Application of fringed iris MYC2 gene as positive regulatory factor in promoting removal of plant ecological dormancy

Abstract

The invention discloses application of a fringed iris MYC2 gene as a positive regulatory factor in promoting removal of ecological dormancy of plants, and relates to the technical field of biology. According to the invention, a homologous gene transient overexpression method is adopted for the first time, the important effect of the MYC2 gene on the removal of ecological dormancy is proved in fringed iris, the gene is determined to be positioned in a cell nucleus, and the expression quantity is increased along with the removal of ecological dormancy; when the fringed iris IjMYC2 gene is over-expressed, the ecological dormancy release and germination of the plant can be promoted.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of the butterfly flower MYC2 gene as a positive regulatory factor in promoting the release of plant ecological dormancy. Background Art

[0002] Plant overwintering dormancy is a complex biological process, which can be divided into two types: physiological dormancy and ecological dormancy. Among them, physiological dormancy refers to the cessation of growth of the growing point due to endogenous signals, and even in a suitable environment, it is impossible to break dormancy and resume growth immediately; ecological dormancy refers to the growth point has recovered its growth ability, but the growth is stagnant due to environmental restrictions (Lang GA, Early JD, Martin GC, Darnell RL. Endo-, para-, and ecodormancy: physiological terminology and classification for dormancy research [J]. Hortscience, 1987, 371-377). At present, the research on plant overwintering dormancy is mostly concentrated in dicotyledons, which generally have typical physiological dormancy and ecological dormancy processes. However, in monocots represented by rhizomatous iris, there is only ecological dormancy in its winter dormancy, and growth can be quickly resumed when the environment is suitable, showing dormancy characteristics different from those of the above-mentioned dicots (Li D., Shao L., Zhang J., et al. MADS-box transcription factors determine the duration of temporary winter dormancy in closely related evergreen and deciduous Iris spp[J]. Journal of Experimental Botany, 2022, 73(5), 1429-1449). Therefore, it is of great significance to explore the key genes that regulate the ecological dormancy of rhizomatous iris and use gene editing technology to achieve directed mutations to obtain germplasm resources that tolerate low temperatures in winter and continue to grow, which is of great significance for cultivating new varieties of rhizomatous iris with long ornamental periods.

[0003] Iris japonica is a perennial perennial flower of the genus Iridaceae. It has fresh and elegant colors, peculiar flower shapes, and is evergreen all year round. It is mainly distributed in subtropical and temperate regions and is a good evergreen ground cover plant for viewing flowers. It can adapt to warm or shady environments and is particularly sensitive to late frost and severe cold. Under suitable conditions, Iris japonica has a long growth period and can show the characteristics of evergreen in winter. However, low temperatures in winter and early spring can significantly inhibit its growth, thereby shortening its viewing period and affecting its winter greening effect. Therefore, exploring the key genes that promote the release of Iris japonica ecological dormancy and homologously overexpressing these key genes in Iris japonica can not only promote the analysis of related gene functions and enrich existing dormancy research theories, but also effectively extend the viewing period and greening effect of Iris japonica, thereby promoting the development and gardening application of Iris japonica resources.

[0004] MYC2 (Myelocytomatosis proteins 2) is a member of the bHLH transcription factor family and a core transcription factor in the jasmonic acid signal transduction pathway (Shen Qian, Lu Xu, Zhang Ling, et al. Research progress on the function of MYC2 transcription factors in plants [J]. Journal of Shanghai Jiaotong University: Agricultural Science Edition, 2012, 30 (6): 7.). Studies have shown that MYC2 homologous genes are widely present in plants and play an important role in regulating plant growth and development, defense response, etc. (Pires N, Dolan l. Origin and diversification of basic helix-loop helix proteins in plants [J]. Molecular biology and evolution, 2010, 27 (4): 862-874.). However, in the field of dormancy, as a perennial monocot, the genetic composition and dormancy characteristics of butterfly flowers are quite different from those of dicot dormant plants. Therefore, there is no research report on the regulation of ecological dormancy of iris plants by MYC2, and it is also unclear whether MYC2 plays a role in bud dormancy of monocot plants.

