Use of gras11 in modulating plant terpenoid synthesis and / or glandular hair development

Abstract

The present application relates to the field of genetic engineering technology, and particularly relates to the application of GRAS11 in regulating plant terpenoid synthesis and / or glandular hair development. Solanum lycopersicum The present application firstly amplifies a transcription factor GRAS11 in tomato (Lycopersicon esculentum Mill.) which is specifically expressed in leaf and stem glandular hair. Overexpression of GRAS11 can significantly enhance the expression level of terpenoid synthase and upstream precursor synthesis pathway genes in leaf and other tissues, thereby increasing the yield of terpenoids and increasing the volume of glandular hair. The present application provides favorable values for research and application in genetic engineering improvement of crop nutritional quality, enhancement of crop pest and disease resistance, etc.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of GRAS11 in regulating the synthesis of plant terpenoids and / or the development of glandular trichomes. Background Technology

[0002] Terpenes are the largest family of plant secondary metabolites, with an estimated 80,000 structures. Based on the number of five-carbon units in their core skeleton, terpenes are classified into hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, disesquiterpenes, and triterpenes. Terpenes are widely found in plants and play important roles in resisting pathogens and phytophagous insects, as well as attracting insect pollinators. Some terpenes are also used as pharmaceuticals, insecticides, and fragrances, possessing significant economic value. Enhancing the biosynthetic pathways of endogenous terpenes in plants and increasing compound yield is of great importance for improving crop quality and enhancing crop resistance to pests and diseases.

[0003] In plant cells, the precursor compounds isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), synthesized via the mevalonate pathway (MVA) in the cytoplasm and the 2-C-methyl-D-erythritol-4-phosphate pathway (MEP) in the plastids, are converted into geranyl diphosphate (GPP, C 10 ), farnesyl diphosphate(FPP,C 15 ) or geranylgeranyl diphosphate (GGPP,C 20 Tomatoes synthesize and store a variety of volatile monoterpenes and sesquiterpenes in the glandular trichomes on the surface of leaves, stems, flowers, and young fruits. The tomato genome contains 52 TPS-encoding genes, of which the functions of 34 genes have been identified, including one isopentenyl synthase, 10 monoterpenes synthases, 17 sesquiterpenes synthases, and 6 diterpenes synthases. TPS5 (linalool synthase), TPS12 (caryophyllene synthase), and TPS20 (phellandrene synthase) are the main terpenes synthases catalyzing the synthesis of monoterpenes and sesquiterpenes in tomato leaves.

[0004] Transcription factors play a crucial role in regulating terpene metabolism in plants. A small number of transcription factors have been identified in tomato, such as the zinc finger protein EOT1, specifically expressed in glandular trichomes, which binds to the promoter of TPS5 to regulate its expression; WRKY73, which can activate the transcription of TPS3, TPS5, and TPS7 genes; and the bHLH transcription factor MYC1, which can promote the synthesis of monoterpenes in leaf and stem glandular trichomes and inhibit the biosynthesis of sesquiterpenes. The regulatory effects of these transcription factors on terpene metabolism are relatively weak, primarily through the regulation of TPS gene expression. Their regulatory roles in upstream precursor synthesis pathways (MEP and MVA pathways) remain unclear, and no applications of these transcription factors in regulating the synthesis and metabolism of plant terpenes using genetic engineering techniques have been reported.

[0005] GRAS family proteins are a class of plant-specific transcription factors. They typically consist of 360-850 amino acid residues and contain domains such as LHR I, LHR II, VHIID, PFYRE, and SAW. GRAS proteins are widely involved in the regulation of plant growth and development, including gibberellin signaling, root and shoot differentiation, light signaling, male gamete development, starch biosynthesis, root nodule formation, and drought and salt stress. The tomato genome contains 54 GRAS proteins, divided into 13 subfamilies. Besides DELLA (also called PROCERA), which is involved in gibberellin signaling, some GRAS genes have been found to be involved in tomato fruit development and interactions with environmental factors. Currently, there are no reports of GRAS transcription factors regulating plant terpene synthesis and metabolism or glandular trichome development. Summary of the Invention

[0006] The main objective of this invention is to provide the application of GRAS11 in regulating the synthesis of terpenoids and / or the development of glandular trichomes in plants. This invention discovers that GRAS11, a transcription factor specifically expressed in tomato glandular trichomes, is a key regulator of terpenoid biosynthesis. Overexpression or inhibition of GRAS11 can significantly increase or decrease the content of terpenoids.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides the application of GRAS11 in regulating the synthesis of plant terpenoids and / or the development of glandular trichomes.

[0009] Furthermore, the nucleotide sequence encoding the GRAS11 gene is shown in SEQ ID NO.1; the amino acid sequence of GRAS11 is shown in SEQ ID NO.2.

