Application of geranyl and geranyl pyrophosphate synthetase gene PmGGPPS14 of masson pine

Abstract

The invention discloses application of a masson pine geranyl and geranyl pyrophosphate synthetase gene PmGGPPS14, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the geranyl and geranyl pyrophosphate synthetase gene PmGGPPS14 of the masson pine is as shown in SEQ ID NO. 1. The amino acid sequence of the expression protein of the PmGGPPS14 is as shown in SEQ ID NO. 2. The invention finds that PmGGPPS14 can promote accumulation of photosynthetic pigments so as to promote plant development and increase leaf area. Overexpression of PmGGPPS14 can improve expression of partial genes in a GGPP downstream pathway, and plays an important role in synthesis of diterpenoid resin acid. The PmGGPPS14 gene provided by the invention is helpful for deep research on the functions of the PmGGPPS14 gene of the pinus massoniana, and provides a molecular means and basis for breeding work for improving synthesis of terpenoid compounds of the pinus massoniana.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the pine resin pyrophosphate synthase gene PmGGPPS14. Background Technology

[0002] Masson pine (Pinus massoniana Lamb.) is a perennial evergreen tree species belonging to the genus Pinus in the family Pinaceae. It is a pioneer species for afforestation of barren hills in southern my country, characterized by rapid growth, strong adaptability, and tolerance to drought and poor soil. The long, fine fibers of Masson pine wood make it ideal for papermaking, making it a major pulpwood species in my country. Furthermore, Masson pine is a major resin-producing species, accounting for over 70% of China's annual resin production. Its resin can be used to extract turpentine and rosin, with rosin being a major export commodity of my country's forestry industry, generating significant economic and ecological value. However, low resin yield is a significant factor restricting the development of the Masson pine resin industry. Therefore, selecting high-yielding varieties and establishing high-yielding plantations are effective measures to increase resin production. In addition, pine resin plays an important role in resisting biological stresses.

[0003] Conifers are rich in terpenoids, and their biosynthesis can be simply divided into three stages. The first stage involves the formation of isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP), common precursors of terpenoids. The second stage involves the synthesis of various direct precursors of terpenoids, such as geranyl diphosphate (GPP), farnesyldiphosphate (FPP), and geranylgeranyl diphosphate (GGPP). The third stage involves the formation and modification of terpenoids. GGPP in the second stage is a key precursor in the terpenoid metabolic pathway. GGPP can synthesize gibberellin (GA), abscisic acid (ABA), strigolactones (SL), pigments (plant-based side chains of carotenoids and chlorophyll), vitamins (plant-based side chains of vitamin A and vitamin E in carotene), and diterpenoid phytoalexins involved in plant defense mechanisms against pathogens. These metabolic branches compete for GGPP, forming a complex regulatory network. The distribution of GGPP within organisms plays a vital role in their growth and development.

[0004] Therefore, discovering and applying GGPP-related synthetic genes or proteins could provide an effective molecular approach and basis for targeted breeding work to improve the growth and development of Masson pine leaves and the synthesis of terpenoid compounds. Summary of the Invention

[0005] The purpose of this invention is to provide a pine resin-geranyl pyrophosphate synthase gene PmGGPPS14 that meets the requirements of use; another purpose of this invention is to provide an expression protein of the pine resin-geranyl pyrophosphate synthase gene PmGGPPS14; and yet another purpose of this invention is to provide the application of the above-mentioned pine resin-geranyl pyrophosphate synthase gene PmGGPPS14.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for cultivating plants with increased leaf area and / or higher diterpenoid content, comprising overexpressing or increasing the activity of the pine resinyl pyrophosphate synthase gene PmGGPPS14 in the plant, wherein the amino acid sequence of the protein encoded by the PmGGPPS14 gene is shown in SEQ ID NO.2.

[0008] Furthermore, the plant is a coniferous tree or a model plant, preferably pine, tobacco or Arabidopsis thaliana.

[0009] Furthermore, the method includes the following steps:

[0010] S1: Construct an expression vector containing the PmGGPPS14 gene;

[0011] S2: Transform the expression vector into plant receptor material;

[0012] S3: Screen and cultivate transgenic plants with increased leaf area and / or higher content of diterpenoids.

[0013] Furthermore, the nucleotide sequence of the PmGGPPS14 gene is shown in SEQ ID NO.1.

[0014] Furthermore, the method for constructing the expression vector is as follows: the PmGGPPS14 gene is linked to a plant functional promoter to form an expression cassette, and then inserted into the vector backbone.

[0015] Furthermore, the plant functional promoter is the cauliflower mosaic virus 35S promoter.

[0016] Furthermore, the transformation step is achieved through Agrobacterium-mediated transformation, gene gun transformation, or vacuum permeation transformation.

[0017] A method for cultivating a model plant with slow needle development and reduced diterpene resin acid content involves inhibiting or silencing the expression of the pine resinyl pyrophosphate synthase gene PmGGPPS14 in the plant, the amino acid sequence of the protein encoded by the PmGGPPS14 gene being shown in SEQ ID NO.2.

[0018] Furthermore, the method of inhibition or silencing is virus-induced gene silencing technology, and the plant is Masson pine.

[0019] The application of the model plants obtained by the method in screening or validating key genes in plant terpene metabolic pathways.

[0020] An isolated gene for pine resinyl pyrophosphate synthase, PmGGPPS14, has the nucleotide sequence shown in SEQ ID NO.1.

[0021] A geraniol geraniol pyrophosphate synthase protein encoded by the gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0022] A biological material containing the PmGGPPS14 gene, wherein the biological material is an expression cassette, a recombinant vector, a recombinant bacterium, or a transgenic cell line.

