Application of PaTM8 protein and its encoding gene in promoting plant flowering

Abstract

This invention relates to the application of a PaTM8 protein and its encoding gene in promoting plant flowering, belonging to the field of plant genetic engineering technology. The amino acid sequence of the PaTM8 protein is shown in SEQ ID No. 2. This invention experimentally confirms that the PaTM8 gene has the function of promoting plant flowering. This invention also provides the amino acid sequence of the PaTM8 protein, the nucleic acid sequence of the gene encoding the PaTM8 protein, functional primers, an overexpression vector, and genetically engineered bacteria. The PaTM8 gene can be used for the regulation of plant flowering traits, providing new gene resources for the regulation of flowering time in horticultural plants and forest trees, the analysis of flowering mechanisms, and molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of a PaTM8 protein and its encoding gene in promoting plant flowering. Background Technology

[0002] The London plane tree (Platanus spp.) is widely used in urban roadside greening due to its tall stature, good shade effect, strong resistance to adverse conditions, and dust and noise reduction properties, making it one of the most widely used street tree species in my country. However, after its mature cones dry and crack in the following spring and summer, the seed hairs attached to the seeds and a large amount of pollen are scattered almost simultaneously. This not only aggravates urban dust pollution but also easily induces respiratory diseases and allergic reactions. This problem of "fallen fruit and flying pollen" has become a prominent obstacle restricting the continued application and promotion of London plane trees.

[0003] Currently, urban landscaping management mainly addresses this problem through manual pruning, chemical suppression, or mechanical clearing. However, these methods generally suffer from drawbacks such as high cost, short duration of action, significant ecological risks, and difficulty in large-scale long-term implementation. In contrast, breeding new, genetically stable, fruitless (or low-fruiting) sycamore germplasm to reduce pollen and seed hair production at the source is the most fundamental, economical, and sustainable technical approach to solving this problem. However, the breeding of fruitless (or low-fruiting) sycamores has long been constrained by unclear genetic bases of key traits, a lack of clear molecular targets and operable regulatory strategies, and a mature molecular breeding technology system has not yet been established.

[0004] Flowering is a prerequisite for fruit and seed formation, and a foundation for pollen and seed hair production. Therefore, elucidating the flowering regulation mechanism of Platanus orientalis and identifying key genes controlling the flowering transition are crucial for molecular design breeding programs involving no (or few) flowers or fruits. Studies have shown that plant flowering is jointly regulated by environmental factors such as photoperiod and temperature, as well as endogenous factors such as hormone levels and plant age. This regulatory network is extremely complex, involving the synergistic effects of hundreds of genes. Functional analysis of key genes in the flowering regulatory network will help elucidate the molecular mechanisms of flowering transition in plants, providing a theoretical basis and genetic resources for subsequent precise regulation of the flowering process through gene editing, transcriptional regulation, and other technologies. Although several flowering regulatory genes have been identified in model plants and some fruit trees, systematic research on key genes related to flowering regulation and their mechanisms of action in Platanus orientalis remains lacking.

[0005] Therefore, it is urgent to discover and identify key genes related to flowering regulation in sycamore, clarify their functions and mechanisms of action in the flowering transition process, and provide new gene resources and technical support for the analysis of the flowering regulation mechanism of sycamore and the creation of new germplasm without (or with few) flowers or fruits. Summary of the Invention

[0006] Based on the above description, this invention provides an application of the PaTM8 protein and its encoding gene in promoting plant flowering. This gene can promote plant flowering and aims to provide gene resources for the study of the flowering regulation mechanism of sycamore and molecular breeding.

[0007] In view of this, the present invention provides an application of PaTM8 protein in promoting plant flowering, wherein the amino acid sequence of PaTM8 protein is shown in SEQ ID NO.2.

[0008] The present invention also provides the application of a gene encoding the PaTM8 protein in promoting plant flowering, wherein the nucleotide sequence of the gene encoding the PaTM8 protein is shown in SEQ ID No. 1; and the amino acid sequence of the PaTM8 protein is shown in SEQ ID No. 2.

[0009] In some embodiments, the method of promoting plant flowering is to increase the expression level of the gene encoding the PaTM8 protein in the plant.

