Hybrid liriodendron adenosine methionine decarboxylase gene, adenosine methionine decarboxylase, plant expression vector and application

Abstract

The invention relates to a liriodendron hybrids LhSAMDC gene and application thereof, and belongs to the technical field of gene engineering. The CDS nucleotide sequence of the LhSAMDC gene disclosed by the invention is as shown in SEQ ID NO.1, and adenosine methionine decarboxylase coded by the LhSAMDC gene plays a key role in plant polyamine metabolism. The invention also provides a primer pair (SEQ ID NO.2 and SEQ ID NO.3) for amplifying the gene and a plant expression vector containing the gene. Transgenic arabidopsis thaliana experiments show that the overexpression of the LhSAMDC gene can significantly improve the biomass, plant height, leaf SPAD value and stress resistance of the plant. The invention provides a new technical means for plant breeding, can be used for cultivating new plant varieties with stronger growth vigor, higher biomass and better stress resistance, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a Liriodendron hybrids adenosylmethionine decarboxylase gene, adenosylmethionine decarboxylase, a plant expression vector and applications thereof. Background Art

[0002] The genus Liriodendron belongs to Magnoliaceae and contains two main tree species: Liriodendron chinense Sarg. in Asia and Liriodendron tulipifera L. in North America. Liriodendron, also known as Chinese tulip tree, is a second-class protected tree species in China. It has the characteristics of fast growth, straight trunk, excellent wood quality, beautiful flowers and leaves, few pests and diseases, and strong anti-pollution ability, and has both wood use, ornamental and ecological values. Liriodendron hybrids (L.chinense Sarg.×L.tulipifera L.) is obtained by crossing Liriodendron chinense Sarg. as the female parent and Liriodendron tulipifera L. as the male parent. Compared with the parents, Liriodendron hybrids shows significant heterosis, including stronger fast growth, stress resistance and adaptability. It is an excellent woody tree species and carbon sink tree species, and has broad prospects for development and utilization.

[0003] Adenosylmethionine decarboxylase (SAMDC) is a key enzyme in plant polyamine metabolism, which can catalyze adenosylmethionine (SAM) to form decarboxylated SAM, thus participating in the regulation of plant growth and development and stress responses. Research shows that the SAMDC gene may play an important role in plants' response to abiotic stresses. However, current research on the SAMDC gene mostly focuses on model plants or a few economic crops, and the functional research on the SAMDC gene in forest trees, especially in the genus Liriodendron, is still relatively limited, and its specific mechanism in plant stress resistance and yield improvement has not been fully revealed. Moreover, in the prior art, methods for improving plant yield and stress resistance mostly rely on traditional breeding, fertilization or exogenous substance treatment. These methods are often costly, have unstable effects, and are difficult to take into account multiple stress conditions at the same time. Therefore, how to obtain a novel SAMDC gene and develop a new technology based on this gene that can comprehensively improve plant yield and stress resistance has become an urgent technical problem to be solved. Summary of the Invention

[0004] The purpose of the present invention is to provide a Liriodendron hybrids LhSAMDC gene, its expression vector and applications thereof. The Liriodendron hybrids LhSAMDC gene of the present invention can improve the yield and stress resistance of plants.

[0005] The present invention provides a Liriodendron hybrids LhSAMDC gene, and the CDS nucleotide sequence of the gene is as shown in SEQ ID NO.1.

[0006] The present invention also provides an amplification primer pair for the LhSAMDC gene of Liriodendron hybrids, wherein the nucleotide sequence of the upstream primer of the amplification primer pair is as shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.3.

[0007] The present invention also provides a plant expression vector, which contains the LhSAMDC gene of Liriodendron hybrids described in claim 1.

[0008] Preferably, the basic 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 as shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.5.

[0010] The present invention also provides an S-adenosylmethionine decarboxylase of Liriodendron hybrids, which is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0011] The present invention also provides the application of the LhSAMDC gene of Liriodendron hybrids described in the above technical solution, or the plant expression vector described in the above technical solution, or the S-adenosylmethionine decarboxylase of Liriodendron hybrids described in the above technical solution in increasing plant yield.