[0023] The application of the gene or protein in regulating plant responses to abiotic stresses, wherein the abiotic stresses include at least one of methyl jasmonate, salicylic acid, abscisic acid, gibberellin, ethephon, hydrogen peroxide, sodium chloride, osmotic stress, and drought.

[0024] Application of overexpression of the PmGGPPS14 gene in breeding Masson pine varieties with improved needle development and / or increased diterpene yield.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1) This application reveals and experimentally confirms for the first time two important plant physiological and biochemical functions of the PmGGPPS14 gene in *Pinus massoniana*: firstly, it directly promotes the growth and development of leaves (needles); secondly, it efficiently drives the biosynthesis of diterpenoid compounds. In *Pinus massoniana* seedlings with the PmGGPPS14 gene silenced, the needles not only showed obvious yellowing, but their length was also significantly reduced by 16.6%, and the overall plant height decreased by 21.5%, directly demonstrating the crucial role of this gene in the vegetative growth of conifers. More notably, gene silencing led to a sharp decrease in the content of various diterpenoid resin acids, including isopyric acid and sedaridin, with sedaridin decreasing by as much as 67%. These results indicate that positive regulation of PmGGPPS14 can simultaneously enhance both wood (biomass) and resin (diterpenoid resin) production in *Pinus massoniana*.

[0027] 2) This application demonstrates that by genetically transforming Arabidopsis thaliana, overexpressing the PmGGPPS14 gene in Arabidopsis thaliana produces a dominant phenotype with significantly larger leaf area and greener color, and completely recovers from the stunted growth and yellowing leaf mutations caused by endogenous GGPPS deficiency. This indicates that this gene can serve as an excellent genetic resource for improving crop plant architecture and increasing biomass yield.

[0028] 3) This invention analyzed the expression levels of the PmGGPPS14 gene in different tissues of *Pinus massoniana* and in leaves under different stresses using qRT-PCR technology. The results showed that PmGGPPS14 was highly expressed in the needles of *Pinus massoniana* and responded to abiotic stresses and hormone treatments. It broadly responded to various biotic and abiotic stress signals, including methyl jasmonate, salicylic acid, drought, and salt stress. The increased expression level of PmGGPPS14 is an important component of plant stress resistance. This indicates that utilizing this gene may not only increase plant biomass but also enhance the overall tolerance of forest trees or crops to complex environmental stresses, thereby improving planting stability.

[0029] In summary, this invention provides an effective molecular approach and gene resource for breeding work that improves leaf growth and development and terpene compound synthesis in plants such as Masson pine. Attached Figure Description

[0030] Figure 1 This is a 1.0% agarose gel electrophoresis image of total RNA from Pinus massoniana, with different lanes representing replicates;

[0031] Figure 2 This is a PCR result of a clone of the PmGGPPS14 gene with its complete open reading frame.

[0032] Figure 3 This is a graph showing the relative expression levels of the PmGGPPS14 gene in different tissues; in the graph, different letters indicate significant differences; FC: female cones; MC: male cones; C: cones; YS: young stems; OS: old stems; R: roots; N: needles;

[0033] Figure 4-1 This is a graph showing the relative expression levels of the PmGGPPS14 gene under different hormone treatments; different letters in the graph indicate significant differences (p < 0.05).

[0034] Figure 4-2 This is a graph showing the relative expression levels of the PmGGPPS14 gene under different environmental treatments; different letters in the graph indicate significant differences (p < 0.05).

[0035] Figure 5 This is a recombinant protein expression analysis and protein purification analysis; 1 indicates protein precipitation; 2 indicates supernatant; 3 indicates flow-through buffer; 4 indicates washing; 5 indicates purified protein.

[0036] Figure 6 This is a diagram showing the principle and results of the E. coli pigment complementation experiment; (A) The pAC-94N vector contains the carotenoid biosynthesis gene but does not contain GGPPS. When co-expressed with GGPPS, this pathway is supplemented, thereby producing β-carotene; (B) The three on the left are negative controls, pET28a and pAC94N co-transformed; the three on the right are the results of PmGGPPS14 and pAC94N co-transformation.

[0037] Figure 7-1 The VIGS results of PmGGPPS14 in Masson pine show the phenotypic characteristics of PmGGPPS14 gene-silenced Masson pine seedlings; scale bar is 2 cm.

[0038] Figure 7-2 The VIGS results of PmGGPPS14 in Masson pine show the needle phenotypic characteristics of PmGGPPS14 gene silencing. The scale bar is 2 cm.

[0039] Figure 7-3 The image shows the VIGS results of PmGGPPS14 in Masson pine; (C) represents the relative expression level of PmGGPPS14 in the experimental group (pTRV2::PmGGPPS14); (D) represents the diameter of needles in the control and experimental groups; (E) represents the length of needles in the control and experimental groups; and (F) represents the height of seedlings in the control and experimental groups.

[0040] Figure 7-4 The VIGS results of Masson pine PmGGPPS14 show the relative expression levels of downstream GGPPS genes in Masson pine pTRV2-PmGGPPS14 plants; p < 0.05; p < 0.01; (p < 0.001).

[0041] Figure 7-5 The VIGS results of PmGGPPS14 from Masson pine show the relative content of diterpene resin acids in Masson pine. p < 0.05; p < 0.01; (p < 0.001).

[0042] Figure 8-1 The graph shows the overexpression analysis of PmGGPPS14 in Arabidopsis thaliana, confirming the expression of PmGGPPS14 through PCR analysis.

[0043] Figure 8-2The diagram shows the differences in developmental rates among wild-type (WT), atggpps11, PmGGPPS14 overexpression (OE), and atggpps11 / PmGGPPS14 overexpression (OE) plants in Arabidopsis thaliana.