[0010] The present invention also provides an application of an overexpression vector in promoting plant flowering, wherein the overexpression vector contains the gene encoding the PaTM8 protein described above.

[0011] This invention also provides the application of a genetically engineered bacterium in promoting plant flowering, wherein the genetically engineered bacterium comprises the above-mentioned overexpression vector; or, The genome of the genetically engineered bacteria integrates the gene encoding the PaTM8 protein.

[0012] The present invention also provides a method for promoting plant flowering, comprising the following steps: S10. Construct the above-mentioned overexpression vector; S20. The overexpression vector was introduced into plants using Agrobacterium-mediated transformation, and positive transgenic lines were screened using hygromycin.

[0013] In some embodiments, step S10 includes a step of amplifying the gene encoding the PaTM8 protein, wherein the primers for gene amplification include a forward primer P1 and a reverse primer P2, the sequence of the forward primer P1 is shown in SEQ ID No. 3, and the sequence of the reverse primer P2 is shown in SEQ ID No. 4.

[0014] In some embodiments, step S10 includes the following steps: ligating the gene encoding the PaTM8 protein into the pCAMBIA1305 vector using homologous recombination; The primer sequences with specific homologous arms include forward primer P3 and reverse primer P4. The sequence of forward primer P3 is shown in SEQ ID No. 5, and the sequence of reverse primer P4 is shown in SEQ ID No. 6.

[0015] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: (1) This application identified the function of the PaTM8 gene of Platanus orientalis in regulating plant flowering, and the experiment proved that it has a clear phenotypic regulatory role in transgenic plants; (2) This application provides the nucleic acid sequence of the PaTM8 gene and the amino acid sequence of the protein encoded by the gene, functional primers and overexpression vectors, providing new gene resources and technical foundation for the study of plant flowering regulation mechanisms and molecular breeding. Attached Figure Description

[0016] Figure 1 This is a phylogenetic analysis diagram of the PaTM8 protein in Example 1 of the present invention.

[0017] Figure 2 The images show wild-type Arabidopsis thaliana (WT) and PaTM8-overexpressing Arabidopsis thaliana (OE3, OE7, and OE8) 27 days after sowing in Example 3 of this invention.

[0018] Figure 3 This is a comparison of flowering time between wild-type Arabidopsis thaliana (WT) and PaTM8-overexpressing Arabidopsis thaliana (OE3, OE7, and OE8) in Example 3 of the present invention.

[0019] Figure 4 This is a comparative chart showing the number of rosette leaves at flowering time of wild-type Arabidopsis thaliana (WT) and PaTM8-overexpressing Arabidopsis thaliana (OE3, OE7, and OE8) in Example 3 of the present invention. Detailed Implementation

[0020] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0021] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0022] This invention provides the application of PaTM8 protein in promoting plant flowering, wherein the amino acid sequence of PaTM8 protein is shown in SEQ ID NO.2.

[0023] Specifically, the amino acid sequence of the PaTM8 protein, SEQ ID NO.2, is as follows: MGRGKVELKRIENTTNRQVTFSKRRNGLLKKAFELSILCDAEVALVVFSPSGKAYQFASHDMDRTIVRYRNEVGLPESNNQPSRTIGFWRTEIKESRKSVETLETRLKHMAGEDLLSLGMKELTQLERQLKTGVERIRSRKRRLISEHINLLKRNQKALQDENTRIQKMLHEASVRSRVSEVTCNAFQRYN.

[0024] The present invention also provides the application of a gene encoding the PaTM8 protein in promoting plant flowering, wherein the nucleotide sequence of the gene encoding the PaTM8 protein is shown in SEQ ID No. 1; and the amino acid sequence of the PaTM8 protein is shown in SEQ ID No. 2.

[0025] This application identifies the function of the PaTM8 gene (encoding the PaTM8 protein) in promoting flowering in Platanus orientalis. Experiments have demonstrated that it has a clear phenotypic regulatory role in transgenic plants: by increasing the expression level of the gene encoding the PaTM8 protein in plants, the time required for flowering can be reduced, that is, it can promote flowering.

[0026] Specifically, the nucleotide sequence of the gene encoding the PaTM8 protein, SEQ ID NO.1, is as follows: .

