PsbHLH35 gene, encoded protein and application of PsbHLH35 gene in peony somatic embryogenesis

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly provides a PsbHLH35 gene, encoding protein and application of the PsbHLH35 gene in peony somatic embryogenesis. The invention relates to a PsbHLH35 gene, wherein the PsbHLH35 gene comprises a nucleotide sequence as shown in SEQ ID NO. 1. The encoding protein is obtained by encoding the PsbHLH35 gene, and the encoding protein comprises an amino acid sequence as shown in SEQ ID NO. 2. The application method of the PsbHLH35 gene in peony somatic embryogenesis comprises the following steps: S1) cloning the PsbHLH35 gene to a carrier to construct a PsbHLH35 gene overexpression vector, and S2) transforming the PsbHLH35 gene overexpression vector into peony. The PsbHLH35 gene has a significant promotion effect on peony somatic embryogenesis.

Description

Technical Field

[0001] The present application belongs to the technical field of plant molecular biology, and in particular relates to a PsbHLH35 gene and encoded protein and their application in peony somatic embryogenesis. Background Art

[0002] Peony (Paeonia Sect. Moutan) has important cultural, ornamental and medicinal values. Researching and applying tissue culture technology and establishing an efficient breeding and propagation technology system have gradually become an inevitable requirement for promoting the industrialization of peony. Plant in vitro meristem nodule plant regeneration and embryo culture are two important tissue culture technologies, which have broad application prospects in the propagation of excellent asexual lines and breeding practice.

[0003] At present, the research on peony tissue culture regeneration system is mainly divided into two pathways: 1. Direct regeneration pathway in which explants are induced to directly form seedlings; 2. Indirect regeneration pathway in which callus tissue is induced by explants and callus tissue is redifferentiated to form clustered buds, namely, somatic embryo regeneration pathway. Among them, the direct regeneration pathway of peony mainly uses seed embryos and bulbils to directly induce seedlings (Wang Zheng et al., 2018; Huang Sujiao, 2020; Yin Xiu et al., 2021). However, the rooting rate and transplant survival rate of test tube seedlings obtained by the direct regeneration pathway have not been effectively solved so far, making it difficult to apply in large-scale production (Shang et al., 2017; Lian Xiaofang et al., 2020; Zhang Runlong et al., 2021). Somatic embryos have the same ability as zygotic embryos to germinate and form complete plants (Karami et al., 2020; Zhang Meng et al., 2021). Therefore, the somatic embryo regeneration pathway should be one of the effective ways to solve the above problems.

[0004] The study of peony somatic embryo regeneration pathways began in 1969, when Demoise and Partanen first used zygotic embryos as explants to induce callus. Subsequently, related researchers have successfully achieved callus induction using explants such as anthers, filaments, petals, stem segments, petioles, and leaves (Beruto et al., 2004; Zhu Xiangtao et al., 2012; Meng Xinya et al., 2018; Du Yiming et al., 2020; Zhang Runlong et al., 2021; Zhu et al., 2022), but there are few reports on the induction of embryonic callus and the occurrence of clustered seedlings.

[0005] (Wang Zheng, 2010) used petioles as explants to preliminarily construct an indirect regeneration system for peony somatic embryos. It was found that the induction rate of embryonic callus was only 25-40%, with low transformation efficiency and poor callus differentiation and development. Zhu Xiangtao et al. (2012) used 'Fengdan' seed embryos for embryonic induction and obtained an embryonic callus induction rate of 38.33%. At the same time, during the embryonic callus formation of peony, there are "bottleneck" problems such as the early occurrence rate of somatic embryos such as spherical embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledon-shaped embryos, which are gradually reduced, the degree of uniformity is low, and the difficulty of inducing clustered buds is high (Shen et al., 2012; Zhu et al., 2018), which further increases the difficulty of establishing a mature peony somatic embryo regeneration system and seriously restricts the construction of the peony regeneration system (Wang Zheng 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] The third aspect of the present application provides a method for applying the PsbHLH35 gene in peony somatic embryogenesis, comprising the following steps: S1) Clone the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector; S2) transforming the PsbHLH35 gene overexpression vector into tree peony.

[0011] Furthermore, it also includes one or more of the following technical features: S11 In S1), the PsbHLH35 gene comprises the nucleotide sequence shown in SEQ ID NO.1; S12 In S1), the vector is a plant protein overexpression vector; S22 In S2), the transformation is carried out by Agrobacterium transformation method.

[0012] Furthermore, the plant protein overexpression vector is pCAMBIA1302.