[0044] Figure 8-3 This is a phenotypic observation of 3-week-old Arabidopsis plants, which is an analysis of the overexpression of PmGGPPS14 in Arabidopsis.

[0045] Figure 8-4 (A) Leaf morphology phenotype of 3-week-old Arabidopsis plants, showing the overexpression analysis of PmGGPPS14 in Arabidopsis thaliana; (D) Comparison of leaf phenotypes between the control and experimental groups; (E) Quantitative comparison of leaf area between the control and experimental groups.

[0046] Figure 8-5 This is a graph analyzing the overexpression of PmGGPPS14 in Arabidopsis thaliana, showing the expression levels of downstream genes of GGPPS in wild-type Arabidopsis thaliana lines overexpressing PmGGPPS14 (OE). p < 0.05; p < 0.01; p < 0.001). Detailed Implementation

[0047] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. Unless otherwise specified, % in the following embodiments refers to mass percentage content. The quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged. The test method was Duncan's test.

[0048] Example 1: Obtaining the open reading frame of the PmGGPPS14 gene from Masson pine

[0049] Seven-year-old Masson pine needle tissue was collected, and total RNA was extracted using a plant total RNA extraction kit (TIANGEN). cDNA was synthesized using a reverse transcription kit (Vazyme), following the kit's instructions. Appropriate primers were designed for PCR. The PCR amplification products were detected by electrophoresis and recovered, then ligated into the cloning vector pEASY (TransGen) and transformed into competent E. coli T1 cells. After antibiotic screening and colony PCR identification, positive bacteria were sequenced, and the ligated cloning vector was named pEASY-PmGGPPS14. The ORF sequence of the protein encoded by the PmGGPPS14 gene was obtained. The coding region of PmGGPPS14 was translated into an amino acid sequence using ORFfinder.

[0050] The specific steps are as follows:

[0051] (1) Extraction of total RNA

[0052] RNA was extracted using the Novizan Plant Total RNA Extraction Kit. Before extraction, the mortar and pestle were wrapped in aluminum foil and baked in an oven at 180°C for approximately 4 hours. The sample was then ground into powder in liquid nitrogen, and RNA was extracted according to the kit instructions. The powder was stored at -80°C. The 1.0% agarose gel electrophoresis results of total RNA from *Pinus massoniana* leaves are shown below. Figure 1 As shown, the bands are relatively clear; the absorbance and OD of total RNA were measured. 260 / OD 280 The value is 2.32, OD 260 / OD 230 With a value of 2.09, it can be used for gene cloning.

[0053] (2) Acquisition of cDNA

[0054] Using the ClonExpress-II One Step Cloning Kit (Vazyme), cDNA was reverse transcribed from the extracted RNA.

[0055] The specific process is as follows:

[0056] 1) Prepare the following mixture in an RNase-free centrifuge tube: 1 μg Total RNA, 3 μL 5 x g DNA digester Mix, RNase-free ddH2O to 15 μL, mix thoroughly by pipetting, centrifuge to the bottom of the tube using a short-circuit centrifuge, and incubate at 42°C for 2 min. This process should be performed on ice.

[0057] 2) Add 5 μL of 4×Hifair III SuperMix plus to the mixture from the previous step, and gently mix by pipetting. Incubate at 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min.

[0058] (3) Cloning of the target gene

[0059] Based on the PmGGPPS14 gene sequence obtained from the transcriptome sequencing of *Pinus massoniana*, specific primers were designed using Primer 5.0 to clone the open reading frame of the PmGGPPS14 gene. Then, the gene was ligated into a vector, transformed into *E. coli*, and the target sequence was sequenced and analyzed to confirm it as the target gene. Figure 2 ).

[0060] The PmGGPPS14 ORF cloning primers are:

[0061] PmGGPPS14-ORF-F: 5'-ATGAGTATCATATTTTTCAATCAGTG-3';

[0062] PmGGPPS14-ORF-R: 5'-CTAATTCTGTCTGTGAGCAATG-3'.

[0063] The PCR reaction system (50 μL) consisted of: 2 μL Forward primer (10 μM / L), 2 μL Reverse primer (10 μM / L), 2 μL Template cDNA (100 ng / μL), 25 μL 2×Taq PCR MASTER Mix, and 19 μL ddH2O.

[0064] The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 58℃ annealing for 10 s, 72℃ extension for 15 s, 35 cycles, and 72℃ extension for 5 min.

[0065] (4) Ligation and Transformation: Add 4 µl of the product to a centrifuge tube, then add 1 µl of Blunt vector, mix, and incubate at room temperature for 15 min. After the reaction, add 50 µl of freshly thawed DH5α competent cells, mix well, and incubate on ice for 30 min. Place the ligation product in a 42℃ metal bath for 40 s, then immediately place it on ice for 2 min. Add 250 µl of LB liquid medium (without Kan) equilibrated to room temperature, incubate at 37℃ for 1 h at 200 rpm. Centrifuge at 10000 rpm for 30 s, discard 150 µl of the supernatant, and mix the remaining culture medium by pipetting. Spread the mixture onto Kan-resistant medium (the medium was pre-incubated at 37℃ for 1 h) and incubate overnight at 37℃. Positive clones were detected and sent to Qingke Biotechnology for sequencing.

[0066] The nucleotide sequence of PmGGPPS14 obtained by sequencing is shown in SEQ ID NO.1; the amino acid sequence is shown in SEQ ID NO.2.

[0067] Example 2: Expression levels of PmGGPPS14 in different tissues and under different stress treatments in Pinus massoniana.