[0027] Furthermore, the amino acid sequence of the PaTM8 protein is shown in SEQ ID No. 2.

[0028] Furthermore, a method to promote plant flowering is to increase the expression level of the gene encoding the PaTM8 protein in the plant.

[0029] The present invention also provides an application of an overexpression vector in promoting plant flowering, wherein the overexpression vector contains the gene encoding the PaTM8 protein described above.

[0030] In this invention, the "overexpression vector" refers to a recombinant expression vector containing a strong promoter operatively linked to the target gene and optional enhancer elements, capable of driving the transcription and translation levels of the target gene in host cells to be significantly higher than the endogenous or conventional expression levels of the gene. It is understood that, in this embodiment, the target gene is the gene encoding the PaTM8 protein described above.

[0031] The present invention also provides the application of genetically engineered bacteria in promoting plant flowering, wherein the genetically engineered bacteria comprises the above-mentioned overexpression vector.

[0032] The present invention also provides the application of a genetically engineered bacterium in promoting plant flowering, wherein the genome of the genetically engineered bacterium integrates the gene encoding the PaTM8 protein.

[0033] In this invention, the "genetically engineered bacteria" refers to an engineered microbial strain into which a foreign target gene is introduced through recombinant DNA technology into a host microorganism (such as Escherichia coli, yeast, Bacillus, etc.), enabling it to be stably inherited and expressed, thereby obtaining a specific biological function or product production capability. It is understood that, in this embodiment, the foreign target gene is the gene encoding the PaTM8 protein mentioned above.

[0034] The present invention also provides a method for promoting plant flowering, comprising the following steps: S10. Construct the above-mentioned overexpression vector; S20. The overexpression vector was introduced into plants using Agrobacterium-mediated transformation, and positive transgenic lines were screened using hygromycin.

[0035] Furthermore, step S10 includes the step of amplifying the gene encoding the PaTM8 protein, wherein the primers for gene amplification include a forward primer P1 and a reverse primer P2, the sequence of the forward primer P1 is shown in SEQ ID No. 3, and the sequence of the reverse primer P2 is shown in SEQ ID No. 4.

[0036] Specifically, the sequence of the forward primer P1, SEQ ID NO.3, is: 5'-TTGACGTGTAATTCTTCCCTGT-3'.

[0037] The sequence of reverse primer P2, SEQ ID NO.4, is: 5'-TCAGCTTTATCAAGTGATCCGG-3'.

[0038] Furthermore, step S10 includes the following steps: using homologous recombination to ligate the gene encoding the PaTM8 protein into the pCAMBIA1305 vector; The primer sequences with specific homologous arms include forward primer P3 and reverse primer P4. The sequence of forward primer P3 is shown in SEQ ID No. 5, and the sequence of reverse primer P4 is shown in SEQ ID No. 6.

[0039] Specifically, the sequence of the forward primer P3, SEQ ID NO. 5, is: 5'-tacgaattcgagctcggtaccATGGGGAGAGGAAAAGTAGA-3'.

[0040] The sequence of reverse primer P4, SEQ ID NO. 6, is: 5'-tctagaggatccccgggtaccGTTATACCTTTGAAATGCAT-3'.

[0041] Furthermore, the Agrobacterium is GV3101.

[0042] The main components of the culture medium used in this application are as follows: LB liquid medium: 10 g / L peptone + 5 g / L yeast extract + 10 g / L NaCl + 100 mg / L kanamycin; Arabidopsis thaliana selection medium: basal MS medium + 40 mg / L hygromycin + 50 mg / L cephalosporin Example 1 Identification and Cloning of the PaTM8 Gene This embodiment aims to illustrate how to identify and clone the PaTM8 gene of Platanus orientalis.

[0043] The specific operation steps of this embodiment are as follows: 1. A candidate gene (PaA3G235220) annotated as TM8-like was obtained from the genome of *Platanus chinensis*. To clarify its phylogenetic classification and homology, MADS-box protein sequences from *Arabidopsis thaliana* and *Vitis vinifera* were obtained from the Phytozome database as reference sequences. Multiple sequence alignment of PaA3G235220 with the reference sequences was performed, and a phylogenetic tree was constructed based on the alignment results. Phylogenetic analysis was conducted using the maximum likelihood (ML) method, and branch confidence was tested using 1000 bootstrap replicates.

