Psbhlh35 gene and coding protein and application thereof in somatic embryogenesis of tree peony

Abstract

This application belongs to the field of plant molecular biology technology and specifically provides a PsbHLH35 gene and encoded protein, and their use in tree peony somatic embryogenesis. A PsbHLH35 gene, comprising the nucleotide sequence shown in SEQ ID NO. 1. A protein encoded by the PsbHLH35 gene, comprising the amino acid sequence shown in SEQ ID NO. 2. A method for using the PsbHLH35 gene in tree peony somatic embryogenesis, comprising the following steps: S1) cloning the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector; S2) transforming the PsbHLH35 gene overexpression vector into tree peony. The PsbHLH35 gene of this application has a significant promoting effect on tree peony somatic embryogenesis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant molecular biology, and particularly relates to a PsbHLH35 gene and a coding protein and application of the PsbHLH35 gene and the coding protein in somatic embryogenesis of peony. BACKGROUND

[0002] Peony (Paeonia Sect. Moutan) has important cultural, ornamental and medicinal values. Research and application of tissue culture technology to establish an efficient breeding and propagation technology system has gradually become an inevitable requirement for promoting the industrialization development of peony. In vitro meristematic nodule plant regeneration and embryo culture are two important tissue culture technologies, which have broad application prospects in the expansion of excellent clones and breeding practices.

[0003] At present, the research on peony tissue culture regeneration system mainly includes two ways: 1. Direct regeneration way of inducing direct seedlings from explants; 2. Indirect regeneration way of forming multiple shoots from callus induced by explants, i.e. somatic embryo regeneration way. Among them, the direct regeneration way of peony mainly induces seed embryos and scale buds to form seedlings (Wang et al., 2018; Huang et al., 2020; Yin et al., 2021). However, the problems such as low rooting rate and transplanting survival rate of test-tube seedlings have not been effectively solved by the direct regeneration way, which is difficult to be applied in large-scale production (Shang et al., 2017; Lian et al., 2020; Zhang et al., 2021). Therefore, the somatic embryo regeneration way should be one of the effective ways to solve the above problems, because the somatic embryo has the same ability to germinate and form a complete plant as the zygotic embryo (Karami et al., 2020; Zhang et al., 2021).

[0004] The research on peony somatic embryo regeneration way started in 1969, when Demoise and Partanen first used zygotic embryos as explants to induce callus. Subsequently, researchers have successfully induced callus using anther, filament, petal, stem segment, petiole, leaf and other explants (Beruto et al., 2004; Zhu et al., 2012; Meng et al., 2018; Du et al., 2020; Zhang et al., 2021; Zhu et al., 2022). However, there are few reports on the induction of embryogenic callus and the occurrence of multiple shoots.

[0005] Wang Zheng (2010) initially established an indirect somatic embryo regeneration system for tree peonies using petioles as explants. They found that the embryonic callus induction rate was only 25-40%, indicating low conversion efficiency and poor callus differentiation and development. Zhu Xiangtao et al. (2012) used 'Fengdan' seed embryos for embryonic induction, achieving an embryonic callus induction rate of 38.33%. Furthermore, during the embryonic callus induction process in tree peonies, there are bottleneck issues such as the gradual decrease in the early initiation rate of somatic embryos (globally, heart-shaped, torpedo-shaped, and cotyledon-shaped embryos), low uniformity, and difficulty in inducing clustered buds (Shen et al., 2012; Zhu et al., 2018). This further complicates the establishment of a mature tree peonies somatic embryo regeneration system and severely restricts the development of such a system (Wang et al., 2018; Wen Shusheng et al., 2018; Song et al., 2022).

[0006] Although some progress has been made in peony somatic embryo regeneration technology, there are still problems such as low induction rate and difficulty in callus transformation. Therefore, it is urgent to develop new peony somatic embryo regeneration methods. Summary of the Invention

[0007] In view of the above problems, in order to further improve the bottleneck problems of peony somatic embryogenesis, the present application provides a PsbHLH35 gene and encoded protein and their application in peony somatic embryogenesis.

[0008] In a first aspect, the present application provides a PsbHLH35 gene, wherein the PsbHLH35 gene comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0009] The second aspect of the present application provides a coded protein, which is encoded by the above-mentioned PsbHLH35 gene; preferably, the coded protein comprises the amino acid sequence shown in SEQ ID NO.2.