[0068] In this embodiment, three 15-year-old clonal Masson pine plants were selected. Female and male cones, cones, young stems, old stems, roots, and needles were collected. RNA was extracted and reverse transcribed into cDNA, and the relative expression level of the PmGGPPS14 gene was analyzed. In addition, three two-year-old Masson pine seedlings of similar growth size were selected and subjected to ten abiotic stresses and hormone treatments, including 15% PEG. 6000Osmotic stress, mechanical damage, 10 mM H2O2, 100 µM methyl jasmonate (MeJA), 500 µM ethephon, and 1 mM salicylic acid (SA). The osmotic stress treatment involved immersing the plant roots in 15% PEG. 6000 In the liquid treatment, mechanical damage involved cutting each needle bundle in half its length. Other treatments involved spraying the liquid onto various parts of the plant. Needle samples were collected at 0h, 3h, 6h, 12h, and 24h, frozen in liquid nitrogen, and stored at -80℃. The untreated 0h samples served as controls. RNA was extracted and cDNA was synthesized from all samples to analyze the relative expression levels of the PmGGPPS14 gene. Each experiment had three biological and technical replicates, and Duncan's test was used to analyze the significance of differences in expression levels compared to the control group.

[0069] The specific steps are as follows:

[0070] (1) Acquisition of cDNA

[0071] The methods for RNA extraction and first-strand cDNA synthesis are the same as in Examples 1(1) and (2). The cDNA was diluted 10-fold after reverse transcription.

[0072] (2) qRT-PCR analysis

[0073] The *Pinus massoniana* TUA (KM496535.1) gene was selected as the internal control gene. PmGGPPS14-specific primers were designed using Primer 5.0 software, with amplification lengths of 100-200 bp. The relevant primer sequences are shown in Appendix 1. The relative expression levels of the PmGGPPS14 gene were analyzed according to the instructions of the ChamQ™ SYBR qPCRMaster Mix kit, and the significance of differences between each expression level and the control group was analyzed using Duncan's test.

[0074] The qRT-PCR primers are:

[0075] TUA-F: 5'-CAAACTTGGTCCCGTATCCTC-3';

[0076] TUA-R: 5'-CACAGAAAGCTGCTCATGGTAA-3';

[0077] qPCR-PmGGPPS14-F: 5'-GCATTGGGCTGATGTTCC-3';

[0078] qPCR-PmGGPPS14-R: 5'-ATCCCTTGCTTGCCTGTT-3';

[0079] The qRT-PCR reaction system (10 μL) is: 0.4 μL Forward primer (10 μM / L), 0.4 μL Reverseprimer (10 μM / L), 1 μL Template cDNA, 5 μL SYBR Green Mix, and 3.2 μL ddH2O.

[0080] The qRT-PCR reaction program was: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 30 s, for 40 cycles.

[0081] The expression levels of the PmGGPPS14 gene in different tissues of *Pinus massoniana* and in leaves under different treatments were analyzed by qRT-PCR. The results showed that PmGGPPS14 expression was highest in the needles of *Pinus massoniana* and responded to some abiotic stresses and hormone treatments. Figure 3 , Figure 4-1 and Figure 4-2 ).

[0082] Example 3: Prokaryotic expression of the PmGGPPS14 gene from Pinus massoniana and pigment complementation experiment in Escherichia coli

[0083] (1) Construction of pET28a-PmGGPPS14 vector

[0084] 1) Ligation and transformation of target fragment and vector

[0085] Using the pET28a vector as the prokaryotic expression vector, primers were designed based on the ORF sequences of the pET28a vector and the PmGGPPS14 gene. The restriction enzyme sites were BamHI and EcoRI. The pET28a vector was double-digested with these enzymes, and the target fragment containing the restriction sites was ligated to the vector to construct the recombinant plasmid pET28a-PmGGPPS14. The ligation product was transformed into *E. coli* DH5α competent cells. Positive clones were obtained through kanamycin resistance selection, and sequencing verified the correctness of the inserted sequence. The correctly sequenced recombinant plasmid was extracted and transformed into *E. coli* BL21(DE3) competent cells for heterologous expression.

[0086] The primers are:

[0087] pET28a-PmGGPPS14 F: 5'-cagcaaatgggtcgcggatccATGAGTATCATATTTTTCAATCAGTGTGTT-3';

[0088] pET28a-PmGGPPS14 R: 5'-ttgtcgacggagctcgaattcATTCTGTCTGTGAGCAATGTAATCAGC-3'.

[0089] 2) Recombinant plasmid was transformed into Escherichia coli BL21(DE3)

[0090] Take 50 µl of thawed BL21(DE3) competent cells from ice, add 5 µl of pET28a-PmGGPPS14 recombinant plasmid and mix gently. Incubate on ice for 25 minutes. Heat shock at 42°C for 45 seconds, then incubate on ice for 2 minutes. Add 700 µl of antibiotic-free LB liquid medium to a centrifuge tube, place in a shaker at 37°C and 200 rpm for 60 minutes to recover. Centrifuge at 5000 rpm for 1 minute to collect the bacteria, resuspend 200 µl of the supernatant by pipetting and spread onto LB solid medium containing Amp, and incubate overnight at 37°C upside down. Protein induction is performed after positive detection.

[0091] (2) Prokaryotic expression analysis and protein purification

[0092] 1) IPTG-induced protein expression

[0093] Pick a single clone of pET28a-PmGGPPS14 and add it to 5 ml of LB liquid medium containing 50 mg / L Kan. Incubate at 37°C and shake at 200 rpm until OD is reached. 600 =0.6, add 0.2 mmol / L IPTG, incubate at 18℃, 200 rpm for 20 h. Centrifuge all bacterial cultures at 6000 rpm for 5 min to collect the bacteria, and discard the supernatant. Resuspend the bacterial cells in 20 mL of lysis buffer (50 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole, pH 8.0) and 200 μL of 100 mM PMSF, and sonicate in an ice bath beaker (3 s sonication, 3 s interval, 300 W) until the bacterial culture is clear; centrifuge the bacterial culture at 4℃, 13,000 rpm for 10 min, and collect the supernatant.

