A hybrid tulip tree adenosine methionine decarboxylase gene, adenosine methionine decarboxylase, plant expression vector, and applications.

Abstract

This invention relates to a hybrid tulip tree LhSAMDC gene and its applications, belonging to the field of genetic engineering technology. The CDS nucleotide sequence of the LhSAMDC gene described in this invention is shown in SEQ ID NO.1, and the encoded adenosine methionine decarboxylase plays a key role in plant polyamine metabolism. This invention also provides primer pairs (SEQ ID NO.2 and SEQ ID NO.3) for amplifying this gene and a plant expression vector containing this gene. Experiments using transgenic Arabidopsis thaliana show that overexpression of the LhSAMDC gene significantly increases plant biomass, plant height, leaf SPAD value, and stress resistance. This invention provides a new technical means for plant breeding, which can be used to cultivate new plant varieties with stronger growth vigor, higher biomass, and better stress resistance, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a hybrid tulip tree adenosine methionine decarboxylase gene, adenosine methionine decarboxylase, a plant expression vector, and its applications. Background Technology

[0002] *Liriodendron*, belonging to the Magnoliaceae family, comprises two main species: the Asian tulip tree (*Liriodendron chinense* Sarg.) and the North American tulip tree (*Liriodendron tulipifera* L.). Also known as the Chinese tulip tree, it is a Class II protected tree species in my country, characterized by rapid growth, straight trunks, excellent wood quality, beautiful flowers and leaves, few pests and diseases, and strong pollution resistance, possessing value for timber, ornamental, and ecological purposes. The hybrid tulip tree (*L. chinense* Sarg. × *L. tulipifera* L.) is obtained by crossing *Liriodendron chinense* as the female parent and *Liriodendron tulipifera* as the male parent. Compared to its parents, the hybrid tulip tree exhibits significant hybrid vigor, including stronger rapid growth, stress resistance, and adaptability, making it an excellent timber and carbon sink species with broad development and utilization prospects.

[0003] S-adenosylmethionine decarboxylase (SAMDC) is a key enzyme in plant polyamine metabolism, catalyzing the formation of decarboxylated SAM from SAM, thereby participating in the regulation of plant growth, development, and stress responses. Studies have shown that SAMDC genes may play an important role in plant responses to abiotic stresses. However, current research on SAMDC genes has largely focused on model plants or a few economic crops, with limited functional studies on SAMDC genes in forest trees, especially in the genus *Liriodendron*. Their specific mechanisms of action in plant stress resistance and yield enhancement have not been fully elucidated. Furthermore, existing technologies for improving plant yield and stress resistance largely rely on traditional breeding, fertilization, or exogenous substance treatments. These methods are often costly, have unstable effects, and are difficult to simultaneously address multiple stress conditions. Therefore, obtaining a novel SAMDC gene and developing a new technology based on it to comprehensively improve plant yield and stress resistance has become an urgent technical problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a hybrid tulip tree LhSAMDC gene, its expression vector, and its applications. The hybrid tulip tree LhSAMDC gene described in this invention can improve plant yield and stress resistance.

[0005] This invention provides a hybrid tulip tree LhSAMDC gene, the CDS nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides a primer pair for amplifying the LhSAMDC gene of hybrid tulip tree, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0007] The present invention also provides a plant expression vector containing the LhSAMDC gene of the hybrid tulip tree as described in claim 1.

[0008] Preferably, the base plasmid for constructing the plant expression vector includes the pCAMBIA3301 vector.

[0009] Preferably, the nucleotide sequence of the upstream primer of the primer pair for constructing the plant expression vector is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

[0010] The present invention also provides a hybrid tulip tree adenosylmethionine decarboxylase, wherein the hybrid tulip tree adenosylmethionine decarboxylase is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0011] This invention also provides the application of the hybrid tulip tree LhSAMDC gene described in the above-mentioned technical solution, or the plant expression vector described in the above-mentioned technical solution, or the hybrid tulip tree adenosine methionine decarboxylase described in the above-mentioned technical solution in improving plant yield.

[0012] The present invention also provides the application of the hybrid tulip tree LhSAMDC gene described in the above technical solution, or the plant expression vector described in the above technical solution, or the hybrid tulip tree adenosine methionine decarboxylase described in the above technical solution in the creation of plant germplasm.