[0010] A third aspect of the present application provides a method for using the PsbHLH35 gene in peony somatic embryogenesis, comprising the following steps:

[0011] S1) Clone the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector;

[0012] S2) Transforming tree peonies with the PsbHLH35 gene overexpression vector.

[0013] Furthermore, it also includes one or more of the following technical features:

[0014] S11 In S1), the PsbHLH35 gene comprises the nucleotide sequence shown in SEQ ID NO. 1;

[0015] S12 in S1), the vector is a plant protein overexpression vector;

[0016] S22 in S2), the transformation adopts Agrobacterium transformation method.

[0017] Further, the plant protein overexpression vector is pCAMBIA1302.

[0018] The fourth aspect of the present application provides a biological material related to the PsbHLH35 gene, which comprises any one of the following: a) a recombinant expression vector containing the PsbHLH35 gene described above; b) a bioengineering bacteria containing the PsbHLH35 gene described above, or a bioengineering bacteria containing the recombinant expression vector shown in a).

[0019] The fifth aspect of the present application provides a use of a biological material related to the PsbHLH35 gene in any one of the following: 1) regulating the somatic embryogenesis of Paeonia suffruticosa; 2) regulating the hormone content in the somatic embryogenesis process of Paeonia suffruticosa; 3) improving the occurrence efficiency of Paeonia suffruticosa embryogenic callus; 4) Paeonia suffruticosa breeding or germplasm improvement; 5) preparing a Paeonia suffruticosa breeding related kit and / or expression cassette; 6) preparing a transgenic Paeonia suffruticosa; 7) preparing a transgenic Arabidopsis.

[0020] The sixth aspect of the present application provides an overexpression vector comprising the PsbHLH35 gene described above; preferably, the overexpression vector is obtained by cloning the PsbHLH35 gene described above into a plant protein overexpression vector; more preferably, the plant protein overexpression vector is pCAMBIA1302.

[0021] The seventh aspect of the present application provides an explant comprising the overexpression vector described above.

[0022] The eighth aspect of the present application provides a Paeonia suffruticosa somatic embryo indirect regeneration method, which cultivates Paeonia suffruticosa somatic embryo callus containing the PsbHLH35 gene described above, and obtains Paeonia suffruticosa somatic embryo regeneration and differentiation of multiple shoots.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] 1. The present application develops Paeonia suffruticosa transcription factor PsbHLH35, which is located in the nucleus. The PsbHLH35 overexpression transformed Arabidopsis plant is constructed, which makes the transgenic Arabidopsis grow rapidly, the plant type is slightly larger, 2 more true leaves than the wild type, and promotes the growth of the main root.

[0025] 2, The application constructs PshHLH35 overexpression transformed peony callus, significantly improves the occurrence efficiency of peony embryogenic callus. The use of transcription factor PshHLH35 significantly promotes the expression level of peony somatic embryogenesis related genes, and effectively regulates the hormone content in the process of peony somatic embryogenesis. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a schematic diagram for predicting and analyzing the signal peptide of the protein encoded by the PshHLH35 gene of the application;

[0027] Figure 2 It is a schematic diagram for analyzing the transmembrane domain of the protein encoded by the PshHLH35 gene of the application;

[0028] Figure 3 It is a schematic diagram for GFP-PshHLH35 transformed DH5a colony PCR (M, DL2000 DNA Maker; 1-2, GFP-PshHLH35 colony PCR);

[0029] Figure 4 It is a schematic diagram for GFP-PshHLH35 transformed GV3101 colony PCR (M, 250bp DNA Ladder; 1-6 GFP-PshHLH35 colony PCR);

[0030] Figure 5 It is a schematic diagram for subcellular localization of the application PshHLH35 (A, All; B, DAPI; C, FITC; D, TRITC; E, TD).

[0031] Figure 6 It is a schematic diagram for the expression amount of PshHLH35 in different parts of peony in the application;

[0032] Figure 7 It is a schematic diagram for pCAMBIA1302-PshHLH35 transformed DH5a colony PCR (M, DL2000 DNA Maker; 1-2, pCAMBIA1302-PshHLH35 colony PCR);

[0033] Figure 8 It is a schematic diagram for pCAMBIA1302-PshHLH35 transformed GV3101 colony PCR (M, DL2000 DNA Maker; 1-8, pCAMBIA1302-PshHLH35 colony PCR);

[0034] Figure 9 It is a schematic diagram for positive screening of transgenic Arabidopsis T0 generation seeds in the application (A, Col-0; B, pCAMBIA1302-PshHLH35).