[0013] The fourth aspect of the present application provides a biological material related to the PsbHLH35 gene, which includes any one of the following: a) a recombinant expression vector containing the above-mentioned PsbHLH35 gene; b) a bioengineering bacterium containing the above-mentioned PsbHLH35 gene, or a bioengineering bacterium containing the recombinant expression vector shown in a).

[0014] The fifth aspect of the present application provides a use of a biological material related to the PsbHLH35 gene in any of the following: 1) regulating peony somatic embryogenesis; 2) regulating the hormone content during peony somatic embryogenesis; 3) improving the efficiency of peony embryonic callus; 4) peony breeding or germplasm improvement; 5) preparing peony breeding-related kits and / or expression cassettes; 6) preparing transgenic peonies; 7) preparing transgenic Arabidopsis thaliana.

[0015] The sixth aspect of the present application provides an overexpression vector comprising the above-mentioned PsbHLH35 gene; preferably, the overexpression vector is obtained by cloning the above-mentioned PsbHLH35 gene into a plant protein overexpression vector; more preferably, the plant protein overexpression vector is pCAMBIA1302.

[0016] The seventh aspect of the present application provides an explant comprising the above-mentioned overexpression vector.

[0017] In an eighth aspect, the present application provides a method for indirect regeneration of peony somatic embryos, which cultivates peony somatic embryo callus containing the above-mentioned PsbHLH35 gene to obtain peony somatic embryo regeneration and differentiation cluster buds.

[0018] Compared with the prior art, this application has the following beneficial effects: 1. This application developed a peony transcription factor PsbHLH35, which is localized in the cell nucleus. PsbHLH35 overexpression-transformed Arabidopsis plants were constructed, which made the transgenic Arabidopsis grow rapidly, with a slightly larger plant shape, 2 more true leaves than the wild type, and promoted the growth of the taproot.

[0019] 2. This application constructs PsbHLH35 overexpression-transformed peony callus tissue, which significantly improves the efficiency of peony embryogenic callus. The use of transcription factor PsbHLH35 significantly promotes the expression level of genes related to peony somatic embryogenesis and effectively regulates the hormone content during peony somatic embryogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a schematic diagram of the prediction and analysis of the signal peptide of the protein encoded by the PsbHLH35 gene in this application; Figure 2 This is a schematic diagram of the analysis of the transmembrane domain of the protein encoded by the PsbHLH35 gene in this application; Figure 3 Schematic diagram of GFP-PsbHLH35 transformed DH5α colony PCR for this application (M, DL2000 DNA Maker; 1-2, GFP-PsbHLH35 colony PCR); Figure 4 Schematic diagram of GFP-PsbHLH35 transformed GV3101 colony PCR for this application (M, 250 bp DNA Ladder; 1-6 GFP-PsbHLH35 colony PCR); Figure 5 Please provide a schematic diagram of the subcellular localization of PsbHLH35 (A, All; B, DAPI; C, FITC; D, TRITC; E, TD).

[0021] Figure 6 This is a schematic diagram of the expression level of PsbHLH35 in different parts of peony in this application; Figure 7 Schematic diagram of colony PCR of DH5α transformed with pCAMBIA1302-PsbHLH35 for this application (M, DL2000 DNAMaker; 1-2, colony PCR of pCAMBIA1302-PsbHLH35); Figure 8 Schematic diagram of colony PCR of GV3101 transformed with pCAMBIA1302-PsbHLH35 for this application (M, DL2000 DNAMaker; 1-8, colony PCR of pCAMBIA1302-PsbHLH35); Fig. 9 Schematic diagram of positive screening of T0 generation seeds of transgenic Arabidopsis thaliana for this application (A, Col-0; B, pCAMBIA1302-PsbHLH35); Fig.10 This is a schematic diagram of semi-quantitative PCR detection of T1 generation transgenic Arabidopsis thaliana in this application; Fig.11 Schematic diagram of the phenotype of the transgenic Arabidopsis T2 plants of this application (A, Col-0; B, pCAMBIA1302-PsbHLH35); Fig.12 Schematic diagram of colony PCR of LBA4404 transformed with pCAMBIA1302-PsbHLH35 in this application (M, 250 bp DNA Ladder; 1-4, colony PCR of pCAMBIA1302-PsbHLH35); Fig.13 Schematic diagram of PsbHLH35 overexpression transformation of peony callus in this application (A is co-cultivation; B is somatic embryo induction); Fig.14 This is a schematic diagram of RT-PCR positive identification of PsbHLH35 overexpression transgenic callus in this application; Fig.15 Schematic diagram of the observation of callus sections of different transgenic peonies in this application (pCAMBIA1302 on the left; 35s:PsbHLH35 on the right, black arrows indicate embryonic callus cell clusters); Fig.16 This is a schematic diagram of the analysis of the expression levels of key genes for somatic embryogenesis in the callus of PsbHLH35 transgenic peony in this application; Fig.17 This is a schematic diagram of the measurement data of hormone content in callus tissue of PsbHLH35 transgenic peony in this application. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0023] The inventors of the present application have found through a large number of experiments that the PsbHLH35 gene can play a key regulatory role in the process of peony somatic embryogenesis, and can maintain high levels of IAA, BL, ABA, ACC, and low levels of BARP and 5DS in the late stage of embryonic cell establishment.