[0012] The present invention also provides the application of the LhSAMDC gene of Liriodendron hybrids described in the above technical solution, or the plant expression vector described in the above technical solution, or the S-adenosylmethionine decarboxylase of Liriodendron hybrids described in the above technical solution in creating plant germplasm;

[0013] The creation of plant germplasm includes any one or two or more of the following as shown in ① to ⑤:

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

[0015] ② Creating plant germplasm with higher biomass;

[0016] ③ Creating plant germplasm with more robust plant types;

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

[0018] ⑤ Creating plant germplasm with higher spad values.

[0019] The present invention also provides the application of the LhSAMDC gene of Liriodendron hybrids described in the above technical solution, or the plant expression vector described in the above technical solution, or the S-adenosylmethionine decarboxylase of Liriodendron hybrids described in the above technical solution in enhancing plant stress resistance.

[0020] Preferably, enhancing the stress resistance of plants includes enhancing the drought stress tolerance ability of plants and / or enhancing the waterlogging stress tolerance ability of plants.

[0021] The present invention provides the LhSAMDC gene of Liriodendron hybrids. Through carrying out the drought stress experiment and transcriptome analysis on the seedlings of Liriodendron hybrids, the key differentially expressed genes during the drought stress process were screened out, and the LhSAMDC gene was successfully cloned. Further, an overexpression vector of the LhSAMDC gene was constructed, transformed into Arabidopsis thaliana, and the T3 generation seeds were obtained through multiple generations of cultivation. Phenotypic observation shows that compared with the wild-type Arabidopsis thaliana, the biomass of the transgenic plants is significantly increased, and the resistance to waterlogging and drought stress is significantly enhanced, indicating 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 germplasms with stronger growth potential, higher biomass, more robust plant types, more developed vegetative organs, higher SPAD values, and stronger stress resistance. By utilizing the LhSAMDC gene, the present invention provides a new technical means for plant breeding, which can significantly improve the growth performance and stress resistance of plants and has broad application prospects. Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is the PCR amplification result diagram of the CDS nucleotide sequence of the LhSAMDC gene provided by the present invention; among them, on the left is the Marker band, from top to bottom in turn: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; on the right, the two are the amplification sample bands of the CDS nucleotide sequence of the LhSAMDC gene;

[0024] Figure 2 It is the PCR identification result diagram of the positive bacterial liquid provided by the present invention; among them, on the left is the positive bacterial liquid band; on the right is the Marker band, from top to bottom in turn: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp;

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

[0026] Figure 4Comparison chart of basal widths between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants provided by the present invention;

[0027] Figure 5 Comparison chart of spad values between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants provided by the present invention;

[0028] Figure 6 Comparison chart of plant height differences between LhSAMDC-overexpressing Arabidopsis thaliana and wild-type plants provided by the present invention;

[0029] Figure 7 Phenotypic change diagram of waterlogging stress of LhSAMDC-overexpressing Arabidopsis thaliana provided by the present invention;

[0030] Figure 8 Phenotypic change diagram of drought stress of LhSAMDC-overexpressing Arabidopsis thaliana provided by the present invention. Detailed implementation manners

[0031] The present invention provides a Liriodendron hybrid LhSAMDC gene, and the CDS nucleotide sequence of the gene is shown as SEQ ID NO.1:

[0032]

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

[0034]

[0035] The present invention also provides an amplification primer pair for the LhSAMDC gene of Liriodendron hybrids, and the nucleotide sequence of the upstream primer of the amplification primer pair is shown as SEQ ID NO.2:

[0036] 5’-ATGGCCTTCCCTGTCTCTGCC-3’;

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

[0038] 5’-TTATTCTTCTTCTTCTTCCTCTTCTTTCCAGCATTTG-3’.