[0035] Figure 10 Schematic diagram of semi-quantitative PCR detection of T1 generation transgenic Arabidopsis thaliana in the application;

[0036] Figure 11 Schematic diagram of T2 generation transgenic Arabidopsis thaliana phenotype in the application (A, Col-0; B, pCAMBIA1302-PsbHLH35);

[0037] Figure 12 Schematic diagram of pCAMBIA1302-PsbHLH35 transformed LBA4404 colony PCR in the application (M, 250bp DNA Ladder; 1-4, pCAMBIA1302-PsbHLH35 colony PCR);

[0038] Figure 13 Schematic diagram of PsbHLH35 overexpression transformed Paeonia suffruticosa callus in the application (A is co-culture; B is somatic embryo induction);

[0039] Figure 14 Schematic diagram of PsbHLH35 overexpression transgenic callus RT-PCR positive identification in the application;

[0040] Figure 15 Schematic diagram of different transgenic Paeonia suffruticosa callus section observation in the application (left is pCAMBIA1302; right is 35s: PsbHLH35, black arrow is embryogenic callus cell mass);

[0041] Figure 16 Schematic diagram of PsbHLH35 transgenic Paeonia suffruticosa callus somatic embryogenesis key gene expression level analysis in the application;

[0042] Figure 17 Schematic diagram of determination data of hormone content in PsbHLH35 transgenic Paeonia suffruticosa callus in the application. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0044] The present inventors have found through a large number of experiments that the PsbHLH35 gene can play a relatively key regulatory role in Paeonia suffruticosa somatic embryogenesis, and can maintain high levels of IAA, BL, ABA, ACC and low levels of BARP and 5DS in the later stage of embryogenic cell establishment.

[0045] 本申请首先提供PsbHLH35基因及其编码蛋白,PsbHLH35基因包括如SEQ ID NO.1所示的核苷酸序列,为CDS序列。

[0046] SEQ ID NO.1:

[0047] ATGGATGATAACATTACTGAAGAATACAAGCTGTGGTGGGAAACCAAGATGTTCCTCCAAACCGAGGAGCTTGACAGGATGTTCCTCCAATCTGAAGAGCCCGACGGTTGGGGACTAGACGAGGCATTTTCTGGTTACTATGATTCGAGCTCGCCAGACGGAGCAGCGACGTCGCCGGCATCTAAGAACATTTTGTCAGAGAGAAACCGGAGGAAGAAGCTGAATGATAGACTCTATACACTTAGATCGGTGGTCCCTAACATTACTAAGATGGACAAAGCATCAATAATCAAAGACGCGATCGAGTATATCCAAGAGTTGCATCAACAAGAGAAGAGAATCCAAGCTGAAATATCACAACTGGAGTCAACAAAGAAGAATTTTGATGGTTTTGATCAGTCGGAGCAACAGGTTACACCATCATTGAGATCCAAGAAAAAAAGAACGGATCAGGTGCCAAGTTATCAAGAATCAACAACTTCTCCCATTGAGCTGCTTGAAATACGGGTGTCTTACATGGGAGAGAAGACGGTGGTAGTGAGCTTGACATGTAGTAAAAGAAGGGACACAATGGTAAAACTTTGTGAGGTTTTTGAATCATTGAAGCTCAAAATTATTACTGCCAATATCACTGCTTTTTCTGGAAGGCTTTTGAAAACGGTCTTTCTTGAGGCAGATGAAGAGGAGAAAGATCAGTTGAAGGCAAGGATTGAGACAGCCATAGCAGCTCTTAATGATCCACCGAGCCCTATGAGCATCTAA

[0048] The encoded protein is obtained by encoding the above-mentioned PsbHLH35 gene. The encoding refers to the gene sequence guiding the synthesis of a specific protein, including DNA transcription, mRNA splicing, mRNA processing, translation, protein folding, post-modification and other processes.

[0049] In a specific embodiment of the present application, the encoded protein includes the amino acid sequence shown in SEQ ID NO. 2, is a non-secretory protein, does not belong to the transmembrane protein class, is an unstable hydrophobic liposoluble protein, and is an acidic protein.

