During the experimental period (60-80d), the growth of seedling height of the OE line was significantly greater than that of the WT plant, and the growth of seedling height of the WT plant was significantly greater than that of the RE line; there were differences in the growth of ground diameter among the OE, WT, and RE lines, but the differences were not significant.

[0088] 3.2 Biomass determination

[0089] After the experiment (80 days), the wild-type and transgenic poplar plants were harvested, the soil was carefully washed off with tap water, and then the water was wiped off and decomposed into three parts: roots, stems, and leaves. They were sterilized in an oven at 105°C for 2 hours and then dried at 60°C to constant weight. The dry weights of roots, stems, and leaves were weighed respectively, and the differences in biomass of different strains were compared. The results are shown in Table 2. The formula for calculating the root-to-crown ratio is: root-to-crown ratio = root dry weight / (stem dry weight + leaf dry weight). The formula for calculating the total biomass is: 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 by faster and more robust plant growth, which was consistent with 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 3 replicates / treatment.

[0095] Figure 4The actual leaves of wild-type poplar and transgenic lines (left) and the SPAD values ​​of chlorophyll content (right) are shown. The results show that the chlorophyll content of OE lines (OE5, OE32, OE33) is significantly lower than that of WT plants, while the RE lines (RE40, RE45, RE72) are 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 cause the change in leaf color of OE plants.

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

[0097] When the transplanted seedlings grew to the 60th, 70th, and 80th day, the internode lengths of OE5, OE32, OE33, WT, RE40, RE45, and RE72 plants were measured with a vernier caliper, and the number of fully expanded leaves was counted. During the measurement, the stem segment between two adjacent leaves was regarded as an internode, and the 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. At the same time, a curve graph was drawn for the length of the 1st to 5th internodes at the top at the three time points to explore whether the difference in seedling height was caused by an increase in the number of internodes or an increase in internode length.

[0098] Figure 5 The leaf number (Figure A) and average internode length (Figure B) of wild-type poplar and transgenic lines at 60d, 70d and 80d after transplantation are shown. The results showed that the leaf number and average internode length of OE lines were significantly higher than WT, and WT was higher than RE lines. Figure 5 The internode growth curves of wild-type poplar and transgenic lines at 60 days (Figure C), 70 days (Figure D) and 80 days (Figure E) after transplantation are also shown. The results showed that the slope of the OE line was higher than that of the WT, and the slope of the WT was higher than that of the RE line, indicating that the difference in seedling height between the OE overexpression line, the WT and the RE suppression expression line was mainly caused by the increase in 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 60d (A), 70d (B) and 80d (C) after transplantation is shown. It can be seen that there are differences in the Pn values ​​of OE overexpression lines, WT and RE suppression expression lines. The Pn values ​​of OE lines are significantly higher than those of WT lines, and the Pn values ​​of WT are significantly higher than those of RE lines, indicating that the OE lines have better photosynthetic performance, which is conducive to their rapid growth and accumulation of biomass.

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

[0103] The stem segment between the 4th and 6th leaves fully expanded 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 the kits of Suzhou Grace Biotechnology Co., Ltd.

[0104] Figure 7 The results of lignin, cellulose and hemicellulose content determination of wild-type poplar and transgenic lines are shown. It shows that the lignin content of OE plants is higher than that of WT plants and RE plants. The lignin content of OE plants is 56.97% higher than that of WT plants, while that of RE plants is 32.02% lower than that of WT plants. The cellulose content of OE plants is higher than that of WT plants and RE plants. The cellulose of OE plants is 28.45% higher than that of WT plants, while that of RE plants is 30.01% lower than that of WT plants. The hemicellulose content of OE plants is significantly higher than that of WT plants, which is 43.19% higher than that of WT plants, while there is no significant difference in hemicellulose content between WT and RE lines.

[0105] 3.7. Determination of hormone content

[0106] The roots (young and tender fine roots), stem tips (stem segments between the 1st and 3rd leaves at the top of the plant that are fully expanded) and leaves (the 3rd leaf at the top of the plant that is fully expanded) of OE5, OE32, OE33, WT, RE40, RE45 and RE72 plants transplanted for 80 days were taken and the contents of auxin indoleacetic acid (IAA, catalog number YX-E21770), gibberellin (GA3, catalog number YX-E21766) and cytokinin (CTK, catalog number YX-22136P) were determined by enzyme-linked immunosorbent assay (ELISA kit produced by Shanghai Youxuan Biotechnology Co., Ltd.). The results are shown in Table 1. Figure 8-Figure 10 .