[0024] The present application first provides a PsbHLH35 gene and a protein encoded therein. The PsbHLH35 gene includes a nucleotide sequence as shown in SEQ ID NO.1, which is a CDS sequence.

[0025] SEQ ID NO.1: ATGGATGATAACATTACTGAAGAATACAAGCTGTGGTGGGAAACCAAGATGTTCCTCCAAACCGAGGAGCTTGACAGGATGTTCCTCCAATCTGAAGAGCCCGACGGTTGGGGACTAGACGAGGCATTTTCTGGTTACTATGATTCGAGCTCGCCAGACGGAGCAGCGACGTCGCCGGCATCTAAGAACA TTTTGTCAGAGAGAAACCGGAGGAAGAAGCTGAATGATAGACTCTATACACTTAGATCGGTGGTCCCTAACATTACTAAGATGGACAAAGCATCAATAATCAAAGACGCGATCGAGTATATCCAAGAGTTGCATCAACAAGAGAAGAGAATCCAAGCTGAAATATCACAACTGGAGTCAACAAAGAAGAAT TTTGATGGTTTTGATCAGTCGGAGCAACAGGTTACACCATCATTGAGATCCAAGAAAAAAAGAACGGATCAGGTGCCAAGTTATCAAGAATCAACAACTTCTCCCATTGAGCTGCTTGAAATACGGGTGTCTTACATGGGAGAGAAGACGGTGGTAGTGAGCTTGACATGTAGTAAAAGAAGGGACACAA TGGTAAAACTTTGTGAGGTTTTTGAATCATTGAAGCTCAAAATTATTACTGCCAATATCACTGCTTTTTCTGGAAGGCTTTTGAAAACGGTCTTTCTTGAGGCAGATGAAGAGGAGAAAGATCAGTTGAAGGCAAGGATTGAGACAGCCATAGCAGCTCTTAATGATCCACCGAGCCCTATGAGCATCTAA The encoded protein is obtained by encoding the above-mentioned PsbHLH35 gene, and 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.

[0026] In a specific embodiment of the present application, the encoded protein includes an amino acid sequence as 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.

[0027] SEQ ID NO.2: MDDNITEEYKLWWETKMFLQTEELDRMFLQSEEPDGWGLDEAFSGYYDSSSPDGAATSPASKNILSERNRRKKLNDRLYTLRSVVPNITKMDKASIIKDAIEYIQELHQQEKRIQAEISQLESTK KNFDGFDQSEQQVTPSLRSKKKRTDQVPSYQESTTSPIELLEIRVSYMGEKTVVVSLTCSKRRDTMVKLCEVFESLKLKIITANITAFSGRLLKTVFLEADEEEKDQLKARIETAIAALNDPPSP The present application provides a method for applying the PsbHLH35 gene in peony somatic embryogenesis, comprising the following steps: S1) Clone the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector; S2) transforming the PsbHLH35 gene overexpression vector into tree peony.

[0028] Furthermore, it also includes one or more of the following technical features: S11 In S1), the PsbHLH35 gene comprises the nucleotide sequence shown in SEQ ID NO.1; S12 In S1), the vector is a plant protein overexpression vector; S22 In S2), the transformation is carried out by Agrobacterium transformation method.

[0029] 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 realizing 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.

[0030] In the specific embodiments of the present application, "transformation" refers to the process of introducing foreign genetic material (such as DNA fragments) into cells or organisms. This process can be spontaneous in nature or completed through laboratory technical operations. There are many methods of transformation, including chemical methods, electroporation methods, gene gun methods, viral vector methods, Agrobacterium-mediated methods, and liposome-mediated methods. Through these technical means, specific gene fragments can be introduced into target cells, thereby changing their genetic characteristics or giving them new functions.