[0044] like Figure 1 As shown, phylogenetic analysis revealed that *Platanus multifiliis* PaA3G235220 and *Vitrus thunbergii* VIT_217s0000g01230 of the TM8 subfamily clustered into the same clade, indicating a close phylogenetic relationship. However, no highly homologous corresponding gene was found in *Arabidopsis thaliana*. Based on phylogenetic and sequence homology analysis, this gene belongs to the TM8 class of MADS-box genes, and therefore it was named PaTM8.

[0045] 2. Design specific amplification primers for the homologous sequence of the PaTM8 gene: forward primer P1: 5'-TTGACGTGTAATTCTTCCCTGT-3' (SEQ ID No. 3); reverse primer P2: 5'-TCAGCTTTATCAAGTGATCCGG-3' (SEQ ID No. 4).

[0046] Using cDNA from the fruit of Platanus orientalis as a template, the cDNA coding region fragment of the PaTM8 gene was obtained by PCR amplification using specific amplification primers P1 / P2.

[0047] The PCR amplification reaction system is as follows: 1 μL cDNA template, 0.5 μL 10 mmol / L forward primer, 0.5 μL 10 mmol / L reverse primer, 5 μL 2×Taq enzyme Mix, and 3 μL ddH2O.

[0048] The PCR amplification program is as follows: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 20 s, 35 cycles, and a final extension at 72℃ for 10 min.

[0049] The amplified product was purified by agarose gel electrophoresis, ligated into the pMD®18-T vector, and transformed into *E. coli* DH5α for positive detection. Positive clones were selected, and plasmids were extracted for sequencing verification. The inserted fragment was confirmed to be the full-length sequence of the PaTM8 gene. The correctly sequenced plasmid was named PaTM8-T. The full-length CDS sequence of the PaTM8 gene is shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2.

[0050] The nucleotide sequence of the PaTM8 gene is SEQ ID No. 1: .

[0051] The amino acid sequence encoded by the PaTM8 gene is SEQ ID No. 2: MGRGKVELKRIENTTNRQVTFSKRRNGLLKKAFELSILCDAEVALVVFSPSGKAYQFASHDMDRTIVRYRNEVGLPESNNQPSRTIGFWRTEIKESRKSVETLETRLKHMAGEDLLSLGMKELTQLERQLKTGVERIRSRKRRLISEHINLLKRNQKALQDENTRIQKMLHEASVRSRVSEVTCNAFQRYN.

[0052] Example 2 Construction and transformation of PaTM8 gene overexpression vector into Arabidopsis thaliana To clarify the biological function of the PaTM8 gene in plants, this embodiment constructs a plant overexpression vector for this gene and introduces it into Arabidopsis thaliana to observe phenotypic changes in transgenic plants. The specific steps are as follows: 1. Using the PaTM8-T plasmid obtained in Example 1 as a template, PCR amplification was performed using specific primers containing homologous arms of the pCAMBIA1305 vector. Homologous recombination sequences matching the plant expression vector pCAMBIA1305 were introduced at both ends of the PaTM8 gene. The DNA sequences of the primer pairs are shown below: forward homologous recombination primer P3: 5'-tacgaattcgagctcggtaccATGGGGAGAGGAAAAGTAGA-3' (SEQ ID No. 5), and reverse homologous recombination primer P4: 5'-tctagaggatccccgggtaccGTTATACCTTTGAAATGCAT-3' (SEQ ID No. 6). In P3, "5'-tacgaattcgagctcggtacc-3'" is the sequence on the pCAMBIA1305 vector, and in P4, "5'-tctagaggatccccgggtacc-3'" is the sequence on the pCAMBIA1305 vector.

[0053] The PCR amplification reaction system is as follows: 1 μL plasmid template, 0.5 μL 10 mmol / L forward primer, 0.5 μL 10 mmol / L reverse primer, 5 μL 2×Taq enzyme Mix, and 3 μL ddH2O.

[0054] The PCR amplification program is as follows: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 20 s, 35 cycles, and a final extension at 72℃ for 10 min.