[0005] Since a mature transgenic system has not yet been established in the genus Iris, the gene transient overexpression technology was used to explore the function of the MYC2 gene in the butterfly flower. This technology can establish a temporary and efficient expression system in cells to obtain a short-term high-level expression of the target gene (Zhao Wenting, Wei Jianhe, Liu Xiaodong, etc. Main methods and application progress of plant transient expression technology [J]. Biotechnology Communications, 2013 (02): 294-300.). In the experimental process, through a large amount of transient transcription and translation of a gene, the target gene can be overexpressed, the corresponding phenotype can be mutated, and the gene function can be reflected by changes in plant phenotypes or physiological indicators. This method can conveniently and quickly study gene function, so that the target gene can be expressed at a high level in a short time, and it does not rely on the establishment of a genetic transformation system. Therefore, the use of this technology can quickly and efficiently verify the function of the key genes of the butterfly flower, providing new insights into its dormancy regulation mechanism. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention proposes a new application of the butterfly flower MYC2 gene as a positive regulatory factor in promoting the release of plant ecological dormancy, providing theoretical support for the cultivation of new varieties of evergreen ornamental flowering ground covers.

[0007] The present invention provides an application of the butterfly flower MYC2 gene as a positive regulatory factor in promoting the release of plant ecological dormancy. The butterfly flower MYC2 gene is the IjMYC2 gene, and the protein coding region sequence of the IjMYC2 gene is shown in SEQ ID No.1.

[0008] The present invention also provides a method for promoting the release of plant ecological dormancy, wherein tissues or plant organs derived from butterfly flowers are subjected to gene overexpression, the overexpressed gene is the IjMYC2 gene, and then plants are obtained, wherein the nucleotide sequence of the protein coding region of the IjMYC2 gene is shown in SEQ ID No.1.

[0009] Gene overexpression uses transient overexpression technology. When the gene is overexpressed, after constructing a recombinant plasmid for gene overexpression, the recombinant plasmid is introduced into the tissue or plant organ of the butterfly flower source through the Agrobacterium transformation method. The plasmid backbone used when constructing the recombinant plasmid is the pHB-YFP vector. The Agrobacterium is GV3101.

[0010] Specifically, when transient overexpression is performed, the coding region sequence (CDS) of the target gene except the stop codon is first cloned into a corresponding plasmid or viral vector to construct a recombinant plasmid for gene overexpression, and then the recombinant plasmid is introduced into the butterfly flower related tissue or plant organ by Agrobacterium transformation. In the process of constructing the recombinant plasmid, the pHB-YFP vector is used as a plasmid backbone and introduced into GV3101 Agrobacterium for transfection.

[0011] Principle of the invention:

[0012] Plant transient gene expression is the use of several different types of viruses or bacteria, such as Agrobacterium, Tobacco Mosaic Virus and soil bacteria, to introduce foreign genes into plant cells and express the target protein in a short time. The basic operation process includes: inserting foreign genes into vectors, then transforming the vectors into viruses or bacteria, and finally introducing these viruses or bacteria into plant cells by infection or injection, so as to achieve the expression of foreign genes. Compared with transgenic plant technology, plant transient expression system has the advantages of short expression time, high transformation efficiency, low mutation and genetic stability. It has broad application prospects in production such as plant gene function research.

[0013] Although transient overexpression can work in some dicotyledonous model plants, the mode of action of this system does not work for all plants (especially monocotyledons), and its application is limited by the affinity between species and Agrobacterium. At present, transient overexpression technology has not yet formed a set of systematic and mature methods, and existing reports have only been successful in a few plants, such as tobacco, tomato, Arabidopsis, etc., and many inoculation conditions and steps need to be further optimized. Before the present invention was proposed, researchers in this field generally believed that transient overexpression was difficult to apply to monocotyledons, especially Iris plants with strong disease resistance, for the above reasons.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention, for the first time, confirmed the important role of the MYC2 gene in the release of ecological dormancy in Butterfly Flower by the method of transient overexpression of homologous genes, and determined that the gene is located in the cell nucleus and the expression level increases with the release of ecological dormancy; when the Butterfly Flower IjMYC2 gene is overexpressed, the release of ecological dormancy and germination of the plant can be promoted.