[0013] The created plant germplasm includes any one or more of the following: ① to ⑤:

[0014] ① Create plant germplasm with stronger growth potential;

[0015] ② Create plant germplasm with higher biomass;

[0016] ③ Create plant germplasm with a more robust plant structure;

[0017] ④ Create plant germplasm with more developed vegetative organs;

[0018] ⑤ Create plant germplasm with higher SPAD values.

[0019] This invention also provides the application of the hybrid tulip tree LhSAMDC gene described in the above-mentioned technical solution, or the plant expression vector described in the above-mentioned technical solution, or the hybrid tulip tree adenosine methionine decarboxylase described in the above-mentioned technical solution in enhancing plant stress resistance.

[0020] Preferably, the enhancement of plant stress resistance includes enhancing the plant's ability to resist drought stress and / or enhancing the plant's ability to resist flooding stress.

[0021] This invention provides the LhSAMDC gene of hybrid tulip tree. Through drought stress experiments and transcriptome analysis of hybrid tulip tree seedlings, this invention screened key differentially expressed genes during drought stress and successfully cloned the LhSAMDC gene. Furthermore, an overexpression vector of the LhSAMDC gene was constructed, transformed into Arabidopsis thaliana, and after multiple generations of planting, T3 generation seeds were obtained. Phenotypic observation showed that compared with wild-type Arabidopsis thaliana, the biomass of transgenic plants was significantly increased, and their resistance to flooding and drought stress was significantly enhanced. This indicates that the LhSAMDC gene can effectively promote plant growth and enhance its stress resistance, and has important application value in molecular breeding. Specifically, it can be used to create plant germplasm with stronger growth potential, higher biomass, more robust plant type, more developed vegetative organs, higher SPAD value, and stronger stress resistance. By utilizing the LhSAMDC gene, this invention provides a new technical means for plant breeding, which can significantly improve plant growth performance and stress resistance, and has broad application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The image shows the PCR amplification results of the LhSAMDC gene CDS nucleotide sequence provided by this invention; the left side shows the marker bands, from top to bottom: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; the two right sides show the sample bands of the LhSAMDC gene CDS nucleotide sequence amplification.

[0024] Figure 2 The image shows the PCR identification results of positive bacterial culture provided by this invention; the left side shows the positive bacterial culture band; the right side shows the marker band, which are from top to bottom as follows: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

[0025] Figure 3 This is a comparison diagram of the differences between leaves of LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants provided by the present invention;

[0026] Figure 4A comparison diagram of the base width of LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants provided by this invention;

[0027] Figure 5 This is a comparison chart of the SPAD values ​​of LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants provided by the present invention;

[0028] Figure 6 This is a comparison diagram of the plant height difference between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants provided by the present invention;

[0029] Figure 7 The diagram showing the phenotypic changes of Arabidopsis thaliana overexpressing LhSAMDC under flooding stress provided by this invention;

[0030] Figure 8 The diagram shows the phenotypic changes of Arabidopsis thaliana overexpressing LhSAMDC under drought stress, as provided in this invention. Detailed Implementation

[0031] This invention provides a hybrid tulip tree LhSAMDC gene, the CDS nucleotide sequence of which is shown in SEQ ID NO.1:

[0032]

[0033] In a specific embodiment, the sequence of the hybrid tulip tree LhSAMDC gene is shown in SEQ ID NO.6:

[0034]

[0035] This invention also provides a primer pair for amplifying the LhSAMDC gene of hybrid tulip tree, the nucleotide sequence of the upstream primer of the amplification primer pair being shown in SEQ ID NO.2:

[0036] 5'-ATGGCCTTCCCTGTCTCTGCC-3';

[0037] The nucleotide sequence of the downstream primer of the amplification primer pair is shown in SEQ ID NO.3:

[0038] 5'-TTATTCTTCTTCTTCTCCTTCTTTCCAGCATTTG-3'.