[0010] This invention provides the use of a product that promotes high or overexpression of GRAS11 in at least one of the following (a)-(e):

[0011] (a) Application in increasing the content of terpenoids in plants;

[0012] (b) Application in improving the expression level of TPS genes in plants;

[0013] (c) Application in improving gene expression levels in the MVA pathway;

[0014] (d) Application in improving gene expression levels in the MEP pathway;

[0015] (e) Application in promoting the development of plant glandular hairs and increasing the volume of plant leaf glandular hairs.

[0016] Furthermore, the product includes a methyljasmonic acid inducer; recombinant plasmids used for overexpressing GRAS11; Agrobacterium; and reagents used for plant transgenics.

[0017] Furthermore, the recombinant plasmid is pCAMBIA-GRAS11, which is prepared by ligating the amplified GRAS11 fragment with the plasmid pCAMBIA2301.

[0018] This invention provides the use of products that inhibit or silence GRAS11 expression in at least one of the following (a')-(d'):

[0019] (a') Application in inhibiting the synthesis of plant terpenoids;

[0020] (b') Application in suppressing TPS gene expression levels in plants;

[0021] (c') Application in inhibiting gene expression levels in the MVA pathway;

[0022] (d') Application in inhibiting gene expression levels in the MEP pathway.

[0023] Furthermore, products that inhibit GRAS11 expression include: gibberellin inhibitors; reagents and plasmids that induce frameshift mutations in the GRAS11 gene.

[0024] Furthermore, the product for silencing GRAS11 expression is Agrobacterium carrying the pTRV1 and pTRV2-GRAS11 plasmids; the pTRV2-GRAS11 plasmid is prepared by ligating the amplified GRAS11 fragment with the pTRV2 plasmid.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention first amplifies the transcription factor GRAS11 in tomato (Solanum lycopersicum L.), which is specifically expressed in the glandular trichomes of leaves and stems. Overexpression of GRAS11 significantly enhances the expression levels of terpene synthases and upstream precursor synthesis pathway genes in leaves and other tissues, thereby increasing the yield of terpenoids and promoting glandular trichome development and volume. This invention provides valuable insights for research and applications in genetic engineering to improve crop nutritional quality and enhance crop resistance to pests and diseases. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 GRAS11 sequence characteristics. (A) Phylogenetic analysis of GRAS family proteins in tomato and Arabidopsis thaliana; (B) Sequence alignment of GRAS11 with Arabidopsis thaliana SCL3. The red lines represent the LRI, VHIID, LRII, PFYRE, and SAW domains.

[0029] Figure 2 Expression characteristics of GRAS11 in tomato. (A) Expression characteristics of GRAS11 in various tomato tissues (roots, hypocotyls, and cotyledons from 10-day-old seedlings; leaves and stems from 6-week-old plants; flowers and fruits at different developmental stages from 3-month-old plants); (B) Expression levels of GRAS11 in tomato glandular trichomes and leaves; (C) Methyljasmonic acid can induce GRAS11 expression; (D) Gibberellin inhibits GRAS11 expression. ** indicates statistically significant differences (t-test, p<0.01).

[0030] Figure 3 GRAS11 silencing affects terpene metabolism in tomato (variety: LA4024). (AB) GC-MS analysis showed that the content of volatile terpenes was significantly reduced in GRAS11-silenced plants; (C) β-phellandrene content was significantly reduced in VIGS plants; (D) β-caryophyllene content was significantly reduced in VIGS plants; (E) α-humulene content was significantly reduced in VIGS plants; (F) GRAS11 expression level was significantly reduced in VIGS plants; (G) TPS20 expression level was significantly reduced in VIGS plants; (H) TPS12 expression level was significantly reduced in VIGS plants. * indicates statistically significant difference (t-test, p<0.05). ** indicates extremely statistically significant difference (t-test, p<0.01).

[0031] Figure 4 GRAS11 silencing affects terpene metabolism in tomato (variety: Micro-Tom). (AB) GC-MS analysis showed that the content of volatile terpenes was significantly reduced in GRAS11-silenced plants; (C) β-ocimene content was significantly reduced in VIGS plants; (D) (-)-β-pinene content was significantly reduced in VIGS plants; (E) β-caryophyllene content was significantly reduced in VIGS plants; (F) α-humulene content was significantly reduced in VIGS plants; (G) GRAS11 expression level was significantly reduced in VIGS plants; (H) TPS5 expression level was significantly reduced in VIGS plants; (I) TPS12 expression level was significantly reduced in VIGS plants. * indicates statistically significant difference (t-test, p<0.05); ** indicates extremely significant difference (t-test, p<0.01).