[0016] (2) A mutant of the butterfly flower with overexpression of the IjMYC2 gene was obtained by transient overexpression technology. The results showed that the germination rate and growth rate of the mutant were significantly better than those of the wild type under recovery growth conditions, and its gene expression level was significantly increased, thereby promoting the release of the ecological dormancy of the butterfly flower. This discovery not only enriches the understanding of the function of the MYC2 gene, clarifies its role in releasing ecological dormancy in monocotyledonous perennial plants, but also provides theoretical support for the cultivation of new varieties of evergreen ornamental flowering ground covers.

[0017] (3) The depth and breadth of research on the MYC2 gene and its regulatory pathway in the prior art vary greatly between monocots and dicots. After dormancy is established, dicot dormant plants can generally transition from physiological dormancy to ecological dormancy through transient overexpression. As a monocot, the butterfly flower only exhibits ecological dormancy during the winter. The mutant that overexpresses the butterfly flower MYC2 gene can promote the plant to break the ecological dormancy state and continue to grow compared to the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the result diagram of MYC2 system occurrence analysis and conservative domain analysis diagram of butterfly flower and monocotyledonous and dicotyledonous representative plants in Example 1.

[0019] Figure 2 This is a graph showing the expression level changes of the IjMYC2 gene in the Butterfly Flower transcriptome in Example 2 in the stem apical meristem of Butterfly Flower at different dormancy periods.

[0020] Figure 3 This is a diagram showing the subcellular localization detection results of the IjMYC2 gene in Example 3.

[0021] Figure 4 These are growth phenotype diagrams and plant height change diagrams of the wild-type butterfly flower under the restored growth conditions in Example 5 and the butterfly flower with IjMYC2 gene overexpression obtained by transient overexpression technology.

[0022] Figure 5 This is a graph showing the results of detecting changes in the expression levels of IjMYC2 and the dormancy key gene IjSVP in the wild-type butterfly flowers and the butterfly flowers overexpressing the IjMYC2 gene under the recovery growth conditions in Example 6. DETAILED DESCRIPTION

[0023] The following embodiments of the present invention and the accompanying drawings are described in detail. The present embodiment is implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following embodiments. If the specific conditions are not specified in the embodiment, the conditions recommended by the normal conditions or manufacturers are carried out. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased from the market. The annual plant of butterfly flower used is purchased from Hangzhou Tianjing Aquatic Botanical Garden.

[0024] The primer sequences involved in the examples are shown in Table 1.

[0025] Table 1

[0026]

[0027] Example 1: Phylogenetic and Conserved Domain Analysis of MYC2 in Pansy and Representative Monocotyledonous and Dicotyledonous Plants

[0028] In order to clarify the phylogenetic relationship of MYC2 gene in monocotyledons and dicotyledons and confirm whether related genes contain its conserved domain, the phylogenetic tree of MYC2 protein was constructed using MEGA7.0 with the neighbor-joining method, and the gene position was analyzed using TBtools.

[0029] The MYC2 protein sequence of the butterfly flower in the evolutionary tree is derived from the transcriptome data sequenced by our laboratory, the genome information of species such as pear (Pyrusspp), peach (Prunuspersica), poplar (Populus trichocarpa), grape (Vitis vinifera), kiwi (Actinidia chinensis) is derived from Phytozome v13, and the genome information of species such as rice (Oryza sativa) and corn (Zea Mays) is derived from Ensembl Plants. The Arabidopsis AtMYC2 protein was selected as the object, and Blastp was used to search for protein sequences with high sequence consistency in the above species, and the amino acid sequence was aligned using the NCBI website. Then, MEGA7.0 was used to construct the phylogenetic tree of the MYC2 protein using the neighbor-joining method, and the protein conserved domain was predicted.

[0030] The results are as follows Figure 1 As shown, from the perspective of phylogenetic analysis, the MYC2 subfamily of dicotyledonous plants can be divided into three evolutionary branches, among which the MYC2 genes of pear, kiwifruit, and poplar are co-located on one branch, and are also clustered with the MYC2 genes of peach and grape. Unlike dicotyledonous plants, the MYC2 subfamily of monocotyledonous plants has two branches, among which the MYC2 of rice and corn are clustered together, while the MYC2 genes of the monocotyledonous ornamental flower butterfly flower are clustered into one branch, with a total of 7 transcripts, namely IJ.PB50840, IJ.PB21209, IJ.PB50797, IJ.PB24067, IJ.PB25449, IJ.PB21618, and IJ.PB48227. Example 2: Expression of the IjMYC2 gene in the stem apical meristem of butterfly flower in different winter months

[0031] The specific method is as follows:

[0032] (1) Shoot tip meristem samples of healthy one-year-old plants were collected during the autumn growth period, dormancy induction period, dormancy period, dormancy release period and spring growth period, and RNA was extracted and reverse transcribed into cDNA.