[0039] This invention also provides a plant expression vector containing the LhSAMDC gene of the hybrid tulip tree as described in claim 1. In a specific embodiment, the plant expression vector can induce LhSAMDC gene expression. In a specific embodiment, the plant expression vector is an LhSAMDC gene overexpression vector. In a specific embodiment, the basic plasmid for constructing the plant expression vector includes the pCAMBIA3301 vector. In a specific embodiment, the plant expression vector is formed by linking the pCAMBIA3301 vector and the LhSAMDC gene. In a specific embodiment, the construction of the plant expression vector includes the following steps: using hybrid tulip tree cDNA as a template, amplifying the LhSAMDC gene fragment carrying the homologous complementary sequence of the pCAMBIA3301 vector by PCR; digesting and purifying the pCAMBIA3301 vector plasmid with enzymes; and linking the LhSAMDC gene with the pCAMBIA3301 vector to obtain the plant expression vector. In a specific embodiment, the LhSAMDC gene fragment carrying the homologous complementary sequence of the pCAMBIA3301 vector can be obtained by introducing homologous complementary sequences flanking the Sac I and Xba I restriction sites on the pCAMBIA3301 vector at both ends of the LhSAMDC gene sequence as PCR primers. In a specific embodiment, the pCAMBIA3301 vector is double-digested with Sac I and Xba I. In a specific embodiment, the ligation of the LhSAMDC gene with the pCAMBIA3301 vector can be performed using the infusion method. In a specific embodiment, the nucleotide sequence of the upstream primer of the primer pair for constructing the plant expression vector is shown in SEQ ID NO.4:

[0040] 5'-GAGAACACGGGGGACGAGCTCATGGCCTTCCCTGTCTCTGCC-3';

[0041] The nucleotide sequence of the downstream primer of the primer pair used to construct the plant expression vector is shown in SEQ ID NO.5:

[0042] 5'-AAGATCTTCGTCGACTCTAGATTATTCTTCTTCTTCTTCCTCTTCTTT CCAGCATTTG-3'.

[0043] This invention also provides a hybrid tulip tree S-adenosylmethionine decarboxylase, which is encoded by a gene with the nucleotide sequence shown in SEQ ID NO.1. The amino acid sequence of the hybrid tulip tree S-adenosylmethionine decarboxylase of this invention is shown in SEQ ID NO.7.

[0044] MAFPVSAIGFEGYEKRLEISFFKPFIFADPQGKGLRSLSKSQLDEILKPAECTIVSSLSNDHVDSYVLSESSLFVHPYRIIMKTCGTTKLLLSIPPILDLAKSLSLSVKAVTYTRGSFIFPGAQSFPHRSFSEEVTILNDHFGNLGSGGNAYVMSSLVESQQWHIYSACAASAAEDDG PLCTLEMCMTGLDRKQASVFYKTQTSSAAEMTSASGIRKILPASDICDFEFDPCGYSMNSIEGAAISTIHVTPEDGFSYASFEAAGYNAKDVDLGELVERVLACFQPAEFSIAIHAVGREMGWVGEELNDVMGYVCGKRSMQELGEGGCVVYQSYKAGGCRSPRSILKCWKEEEEEEE.

[0045] In a specific embodiment, the hybrid tulip tree adenosylmethionine decarboxylase can be expressed in other plants besides the hybrid tulip tree using a plant expression vector. In a specific embodiment, the other plants include Arabidopsis thaliana.

[0046] This invention also provides the application of the hybrid tulip tree LhSAMDC gene, the plant expression vector, or the hybrid tulip tree S-adenosylmethionine decarboxylase described in the above-mentioned technical solutions in improving plant yield. In specific embodiments, improving plant yield includes one or more of the following: enhancing plant growth vigor, increasing plant biomass, improving plant structure, developing plant vegetative organs, and increasing the plant SPAD value.

[0047] The present invention also provides the application of the hybrid tulip tree LhSAMDC gene described in the above technical solution, or the plant expression vector described in the above technical solution, or the hybrid tulip tree adenosine methionine decarboxylase described in the above technical solution in the creation of plant germplasm.

[0048] The created plant germplasm includes any one or more of the following: ① to ⑤:

[0049] ① Create plant germplasm with stronger growth potential;

[0050] ② Create plant germplasm with higher biomass;

[0051] ③ Create plant germplasm with a more robust plant structure;

[0052] ④ Create plant germplasm with more developed vegetative organs;

[0053] ⑤ Create plant germplasm with higher SPAD values.

[0054] In a specific embodiment, the creation of plant germplasm includes the cultivation of new plant varieties.