[0031] In the specific embodiment of the present application, the transformation adopts Agrobacterium transformation method, and the method for transforming Agrobacterium can be 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 method can be adopted. Freeze-thaw method increases the permeability of bacterial cell membrane by alternating freezing and thawing process, promotes the absorption of exogenous DNA. Electroporation method is to apply electric field to form short-term micropores on bacterial cell membrane, so that DNA fragments can pass through cell membrane and enter cells. After completing the transformation, by culturing Agrobacterium in a culture medium containing appropriate antibiotics, the strains successfully transformed can be screened out.

[0032] The present application also provides an explant, wherein the above-mentioned overexpression vector is transformed into the explant. An explant refers to a tissue fragment cut from an organism for tissue culture or cell culture. In plant tissue culture, an explant is a starting material used in the culture process, which can be any part of a plant, such as a leaf, stem segment, root tip, anther, embryo, etc. The explant is used to start the culture process, with the purpose of inducing tissue differentiation, proliferation or regeneration to form a new plant body or cell line.

[0033] Example The application of the PsbHLH35 gene in peony somatic embryogenesis of this embodiment includes the following experiments: PsbHLH35 encoding amino acid analysis of this example: According to the cloned CDS sequence of the 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 prediction analysis of transmembrane domains revealed that PsbHLH35 protein does not have a transmembrane domain and does not belong to the transmembrane protein class (see for details). Figure 2). The physicochemical properties of the amino acid sequence encoded by the PsbHLH35 gene were analyzed online, and it was found that the relative molecular mass of the protein encoded by the PsbHLH35 gene was 29.04 kDa, the instability index was 77.92, the fat solubility index was 79.80, and the total average hydrophilicity index was negative, indicating that it was an unstable hydrophobic fat-soluble protein (see Table 1 for details). The isoelectric point of the protein was 5.10, and the number of its acidic amino acid residues was greater than the number of basic amino acid residues, indicating that it was an acidic protein (see Table 1 for details). Table 1 Analysis of the components and physicochemical properties of the nucleotide sequence and amino acid sequence of the peony PsbHLH35 gene Subcellular localization of PsbHLH35 in this example: Design GFP-specific primers with KpnI and XbaI restriction sites for PsbHLH35, construct its GFP expression vector, pick a single strain of 1-2 mm and perform colony PCR to identify the correct band size (see Figure 3 ), sent to Bioengineering for sequencing. The plasmid was extracted from the correct positive strain. The GFP fusion expression vector GFP-PsbHLH35 of PsbHLH35 was successfully constructed for subcellular localization experiment.

[0034] Transform the GFP-PsbHLH35 plasmid into Agrobacterium GV3101, and select a single strain of 1-2 mm and verify the correctness by colony PCR (see Figure 4 The correct positive strain was cultured with two-step shaking method until OD600 = 1.0, then injected to infect the lower epidermis of tobacco leaves, and observed by laser confocal microscopy after 3 days of culture (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 its fluorescence signal completely coincides in the overlapping field, thus proving that the PsbHLH35 protein is subcellularly localized in the nucleus.

[0035] PsbHLH35 tissue-specific expression analysis in this example: Select the well-growing peony 'Fengdanbai' seedlings, when they set seeds in mid-to-late June of the same year, directly extract the total RNA from their roots, stems, leaves, flowers, and seeds, and reverse transcribe 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.

[0036] The experiment of transforming Arabidopsis thaliana with the PsbHLH35 overexpression vector in this example is as follows: Design specific primers with NcoI and SpeI restriction sites for PsbHLH35 to amplify the PsbHLH35 sequence, recover and purify it, and then double-digest it with the overexpression vector pCAMBIA1302. Then, use T4 ligase to connect overnight to construct its overexpression vector. Pick out a single strain of 1-2 mm and identify it as correct by colony PCR (see Figure 7 ), sent to Bioengineering for sequencing, the result was correct, indicating that the pCAMBIA1302-PsbHLH35 overexpression vector was successfully constructed. The plasmid was extracted from the positive strain.

[0037] 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 band was correct (see Figure 8 The correct positive strain was cultured with two-step shaking method until OD600 = 1.0, and the wild-type Arabidopsis thaliana Col-0 was infected by inflorescence infection method. The T0 generation seeds were collected and planted in positive screening medium for positive screening (see Fig. 9 ), the proportion of positive plants was relatively low (5 plants), and the positive plants were continued to be cultured and T1 generation seeds were collected. In order 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. AtEF1α was used as the internal reference gene for semi-quantitative PCR detection, such as Fig.10 As shown in the figure, 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, and not in wild-type Arabidopsis Col-0. This indicates that Peony PsbHLH35 has been successfully transferred into Arabidopsis and continued to be cultured to the T2 generation of plants for subsequent related research. Fig.11 As shown, the transgenic plants grew faster than the wild type, were slightly larger, had two more true leaves than the wild type, but the root system tended to grow as a taproot with fewer lateral roots.