[0055] The plant expression vector pCAMBIA1305 was linearized by KpnI digestion, and the linearized vector fragment was recovered. The linearized vector was ligated with the PaTM8 PCR product containing homologous arms according to the ClonExpress II One Step Cloning Kit instructions to construct the recombinant expression vector 35S::PaTM8. The recombinant plasmid was transformed into *E. coli* DH5α competent cells, plated on selection medium, and positive clones were picked for PCR identification and sequencing verification. After confirming the sequence was correct, the recombinant expression vector was introduced into *Agrobacterium* GV3101 competent cells for subsequent *Arabidopsis thaliana* genetic transformation.

[0056] 2. Genetic transformation of Arabidopsis thaliana was carried out using the flower immersion transformation method. The specific steps are as follows: 1) Inoculate a single colony of Agrobacterium containing the target gene fragment onto 5 mL of LB liquid medium and incubate at 28°C and 200 rpm until OD. 600nm The value is 0.6-0.8. Take 1 mL of the above bacterial culture and transfer it to 50 mL of LB liquid medium. Incubate at 28℃ and 200 rpm until the OD value reaches 0.6-0.8.600nm The OD value was 0.6-0.8. Bacterial cells in the LB culture were collected by centrifugation at 5000 rpm and resuspended in a resuspension solution (5% sucrose + Arabidopsis thaliana conversion aid) until OD=0.8. Arabidopsis inflorescences were immersed in the resuspension solution for 1 min and cultured in the dark for 24 h. Afterwards, the plant surface was gently rinsed with water to remove any remaining bacterial solution, and the formed pods were removed. The plants were allowed to continue growing normally until seeds were harvested to obtain T0 generation seeds.

[0057] 2) After surface sterilization, T0 generation seeds were sown on Arabidopsis selection medium and vernalized at 4 ℃ for 3 days. They were then cultured in a growth chamber (23 ℃, 16 h light, 8 h dark) for approximately 10 days. Seedlings that grew normally on the selection medium were identified as T1 generation positive plants and transplanted to nutrient soil for further cultivation. After the plants matured, genomic DNA was extracted for PCR identification, confirming stable integration of the target gene PaTM8.

[0058] 3) T1 generation positive plants exhibiting the early flowering phenotype were individually harvested and sown to obtain T2 generation seeds. T2 generation seeds were sown on Arabidopsis selection medium, and the segregation ratio between resistant and susceptible plants was calculated. Single-copy insert lines with a resistance segregation ratio of 3:1 were selected and individually sown to obtain T3 generation seeds. Subsequently, T3 generation plants were screened for resistance, and homozygous lines without resistance segregation were selected for subsequent phenotypic analysis. Three independent homozygous PaTM8 overexpression lines were obtained through screening and named OE3, OE7, and OE8, respectively.

[0059] Example 3 Phenotypic observation of PaTM8 transgenic Arabidopsis thaliana Wild-type Arabidopsis thaliana (WT) seeds and homozygous T3 generation transgenic Arabidopsis thaliana seeds overexpressing PaTM8 obtained in Example 2 were vernalized at 4 ℃ for 3 days, and then sown on basal MS solid medium and cultured at 22 ℃, 16 h light, 8 h dark for 10 days. After the seedlings grew vigorously, they were transplanted into nutrient soil and cultured in a growth chamber (23 ℃, 16 h light / 8 h dark) for further cultivation.

[0060] Flowering time and the number of rosette leaves at flowering were statistically analyzed in wild-type Arabidopsis thaliana (WT) and PaTM8 overexpression lines. Flowering time was defined as the number of days from sowing to when the main inflorescence reached a height of 1 cm; the number of rosette leaves at flowering was defined as the total number of rosette leaves on the plant when the main inflorescence reached a height of 1 cm. Twenty plants from each line were selected for phenotypic investigation. Statistical data are expressed as mean ± standard error (Mean ± SE), and significance analysis was performed.

[0061] Phenotypic observations and photographic records were conducted 27 days after sowing. Figure 2A comparison of wild-type Arabidopsis thaliana (WT) and PaTM8 overexpression lines (OE3, OE7, and OE8) at 27 days after sowing.