[0055] This invention also provides the application of the hybrid tulip tree LhSAMDC gene described in the above-mentioned technical solution, or the plant expression vector described in the above-mentioned technical solution, or the hybrid tulip tree adenosine methionine decarboxylase described in the above-mentioned technical solution in enhancing plant stress resistance.

[0056] In a specific embodiment, enhancing plant stress resistance includes enhancing the plant's ability to resist drought stress and / or enhancing the plant's ability to resist flooding stress.

[0057] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a hybrid tulip tree adenosine methionine decarboxylase gene, adenosine methionine decarboxylase, plant expression vector, and applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0058] The plant material used in the examples, hybrid tulip trees, was obtained from the Jiangxi Academy of Sciences in Nanchang City, Jiangxi Province. In July 2023, the mixed tissue of flowers and leaves from tender spring flower branches was collected, frozen in liquid nitrogen, and stored at -80°C for later use.

[0059] Example 1

[0060] Discovery, cloning, and expression vector construction of the LhSAMDC gene.

[0061] 1) Total RNA extraction from mixed leaves and buds of hybrid tulip tree.

[0062] Take 1g of fresh leaf and bud mixture tissue, grind it into powder rapidly in liquid nitrogen, extract total RNA using a plant RNA extraction kit according to the instructions, and detect it by 1% agarose gel electrophoresis. Store in an ultra-low temperature freezer at -80℃ for later use.

[0063] 2) LhSAMDC gene primer design:

[0064] Download the Liriodendron tulipifera genome data (https: / / www.ncbi.nlm.nih.gov / ), and obtain the LhSAMDC gene sequence as shown in SEQ ID NO.6 from the Liriodendron tulipifera transcriptome database. The full-length ORF sequence of this gene is 1071 bp as shown in SEQ ID NO.1. Upstream and downstream primers were designed at both ends of the ORF, respectively. Specifically, the primers were designed using Primer Premier 5.0 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0065] The primer sequences are as follows:

[0066] LhSAMDC-F1:

[0067] 5'-ATGGCCTTCCCTGTCTCTGCC-3' (SEQ ID NO: 2);

[0068] LhSAMDC-R1:

[0069] 5'-TTATTCTTCTTCTTCTCCTCTTCTTTCCAGCATTTG-3' (SEQ ID NO: 3).

[0070] 3) Cloning of the LhSAMDC gene.

[0071] The synthesis of the first strand of hybrid tulip tree cDNA was performed using a Takara reverse transcription kit, following the kit's recommended system and reaction conditions. After the reaction, amplification was performed using gene-specific PCR primers (LhSAMDC-F and LhSAMDC-R) near the designed CDS region, according to the following reaction system and procedure. The PCR amplification results are shown below. Figure 1 As shown, the PCR products were sequenced. The sequencing results were compared with the sequences obtained from the transcriptome of hybrid tulip tree to verify the correctness of the LhSAMDC gene sequence.

[0072] The PCR reaction system is shown in Table 1:

[0073] Table 1 PCR reaction system

[0074] Components Dosage PCR-GradeWater 15.0μl 2×PCRBuffer for KODFXNeo 25.0μl dNTPMix(10mM) 1.0μl KODFXNeo (1U / ul) 1.0μl cDNA 5.0μl primerF(10X) 1.5μl primerR(10X) 1.5μl <![CDATA[ddH2O]]> Add to a final volume of 50.0 μL

[0075] The PCR reaction procedure is shown in Table 2:

[0076] Table 2 PCR reaction procedures

[0077]

[0078] 4) Construction of plant expression vector for LhSAMDC gene of hybrid tulip tree.

[0079] Homologous complementary sequences flanking the Sac I and Xba I restriction sites on the pCAMBIA3301 vector were introduced at both ends of the LhSAMDC gene sequence as PCR primers. The specific primer sequences for the overexpression vector construction are shown in Table 3. Using hybrid tulip cDNA as a template, the LhSAMDC gene fragment carrying the homologous complementary sequence of the pCAMBIA3301 vector was amplified by PCR, and the amplification product was recovered by gel extraction.

[0080] Table 3 Primers for overexpression vector construction

[0081]

[0082] Enzyme digestion and purification of pCAMBIA3301 vector plasmid.