[0038] The experiment of transforming peony callus with the PsbHLH35 overexpression vector in this example is as follows: The constructed pCAMBIA1302-PsbHLH35 overexpression vector was transformed into Agrobacterium LBA4404, and a single strain of 1-2 mm was selected for colony PCR verification. The band was correct (see Fig.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 the screening medium. After 3 days of screening culture, inoculate it into the somatic embryo induction medium (see Fig.13). Using the uninfected callus and the callus infected with 1302 empty vector as controls, the infected callus was positively identified and verified by qRT-PCR. The results showed that the expression level in the pCAMBIA1302-PsbHLH35 transgenic callus was significantly increased, 14 times higher than that of the control and empty vector (see Fig.14 ), indicating that the PsbHLH35 overexpression vector was successfully transformed into peony callus.

[0039] Observation on the morphology and anatomy of callus tissues of different transgenic peonies in this example: like Fig.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 promoted somatic embryogenesis in peony.

[0040] Analysis of key gene expression levels in somatic embryogenesis of PsbHLH35 transgenic peony callus in this example: like Fig.16 As shown, in 35s:PsbHLH35 transgenic peony callus, PsbHLH35 overexpression significantly promoted the expression levels of somatic embryogenesis key genes PsSERK and PsABI3, and reduced the expression levels of PsLEC1 and PsAGL15.

[0041] In this example, the hormone content of PsbHLH35 transgenic peony callus was detected: like Fig.17 As shown in the results, in the callus of 35s:PsbHLH35 transgenic peony, ACC and 5DS showed a trend of first increasing and then decreasing, while ABA was just the opposite, decreasing first and then increasing. The content of BARP gradually decreased with the culture time, while BL gradually increased. The change trend of IAA content was a bimodal expression pattern of first increasing, then decreasing, and then increasing. When PsbHLH35 was overexpressed, the endogenous ABA content of peony callus decreased significantly from day 0 to day 15, and suddenly rebounded sharply on day 20. The endogenous IAA content increased significantly on day 0 and day 5 in the early stage and day 20 in the late stage, and was lower than CK in other periods. At the same time, the overexpression of PsbHLH35 can cause the endogenous ACC to increase during somatic embryo induction, but reduce the content of BARP. The overexpression of PsbHLH35 can maintain high levels of IAA, BL, ABA, ACC, and low levels of BARP and 5DS in the late stage of embryonic cell establishment.

[0042] Although the present application has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to 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 protection scope of the present invention.

Claims

1. A PsbHLH35 gene, characterized in that: The PsbHLH35 gene comprises the nucleotide sequence shown in SEQ ID NO.

1.

2. A coding protein, characterized in that: Obtained by the PsbHLH35 gene encoding as described in claim 1; preferably, the encoded protein comprises the amino acid sequence shown in SEQ ID NO.

2.

3. A method for applying the PsbHLH35 gene in peony somatic embryogenesis, characterized in that: The steps include: S1) Clone the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector; S2) transforming the PsbHLH35 gene overexpression vector into tree peony.

4. The application method according to claim 3, characterized in that: It also includes one or more of the following technical features: S11 In S1), the PsbHLH35 gene comprises the nucleotide sequence shown in SEQ ID NO.1; S12 In S1), the vector is a plant protein overexpression vector; S22 In S2), the transformation is carried out by Agrobacterium transformation method.

5. The application method 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) a bioengineering bacterium containing the PsbHLH35 gene as claimed in claim 1, or a bioengineering bacterium containing the recombinant expression vector 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 items, characterized in that: 1) Regulate peony somatic embryogenesis; 2) Regulate hormone content during peony somatic embryogenesis; 3) Improve the efficiency of peony embryonic callus formation; 4) Peony breeding or germplasm improvement; 5) Preparation of peony breeding related kits and / or expression cassettes; 6) Preparation of transgenic peonies; 7) Preparation of transgenic Arabidopsis thaliana.

8. An overexpression vector, characterized in that: It comprises the PsbHLH35 gene as claimed in claim 1; preferably, the overexpression vector is obtained by cloning the PsbHLH35 gene as claimed in claim 1 into a plant protein overexpression vector; more preferably, the plant protein overexpression vector is pCAMBIA1302.

9. An explant, characterized in that: Comprising the overexpression vector as claimed in claim 8.

10. A method for indirect regeneration of peony somatic embryos, characterized in that: The peony somatic embryo callus containing the PsbHLH35 gene as claimed in claim 1 is cultivated to obtain peony somatic embryo regeneration and differentiation clustered buds.

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