[0033] (2) After testing the cDNA quality, the SMARTer PCR cDNA Synthesis Kit was used to synthesize cDNA, and the Iso-seq libraries of 1-2 kb, 2-3 kb, and 3-6 kb were constructed after Blue Pippin fractionation, and sequenced using the PacBio RS II platform. At the same time, Illumina libraries were constructed for the above samples, and NGS data were generated using the Illumina Xten platform.

[0034] The relevant raw data were uploaded to the NCBI SRA database BioProject PRJNA486414. SMRT data were processed by SMRT Analysis v2.3.0 software, and the ICE algorithm was used to identify and correct FL and NFL cDNAs. The NGS data were further corrected and de-redundant to generate high-quality transcriptomes. The NGS data were de-linked, low-quality, and "N"-containing base sequences, and aligned to the FL transcriptome using Bowtie 2. The expression level was calculated using RSEM, and the results were expressed in FPKM.

[0035] (3) Use BLAST tools to annotate multiple databases (COG, eggNOG, KEGG, Pfam, Swiss-Prot, Nr and GO); predict open reading frames (ORFs) using TransDecoder, and screen out complete coding sequences (CDS). Locate the MYC2 gene in the annotation results, and calculate its FPKM value at different developmental stages.

[0036] (4) Based on the expression level of the MYC2 gene in the stem apical meristem during the critical period, the function of the gene in ecological dormancy was predicted. Among them, the expression level of IJ.PB48227 increased with the release of dormancy, and the expression level was always high. Therefore, it was speculated that it may play a role in promoting the release of dormancy in Butterfly Flower. This gene was selected for subsequent functional verification analysis.

[0037] The results are as follows Figure 2 As shown, the expression level of IjMYC2 gene varies in different winter months, but the gene expression level generally turns during the hibernation dormancy induction period and the dormancy release period, and then gradually recovers with the release of dormancy.

[0038] Example 3: Full-length cloning and subcellular localization of the MYC2 gene

[0039] First, using the NCBI website, full-length cloning primers were designed based on the transcript conserved region of the target gene IJ.PB48227 annotated in the transcriptome data of Pansy, and used to amplify the full-length coding sequence. IjMYC2 full-length cloning primers IjMYC2-F and IjMYC2-R.

[0040] Total RNA was extracted from the stem tip of the butterfly flower, and the total RNA of the butterfly flower was reverse transcribed into cDNA using the TAKARA first-strand cDNA synthesis kit (Cat#6210A). The IjMYC2 gene fragment was obtained by PCR amplification using a high-fidelity enzyme. The PCR product was obtained by agarose gel electrophoresis and gel excision recovery, and then connected to the 5min pEASY-Blunt Zero Cloning Vector (Novozyme), and the ligation product was transferred into Escherichia coli DH5a. The single clone was screened and picked on the resistance medium, and the positive clone was identified by PCR using the universal primer M13 provided by the vector and the correctness of the PCR product was confirmed by sequencing.

[0041] Subsequently, tobacco leaves were used to perform subcellular localization of the IjMYC2 gene. According to the nucleotide sequence of the IjMYC2 gene coding region verified by sequencing, specific primers with homology arms were designed to amplify the full-length coding sequence except the stop codon. The nucleotide sequence of the IjMYC2 gene coding region verified by sequencing is shown in SEQ ID No.1 (the encoded amino acid sequence is shown in SEQ ID No.2), with a length of 1992bp. At the same time, the recognition bases and protective base sequences CTGCAG and ACTAGT of the restriction endonucleases PstI and SpeI were added to the 5' ends of the forward and reverse primers, respectively. The IjMYC2 fragment cloning primers are IjMYC2-YFP-F and IjMYC2-YFP-R.