[0083] The pCAMBIA3301 plasmid was double-digested with Sac I and Xba I. The digestion reaction was carried out overnight at 37°C. The digestion reaction system is shown in Table 4.

[0084] Table 4 Enzyme digestion reaction system

[0085] Components Dosage 10×BufferTango(ThermoScientific) 12.0ul pCAMBIA3301-35S-NOS 46.0μl SacI 1.0μl XbaI 1.0μl <![CDATA[ddH2O]]> Add to 60.0 μL

[0086] (3) Ligation of LhSAMDC gene with pCAMBIA3301 vector

[0087] The LhSAMDC gene fragment was ligated with the pCAMBIA3301 enzyme digestion product using the infusion method. The reaction conditions were 50℃ for 30 min, and the ligation system is shown in Table 5.

[0088] Table 5 Connection Reaction System

[0089] Components Dosage 2xGenRecAssemblyMasterMix 5μL Target gene 3μL carrier 2μL

[0090] After incubation, the reaction system was quickly transferred to ice and placed for 5 minutes to terminate the reaction and stabilize the product.

[0091] The coding region sequence of the LhSAMDC gene was introduced into the pCAMBIA3301 vector to construct the overexpression plant vector pCAMBIA3301-LhSAMDC, which was then transformed into E. coli. PCR detection was performed on positive colonies, and the results are as follows: Figure 2 As shown, take the plasmid with the correct PCR product band size for sequencing, compare the sequencing results, and save the plasmid with completely correct sequencing results for later use.

[0092] Example 2

[0093] Agrobacterium-mediated genetic transformation of Arabidopsis thaliana and functional analysis.

[0094] 1) Sow wild-type Arabidopsis thaliana seeds (Arabidopsis thaliana Colombianis) in pre-watered nutrient soil (organic matter: vermiculite: perlite = 3:1:1), cover with plastic wrap to keep moist, and remove the film after the seeds germinate. The culture environment in the artificial climate chamber is 26℃ with a light time of 14h, and culture until flowering.

[0095] 2) Escherichia coli transformation.

[0096] Take 80 μL of DH5α competent cells, add 10 μL of ligation product, mix well with a pipette, and incubate on ice for 30 min. After the ice incubation, heat shock in a 42℃ water bath for 90 s, and then quickly cool on ice for 2 min. Add 500 μL of LB liquid medium to the tube, mix well, and incubate at 37℃ for 1 h with shaking at 160 rpm. After the shaking culture, spread 100 μL of bacterial culture onto LB solid medium containing 50 mg / L ampicillin, and incubate upside down at 37℃ for 12 h to 16 h.

[0097] 3) Identification of fusion expression vectors.

[0098] Single colonies were randomly picked from the transformation medium and expanded. PCR identification of the bacterial culture was performed using universal primers for the pCAMBIA3301 vector. The PCR reaction system and procedure are as follows. The results were detected by 1% agarose gel electrophoresis. The PCR-positive bacterial cultures were preserved and sequenced. The sequencing results are shown in SEQ ID NO. 8.

[0099]

[0100] The reaction system is shown in Table 6:

[0101] Table 6 PCR reaction system

[0102] Components Dosage 2×TSINGKEMasterMix 25.0ul Bacterial template 5.0μl Forward primer P1 2.0μl Reverse primer P2 2.0μl <![CDATA[ddH2O]]> 21.0μl

[0103] The reaction conditions are shown in Table 7:

[0104] Table 7 PCR reaction conditions

[0105]

[0106]

[0107] (1) Preparation of Agrobacterium.

[0108] Add 1 μL of plasmid (pCAMBIA3301-LhSAMDC) to 50 μL of GV3101 Agrobacterium competent cells, mix thoroughly, and transfer to an electroporation cuvette. After electroporation, add 1 mL of LB (Luria-Bertani) liquid medium, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate at 30℃ and 180 rpm for 30 min on a shaker. Spread 50 μL of the activated Agrobacterium culture evenly onto LB solid medium and incubate in the dark at 28℃ for 48 h. Pick single-clone transformants for expansion culture. Using the Agrobacterium culture as a template, perform PCR detection using universal primers for the pCAMBIA3301 vector. Store the Agrobacterium culture with correct results (pCAMBIA1301-LhSAMDC).