[0094] 2) Detection of target protein by polyacrylamide gel electrophoresis (SDS-PAGE)

[0095] Gel preparation: Place the glass plate into the gel mold, prepare 10ml of 10% separating gel according to the instructions, immediately pour it into the mold, add distilled water until it is level with the glass, wait 30-40 minutes until the separating gel solidifies, pour off the distilled water and absorb the remaining water with filter paper, then prepare 4ml of 5% stacking gel, pour it into the mold, insert the comb, let it stand for about 40 minutes, and then put it into the electrophoresis tank to wait for gel running.

[0096] Prepare 5×Tris-glycine electrophoresis buffer: Add 0.125 mol / L Tris, 1.25 mol / L glycine, and 0.5% SDS to deionized water and store at room temperature.

[0097] After spotting the sample, set the voltage to 160V and the electrophoresis time to 50 minutes. Then, remove the gel, rinse it thoroughly, and stain it with Coomassie Brilliant Blue Ultrafast Staining Solution for 30-40 minutes. Finally, destain with water until the protein bands are clearly visible, and take photos for recording. Figure 5 ).

[0098] 3) Protein purification

[0099] Prepare the protein supernatant and pass it through a pre-packed Ni-NTA purification resin column. Wash away contaminating proteins with a buffer containing 10 mM imidazole (50 mM NaH2PO4, 500 mM NaCl, pH 8.0), and then elute the target protein using a gradient of elution buffer containing 500 mM imidazole. Collect the elution fractions and perform SDS-PAGE electrophoresis analysis on each eluent.

[0100] (3) Escherichia coli pigment complementation experiment

[0101] The catalytic activity of the PmGGPPS14 gene was evaluated using a bacterial pigment complementation assay.

[0102] 1) Plasmid transformation

[0103] The pAC-94N plasmid carrying multiple genes involved in β-carotene biosynthesis will be used. Figure 6 A) The recombinant vector pET28a-PmGGPPS14 was co-transformed into Escherichia coli BL21(DE3) competent cells, following the same steps as in (1) of Example 3.

[0104] 2) Carotene Expression and Observation

[0105] The transformed *E. coli* were first incubated overnight at 37°C. After colony formation, they were transferred to an 18°C ​​incubator for PmGGPPS14 protein expression. If the bacteria exhibited GGPPS enzyme catalytic activity, geranylgeranyl diphosphate (GGPP) would be generated, resulting in a distinct orange color and accumulation of β-carotene in the bacterial colonies. Results are as follows... Figure 6 As shown in B.

[0106] Example 4: Silencing of the PmGGPPS14 gene in Masson pine seedlings using VIGS technology.

[0107] The pTRV2-PmGGPPS14 recombinant vector was constructed, and Masson pine seedlings were treated by vacuum permeation. The differences between wild-type and silenced Masson pine were compared. The content of terpenoid metabolites and gene expression levels in downstream pathways of silenced Masson pine were measured to verify the function of PmGGPPS14.

[0108] (1) Construction of pTRV2-PmGGPPS14 vector

[0109] Using tobacco brittle virus pTRV2 as a vector, a specific fragment of the PmGGPPS14 gene was amplified by PCR and directionally inserted into the EcoRI and BamHI sites of pTRV2. After ligation, the fragment was transformed into *E. coli* DH5α competent cells, and positive clones were confirmed by colony PCR and sequencing. The recombinant plasmid was extracted and transformed into *Agrobacterium* GV3101. Primers are as follows:

[0110] pTRV2-PmGGPPS14-F: 5'-aaggttaccgaattctctagaGATGATTTGCCTTGTATGGACAAT-3'

[0111] pTRV2-PmGGPPS14-R: 5'-cgtgagctcggtaccggatccGTCAAGAATGTCATCAACAACCTGG-3'

[0112] Agrobacterium GV3101 containing the recombinant plasmid was inoculated into LB liquid medium, and 50 mg / L kanamycin and 25 mg / L rifampin were added. The medium was then incubated at 28°C with shaking until the OD reached its limit. 600 Centrifuge at approximately 1.0 rpm for 5 min at 6000 rpm, collect the bacterial cells, and resuspend them in a buffer solution (pH 5.6) containing 10 mM 2-morpholinoethanesulfonic acid, 200 μM acetylsuccinone, and 10 mM MgCl₂. Resuspend the cells containing pTRV1 in the same manner. Then, mix the pTRV1 and pTRV2-PmGGPPS14 resuspension 1:1 and bring to OD₂O₅. 600 =1. After resuspending the Agrobacterium tumefaciens culture, let it stand in the dark at room temperature for 3 hours, then add 0.1% (v / v) of Silwet L-77.

[0113] Six-month-old Masson pine seedlings were immersed in Agrobacterium-containing solution under vacuum at a pressure of 0.08 MPa for 5 minutes. Residual Agrobacterium was washed away with sterile water, and the seedlings were then planted in soil. The expression levels of target genes, PmCPS (ent-copalyl diphosphate synthase), PmGGR (geranylgeranyl reductase), PmKAO (ent-kaurenoic acid oxidase), PmPSY (phytoene synthase), PmLYCb (lycopene β-cyclase), PmSPS (solanesyl diphosphate synthase), and PmKS (ent-kaurene synthase), were assessed using RT-qPCR. The accumulation of terpenoid compounds in silenced Masson pine was also quantified. The content of diterpene resin acids was determined by UPLC-MS / MS.