[0042] Subcellular localization uses pHB-YFP expression vector. Total RNA was extracted from the stem tip of Pansy, and the total RNA of Pansy was reverse transcribed into cDNA using TAKARA first-strand cDNA synthesis kit (Cat#6210A). The IjMYC2 gene fragment containing restriction sites and protective bases was obtained by PCR amplification using high-fidelity enzymes. The pHB-YFP plasmid was digested with PstI and SpeI at 37°C for 30 minutes, and the PCR product was obtained by electrophoresis and gel cutting. II One Step Cloning Kit (Novozyme) was used to obtain the recombinant product of the target fragment and the linearized pHB-YFP vector, which was transformed into competent Escherichia coli DH5α. After culturing in the dark at 37°C overnight, a single clone was picked out, and PCR identification was performed with detection primers IjMYC2-YFP-F and IjMYC2-YFP-R to obtain positive clones. The correctness of the PCR product was confirmed by sequencing, and Escherichia coli containing 35S::IjMYC2-YFP was obtained.

[0043] Use the correctly sequenced Escherichia coli to extract its plasmid by shaking, and transfer the target plasmid (35S::IjMYC2-YFP) into Agrobacterium GV3101. Pick the positive clone in 5mL liquid LB medium (containing 50mg / L kanamycin and 50mg / L rifampicin), 28℃, 200rpm, shake and culture until the logarithmic growth phase. Collect the bacteria by centrifugation at 5000rpm for 5min, resuspend the bacteria in infection solution (containing 10mM MgCl2, 100mM MES with pH=5.6 and 100μM acetosyringone), and inject it into the back of the leaves of transgenic Nicotiana benthamiana containing mCherry nuclear localization signal. After three days of dark culture, observe the fluorescence and take pictures under a laser confocal microscope (OlymDus FV3000, Japan).

[0044] The results are as follows Figure 3 As shown, the results showed that IjMYC2-YFP protein was localized in the cell nucleus, while free YFP was distributed throughout the cell.

[0045] Example 4: Transient overexpression of the IjMYC2 gene in Pansy to obtain mutants

[0046] In order to clarify the effect of IjMYC2 gene overexpression on the overwintering growth of butterfly flowers, the IjMYC2 gene was homologously overexpressed using transient overexpression technology to obtain mutants.

[0047] Transient overexpression used the Agrobacterium GV3101 vector containing 35S::IjMYC2-YFP obtained by subcellular localization in Example 3. 50 μL of Agrobacterium GV3101 bacterial solution containing YFP and IjMYC2-YFP was added to 5 mL of LB culture solution containing 50 mg / L kanamycin and 50 mg / L rifampicin, and the culture was shaken at 28°C and 200 rpm for 16 hours, and then transferred to 200 mL LB liquid culture medium containing 50 mg / L kanamycin, 50 mg / L rifampicin, 10 mM MES and 20 μM AS, and the culture was shaken at 28°C and 200 rpm for 16 hours; when the OD of the bacterial solution reached 600 When the concentration is about 2.0, centrifuge at 4000rpm and 25℃ for 10 minutes to collect the bacteria. Resuspend the bacteria in 200mL of infection solution containing 10mM MgCl2, 10mM MES, and 200μM AS. Mix the components of each group of bacterial solutions in a volume ratio of 1:1, activate them at 25℃ in the dark for 3 hours, and use a 1mL syringe to inject the leaves of one-year-old butterfly flowers with the same growth: Agrobacterium GV3101 bacterial solution containing IjMYC2-YFP. The plants were then cultured in the dark at 21℃ for 3 days, and then cultured for 1 week under the conditions of a day and night temperature difference of 25℃-18℃, a relative humidity of 60%, and a light cycle of 16h / dark cycle of 8h.

[0048] The IjMYC2 gene was introduced into the leaves of the butterfly flower for transient overexpression using Agrobacterium, which allowed for experimental operations such as growth observation of overexpressed plants and QPCR expression analysis of key genes, opening up a new way to conveniently and quickly study gene functions and characteristics.