[0039] The present invention also provides a plant expression vector, and the plant expression vector contains the LhSAMDC gene of Liriodendron hybrids as claimed in claim 1. In a specific embodiment, the plant expression vector can induce the expression of the LhSAMDC gene. In a specific embodiment, the plant expression vector is an overexpression vector of the LhSAMDC gene. 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 connecting the pCAMBIA3301 vector and the LhSAMDC gene. In a specific embodiment, the construction of the plant expression vector includes the following steps: using the cDNA of Liriodendron hybrids as a template, amplifying the LhSAMDC gene fragment with a homologous complementary sequence of the pCAMBIA3301 vector by PCR; carrying out enzyme digestion and purification on the pCAMBIA3301 vector plasmid; connecting the LhSAMDC gene with the pCAMBIA3301 vector to obtain the plant expression vector. In a specific embodiment, the LhSAMDC gene fragment with a homologous complementary sequence of the pCAMBIA3301 vector can be obtained by introducing homologous complementary sequences on both sides of the Sac I and Xba I enzyme digestion sites on the pCAMBIA3301 vector as PCR primers at both ends of the LhSAMDC gene sequence. In a specific embodiment, the pCAMBIA3301 vector is digested with Sac I and Xba I by double enzyme digestion. In a specific embodiment, the connection of the LhSAMDC gene with the pCAMBIA3301 vector can be carried out by using the infusion fusion method. In a specific embodiment, the nucleotide sequence of the upstream primer of the primer pair for constructing the plant expression vector is shown as SEQ ID NO.4:

[0040] 5’-GAGAACACGGGGGACGAGCTCATGGCCTTCCCTGTCTCTGCC-3’;

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

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

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

[0044] MAFPVSAIGFEGYEKRLEISFFKPFIFADPQGKGLRSLSKSQLDEILKPAECTIVSSLSNDHVDSYVLSESSLFVHPYRIIMKTCGTTKLLLSIPPILDLAKSLSLSVKAVTYTRGSFIFPGAQSFPHRSFSEEVTILNDHFGNLGSGGNAYVMSSLVESQQWHIYSACAASAAEDDGPLCTLEMCMTGLDRKQASVFYKTQTSSAAEMTSASGIRKILPASDICDFEFDPCGYSMNSIEGAAISTIHVTPEDGFSYASFEAAGYNAKDVDLGELVERVLACFQPAEFSIAIHAVGREMGWVGEELNDVMGYVCGKRSMQELGEGGCVVYQSYKAGGCRSPRSILKCWKEEEEEEE 。

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

[0046] The present invention also provides the application of the hybrid tulip tree LhSAMDC gene, the plant expression vector or the hybrid tulip tree S-adenosylmethionine decarboxylase in the above technical solution in increasing plant yield. In a specific embodiment, the increase in plant yield includes one or more of the following: making the plant growth potential stronger, making the plant biomass higher, making the plant plant type more robust, making the plant vegetative organs more developed, and making the plant spad value higher.

[0047] The present invention also provides the application of the LhSAMDC gene of Liriodendron hybrids as described in the above technical solution, or the plant expression vector as described in the above technical solution, or the S-adenosylmethionine decarboxylase of Liriodendron hybrids as described in the above technical solution in creating plant germplasm;

[0048] The creation of plant germplasm includes any one or two or more of the following as shown in ① to ⑤:

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

[0050] ② Creating plant germplasm with higher biomass;

[0051] ③ Creating plant germplasm with more robust plant types;

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

[0053] ⑤ Creating plant germplasm with higher spad values.

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

[0055] The present invention also provides the application of the LhSAMDC gene of Liriodendron hybrids as described in the above technical solution, or the plant expression vector as described in the above technical solution, or the S-adenosylmethionine decarboxylase of Liriodendron hybrids as described in the above technical solution in enhancing plant stress resistance.

[0056] In a specific embodiment, the enhancement of plant stress resistance includes enhancing the ability of plants to resist drought stress and / or enhancing the ability of plants to resist waterlogging stress.

