Application of the MYC2 gene of Pansy as a positive regulatory factor in promoting the release of plant ecological dormancy

Abstract

The present invention discloses the use of the MYC2 gene of Pansy as a positive regulatory factor in promoting the release of plant ecodormancy, relating to the field of biotechnology. The present invention, for the first time, demonstrates the important role of the MYC2 gene in releasing ecodormancy in Pansy by transiently overexpressing a homologous gene. The invention also confirms that the gene is localized in the cell nucleus and that its expression increases with the release of ecodormancy. Overexpression of the MYC2 gene in Pansy promotes the release of plant ecodormancy and germination.

Description

Technical Field

[0001] The present 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 dormancy is a complex biological process that can be divided into two types: physiological dormancy and ecological dormancy. Physiological dormancy refers to the cessation of growth at a growing point due to endogenous signals, and even under suitable conditions, it is impossible to immediately break dormancy and resume growth. Ecological dormancy occurs when a growing point has regained its growth capacity but is restricted by environmental factors (Lang GA, Early JD, Martin GC, Darnell RL. Endo-, para-, and ecodormancy: physiological terminology and classification for dormancy research [J]. Hortscience, 1987, 371-377). Currently, research on plant dormancy has mostly focused on dicotyledons, which generally exhibit typical physiological and ecological dormancy processes. However, in monocots represented by rhizome iris, there is only ecological dormancy in its overwintering 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 important to explore the key genes that regulate the ecological dormancy of rhizome 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 rhizome iris with long ornamental periods.

[0003] Iris japonica is a perennial perennial flower of the genus Iridaceae. It has fresh and elegant colors, unique flower shapes, and is evergreen all year round. It is mainly distributed in subtropical and temperate regions and is a good evergreen ornamental ground cover plant. It can adapt to warm or shady environments and is particularly sensitive to late frosts and severe cold. Under suitable conditions, Iris japonica has a long growth period and can exhibit 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 of Iris plant resources and garden applications.

[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 Jiao Tong University: Agricultural Sciences 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 responses, 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 flower are quite different from those of dicot dormant plants. Therefore, there are currently no reports on the regulation of ecological dormancy of Iris plants by MYC2, and it is still unclear whether MYC2 plays a role in bud dormancy in 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, et al. Main methods and application progress of plant transient expression technology [J]. Biotechnology Letters, 2013 (02): 294-300.). During the experiment, by transiently transcribing and translating a gene in large quantities, the target gene can be overexpressed, causing the corresponding phenotype to mutate, and the gene function can be reflected by changes in plant phenotype or physiological indicators. This method can quickly and conveniently study gene function, allowing the target gene to be expressed at a high level in a short period of time, and does not rely on the establishment of a genetic transformation system. Therefore, using this technology can quickly and efficiently verify the function of key genes in the butterfly flower, providing new insights into its dormancy regulation mechanism. Summary of the Invention

[0006] In response to 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 a tissue or plant organ derived from butterfly flower is subjected to gene overexpression, wherein the overexpressed gene is the IjMYC2 gene, and then a plant is obtained, wherein the nucleotide sequence of the protein coding region of the IjMYC2 gene is shown in SEQ ID No. 1.

[0009] Gene overexpression utilizes transient overexpression technology. A recombinant plasmid for gene overexpression is constructed and then introduced into tissues or plant organs derived from Pansy via Agrobacterium tumefaciens transformation. The plasmid backbone used in constructing the recombinant plasmid is the pHB-YFP vector. The Agrobacterium is GV3101.

[0010] Specifically, for transient overexpression, the coding sequence (CDS) of the target gene, excluding the stop codon, is first cloned into a corresponding plasmid or viral vector to construct a recombinant plasmid for gene overexpression. This recombinant plasmid is then introduced into the relevant tissues or plant organs of Pansy via Agrobacterium transformation. During the construction of the recombinant plasmid, the pHB-YFP vector is used as the plasmid backbone and introduced into GV3101 Agrobacterium for transfection.

[0011] Principle of the invention:

[0012] Transient gene expression in plants utilizes 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 within a short period of time. The basic process involves inserting the foreign gene into a vector, then transforming the vector into a virus or bacterium, and finally introducing the virus or bacteria into plant cells through infection or injection to achieve gene expression. Compared to transgenic plant technology, plant transient expression systems offer advantages such as shorter expression times, higher transformation efficiency, lower mutation rates, and greater genetic stability. They hold broad application prospects in areas 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, due to 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 through 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, it can promote the release of ecological dormancy and germination of plants.

[0016] (2) Using transient overexpression technology, a mutant of the butterfly flower with overexpression of the IjMYC2 gene was obtained. The results showed that the germination rate and growth rate of the mutant plant under recovery growth conditions were significantly better than those of the wild type. At the same time, its gene expression level was significantly increased, thereby promoting the release of the butterfly flower's ecological dormancy. This discovery not only enriches the understanding of the function of the MYC2 gene and clarifies its role in releasing ecological dormancy in monocotyledonous perennials, 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 existing technologies 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. However, as a monocot, the butterfly flower only exhibits ecological dormancy during the winter. Mutants that overexpress the butterfly flower MYC2 gene can promote the plant to break out of ecological dormancy and continue to grow compared to the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the MYC2 phylogenetic analysis results and conserved domain analysis of the butterfly flower and representative monocotyledonous and dicotyledonous 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 stages.