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

[0108] Fig. 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 that of RE lines was lower than that of WT. In the 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. In all OE and RE lines, the GA3 content had a roughly similar trend of change. The GA3 content of OE overexpression lines increased to varying degrees in the aboveground part of the whole plant, but did not change significantly in the underground part. The GA3 content of RE inhibition expression lines increased significantly in the underground part of the whole plant, but did not change significantly in the aboveground part.

[0109] Fig.10 The results showed that in the root system, the CTK content of OE plants was lower than that of WT, and the CTK content of RE plants was higher than that of WT, but the differences were not significant. In the stem apex, the CTK content of OE strains was higher than that of WT, and the CTK content of RE strains was not significantly changed compared with WT. In leaves, the CTK content of OE strains was significantly higher than that of WT and RE strains, while the CTK content of RE strains was not significantly different from that of WT. In all OE and RE strains, the CTK content had a roughly similar trend of change. The CTK content of OE overexpression strains increased to varying degrees in the stem apex and leaves, but the change in the stem apex was not significant, while it increased significantly in the leaves. The CTK content of OE overexpression strains decreased in the root system, but the difference was not significant. The CTK content of RE inhibition expression strains increased in the underground part of the whole plant, but the difference was not significant, and there was basically no change in the stem apex and leaves.

[0110] Auxin is mainly produced at the stem tip and the unfolding young leaves, and is a growth-promoting substance; cytokinin is a hormone that promotes cytoplasmic division, which is beneficial to promote the differentiation and growth of various tissues; gibberellin is a hormone that regulates elongation growth and also promotes cell division. The increase in the levels of these hormones plays an important role in the rapid elongation growth of poplars.

[0111] 3.8. Anatomy of the stem apex

[0112] Anatomical structure of stem apex The stem segments between the first and second fully expanded leaves at the top of OE5 and RE72 plants transplanted 80 days ago were selected and cut into small segments of about 0.5 cm in length using a surgical blade. Semi-thin sections were made to observe the differences in the cell structure of the stem apex. The preparation and observation of semi-thin sections were performed according to the instructions of Technovit 7100 resin semi-thin sections.

[0113] Fig.11The longitudinal sections of the stem tips of wild-type and transgenic poplars are shown in Figure 1. 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 types of cells in the stem tips were counted, and the results are shown in Figure 1. Fig.12 . It can be seen that there are significant differences in the lengths of epidermal cells, phloem cells, and medullary cells among WT, OE5, and RE72. The length of epidermal cells of OE5 plants increased by 97.3% compared with WT, while the length of epidermal cells of RE72 decreased by 21.79% compared with WT; the length of phloem cells of OE5 plants increased by 133.25% compared with WT, while the length of phloem cells of RE decreased by 13.2% compared with WT; the length of medullary cells of OE5 plants increased by 96.69% compared with WT, while the length of medullary cells of RE decreased by 15.11% compared with WT. It can be seen that there are significant differences in the size of stem tip cells in different strains, which may be an important reason for the differences in growth rates of different strains.

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

[0115] The tissue culture seedlings of PdeMYB73 gene overexpression lines (OE4, OE24, OE30), RNAi inhibition 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 culture room at a temperature of (25±2)℃ and a light intensity of 400μmol·m -2 ·s -1 , photoperiod 16h / d. After 6 weeks of growth, the root culture medium was cleaned with clean water and transplanted into nutrient pots. 4 weeks after transplanting, all plants were watered with modified 1 / 2 Hoagland nutrient solution and tap water once a week, 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 robust 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 holding 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 surface kept 5 cm above the soil surface. The flooding treatment lasted for 28 days and the drainage was restored 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 for plant phenotype every day, 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, the morphological characteristics of the plants were observed and recorded every day, including leaf yellowing, wilting and falling, and the number of surviving plants in the flooding treatment was counted at the end of the experiment (PS35). At the end of the experiment, all plants survived, with a survival rate of 100%. Fig.13 The phenotypes of wild-type poplar and transgenic lines at the end of flooding stress treatment (PS28) are shown. It can be seen that the phenotypes of WT and RE lines after flooding stress have obvious changes, including slowing down of leaf expansion and growth rate, yellowing, wilting and falling of leaves, enlarged lenticels, induction of adventitious roots, etc., but the OE line is less affected by flooding stress. At 28 days (PS28) of flooding treatment, leaves of WT and RE lines (RE40, RE45, RE72) fell off seriously, while only a small number of lower leaves of OE lines (OE4, OE24, OE30) turned yellow and fell off. It can be seen that the OE overexpression line has mild symptoms and strong waterlogging resistance; the RE suppression expression line has severe symptoms and poor waterlogging resistance, and the WT is between the two, which shows that increasing the expression of the PdeMYB73 gene can enhance the plant's ability to withstand flooding.