[0109] (2) Agrobacterium infection.

[0110] Agrobacterium was collected and resuspended to prepare an Agrobacterium resuspension with an OD600 of 0.8-1.2. Silwet-77 was added to a concentration of 0.02%. All inflorescences of Arabidopsis thaliana were dipped into the bacterial suspension for 2-3 seconds, sealed with a film to maintain humidity >90%, and incubated in the dark at 25°C for 24 hours. The infection cycle was 7 days, with a total of 3 infections. The infected seedlings were then placed in a 23°C 16h / 8h light / dark culture until seed formation. Mature pods were gently rubbed onto clean white paper, wrapped, and dried at 37°C for 24 hours. After drying, the seeds were sieved through a 60-mesh sieve, and the clean seeds (labeled as T0 generation) were stored at 4°C.

[0111] (3) Screening of transformed Arabidopsis thaliana plants.

[0112] First, the harvested T0 generation seeds were sterilized in 95% ethanol for 10 minutes, followed by sterilization with 75% ethanol for another 10 minutes, and then washed 2 to 3 times with sterile water for 1 minute each time. After washing, the seeds were evenly spread on the appropriate resistance selection medium and placed at 4°C for 2 to 3 days. Then, the plates were removed and placed in an environment of 23°C to 25°C, with a 16-hour light-8-hour dark cycle for 10 to 14 days. The concentration used for selection was Basta 20 mg / L. After selecting surviving seedlings (marked as T1 generation), they were transplanted into nutrient soil and cultured again at 23°C under 16-hour light-8-hour dark conditions. When the Arabidopsis seedlings reached approximately 20 days of growth, genomic DNA was extracted from Arabidopsis using the CTAB method (Cetyltrimethylammonium Bromide) and then detected by PCR. The overexpression vector pCAMBIA3301-LhSAMDC was transformed into Agrobacterium and then into wild-type Arabidopsis thaliana via the flower immersion method. Resistance screening was performed on T0 generation seeds, yielding six T1 generation resistant lines. When the T1 generation Arabidopsis seedlings reached approximately 20 days of growth, DNA-level PCR identification was performed on each line. The results showed that all transgenic plants amplified the LhSAMDC gene fragment, indicating that all transgenic lines were positive. The plants were further cultured to the T3 generation, and the T3 generation transgenic Arabidopsis seeds were collected for subsequent experiments.

[0113] Example 3

[0114] Phenotypic observation of transgenic Arabidopsis thaliana with the LhSAMDC gene.

[0115] After vernalization, T3 generation seeds were evenly sown in pre-watered nutrient soil (organic matter: vermiculite: perlite = 2:1:1). The culture environment was a climate chamber at 24℃ with 16 hours of light / 8 hours of darkness. After 3 days of cultivation covered with plastic wrap, the plastic wrap was removed. When the seeds had two true leaves, the transgenic plants were sprayed with a 20 mg / L BASTA herbicide solution every 2-3 days until the fourth true leaf appeared. Cultivation continued until eight true leaves appeared. Leaf DNA was extracted and positive detection was performed using primers (LhMFT1-F and LhMFT1-R) based on its CDS sequence. The positive rate was 100%. Phenotypic differences between positive transgenic plants and wild-type plants were further observed. The results of phenotypic differences between positive transgenic plants and wild-type plants are shown in Table 8. Figures 3-6 As shown, where, Figure 3 This image shows a comparison of leaf differences between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants. Figure 4 This image shows a comparison of the base width between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants. Figure 5 This is a comparison of the SPAD values ​​between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants. Figure 6This is a comparison of plant height between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants. It can be seen that the transgenic Arabidopsis thaliana plants are taller, with larger leaves, and the base width increased by 28.85% and the SPAD value increased by 6.17% compared to the wild-type plants.

[0116] Table 8. Phenotypic differences between transgenic and wild-type plants

[0117] Grouping Base width / mm SPAD value Wild-type plant CK 34.49±2.91 26.27±0.53 OE transgenic plants 44.44±2.56 27.89±0.31

[0118] Example 4

[0119] Analysis of the water stress resistance of transgenic Arabidopsis thaliana with the LhSAMDC gene.