[0050] SEQ ID NO.2:

[0051] MDDNITEEYKLWWETKMFLQTEELDRMFLQSEEPDGWGLDEAFSGYYDSSSPDGAATSPASKNILSERNRRKKLNDRLYTLRSVVPNITKMDKASIIKDAIEYIQELHQQEKRIQAEISQLESTK KNFDGFDQSEQQVTPSLRSKKKRTDQVPSYQESTTSPIELLEIRVSYMGEKTVVVSLTCSKRRDTMVKLCEVFESLKLKIITANITAFSGRLLKTVFLEADEEEKDQLKARIETAIAALNDPPSP

[0052] The present application provides a method for applying the PsbHLH35 gene in peony somatic embryogenesis, comprising the following steps:

[0053] S1) Clone the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector;

[0054] S2) Transforming tree peonies with the PsbHLH35 gene overexpression vector.

[0055] Furthermore, it also includes one or more of the following technical features:

[0056] S11 In S1), the PsbHLH35 gene comprises the nucleotide sequence shown in SEQ ID NO. 1;

[0057] S12 in S1), wherein the vector is a plant protein overexpression vector;

[0058] S22 In S2), the transformation is carried out using the Agrobacterium transformation method.

[0059] In a specific embodiment of the present application, the vector is a plant protein overexpression vector, which refers to a vector comprising a strong promoter, a multiple cloning site, a selection marker, a terminator and other elements for achieving efficient expression of a specific protein in plant cells. For example, the plant protein overexpression vector can be a pCAMBIA series (such as pCambia1301, pCambia1302, pCambia1300), etc., which only needs to overexpress the target gene. In a specific embodiment of the present application, the plant protein overexpression vector is pCAMBIA1302.

[0060] In the specific embodiments of this application, "transformation" refers to the process of introducing foreign genetic material (e.g., DNA fragments) into cells or organisms. This process can occur spontaneously in nature or through laboratory techniques. Various transformation methods exist, including chemical methods, electroporation, gene guns, viral vectors, Agrobacterium-mediated methods, and liposome-mediated methods. Through these techniques, specific gene fragments can be introduced into target cells, thereby changing their genetic characteristics or imparting new functions.

[0061] In the specific embodiment of the present application, the transformation adopts the Agrobacterium transformation method, and the method for transforming Agrobacterium can be the freeze-thaw method. When carrying out Agrobacterium transformation, conventional Agrobacterium strains can be selected, such as EHA105, GV3101, etc., which have been widely used in plant transformation. When transforming Agrobacterium, freeze-thaw method or electroporation can be adopted. The freeze-thaw method increases the permeability of the bacterial cell membrane by alternating freezing and thawing processes, promoting the absorption of exogenous DNA. The electroporation method is to apply an electric field to form short-term micropores on the bacterial cell membrane, so that DNA fragments are able to pass through the cell membrane and enter the cell. After completing the transformation, by cultivating Agrobacterium in a culture medium containing appropriate antibiotics, the bacterial strain that has been successfully transformed can be screened out.

[0062] The present application also provides an explant transformed with the above-mentioned overexpression vector. An explant refers to a tissue fragment cut from an organism for use in tissue culture or cell culture. In plant tissue culture, an explant is a starting material used in the culture process and can be any part of a plant, such as a leaf, stem segment, root tip, anther, embryo, etc. The explant is used to initiate the culture process with the goal of inducing tissue differentiation, proliferation, or regeneration to form a new plant or cell line.

[0063] Example

[0064] The application of the PsbHLH35 gene in tree peony somatic embryogenesis in this example includes the following experiments:

[0065] PsbHLH35 encoding amino acid analysis in this example:

[0066] Based on the cloned CDS sequence of the tree peony PsbHLH35 gene, online signal peptide prediction analysis showed that the PsbHLH35 protein did not have a signal peptide and was a non-secreted protein (see Figure 1 ). The transmembrane domain prediction analysis revealed that the PsbHLH35 protein does not have a transmembrane domain and does not belong to the transmembrane protein class (see for details). Figure 2 Online analysis of the physicochemical properties of the amino acid sequence encoded by the PsbHLH35 gene revealed that the protein encoded by the PsbHLH35 gene has a relative molecular mass of 29.04 kDa, an instability index of 77.92, a lipid solubility index of 79.80, and a negative overall average hydrophilicity index, indicating an unstable, hydrophobic, liposoluble protein (see Table 1 for details). The protein has an isoelectric point of 5.10, and the number of acidic amino acid residues is greater than the number of basic amino acid residues, indicating that it is an acidic protein (see Table 1 for details).