[0062] Figure 3 The flowering time statistics are for wild-type Arabidopsis thaliana (WT) and PaTM8 overexpression lines (OE3, OE7 and OE8); Figure 4 The results show the number of rosette leaves at flowering time for wild-type Arabidopsis thaliana (WT) and PaTM8 overexpression lines (OE3, OE7 and OE8).

[0063] The results showed that the average flowering time of wild-type Arabidopsis thaliana (WT) was 28.4 days, while the average flowering times of the PaTM8 overexpressing lines OE3, OE7, and OE8 were 23.7 days, 24.5 days, and 24.7 days, respectively, all significantly earlier than those of wild-type plants. Compared with WT, the flowering time of the PaTM8 overexpressing lines was approximately 3.7–4.7 days earlier, indicating that PaTM8 overexpression can promote the transition of Arabidopsis thaliana from vegetative growth to reproductive growth.

[0064] Furthermore, the average number of rosette leaves at flowering time was 11.5 in WT plants, while the average number of rosette leaves at flowering time in PaTM8-overexpressing lines OE3, OE7, and OE8 were 7.9, 8.1, and 7.7, respectively, significantly fewer than in WT. A reduced number of rosette leaves is typically an important phenotypic characteristic for earlier flowering in plants, further illustrating that PaTM8 overexpression promotes the flowering process in Arabidopsis thaliana.

[0065] The above results indicate that the PaTM8 gene has the function of promoting plant flowering. By increasing the expression level of the PaTM8 gene, the vegetative growth period of plants can be significantly shortened and flowering can be promoted, providing a new gene resource for the regulation of plant flowering period and molecular breeding.

[0066] In summary, the technical solution of this application has the following beneficial technical effects: (1) This application identified the function of the PaTM8 gene of Platanus orientalis in regulating plant flowering, and the experiment proved that it has a clear phenotypic regulatory role in transgenic plants; (2) This application provides the nucleic acid sequence of the PaTM8 gene and the amino acid sequence of the protein encoded by the gene, functional primers and overexpression vectors, providing new gene resources and technical foundation for the study of plant flowering regulation mechanisms and molecular breeding.

Claims

1. The application of a PaTM8 protein in promoting plant flowering, characterized in that, The amino acid sequence of the PaTM8 protein is shown in SEQ ID No.

2.

2. The application of a gene encoding the PaTM8 protein in promoting plant flowering, characterized in that, The nucleotide sequence of the gene encoding the PaTM8 protein is shown in SEQ ID No. 1; wherein the amino acid sequence of the PaTM8 protein is the amino acid sequence of the PaTM8 protein according to claim 1.

3. The application of the PaTM8 protein according to claim 2 in promoting plant flowering, characterized in that, The method to promote flowering in plants is to increase the expression level of the gene encoding the PaTM8 protein in the plant.

4. The application of an overexpression vector in promoting plant flowering, characterized in that, The overexpression vector contains the gene encoding the PaTM8 protein as described in claim 2.

5. The application of a genetically engineered bacterium in promoting plant flowering, characterized in that, The genetically engineered bacteria comprises the overexpression vector as described in claim 4; or... The genome of the genetically engineered bacterium integrates the gene encoding the PaTM8 protein as described in claim 2.

6. A method for promoting plant flowering, characterized in that, Includes the following steps: S10. Construct the overexpression vector as described in claim 4; S20. The overexpression vector was introduced into plants using Agrobacterium-mediated transformation, and positive transgenic lines were screened using hygromycin.

7. The method for promoting plant flowering according to claim 6, characterized in that, Step S10 includes the amplification of the gene encoding the PaTM8 protein. The primers for gene amplification include a forward primer P1 and a reverse primer P2. The sequence of the forward primer P1 is shown in SEQ ID No. 3, and the sequence of the reverse primer P2 is shown in SEQ ID No.

4.

8. The method for promoting plant flowering according to claim 6, characterized in that, Step S10 includes the following steps: The gene encoding the PaTM8 protein was ligated into the pCAMBIA1305 vector using homologous recombination. The primer sequences with specific homologous arms include forward primer P3 and reverse primer P4. The sequence of forward primer P3 is shown in SEQ ID No. 5, and the sequence of reverse primer P4 is shown in SEQ ID No. 6.

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