[0057] To further illustrate the present invention, the following describes in detail a S-adenosylmethionine decarboxylase gene, S-adenosylmethionine decarboxylase, plant expression vector and application provided by the present invention with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0058] The plant material Liriodendron hybrids used in the examples was collected from the Jiangxi Academy of Sciences in Nanchang, Jiangxi Province. In July 2023, the mixed leaf and flower tissues of spring young flower branches were collected, frozen with liquid nitrogen and stored at -80 °C for later use.

[0059] Example 1

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

[0061] 1) Extraction of total RNA from the mixed leaves and buds of Liriodendron hybrids.

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

[0063] 2) Design of LhSAMDC gene primers:

[0064] Download the Liriodendron genome data (https: / / www.ncbi.nlm.nih.gov / ), and obtain the LhSAMDC gene sequence from the transcriptome database of Liriodendron hybrids as shown in SEQ ID NO.6. The full-length of this gene contains an ORF sequence of 1071 bp as shown in SEQ ID NO.1. Design upstream and downstream primers at both ends of the ORF. Specifically, use Primer Premier 5.0 software to design primers, and entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize the primers.

[0065] The primer sequences are as follows:

[0066] LhSAMDC-F1:

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

[0068] LhSAMDC-R1:

[0069] 5’-TTATTCTTCTTCTTCTTCCTCTTCTTTCCAGCATTTG-3’ (SEQ ID NO:3).

[0070] 3) Cloning of the LhSAMDC gene.

[0071] The synthesis of the first strand of cDNA from Liriodendron hybrids is completed by using Takara's reverse transcription kit and carried out according to the system and reaction conditions recommended by the kit. After the reaction, use the gene-specific PCR primers (LhSAMDC-F and LhSAMDC-R) near the designed CDS region for amplification according to the following reaction system and procedure, and obtain the PCR amplification result as Figure 1 shown, and sequence the PCR product. Compare the sequencing result with the sequence obtained from the transcriptome of Liriodendron hybrids 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] Component Dosage PCR-Grade Water 15.0 μl 2×PCR Buffer for KOD FX Neo 25.0 μl dNTP Mix (10 mM) 1.0 μl KOD FX Neo (1 U / ul) 1.0 μl cDNA 5.0 μl primer F (10X) 1.5 μl primer R (10X) 1.5 μl <![CDATA[ddH2O]]> Make up to 50.0 μL

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

[0076] Table 2 PCR reaction program

[0077]

[0078] 4) Construction of the plant expression vector of the LhSAMDC gene of hybrid Liriodendron

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

[0080] Table 3 Primers for overexpression vector construction

[0081]

[0082] Restriction enzyme digestion and purification of the pCAMBIA3301 vector plasmid

[0083] The pCAMBIA3301 plasmid was digested with Sac I and Xba I double restriction enzymes. The reaction conditions for restriction enzyme digestion were overnight digestion at 37°C. The reaction system for restriction enzyme digestion is shown in Table 4:

[0084] Table 4 Reaction system for restriction enzyme digestion

[0085] Component Dosage 10×Buffer Tango (Thermo Scientific) 12.0ul pCAMBIA3301-35S-NOS 46.0 μl SacI 1.0 μl XbaI 1.0 μl <![CDATA[ddH2O]]> Make up to 60.0 μL

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

[0087] The LhSAMDC gene fragment and the pCAMBIA3301 restriction enzyme digestion product were ligated using the infusion fusion method. The reaction conditions were incubation at 50°C for 30 min. The ligation system is shown in Table 5:

[0088] Table 5 Ligation reaction system

[0089] Component Dosage 2xGenRec Assembly Master Mix 5 μL Target gene 3 μL Vector 2 μL

[0090] After the incubation ended, the reaction system was quickly transferred to ice and placed for 5 min 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 transformed into Escherichia coli, and the positive colonies were detected by PCR. The detection results are as followsFigure 2 As shown, plasmids with correct PCR product band sizes were selected for sequencing. The sequencing results were compared, and plasmids with completely correct sequencing results were saved for future 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 Columbia) in pre-watered nutrient soil (organic matter: vermiculite: perlite = 3:1:1), cover with plastic wrap to keep moist. After the seeds germinate, remove the film. The culture environment in the artificial climate chamber is 26°C, the light time is 14 h, and culture until flowering.