[0119] On the 0th and 35th days 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. Fig.14 The results of the measurement of the growth of seedling height (left) and ground diameter (right) of wild-type poplar and transgenic strains are shown. The waterlogging resistance coefficient was used to evaluate the effect of flooding stress on various observed indicators of plants. The waterlogging resistance coefficient = flooding treatment / control treatment × 100%. It can be seen from the figure that flooding stress inhibited the growth of seedling height and ground diameter of all plants, but the degree of inhibition varied among different strains. 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 the physiological processes of plant leaves such as photosynthesis, respiration, and transpiration. On the 0th, 7th, 14th, 21st, 28th, and 35th days of the experiment, the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), intercellular CO2 concentration / ambient CO2 concentration (Ci / Ca) and other leaf gas exchange parameters of the control treatment (CK) and flooding treatment (PS) of each strain were measured using a LI-6400 photosynthetic meter (LI-COR Inc., Lincoln, NE, USA), as well as the corresponding saturated water vapor pressure, leaf surface temperature and other surrounding environmental conditions. The measurement time was 9:00-11:00 am, with 3 repetitions / treatment. Before the start of the experiment, the fifth leaf that was fully expanded at the top of the plant was marked as the measurement leaf for photosynthesis indicators. In the later stage, when the leaves fell off severely, the healthy leaf that was fully expanded at the top was used as the measurement leaf. 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. Fig.15 The results of the net photosynthetic rate determination of WT, OE and RE plants under flooding stress are shown. It can be seen that the net photosynthetic rate (Pn) values ​​of the control treatment plants did not change significantly during the experiment, while the Pn values ​​of the flooded plants decreased significantly with the extension of the stress time; but the Pn value of the OE strain was always higher than that of the WT and RE strains. During the rewatering period, the Pn values ​​of all strains increased to varying degrees, but the increase in the Pn value of the RE strain was much less than that of the WT and OE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of Pn 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 strain was significantly smaller than that of the WT and RE strains, 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] Fig.16The results of transpiration rate (Tr) determination of WT, OE and RE plants under flooding stress are shown. It can be seen that the Tr value of the leaves of flooded plants decreased significantly with the increase of flooding stress. At 14 days of flooding, the Tr values ​​of each strain were significantly lower than those of the control treatment; at the 28th day, the Tr values ​​of each strain dropped to the lowest value; the decline of the RE strain was significantly higher than that of the WT and OE strains during the experiment. During the rewatering period, the Tr values ​​of each strain rebounded, but the Tr value of the OE strain increased more than that of the WT and RE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of Tr of each strain was 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] Fig.17 The results of stomatal conductance (Gs) measurement of WT, OE and RE plants under flooding stress are shown. It can be seen that the Gs of the leaves of flooded plants showed a decreasing trend as the degree of flooding stress intensified. The reduction of stomatal conductance will lead to the obstruction of CO2 entering the leaves, thereby causing a decrease in photosynthetic rate. After 1 day of flooding, the Gs values ​​of each strain were significantly lower than those of the control treatment; by the 28th day, the Gs values ​​of each strain had dropped to the lowest value; during the experiment, the Gs value of the OE strain was significantly higher than that of the WT and RE strains. During the re-watering period, the Gs values ​​of each strain rebounded, and the Gs value of the OE strain increased more significantly than that of the WT and RE strains. At the end of flooding (28 days), the waterlogging resistance coefficients of 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] Fig.18The results of the determination of intercellular CO2 concentration / ambient CO2 concentration (Ci / Ca) of WT, OE and RE plants under flooding stress are shown. It can be seen that the Ci / Ca of the leaves of flooded plants continued to decrease with the aggravation of flooding stress. At 14 days after flooding, the Ci / Ca values ​​of each strain were significantly lower than those of the control treatment; by the 28th day, the Ci / Ca values ​​of each strain had dropped to the lowest value; during the experiment, the Ci / Ca value of the OE strain was significantly higher than that of the WT and RE strains. During the rewatering period, the Ci / Ca values ​​of each strain rebounded, but compared with the WT and RE strains, the increase in the Ci / Ca value of the OE strain was greater. 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 utilization rate (R WUEi ) is calculated as: WUEi =Pn / Gs. Fig.19 The calculation results 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 At the end of flooding (28 days), the R WUEi The flood resistance coefficient is ranked as follows: 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 photosynthesis mechanism and physiological status of plants. They reflect the photosynthesis mechanism and photosynthetic physiological status of plants and are regarded as intrinsic probes for studying the relationship between plant photosynthesis and the environment. On the 0th, 7th, 14th, 21st, 28th and 35th days of the experiment, the chlorophyll fluorescence parameters such as initial fluorescence (Fo), variable fluorescence (Fv), maximum fluorescence (Fm), potential activity of photoreaction center PSⅡ (Fv / Fo), maximum photochemical quantum yield (Fv / Fm), effective photochemical quantum yield of PSⅡ (Fv' / Fm'), actual photochemical efficiency of photoreaction center PSⅡ (ФPSⅡ), photosynthetic electron transfer rate (ETR) of photoreaction center PSⅡ, photochemical quenching coefficient (qP), non-photochemical quenching coefficient (NPQ) of leaves of plants in the control treatment (CK) and flooding treatment (PS) of each strain were measured and calculated using LI-6400 photosynthetic meter (LI-COR Inc., Lincoln, NE, USA); among which, qP = (Fm'-Fs) / (Fm'-Fo'), NPQ = Fm / Fm'-1. Before the test, the fifth leaf that is fully expanded at the top of the plant is marked as the measuring leaf for the fluorescence index. When the leaves fall off seriously in the later stage, the healthy leaf that is fully expanded at the top is used as the measuring leaf. Before the test, the plant leaves are dark-adapted for 30 minutes, and then the activation light is turned on to activate the same leaf for 30 minutes, and then the test is carried out, with 3 repetitions / treatment.