[0067] Table 1 Analysis of the components and physicochemical properties of the nucleotide sequence and encoded amino acid sequence of the tree peony PsbHLH35 gene

[0068]

[0069] Subcellular localization of PsbHLH35 in this example:

[0070] GFP-specific primers with KpnI and XbaI restriction sites were designed for PsbHLH35, and its GFP expression vector was constructed. 1-2 mm single strains were selected and the band size was confirmed by colony PCR (see Figure 3 ), and the strain was sent to Bioengineering for sequencing. Plasmids were extracted from the correct positive strain. A GFP fusion expression vector, GFP-PsbHLH35, was successfully constructed for subcellular localization studies.

[0071] Transform the GFP-PsbHLH35 plasmid into Agrobacterium tumefaciens GV3101, and pick a single strain of 1-2 mm and verify its correctness by colony PCR (see Figure 4 The correct positive strain was cultured with shaking in two steps until OD600 = 1.0, then injected to infect the lower epidermis of tobacco leaves. After culturing for 3 days, laser confocal microscopy was used for observation (see Figure 5 ),like Figure 5 As shown, the target protein emits green fluorescence in the nucleus of tobacco leaves, DAPI staining emits blue fluorescence in the nucleus, no fluorescence signal is seen under the chloroplast fluorescence field, and their fluorescence signals completely coincide in the overlapping field, thus proving that the PsbHLH35 protein is subcellularly localized in the nucleus.

[0072] PsbHLH35 tissue-specific expression analysis in this example:

[0073] Selected well-growing peony 'Fengdanbai' seedlings, when they were setting seeds in mid-to-late June of the same year, directly extracted total RNA from their roots, stems, leaves, flowers, and seeds, and reverse transcribed to obtain cDNA from the corresponding parts for RT-PCR. The RT-PCR results are as follows: Figure 6 As shown, the PsbHLH35 gene was highly expressed in peony roots.

[0074] The experiment of transforming Arabidopsis thaliana with the PsbHLH35 overexpression vector in this example is as follows:

[0075] Specific primers with NcoI and SpeI restriction sites were designed to amplify the PsbHLH35 sequence. After recovery and purification, the PsbHLH35 sequence was double-digested with the overexpression vector pCAMBIA1302. The overexpression vector was then ligated overnight with T4 ligase to construct the overexpression vector. A single strain of 1-2 mm was selected and confirmed by colony PCR (see Figure 7 ), sent to Bioengineering for sequencing, and the results were correct, indicating that the pCAMBIA1302-PsbHLH35 overexpression vector was successfully constructed. Plasmids were extracted from the positive strains.

[0076] The constructed pCAMBIA1302-PsbHLH35 overexpression vector was transformed into Agrobacterium GV3101, and a single strain of 1-2 mm was selected for colony PCR verification. The bands were correct (see Figure 8 The correct positive strain was cultured with a two-step shaking method until OD600 = 1.0, and the wild-type Arabidopsis thaliana Col-0 was infected by the inflorescence infection method. The T0 generation seeds were collected and planted on the positive screening medium for positive screening (see for details). Figure 9 ), the proportion of positive plants was relatively low (5 plants), and the positive plants were further cultured and T1 generation seeds were collected. To verify whether peony PsbHLH35 can be normally expressed in Arabidopsis, T1 generation PsbHLH35 positive transgenic Arabidopsis plants were taken, RNA was extracted, and its cDNA was obtained by reverse transcription. Semi-quantitative PCR was performed using AtEF1α as an internal reference gene, as shown in Figure 2. Figure 10 As shown, the reference gene AtEF1α is expressed in both wild-type Arabidopsis Col-0 and PsbHLH35 transgenic Arabidopsis; however, PsbHLH35 is only expressed in PsbHLH35 transgenic Arabidopsis, not in wild-type Arabidopsis Col-0. This indicates that the tree peony PsbHLH35 has been successfully transferred into Arabidopsis and will continue to be cultured to the T2 generation of plants for subsequent related research. Figure 11 As shown, the transgenic plants grew faster than the wild type, were slightly larger, and had two more true leaves than the wild type, but their root system tended to grow as a taproot with fewer lateral roots.