[0095] 2) Escherichia coli transformation.

[0096] Take 80 μL of DH5α competent cells, add 10 μL of the ligation product, mix well with a pipette and incubate on ice for 30 min; after the ice bath, place in a 42°C water bath for heat shock for 90 s, then quickly place on ice for cooling for 2 min; add 500 μL of LB liquid medium to the tube, mix well, place on a shaker at 160 rpm and incubate at 37°C for 1 h; after the shaking culture, take 100 μL of the bacterial solution and spread it on an LB solid medium containing 50 mg / L ampicillin at a final concentration, and incubate inverted at 37°C for 12 h - 16 h.

[0097] 3) Identification of the fusion expression vector.

[0098] Randomly pick single colonies from the transformation medium for enlarged culture, and perform PCR identification on the bacterial solution using the universal primers of the pCAMBIA3301 vector. The PCR reaction system and reaction program are as follows. Detect by 1% agarose gel electrophoresis, save the bacterial solution with positive PCR results and send it for sequencing. The sequencing results are as shown in SEQ ID NO.8:

[0099]

[0100]

[0101] The reaction system is shown in Table 6:

[0102] Table 6 PCR reaction system

[0103] Component Dosage 2×TSINGKE Master Mix 25.0ul Bacterial liquid template 5.0 μl Forward primer P1 2.0 μl Reverse primer P2 2.0 μl <![CDATA[ddH2O]]> 21.0 μl

[0104] The reaction conditions are shown in Table 7:

[0105] Table 7 PCR reaction conditions

[0106]

[0107]

[0108] (1) Agrobacterium preparation.

[0109] Take 1 μL of plasmid (pCAMBIA3301-LhSAMDC) and add it to 50 μL of Agrobacterium tumefaciens GV3101 competent cells. After thorough mixing, transfer it to an electroporation cuvette. After electroporation, add 1 mL of LB (Luria-Bertani) liquid medium, mix well and transfer it to a 1.5 mL centrifuge tube. Incubate it on a shaker at 30 °C and 180 rpm for 30 min. Take 50 μL of the activated Agrobacterium liquid and spread it evenly on the LB solid medium, and incubate it in the dark at 28 °C for 48 h. Pick monoclonal transformants for large-scale culture. Use the Agrobacterium liquid as a template and perform PCR detection with the universal primers of the pCAMBIA3301 vector. Preserve the correct pCAMBIA1301-LhSAMDC Agrobacterium strain.

[0110] (2) Agrobacterium infection.

[0111] Pick Agrobacterium into the resuspension solution to prepare an Agrobacterium resuspension with OD600 = 0.8 - 1.2. Add silwet-77 to a concentration of 0.02%. Dip all the inflorescences of the Arabidopsis thaliana material into the bacterial solution for 2 - 3 s, seal the film to keep the humidity > 90%, and incubate it in the dark at 25 °C for 24 h. The infection cycle is 7 d, and a total of 3 infections are carried out. Place the infected seedlings in a 23 °C incubator with a 16 h / 8 h light / dark cycle until they produce seeds; gently rub the mature pods onto a clean white paper, wrap them up and dry them at 37 °C for 24 h. After drying, sieve them through a 60-mesh sieve, and store the clean seeds (labeled as T0 generation) at 4 °C.

[0112] (3) Screening of transformed Arabidopsis thaliana plants.