[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 recovery 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 wild-type butterfly flowers and IjMYC2 gene-overexpressing butterfly flowers under the recovery growth conditions in Example 6. DETAILED DESCRIPTION

[0023] Below, embodiments of the present invention and accompanying drawing are described in detail. The present embodiment is implemented under the premise of technical solution of the present invention, and detailed implementation method and specific operating process are provided, but protection scope of the present invention is not limited to following embodiment. In the embodiment, those who do not indicate specific conditions are carried out according to the conditions of normal conditions or manufacturer's advice. Reagents used or instruments that do not indicate manufacturers are 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] To clarify the phylogenetic relationship of MYC2 genes in monocotyledons and dicotyledons and to confirm whether related genes contain its conserved domains, a phylogenetic tree of MYC2 protein was constructed using MEGA7.0 with the neighbor-joining method, and gene position analysis was performed using TBtools.

[0029] The MYC2 protein sequence from the butterfly flower in the phylogenetic tree was derived from transcriptome data sequenced in our laboratory. Genome information for species such as pear (Pyrus spp.), peach (Prunus persica), poplar (Populus trichocarpa), grape (Vitis vinifera), and kiwifruit (Actinidia chinensis) was obtained from Phytozome v13, and genome information for species such as rice (Oryza sativa) and maize (Zea mays) was obtained from Ensembl Plants. The Arabidopsis thaliana AtMYC2 protein was selected as the target. Blastp was used to search for protein sequences with high sequence identity in the above species. Amino acid sequence alignment was performed using the NCBI website. A phylogenetic tree of the MYC2 protein was then constructed using the neighbor-joining method using MEGA 7.0, and conserved domains were predicted.

[0030] The results are as follows Figure 1 As shown, from the perspective of phylogeny, the MYC2 subfamily of dicotyledons can be divided into three evolutionary branches, among which the MYC2 genes of pear, kiwifruit, and poplar are located on the same branch, and are also clustered with the MYC2 genes of peach and grape. Unlike dicotyledons, the MYC2 subfamily of monocotyledons 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 apical meristem samples were collected from healthy one-year-old plants during the autumn growth period, dormancy induction period, dormancy period, dormancy release period, and spring growth period. 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. After fractionation using Blue Pippin, 1-2 kb, 2-3 kb, and 3-6 kb Iso-seq libraries were constructed 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 using SMRT Analysis v2.3.0 software, using the ICE algorithm to identify and correct FL and NFL cDNAs. Combined with NGS data, the data were further corrected and de-redundant to generate a high-quality transcriptome. The NGS data were stripped of adapters, low-quality sequences, and sequences containing "N" bases. The data were then aligned to the FL transcriptome using Bowtie 2. Expression levels were calculated using RSEM, and the results are expressed in FPKM.

[0035] (3) BLAST tools were used to annotate multiple databases (COG, eggNOG, KEGG, Pfam, Swiss-Prot, Nr, and GO). TransDecoder was used to predict open reading frames (ORFs) and screen the complete coding sequences (CDSs). The MYC2 gene in the annotation results was located and its FPKM values ​​at different developmental stages were calculated.

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

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

[0038] Example 3: Full-length cloning and subcellular localization of the IjMYC2 gene

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

[0040] Total RNA was extracted from the stem apex of Pansy lilies and reverse-transcribed into cDNA using the TAKARA First-Strand cDNA Synthesis Kit (Cat#6210A). The IjMYC2 gene fragment was amplified by PCR using a high-fidelity enzyme. The PCR product was recovered by agarose gel electrophoresis and ligated with the 5-min pEASY-Blunt Zero Cloning Vector (Novagen). The ligation product was then transformed into Escherichia coli DH5a. Single colonies were screened and isolated on resistance culture medium. Positive clones were identified by PCR using the universal primer M13 provided with 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. Based on 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 for 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 protection 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 was performed using the pHB-YFP expression vector. 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 containing restriction sites and protective bases was obtained by PCR amplification using a high-fidelity enzyme. 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. The target fragment and the linearized pHB-YFP vector were recombined using II One Step Cloning Kit (Novozymes), and the recombinant product was transformed into competent Escherichia coli DH5α. After culturing in the dark at 37°C overnight, single clones were picked and PCR identification was performed using the detection primers IjMYC2-YFP-F and IjMYC2-YFP-R. After obtaining positive clones, the correctness of the PCR product was confirmed by sequencing, and Escherichia coli containing 35S::IjMYC2-YFP was obtained.