[0114] These results indicate that silencing PmGGPPS14 may reduce endogenous GGPP levels in *Pinus massoniana*, leading to decreased expression of downstream genes and negatively impacting diterpenoid resin biosynthesis. Compared to the control group, plants transgenic with pTRV2-PmGGPPS14 exhibited a phenotype with lighter and smaller needles and slower growth. Figure 7-1 , Figure 7-2 Furthermore, the expression level of PmGGPPS14 in pTRV2-PmGGPPS14 plants was significantly lower than that in the control group (). Figure 7-3 C). The needle diameter of pTRV2-PmGGPPS14 plants was reduced, but the difference was not statistically significant (7-3D). The needle length was significantly reduced by 16.6% ( Figure 7-3 E), seedling height was significantly reduced by 21.5% ( Figure 7-3 F). Furthermore, qPCR analysis showed that the expression levels of several key genes in the downstream GGPP metabolic pathway were significantly downregulated (F). Figure 7-4CPS, KS, and KAO are key enzymes in the gibberellin biosynthesis pathway, which regulates plant growth and development by promoting cell division, cell elongation, seed germination, and flower and fruit development. GGR is a key enzyme in chlorophyll biosynthesis; PSY catalyzes the rate-limiting step in carotenoid biosynthesis; SPS is crucial for the biosynthesis of tocopherol and plastoquinone; and LYCb plays a key role in strigolactone biosynthesis. GGPP is a precursor to various terpenoid pathways, including gibberellins, chlorophyll, carotenoids, strigolactones, tocopherol, and plastoquinone. Low expression of PmGGPPS14 may lead to reduced GGPP levels, resulting in decreased levels of the corresponding metabolites, which may explain the delayed development and paler, smaller needles in pTRV2-PmGGPPS14 seedlings. In addition, the levels of several diterpenoid metabolites were significantly reduced, including isopimaric acid, sandaracopiric acid, dehydroabietic acid, 7-oxopimaric acid, and 7-oxoabietic acid. Sandaracopiric acid showed the largest decrease (67% reduction compared to the control group), followed by isopimaric acid (34.8% reduction), while 7-oxoabietic acid showed the smallest decrease (12% reduction). Figure 7-5 These results indicate that silencing PmGGPPS14 may reduce endogenous GGPP levels in Pinus massoniana, leading to decreased expression of downstream pathway genes and negatively impacting the biosynthesis of diterpene resin acids.

[0115] Example 5 Identification and Screening of PmGGPPS14 Transgenic Arabidopsis

[0116] A 35S:PmGGPPS14 expression vector was constructed and transformed into wild-type Arabidopsis thaliana. The differences between T3 generation transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana were compared to observe whether the PmGGPPS14 gene of Pinus massoniana can promote the production of terpenoids and to analyze the function of the PmGGPPS14 gene of Pinus massoniana.

[0117] (1) Construction of pCAMBIA-PmGGPPS14-1302 vector

[0118] The Escherichia coli strain used in this invention is DH5α; the expression vector is pCAMBIA-1302 (preserved in the laboratory of Nanjing Forestry University); the restriction endonuclease and ligase were purchased from Vazyme.

[0119] The specific steps are as follows:

[0120] NcoI and BglII restriction sites were added upstream and downstream of the fragment by PCR, and the fragments were separated by 1.2% agarose gel electrophoresis. The digested products were recovered and purified using the FastPure® Gel DNA Extraction Mini Kit and dissolved in 20 μL of Elution Buffer.

[0121] 1302-PmGGPPS14-F: 5'-acgggggactcttgaccatggATGAGTATCATATTTTTCAATCAGTGTGTT-3';

[0122] 1302-PmGGPPS14-R: 5'-tctcctttactagtcagatctATTCTGTCTGTGAGCAATGTAATCAGC-3'.

[0123] 2) After the PCR product is correctly sequenced, the vector is digested with enzymes, ligated, transformed, positively tested, and then sent for testing. Plasmids with correct sequencing are transformed into Agrobacterium for Arabidopsis transformation. The enzyme digestion, ligation, and transformation system is as follows:

[0124] The double digestion system (50 µl) for the expression vector was: 1 µg pCAMBIA-1302 plasmid, 1 µl QuickCut NcoI, 1 µl QuickCut BglⅡ, 5 µl 10×QuickCut Buffer, and ddH2O up to 50 µl.

[0125] The double digestion program for the expression vector was: 37℃ for 15 min; 85℃ for 20 min.

[0126] The ligation system is as follows (20µl): linearized vector with 0.02 × cloning vector base pairs (ng); insert fragment with 0.04 × insert fragment base pairs (ng); 4µl 5× CE II Buffer; 2µl Exnase II; add ddH2O to 20µl.

[0127] The successfully constructed recombinant vector plasmid was transformed into Agrobacterium competent cells GV3101 using the following transformation method:

[0128] Add 10 μl of recombinant plasmid to 100 μl of Agrobacterium competent cells in an ice-water mixture, gently tap the bottom of the tube to mix, and then place the tube on ice, in liquid nitrogen, in a 37°C water bath, and in an ice bath for 5 min each.

[0129] Add 700 μl of LB liquid culture medium and incubate at 28°C in a shaker for 2.5 h.

[0130] Take approximately 100 μl of the supernatant and spread it onto a solution containing 50 mg·L⁻¹ -1 Kan, 25 mg·L -1 Incubate on LB plates of Rif, upside down in a 28°C incubator for 2 days.

[0131] Select healthy monoclonal colonies for PCR detection. Expand the culture of positive monoclonal colonies, add an equal volume of 50% glycerol to the bacterial culture, and store at -80℃ for subsequent experiments.