[0049] Example 5: Observation of the growth of Pansy lily IjMYC2 overexpressing plants under recovery growth conditions

[0050] The leaves of the butterfly flower mutant and wild-type plants containing the recombinant plasmid were uniformly cut to 10 cm high from the rhizome, and then placed in a growth room with a day and night temperature difference of 25℃-18℃, a relative humidity of 60%, and a light cycle of 16h / dark cycle of 8h for recovery growth culture for 28 days, and the growth changes of the wild-type and IjMYC2 overexpressing plants were recorded.

[0051] Plant height statistics start from the junction of rhizomes and leaves and measure to the highest point of leaf expansion; germination rate refers to the percentage of plants with plant height increase greater than 0.5 cm in a treatment to the number of plants used in the treatment; the number of leaves is based on the number of leaves with green parts greater than 2 / 3; each treatment contains 3 biological replicates. Figure 4 As shown in A, under the recovery growth conditions, the germination rate of the Pansy floribunda IjMYC2-overexpressing plants was higher than that of the wild type control, and the plant height of the Pansy floribunda IjMYC2-overexpressing plants increased faster than that of the wild type.

[0052] Eighteen wild-type butterfly flowers and eighteen butterfly flowers overexpressing the IjMYC2 gene obtained by transient overexpression technology were divided into three groups, and the average plant height of six plants in each group was taken, and then the average plant height of the three groups was taken; each treatment contained six biological replicates. Figure 4 As shown in B, under the recovery growth condition, the height of the MYC2-overexpressing plants of Pansy was faster than that of the wild type. Within 28 days, the former increased by about 10 cm or more, while the latter increased by about 10 cm or less. This proves that overexpressing the MYC2 gene can effectively release the ecological dormancy of Pansy and promote its growth.

[0053] Example 6: Changes in the expression levels of IjMYC2 and dormancy key gene IjSVP in IjMYC2-overexpressing plants of Pansy under recovery growth conditions

[0054] 100 mg of functional leaves of the above-mentioned butterfly flower IjMYC2 overexpressing plant that had been cultured for 28 days of recovery growth were taken to extract total RNA, and the total RNA was reverse transcribed into cDNA. The expression of IjMYC2 and the dormancy key gene IjSVP in the young leaves of butterfly flower was determined according to the method and primer sequences in Example 2. The specific primers of the four genes were IjMYC2-qpcr-F and IjMYC2-qpcr-R, IjSVP-qpcr-F and IjSVP-qpcr-R, respectively.

[0055] like Figure 5 As shown, the expression level of IjMYC2 was significantly increased and the expression level of IjSVP was significantly decreased in the IjMYC2 gene knockout mutant of Pansy, thereby promoting plant germination.

[0056] Based on the above research, the present invention discovered that the IjMYC2 gene positively regulates the ecological dormancy process of butterfly flowers during winter, and its overexpression mutant can promote the release of ecological dormancy compared with the wild type.

Claims

1. Application of the butterfly flower MYC2 gene as a positive regulatory factor in promoting the release of plant ecological dormancy.

2. The use according to claim 1, characterized in that The butterfly flower MYC2 gene is the IjMYC2 gene, and the protein coding region sequence of the IjMYC2 gene is shown in SEQ ID No.

1.

3. A method for promoting the release of plant ecological dormancy, characterized in that: The tissue or plant organ derived from the butterfly flower is subjected to gene overexpression, the overexpressed gene is the IjMYC2 gene, and then the plant is obtained.

4. The method for promoting the release of plant ecological dormancy as claimed in claim 3, characterized in that: The nucleotide sequence of the protein coding region of the IjMYC2 gene is shown in SEQ ID No.

1.

5. The method for promoting the release of plant ecological dormancy as claimed in claim 3, characterized in that: The gene overexpression uses transient overexpression technology.

6. The method for promoting the release of plant ecological dormancy as claimed in claim 5, characterized in that: When the gene is overexpressed, a recombinant plasmid for gene overexpression is constructed, and then the recombinant plasmid is introduced into tissues or plant organs derived from butterfly flowers through the Agrobacterium transformation method.

7. The method for promoting the release of plant ecological dormancy as claimed in claim 6, characterized in that: The plasmid backbone used in constructing the recombinant plasmid was pHB-YFP vector.

8. The method for promoting the release of plant ecological dormancy as claimed in claim 6, characterized in that: The Agrobacterium is GV3101.

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