[0113] First, the harvested T0 generation seeds were disinfected in 95% ethanol for 10 min, then disinfected in 75% ethanol for another 10 min, and then washed with sterile water 2 to 3 times, 1 min each time. After washing, the seeds were evenly spread on the corresponding resistance screening medium and placed at 4 °C for 2 to 3 days. After that, the petri dishes were taken out and placed in an environment of 23 °C to 25 °C, and cultured for 10 to 14 days with a cycle of 16 h of light and 8 h of darkness. The concentration used for screening was: Basta 20 mg / L. After screening out the surviving seedlings (labeled as T1 generation), they were transplanted into nutrient soil and continued to be cultured under the conditions of 23 °C, 16 h of light and 8 h of darkness. When the Arabidopsis seedlings grew to about 20 days old, the genomic DNA of Arabidopsis was extracted using the CTAB method (Cetyltrimethylammonium Bromide) and subjected to PCR detection. The overexpression vector pCAMBIA3301-LhSAMDC was transformed into Agrobacterium, and wild-type Arabidopsis was transformed by the floral dip method. Resistance screening was carried out on the T0 generation seeds, and a total of 6 T1 generation resistant lines were obtained. When the T1 generation Arabidopsis seedlings grew to about 20 days old, PCR identification at the DNA level was carried out on each line. The results showed that all transgenic plants amplified the gene fragment of LhSAMDC, indicating that all transgenic lines were positive. They were continued to be cultured until the T3 generation, and the T3 generation transgenic Arabidopsis seeds were collected for subsequent experiments.

[0114] Example 3

[0115] Phenotypic observation of transgenic Arabidopsis thaliana with the LhSAMDC gene.

[0116] The vernalized T3 generation seeds were evenly sown in the nutrient soil that had been watered thoroughly in advance (organic matter: vermiculite: perlite = 2:1:1). The culture environment was an artificial climate chamber at 24 °C, with 16 h of light / 8 h of darkness. After culturing with a plastic wrap for 3 days, the plastic wrap was removed. When the seeds grew 2 true leaves, the transgenic plants were sprayed with a BASTA herbicide solution at a concentration of 20 mg / L, sprayed once every 2 to 3 days until the fourth true leaf grew. They were continued to be cultured until 8 true leaves grew, and the leaf DNA was extracted and used to perform positive detection with the primers of its CDS sequence (LhMFT1-F and LhMFT1-R). The positive rate was 100%. The phenotypic differences between the positive transgenic plants and the wild type were continued to be observed. The results of the phenotypic differences between the positive transgenic plants and the wild type are shown in Table 8 and Figures 3 to 6 as shown below, where Figure 3 is the comparison diagram of the leaf differences between LhSAMDC overexpressing Arabidopsis thaliana and wild-type plants, Figure 4 is the comparison diagram of the basal widths between LhSAMDC overexpressing Arabidopsis thaliana and wild-type plants, Figure 5 is the comparison diagram of the spad values between LhSAMDC overexpressing Arabidopsis thaliana and wild-type plants, Figure 6This is a comparison chart of the plant height differences between LhSAMDC overexpressing Arabidopsis thaliana and wild-type plants. It can be seen that the plant height of the transgenic Arabidopsis thaliana has increased, the leaves are hypertrophied, the base width has increased by 28.85% compared to the wild-type plants, and the spad value has increased by 6.17%.

[0117] Table 8 Phenotypic differences between transgenic plants and wild types

[0118] Grouping Base width / mm SPAD value Transgenic plant OE 34.49±2.91 26.27±0.53 Wild-type plant CK 44.44±2.56 27.89±0.31

[0119] Example 4

[0120] Analysis of the water stress resistance of transgenic Arabidopsis thaliana with the LhSAMDC gene

[0121] Plant the LhSAMDC overexpressing Arabidopsis thaliana and wild-type Arabidopsis thaliana in nutrient soil respectively. When the seedlings grow to 7-8 leaves, select the plants with good growth and divide them into two groups, namely CK (wild-type plants) and OE (transgenic plants). Conduct waterlogging stress and drought stress treatments respectively. The waterlogging treatment is carried out in a water tank, and the plant phenotypes are observed after 1 day, 3 days, and 7 days of waterlogging stress treatment. For the drought stress treatment, use 20% PEG6000 solution to water the wild-type and transgenic Arabidopsis thaliana, and observe the phenotypes after 1 day, 3 days, and 5 days of stress treatment.