[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. Fig. 20The results of the maximum photochemical efficiency (Fv / Fm) determination 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 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 waterlogging resistance coefficient of Fv / Fm of each strain was 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 activity of PSⅡ

[0137] Fig.21 The results of the determination of the potential activity (Fv / Fo) of PSⅡ in WT, OE and RE plants under flooding stress are shown. From the perspective of flooding degree, there was no significant difference in the Fv / Fo values ​​of the control and the flooding treatment for 7 days; at 14 days of flooding, the Fv / Fo values ​​of each strain decreased, but the decrease in the RE strain was much greater than that of the WT and OE strains, indicating that flooding stress had a smaller inhibitory effect on the Fv / Fo of the WT and OE strains. At 21 days of flooding, the Fv / Fo of each strain decreased significantly, but the decrease in the RE strain was more obvious. At 28 days of flooding, the Fv / Fo values ​​of each strain decreased to the lowest, and the Fv / Fo value of the RE strain was significantly lower than that of the WT and OE strains. During the re-watering period, the Fv / Fo values ​​of all strains rebounded, but the recovery of the Fv / Fo value of RE was less than that of the WT and OE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of each strain Fv / Fo 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 Effective photochemical quantum yield of PSⅡ

[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. Fig. 22The results of Fv' / Fm' value determination of WT, OE and RE plants under flooding stress are shown. Under the control treatment, the Fv' / Fm' values ​​of all plants did not change significantly. During flooding treatment, the Fv' / Fm' values ​​of all strains decreased, and the Fv' / Fm' value of the RE strain decreased more significantly compared with the OE strain. During the drainage recovery period, the Fv' / Fm' values ​​of all strains recovered to a certain extent, but the recovery degree of the Fv' / Fm' value of the RE strain was less than that of the WT and OE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of each strain Fv' / Fm' was 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. Fig.23 The results of ФPSⅡ value determination of WT, OE and RE plants under flooding stress are shown. Under the control treatment, the actual photochemical efficiency (ФPSⅡ) of all plants did not change significantly. During the flooding treatment, the ФPSⅡ values ​​of all strains showed a downward trend, but the ФPSⅡ value of the OE strain was higher than that of the WT, and the WT was higher than that of the RE strain. During the drainage recovery period, the ФPSⅡ values ​​of all strains recovered to a certain extent, but the recovery degree of the ФPSⅡ value of 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 inhibited. 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 is represented by qP, and non-photochemical quenching has two representation methods, namely NPQ or qN. qP reflects the fluorescence quenching caused by the excitation energy being captured by the open PSⅡ reaction center and converted into chemical energy, indicating the ability of PSⅡ to perform photochemical reactions under light adaptation, that is, the proportion of open PSⅡ reaction centers. Non-photochemical quenching (NPQ) is the process by which leaves eliminate excess energy through heat transfer and photoprotection mechanisms under strong light irradiation. A higher NPQ value indicates that the leaves have a stronger tolerance to light damage. Fig.24 and Fig.25 The results of qP and NPQ values ​​of WT, OE and RE plants under flooding stress are shown respectively. Flooding stress significantly affected the qP and NPQ processes of each strain. The qP of each strain decreased significantly under flooding stress. Compared with the WT and RE strains, the qP value of the OE strain was relatively less affected and had higher photosynthetic efficiency. During the drainage recovery period, the qP values ​​of all strains recovered to a certain extent, but