[0043] Correctly sequenced Escherichia coli were shaken to extract the plasmid, and the target plasmid (35S::IjMYC2-YFP) was transformed into Agrobacterium tumefaciens GV3101. Positive clones were selected and cultured in 5 mL of liquid LB medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin) at 28°C, shaking at 200 rpm until the logarithmic growth phase. Bacteria were harvested by centrifugation at 5000 rpm for 5 minutes, resuspended in infection solution (containing 10 mM MgCl2, 100 mM MES (pH 5.6), and 100 μM acetosyringone), and then injected into the dorsal surface of leaves of transgenic Nicotiana benthamiana plants containing the mCherry nuclear localization signal. After three days of incubation in the dark, fluorescence was observed and photographed 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 nucleus, while free YFP was distributed throughout the cell.

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

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

[0047] Transient overexpression was performed using the Agrobacterium GV3101 vector containing 35S::IjMYC2-YFP obtained by subcellular localization in Example 3. 50 μL of the Agrobacterium GV3101 bacterial solution containing YFP and IjMYC2-YFP was added to 5 mL of LB culture medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and the culture was shaken at 28°C and 200 rpm for 16 hours. The culture was then transferred to 200 mL of 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 0. 600 When the concentration is approximately 2.0, collect the cells by centrifugation at 4000 rpm and 25°C for 10 minutes. Resuspend the cells in 200 mL of infection solution containing 10 mM MgCl2, 10 mM MES, and 200 μM AS. Mix the following components of each solution at a 1:1 volume ratio, incubate at 25°C in the dark for 3 hours, and then inject the Agrobacterium GV3101 solution containing IjMYC2-YFP into the leaves of one-year-old butterfly plants using a 1 mL syringe. The plants are then incubated in the dark at 21°C for 3 days, followed by incubation for 1 week under conditions with a diurnal temperature range of 25°C-18°C, 60% relative humidity, and a 16-hour light / 8-hour dark cycle.

[0048] By using Agrobacterium to introduce the IjMYC2 gene into the leaves of the butterfly flower for transient overexpression, experimental operations such as growth observation of overexpressed plants and QPCR expression analysis of key genes can be carried out, opening up a new way to conveniently and quickly study gene functions and characteristics.

[0049] Example 5: Observation of the growth of IjMYC2-overexpressing plants of Pansy 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 chamber 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. The growth changes of the wild-type and IjMYC2 overexpressing plants were recorded.

[0051] Plant height statistics were measured from the junction of the rhizome and the leaves to the highest point of the leaves; the germination rate refers to the percentage of plants with a plant height increase of more than 0.5 cm in a treatment to the total number of plants used in that treatment; the number of leaves is based on the number of leaves with a green part greater than 2 / 3; each treatment contained three biological replicates. Figure 4 As shown in A, under recovery growth conditions, the germination rate of the IjMYC2-overexpressing plants of Pansy was higher than that of the wild type control, and the plant height of the IjMYC2-overexpressing plants of Pansy grew 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, with the average plant height of six plants in each group being taken, and then the average plant height of the three groups was taken; each treatment contained six biological replicates. Figure 4 As shown in Figure B, under recovery conditions, MYC2-overexpressing plants in Pansy rosea grew faster than wild-type plants, increasing by approximately 10 cm over 28 days, while wild-type plants increased by approximately 10 cm. This demonstrates that overexpressing MYC2 can effectively break Pansy rosea's ecological dormancy and promote its growth.

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

[0054] 100 mg of functional leaves of the above-mentioned butterfly flower IjMYC2 overexpressing plant that had recovered growth and cultured for 28 days were used to extract total RNA and 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 for 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 butterfly flower, thereby promoting plant germination.

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

Claims

1. Overexpression of Pansy MYC2 Application of genes in promoting the release of the ecological dormancy of butterfly flowers, the butterfly flowers MYC2 Gene IjMYC2 gene, the IjMYC2 The protein coding region sequence of the gene is shown in SEQ ID No.

1.

2. A method for promoting the release of plant ecological dormancy, characterized in that: The tissue or plant organ derived from butterfly flower is subjected to gene overexpression, and the overexpressed gene is IjMYC2 Gene, and then obtain a plant; the plant is butterfly flower; the IjMYC2 The nucleotide sequence of the protein coding region of the gene is shown in SEQ ID No.

1.

3. The method for promoting the release of plant ecological dormancy according to claim 2, wherein: The gene overexpression uses transient overexpression technology.

4. The method for promoting the release of plant ecological dormancy according to claim 3, wherein: When the gene is over-expressed, a recombinant plasmid for gene over-expression is constructed, and then the recombinant plasmid is introduced into tissues or plant organs derived from butterfly flowers through the Agrobacterium transformation method.

5. The method for promoting the release of plant ecological dormancy according to claim 4, wherein: The plasmid backbone used in constructing the recombinant plasmid was the pHB-YFP vector.

6. The method for promoting the release of plant ecological dormancy according to claim 4, wherein: The Agrobacterium is GV3101.

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