[0122] The results of waterlogging stress of LhSAMDC overexpressing Arabidopsis thaliana are as Figure 7 shown. From the results of the waterlogging stress experiment, it can be seen that compared with the wild-type plants (CK), the Arabidopsis thaliana plants overexpressing LhSAMDC (OE) did not show obvious phenotypic differences under waterlogging stress conditions. Under waterlogging stress, both the Arabidopsis thaliana plants overexpressing LhSAMDC and the wild-type plants showed different degrees of stress responses, and the growth of the overexpressing plants was relatively better, showing a certain waterlogging tolerance ability.

[0123] Drought tolerance analysis of LhSAMDC overexpressing Arabidopsis thaliana

[0124] The results of drought stress of LhSAMDC overexpressing Arabidopsis thaliana are as Figure 8 shown. The results of the drought stress experiment show that significant phenotypic differences also occurred between the Arabidopsis thaliana plants overexpressing LhSAMDC and the wild-type plants under drought stress conditions. During the drought stress process, the growth of the wild-type plants was affected earlier, and severe wilting occurred in the leaves of some plants. The Arabidopsis thaliana plants overexpressing LhSAMDC showed strong drought tolerance under drought stress. The wilting degree of the leaves of these plants was relatively light, and the growth conditions were relatively better. It can be seen that overexpressing the LhSAMDC gene has a positive effect on improving the drought stress resistance of Arabidopsis thaliana.

[0125] Although the above embodiments have described the present invention in detail, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

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

1.

2. An amplification primer pair for the LhSAMDC gene of Liriodendron hybrids, characterized in that, The nucleotide sequence of the upstream primer of the said amplification primer pair is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

3.

3. A plant expression vector, characterized in that, The said plant expression vector contains the LhSAMDC gene of Liriodendron hybrids as claimed in claim 1.

4. The plant expression vector according to claim 3, characterized in that, The basic plasmid for constructing the said plant expression vector includes the pCAMBIA3301 vector.

5. The plant expression vector according to claim 3, wherein The nucleotide sequence of the upstream primer of the primer pair for constructing the said 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 adenosylmethionine decarboxylase, characterized in that, The adenosylmethionine decarboxylase of Liriodendron hybrids is encoded by the gene with the nucleotide sequence shown as SEQ ID NO.

1.

7. Use of the LhSAMDC gene of Liriodendron hybrids as claimed in claim 1, or the plant expression vector as claimed in any one of claims 3 to 5, or the adenosylmethionine decarboxylase of Liriodendron hybrids as claimed in claim 6 in increasing plant yield.

8. Use of the LhSAMDC gene of Liriodendron hybrids as claimed in claim 1, or the plant expression vector as claimed in any one of claims 3 to 5, or the adenosylmethionine decarboxylase of Liriodendron hybrids as claimed in claim 6 in creating plant germplasm; The said creating of plant germplasm includes any one or two or more of ① to ⑤ as follows: ① Creating plant germplasm with stronger growth potential; ② Creating plant germplasm with higher biomass; ③ Creating plant germplasm with more robust plant types; ④ Creating plant germplasm with more developed vegetative organs; ⑤ Creating plant germplasm with higher spad value.

9. Use of the LhSAMDC gene of Liriodendron hybrids as claimed in claim 1, or the plant expression vector as claimed in any one of claims 3 to 5, or the adenosylmethionine decarboxylase of Liriodendron hybrids as claimed in claim 6 in enhancing plant stress resistance.

10. The application according to claim 9, wherein The said enhancing of plant stress resistance includes enhancing the plant's ability to resist drought stress and / or enhancing the plant's ability to resist waterlogging stress.

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