the qP value of the RE strain recovered less than that of the WT and OE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of qP 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 the drainage recovery period, the NPQ of each strain decreased rapidly, and the NPQ of the RE strain decreased more than that of 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. Fig.26The results of ETR determination of WT, OE and RE plants under flooding stress are shown. It can be seen that flooding stress significantly affected the ETR values ​​of each strain. Under flooding stress, the ETR of each strain decreased significantly. By 28 days, the ETR values ​​of all strains dropped to the lowest value, which was significantly different from the control; at this time, the ETR value of the OE strain was higher than that of the WT and RE strains. During the drainage recovery period, the ETR values ​​of all strains rebounded to a certain extent, but the ETR value of the RE strain rebounded less than that of the WT and OE strains. At the end of flooding (28 days), the waterlogging resistance coefficient of the ETR 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 content of chlorophyll

[0147] Chlorophyll is a light energy harvester in photosynthesis, and the amount of chlorophyll directly affects the efficiency of light energy utilization and photosynthesis in plants. On the 0th, 7th, 14th, 21st, 28th and 35th day of the experiment, the 5th fully expanded leaf of the control treatment (CK) and flooding treatment (PS) plants of each strain 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 for each leaf, and the average value was taken as the relative chlorophyll content of the leaf, with 3 replicates / treatment.

[0148] Fig. 27 The results of the relative chlorophyll content determination of WT, OE and RE plants under flooding stress are shown. The relative chlorophyll content of each strain showed a downward trend under flooding stress, but the decline in WT and RE strains was greater. By the 14th day, the SPAD values ​​of each strain did not decrease significantly; by the 28th day, the SPAD values ​​of OE and WT did not decrease significantly, while the SPAD value of RE strain decreased significantly. After drainage was restored, the SPAD values ​​of each strain gradually increased to the control level. At the end of flooding (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 flooding stress, harmful free radicals in plants will increase due to low oxygen or anoxic environment, which will damage the stability of plant cell membranes. The malondialdehyde (MDA) content can reflect the degree of oxidative damage to the cell membrane. The greater the damage, the higher the value; therefore, the MDA content and its waterlogging resistance coefficient are negatively correlated with the waterlogging resistance of plants. The larger the value, the worse the waterlogging resistance of plants. On the 0th, 7th, 28th and 35th day of the experiment, leaves and fine roots of control treatment (CK) and flooding treatment (PS) plants of each strain were collected, and the malondialdehyde (MDA) content was determined by thiobarbituric acid (TBA) method, with 3 replicates / treatment.

[0151] Fig.28 The results of malondialdehyde content determination in leaves and roots of WT, OE and RE plants under flooding stress are shown. As shown in the figure, flooding stress significantly increased the MDA content in leaves and fine roots of each strain. On the 7th day, the MDA content in leaves of each strain increased slightly, and the MDA content in roots did not change significantly; on the 28th day, the MDA content in leaves and roots of each strain increased significantly, and the increase in MDA content in RE strain was significantly greater than that in OE expression strain, and WT was between the two; 7 days after drainage was restored (PS 35d), the MDA content in leaves and roots decreased, and the decrease in RE strain was less than that in 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, the 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 Detection Kit (TTC method, Shanghai Yuanye Biotechnology Co., Ltd., product number R30350), with 3 replicates / treatment.