[0132] (2) Cultivation of Arabidopsis thaliana

[0133] 1) Disinfection of Arabidopsis thaliana seeds: Take an appropriate amount of wild-type Arabidopsis thaliana seeds and put them into a sterilized EP tube. Add an appropriate amount of 75% ethanol and disinfect for 10 minutes, then aspirate the seeds. Add an appropriate amount of 5% sodium hypochlorite and disinfect for 10 minutes, then aspirate the seeds. Add an equal amount of deionized water, shake and wash the seeds, then aspirate the seeds and put them into a new EP tube. Repeat the washing process four times.

[0134] 2) Arabidopsis thaliana cultivation: After sterilization, the seeds were placed in a suspension and evenly sown into 1 / 2 MS medium using a pipette; the medium was sealed and placed in a 4℃ refrigerator for vernalization for 3 days; the vernalized medium was then placed in a 23℃ constant temperature incubator for 7 days; when the Arabidopsis thaliana seedlings had grown 2 true leaves, they were transplanted into the prepared substrate and placed in a light incubator. The light incubator was set with the following parameters: 23℃ constant temperature, light intensity 5LS, humidity 75%, and light duration 16h / d; the seeds were used for transformation before the Arabidopsis thaliana entered its full flowering period.

[0135] (3) Transformation of Agrobacterium

[0136] The recombinant vector was transformed into competent Agrobacterium GV3101 cells. Single colonies were picked and inoculated into LB broth, and cultured at 28°C with shaking for 2 days. Full-length primers were used for colony PCR to screen for positive clones, which were then stored at 4°C for later use. Healthy Arabidopsis thaliana plants were allowed to grow until flowering. Positive clones detected by PCR were cultured to OD200. 600 When the value was 0.8, Arabidopsis flower organ immersion transformation was performed.

[0137] The specific steps are as follows:

[0138] Centrifuge the bacterial culture at 5000 rpm for 5 min, collect the bacterial cells, and suspend them in a 5% sucrose solution;

[0139] 1) Before soaking, add SilwetL-77 at a concentration of 0.05% (500μL / L) and shake to remove foam;

[0140] 2) Soak the aboveground parts of Arabidopsis thaliana in Agrobacterium suspension for 30 seconds, gently shaking during the process;

[0141] 3) Lay the soaked Arabidopsis thaliana flat on a tray, cover with plastic wrap to keep it moist, and seal with aluminum foil to protect it from light for 24 hours;

[0142] 4) Remove the aluminum foil and cultivate under normal conditions. Stop watering when the seeds are mature.

[0143] 5) Collect Arabidopsis thaliana seeds as T1 generation seeds, dry them at 37℃ for one week, and then store them.

[0144] (4) Screening of positive Arabidopsis T1 generation plants transgenic with PmGGPPS14 gene and obtaining T3 generation transgenic seedlings

[0145] 1) PCR detection of T1 generation transgenic Arabidopsis thaliana

[0146] After disinfection, the seeds of Arabidopsis thaliana transformed by Agrobacterium were sown in 1 / 2 MS medium containing hygromycin (25 mg / L). Germination began after 3 days of vernalization, and the seedlings were then transferred to a light-controlled culture room for observation of plant growth. Due to the influence of kanamycin, the seedlings of non-transgenic and control plants gradually yellowed and withered, while the transgenic seedlings grew normally. After approximately 10 days, all transgenic and control plants yellowed and died. Ten plants selected after hygromycin treatment were collected. DNA was extracted from these 10 transgenic Arabidopsis lines using a kit, and PCR detection was performed. Bands (target fragment length 1158 bp) were obtained from T1-1 to T1-10. Figure 8-1 This means that the gene PmGGPPS14 was successfully transferred.

[0147] T1 generation transgenic Arabidopsis thaliana PCR detection upstream primer:

[0148] 5'-ATGGGTTACAGTGGCATGGT-3';

[0149] T1 generation transgenic Arabidopsis thaliana PCR detection downstream primers:

[0150] 5'-GCACAAGCAGTTGGCATCGC-3'.

[0151] The PCR system (50 μL) is: 19 μL ddH2O, 25 μL 2×Taq PCR MASTER Mix, 2 μL Primer-F (10 μM), 2 μL Primer-R (10 μM), and 2 μL DNA.

[0152] The PCR program was as follows: 95℃ for 5 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min; store at 4℃.

[0153] 2) Obtaining T3 generation transgenic Arabidopsis thaliana

[0154] After disinfecting the T1 generation seeds (using the same method as in Example 6(4)1), they were sown in 1 / 2 MS medium containing kanamycin (50 mg / L). After vernalization for 3 days, they were placed in a suitable environment for cultivation (under the same cultivation conditions as above). Then, they were transferred to substrate soil and the seeds were collected as T2 generation transgenic Arabidopsis seeds. The obtained T2 generation transgenic Arabidopsis seeds were cultured in accordance with the above steps. T3 generation homozygous transgenic Arabidopsis seeds and plants were screened, transferred to substrate soil, and placed in a light incubator for cultivation (setting parameters: constant temperature and light at 23℃, light intensity of 5 LS, humidity of 75%, and light duration of 16 h / d).

[0155] (6) Expression analysis of genes related to terpene synthesis in transgenic Arabidopsis thaliana seeds

[0156] After overexpression of PmGGPPS14 in Arabidopsis thaliana, the three transgenic lines with the highest expression levels were selected to evaluate the functional effects of PmGGPPS14 on leaf development and the expression of downstream genes in the terpene biosynthesis pathway. Figure 8-1 The atggpps11 mutant exhibits pale leaf color and delayed development due to reduced AtGGPPS11 levels, consistent with previously reported findings (Ruiz-Sola MÁ, Coman D, Beck G, Barja MV, Colinas M, Graf A, Welsch R, Rütimann P, Büttmann P, Bigler L, Gruissem W, Rodríguez-Concepción M, Vranová E. 2016. Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is ahub isozyme required for the production of most photosynthesis-related isoprenoids. New Phytologist 209(1): 252-264.). Notably, overexpression of PmGGPPS14 can compensate for this defective phenotype ( Figure 8-2 , 8-3 Furthermore, overexpression of PmGGPPS14 in the wild type resulted in larger, greener leaves and accelerated development. The leaf area of ​​the PmGGPPS14-OE line was significantly larger than that of the wild type and the atggpps11 mutant plants. Figure 8-4 D, E).