[0120] LhSAMDC-overexpressing Arabidopsis thaliana and wild-type Arabidopsis thaliana were planted separately in nutrient soil. When the seedlings grew to 7-8 leaves, healthy plants were selected and divided into two groups: CK (wild-type plants) and OE (transgenic plants). Both groups were subjected to waterlogging and drought stress treatments. For waterlogging, the plants were observed after 1, 3, and 7 days of waterlogging stress. For drought stress, both wild-type and transgenic Arabidopsis thaliana were irrigated with a 20% PEG6000 solution, and the phenotypes were observed after 1, 3, and 5 days of drought stress treatment.

[0121] The results of LhSAMDC overexpression in Arabidopsis thaliana under waterlogging stress are as follows: Figure 7 As shown in the results of the flooding stress experiment, the Arabidopsis thaliana plants overexpressing LhSAMDC (OE) did not exhibit significant phenotypic differences compared to wild-type plants (CK) under flooding stress. Under flooding stress, both the LhSAMDC-overexpressing and wild-type Arabidopsis thaliana plants showed varying degrees of stress response, with the overexpressing plants exhibiting relatively better growth and demonstrating a certain degree of flood tolerance.

[0122] Drought resistance analysis of Arabidopsis thaliana overexpressing LhSAMDC

[0123] The results of drought stress in Arabidopsis thaliana overexpressing LhSAMDC are as follows: Figure 8 As shown in the results of the drought stress experiment, Arabidopsis thaliana plants overexpressing LhSAMDC exhibited significant phenotypic differences compared to wild-type plants under drought stress. During drought stress, the growth of wild-type plants was affected earlier, with some plants showing severe leaf wilting. In contrast, Arabidopsis thaliana plants overexpressing LhSAMDC demonstrated stronger drought tolerance under drought stress. These plants showed less leaf wilting and relatively better growth. This indicates that overexpression of the LhSAMDC gene has a positive effect on improving the drought stress resistance of Arabidopsis thaliana.

[0124] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A hybrid Taxodium distichum LhSAMDC gene, characterized in that, The CDS nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. A pair of primers for amplifying a hybridized Taxodium LhSAMDC gene, characterized in that, The nucleotide sequence of the upstream primer of the primer pair for constructing the plant expression vector is shown as SEQ ID NO. 4, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

5.

3. A plant expression vector, characterized in that, The plant expression vector contains the hybrid Liriodendron tulipifera LhSAMDC gene.

4. The plant expression vector of claim 3, wherein, The basic plasmid for constructing the plant expression vector includes a pCAMBIA3301 vector.

5. The plant expression vector of claim 3, wherein, The nucleotide sequence of the upstream primer of the primer pair for constructing the plant expression vector is shown as SEQ ID NO. 4, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

5.

6. A hybrid Liriodendron tulipifera adenosinemethionine decarboxylase, characterized in that, The hybrid Liriodendron tulipifera LhSAMDC gene is encoded by a gene with a nucleotide sequence shown as SEQ ID NO.

1.

7. Use of the hybrid Liriodendron tulipifera LhSAMDC gene of claim 1, the plant expression vector of any one of claims 3-5, or the hybrid Liriodendron tulipifera LhSAMDC gene of claim 6 in improving the yield of Arabidopsis thaliana.

8. Use of the hybrid Liriodendron tulipifera LhSAMDC gene of claim 1, the plant expression vector of any one of claims 3-5, or the hybrid Liriodendron tulipifera LhSAMDC gene of claim 6 in creating an Arabidopsis thaliana germplasm. The created plant germplasm includes any one or more than two of the following: ① creating an Arabidopsis thaliana germplasm with stronger growth vigor; ② creating an Arabidopsis thaliana germplasm with higher biomass; ③ creating an Arabidopsis thaliana germplasm with stronger plant type; ④ creating an Arabidopsis thaliana germplasm with more developed vegetative organs; ⑤ creating an Arabidopsis thaliana germplasm with higher spad value.

9. Use of the hybrid Liriodendron tulipifera LhSAMDC gene of claim 1, the plant expression vector of any one of claims 3-5, or the hybrid Liriodendron tulipifera LhSAMDC gene of claim 6 in improving the stress resistance of Arabidopsis thaliana. The improved stress resistance of Arabidopsis thaliana is improved drought stress resistance and / or improved flooding stress resistance.

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