[0077] The experiment of transforming peony callus with the PsbHLH35 overexpression vector in this example is as follows:

[0078] The constructed pCAMBIA1302-PsbHLH35 overexpression vector was transformed into Agrobacterium tumefaciens LBA4404, and a single strain of 1-2 mm was selected for colony PCR verification. The bands were correct (see Figure 12 ), which can be used for subsequent callus infection. Select positive single strains and use the peony callus genetic transformation system established by the research group in the early stage (Shen Ping, 2014) to infect peony callus, then inoculate it into screening medium. After screening culture for 3 days, inoculate it into somatic embryo induction medium (see Figure 13 ). Using uninfected blank callus and callus infected with 1302 empty vector as controls, qRT-PCR positive identification and verification were performed on the infected callus. The results showed that the expression level of pCAMBIA1302-PsbHLH35 in transgenic callus was significantly increased, 14 times higher than that in the control and empty vector (see for details). Figure 14 ), indicating that the PsbHLH35 overexpression vector was successfully transformed into peony callus.

[0079] Observation on the morphology and anatomy of callus tissues of different transgenic tree peonies in this example:

[0080] like Figure 15 As shown in the figure, the number of embryonic cell clusters in the callus of transgenic 35s:PsbHLH35 was significantly higher than that in the 1302 empty-load treatment, and the cell structure was dense and the morphology was regular, indicating that PsbHLH35 has a promoting effect on somatic embryogenesis in tree peony.

[0081] In this example, the expression levels of key genes for somatic embryogenesis in PsbHLH35 transgenic peony callus were analyzed:

[0082] like Figure 16 As shown in the results, in 35s:PsbHLH35 transgenic peony callus, overexpression of PsbHLH35 significantly promoted the expression levels of key somatic embryogenesis genes PsSERK and PsABI3, and reduced the expression levels of PsLEC1 and PsAGL15.

[0083] In this example, the hormone content of PsbHLH35 transgenic peony callus was detected:

[0084] like Figure 17As shown, in 35s:PsbHLH35 transgenic peony calli, ACC and 5DS levels initially increased and then decreased, while ABA showed the opposite pattern, decreasing and then increasing. BARP content gradually decreased over time, while BL gradually increased. IAA content exhibited a bimodal expression pattern, initially increasing, then decreasing, and then increasing again. Overexpression of PsbHLH35 significantly reduced endogenous ABA content in peony calli from days 0 to 15, with a sudden and significant rebound on day 20. Endogenous IAA content significantly increased on days 0 and 5 in the early stage and on day 20 in the late stage, remaining lower than that in CK at all other times. Furthermore, overexpression of PsbHLH35 caused an increase in endogenous ACC during somatic embryo induction, but reduced BARP content. Overexpression of PsbHLH35 maintained high levels of IAA, BL, ABA, and ACC, as well as low levels of BARP and 5DS, during the late stages of embryonic cell establishment.

[0085] Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the present invention.

Claims

1. A PsbHLH35 gene, characterized in that: The CDS sequence of the PsbHLH35 gene is shown in SEQ ID NO.

1.

2. A coding protein, characterized in that: The protein is encoded by the PsbHLH35 gene according to claim 1.

3. An application of the PsbHLH35 gene according to claim 1 in promoting somatic embryogenesis of tree peony, characterized in that: The steps include: S1) Clone the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector; S2) Transforming tree peonies with the PsbHLH35 gene overexpression vector.

4. The use according to claim 3, characterized in that: In S1), the vector is a plant protein overexpression vector; in S2), the transformation is carried out by Agrobacterium transformation.

5. The use according to claim 4, characterized in that: The plant protein overexpression vector is pCAMBIA1302.

6. The biological material related to the PsbHLH35 gene according to claim 1, characterized in that: The biological material includes any one of the following: a) a recombinant expression vector containing the PsbHLH35 gene as claimed in claim 1; b) an engineered bacterium containing the PsbHLH35 gene as claimed in claim 1, or an engineered bacterium containing the recombinant expression vector as shown in a).

7. Use of the PsbHLH35 gene according to claim 1 or the biomaterial according to claim 6 in any of the following: 1) Promote somatic embryogenesis in tree peony; 2) Regulate hormone levels during tree peony somatic embryogenesis; 3) Improve the efficiency of embryonic callus formation in tree peony; 4) Prepare transgenic Arabidopsis thaliana.

8. An overexpression vector, characterized in that: Comprising the PsbHLH35 gene according to claim 1.

9. A method for indirect regeneration of tree peony somatic embryos, characterized by: The peony somatic embryo callus containing the PsbHLH35 gene as claimed in claim 1 is cultivated to obtain clustered buds regenerated and differentiated from the peony somatic embryo.

Images