[0157] In addition, qPCR analysis was performed to analyze the expression of other genes in the terpene synthesis pathway, such as 1-deoxy-D-xyulose 5-phosphate reductase (PmSPS), 4-hydroxy-3-methyl-2-phenyl diphosphate reductase (PmCPS), phytopenic acid synthase (PmPSY), terpene synthase (PmKAO), 3-hydroxy-3-methylglutaryl-CoA reductase (PmKS), mevalonate kinase (PmLYCb), and PmGGR. Arabidopsis thaliana Actin2 was used as an internal reference gene. Specific primers for each gene were designed using Primer 5.0, and the amplification length of each target fragment was 150-200 bp. The methods and procedures were the same as in Example 2 (2).

[0158] The primer sequences are as follows:

[0159] AtActin-F: 5'-GGTAACATTGTGCTCAGTGGTGG-3';

[0160] AtActin-R: 5'-AACGACCTTAATCTTCATGCTGC-3';

[0161] AtGGPPS11-qpcrF:5'-GGAAAACCGACCAACCACA-3';

[0162] AtGGPPS11-qpcrR: 5'-CGACTTGACCCGCCACTAA-3';

[0163] AtKao2-qpcrF:5'-AAGATGGGAGGGATACACAC-3';

[0164] AtKao2-qpcrR: 5'-AGGAGGAAATGATGAAGAAAA-3';

[0165] AtGGR-qpcrF: 5'-ATTTCAGGAGAGGATTAGGATTC-3';

[0166] AtGGR-qpcrR: 5'-CTACATGGTCGCACTTAGGG-3';

[0167] AtGA3ox2-qpcrF: 5'-CGCCATACGACTAAACCACTAC-3';

[0168] AtGA3ox2-qpcrR: 5'-GACCACGAGGACACCAGGA-3';

[0169] AtGA3ox1-qpcrF:5'-TGGGGTCAGCGAAGAAGAC-3';

[0170] AtGA3ox1-qpcrR:5'-GGTACAGAATGGTTAGGAGGGT-3';

[0171] AtCPS-qpcrF:5'-GAGAAACCTAACGGACGGGG-3';

[0172] AtCPS-qpcrR:5'-GCAAGGGTATTGATGAGACGA-3';

[0173] AtLYCb-qpcrF: 5'-AACGGCGTAGACAGAGGGAGR-3';

[0174] AtLYCb-qpcrF: 5'-GAAGCGTGTGAGAAGAGCGA-3';

[0175] AtSPS2-qpcrF:5'-GCGCTAGAGAATGAGCCAAG-3';

[0176] AtSPS2-qpcr:5'-CAAGAGCCGATCTGAAAACCA-3';

[0177] AtSPS1-qpcrF:5'-TTTATCGATTGTTGGTGCGG-3';

[0178] AtSPS1-qpcrR: 5'-TTCTTGTTGTTAGTTCCTTTAGGC-3'.

[0179] The results showed that in PmGGPPS14-OE plants, the expression levels of key downstream genes in the GGPP metabolic pathway were significantly upregulated, including AtGGPPS11, AtGGR, AtSPS1, AtSPS2, and AtKAO2. Figure 8-5 These results indicate that PmGGPPS14 is involved in the biosynthesis of terpenoids and the regulation of photosynthetic pigment accumulation, thereby affecting leaf development.

[0180] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for cultivating plants with increased leaf area and / or higher content of diterpenoid compounds, characterized in that, Overexpression or enhancement of the activity of the pine resinyl pyrophosphate synthase gene PmGGPPS14 in plants, wherein the amino acid sequence of the protein encoded by the PmGGPPS14 gene is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, The plant is a coniferous tree or a model plant, preferably a Masson pine, tobacco, or Arabidopsis thaliana.

3. The method according to claim 1 or 2, characterized in that, Includes the following steps: S1: Construct an expression vector containing the PmGGPPS14 gene; S2: Transform the expression vector into plant receptor material; S3: Screen and cultivate transgenic plants with increased leaf area and / or higher content of diterpenoids.

4. A method for cultivating a model plant with slow needle development and reduced diterpene resin acid content, characterized in that, The expression of the geranium pyrophosphate synthase gene PmGGPPS14 in the plant was inhibited or silenced. The amino acid sequence of the protein encoded by the PmGGPPS14 gene is shown in SEQ ID NO.

2. The method of inhibition or silencing is virus-induced gene silencing technology. The plant is Pinus massoniana.

5. The application of the model plant obtained by the method of claim 4 in screening or validating key genes in plant terpene metabolic pathways.

6. An isolated pine resin-based gerany-based pyrophosphate synthase gene, PmGGPPS14, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

7. The protein encoded by the gene according to claim 6, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

8. A biomaterial containing the PmGGPPS14 gene as described in claim 6, characterized in that, The biological material is an expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line.

9. The use of the gene of claim 6 or the protein of claim 7 in regulating plant responses to abiotic stress, characterized in that, The abiotic stresses include at least one of methyl jasmonate, salicylic acid, abscisic acid, gibberellin, ethephon, hydrogen peroxide, sodium chloride, osmotic stress, and drought.

10. The application of overexpression of the PmGGPPS14 gene as described in claim 6 in the breeding of Masson pine varieties with improved needle development and / or increased diterpene yield.

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