[0154] Fig.29The results of root activity determination of WT, OE and RE plants under flooding stress are shown. As the flooding stress time increases, the root activity values ​​of each strain continue to decrease, indicating that flooding stress will cause a serious reduction in the root activity of plants and affect the normal growth of plants. On the 7th day of flooding, the root activity of each strain decreased, but the difference with the control was not significant. On the 28th day, the root activity of each strain dropped to the lowest level, significantly lower than that of the control plants, but the decline in the OE strain was less than that of the WT and RE strains. During the drainage recovery period, the root activity values ​​of each strain recovered to varying degrees. At the end of flooding (28 days), the root activity resistance coefficient of each strain was ranked as follows: OE4 (63.58%) > OE24 (62.87%) > OE30 (59.43%) > WT (48.86%) > RE72 (46.02%) > RE40 (44.69%) > RE45 (44.45%). It can be seen that the OE strain was less affected by flooding stress, indicating that it has stronger flooding resistance.

[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, and seedling height growth and ground diameter growth at the end of the experiment (35 days) as indicators. The results are shown in Fig.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: 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) were used as indicators for membership function analysis. The results are shown in Table 3, showing that the waterlogging resistance of each strain is ranked as follows: OE4>OE30>OE24>WT>RE72>RE40>RE45, indicating that the waterlogging resistance of the OE strain is stronger than that of the RE strain, and the waterlogging resistance of WT is between OE and RE. The results are consistent with the cluster analysis.

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

[0159]

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

[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 protection scope of the present invention.

Claims

1. Application of PdeMYB73 gene in regulating poplar growth performance, characterized in that: The nucleotide sequence of the PdeMYB73 gene is shown in SEQ ID NO.

1.

2. The use of the PdeMYB73 gene in regulating the growth performance of poplar according to claim 1, characterized in that: The poplar growth performance includes at least one of the following (1)-(6): (1) Growth of seedling height or ground diameter; (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.

3. The use of the PdeMYB73 gene in regulating the growth performance of poplar according to claim 1, characterized in that: The regulation is positive regulation or negative regulation, wherein: 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 expression of the PdeMYB73 gene; Negatively regulating the growth performance of poplars includes the following steps: constructing a PdeMYB73 gene inhibition expression vector, transforming wild-type poplars, and cultivating transgenic poplars with reduced expression of the PdeMYB73 gene.

4. The use of the PdeMYB73 gene in regulating the growth performance of poplar according to claim 3, 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 primer pairs PdeMYB73-OE-F and PdeMYB73-OE-R to obtain the CDS sequence of the PdeMYB73 gene, and the PdeMYB73 gene sequence was inserted into the pKGW-RR-MGW expression vector by 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.

5. The use of the PdeMYB73 gene in regulating the growth performance of poplar according to claim 3, characterized in that: The construction of the PdeMYB73 gene suppression expression vector includes the following steps: Using poplar cDNA as a template, PCR amplification was performed using the PdeMYB73-RE-F and PdeMYB73-RE-R primer pairs to obtain the PdeMYB73 gene RNAi sequence, and the RNAi sequence was inserted into the pK7GWIWG2 (II) expression vector through 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.

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

7. Application of the PdeMYB73 gene in improving poplar resistance to flooding stress, characterized in that: The nucleotide sequence of the PdeMYB73 gene is shown in SEQ ID NO.

1.

8. The use of the PdeMYB73 gene according to claim 7 in improving the resistance of poplar to flooding stress, characterized in that: The poplar tree's resistance to flooding stress includes at least one of the following (1)-(5): (1) Growth of seedling height or ground diameter under waterlogging stress; (2) leaf gas exchange parameters under flooding 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 flooding stress, wherein the chlorophyll fluorescence parameters include 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 waterlogging stress.

9. 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, wild-type poplars were transformed, and transgenic poplars with enhanced PdeMYB73 gene expression were obtained by cultivation.

10. The method for breeding fast-growing and / or flood-resistant poplars according to claim 9, 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 primer pairs PdeMYB73-OE-F and PdeMYB73-OE-R to obtain the CDS sequence of the PdeMYB73 gene, and the PdeMYB73 gene sequence was inserted into the pKGW-RR-MGW expression vector by 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.

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