[0032] Figure 5 Overexpression of GRAS11 enhances terpene synthesis and metabolism. (A) Increased β-ocimene content in transgenic lines; (B) Increased (-)-β-pinene content in transgenic lines; (C) Increased β-(E)-caryophyllene content in transgenic lines; (D) Increased α-humulene content in transgenic lines; (E) Increased TPS5 gene expression level in transgenic lines; (F) Increased TPS12 gene expression level in transgenic lines. * indicates statistically significant differences (t-test, p<0.05);

[0033] ** indicates a highly significant statistical difference (t-test, p<0.01).

[0034] Figure 6 Overexpression of GRAS11 increases the expression levels of genes in the MEP and MVA pathways.

[0035] ** indicates a highly significant statistical difference (t-test, p<0.01).

[0036] Figure 7 Analysis of GRAS11 mutants. (A)gras11-1 - / - and gras11-2 - / - The GRAS11 gene is mutated in the mutant; (B)gras11-1 - / - and gras11-2 - / - The β-ocimene content in the leaves of the mutant was significantly reduced; (C)gras11-1 - / - and gras11-2 - / -The content of (-)-β-pinene in the leaves of the mutant was significantly reduced; (D)gras11-1 - / - and gras11-2 - / - The content of β-(E)-caryophyllene in the leaves of the mutant was significantly reduced; (E)gras11-1 - / - and gras11-2 - / - The α-humulene content in the leaves of the mutant was significantly reduced; (F)gras11-1 - / - and gras11-2 - / - The expression level of TPS5 in the leaves of the mutant was significantly reduced; (G)gras11-1 - / - and gras11-2 - / - The expression level of TPS12 in the mutant leaves was significantly reduced. * indicates statistically significant difference (t-test, p<0.05); ** indicates extremely significant difference (t-test, p<0.01).

[0037] Figure 8 Mutations in the GRAS11 gene attenuate gene expression levels in the MEP and MVA pathways. * indicates statistically significant differences (t-test, p<0.05); ** indicates extremely significant differences (t-test, p<0.01).

[0038] Figure 9 Overexpression of GRAS11 increased glandular trichome volume (diameter). (A) Glandular trichomes on transgenic tomato leaves and stems; (B) Statistical results of glandular trichome diameter in tomato leaves; (C) Statistical results of glandular trichome diameter in tomato stems. a and b indicate that, according to One-Way ANOVA and Tukey HSD statistical analysis, they can be divided into two groups with extremely significant differences (p<0.01). Detailed Implementation

[0039] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0041] Through in-depth research, the inventors discovered that GRAS11, a transcription factor specifically expressed in tomato glandular trichomes, is a key regulator of terpene biosynthesis. Overexpression of GRAS11 in tomatoes can significantly increase the expression levels of MVA, MEP, and TPS genes, thereby increasing the content of volatile monoterpenes and sesquiterpenes, and increasing the diameter (volume) of glandular trichomes.

[0042] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd edition). rd As described in Cold Spring Harbor Laboratory (2001) and Plant Molecular Biology-A Laboratory Manual (Springer-Verlag, 1997), or as recommended by the manufacturer.

[0043] Example 1: Isolation and Sequence Analysis of the GRAS11 Gene Fragment

[0044] Tomato leaf material was thoroughly ground in liquid nitrogen and transferred to a 2ml centrifuge tube. 1ml of Trizol (Invitrogen, Cat. 15596-018) was added and mixed well. The mixture was incubated at room temperature for 5 minutes. The mixture was centrifuged at 12000 rpm for 10 minutes, and the precipitate was discarded. 0.2ml of chloroform was added to the supernatant, mixed well, and centrifuged at 12000 rpm for 10 minutes. 0.5ml of isopropanol was added to the supernatant to precipitate RNA. The mixture was centrifuged at 12000 rpm for 10 minutes. The precipitate was washed with 75% ethanol, dried under vacuum, and dissolved in 50μl of water.

[0045] cDNA was synthesized using an RNA PCR system (TaKaRa, Cat.DRR019A). The reaction solution consisted of: 10×RT buffer: 1 μl; dNTP: 1 μl; MgCl2: 2 μl; Oligo dT: 0.5 μl; RNA: 1 μl; RNase inhibitor: 0.5 μl; AMV reverse transcriptase: 0.5 μl; H2O: 4 μl. The reaction conditions were: 42℃ for 30 min, followed by 85℃ for 5 min.

[0046] use PCR amplification was performed using FastPfu DNA Polymerase (Cat.AP221-01). The forward primer was 5'-ATGTTACAAGATGATGGTTCTTCA-3', and the reverse primer was 5'-TCACTTCCTACATCGCCAAG-3'. PCR conditions were: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 60 s, 35 cycles; extension at 72℃ for 5 min. PCR products were detected by 1% agarose gel electrophoresis. Agarose gel fragment of approximately 1100 bp was excised and analyzed. The QuickGel Extraction Kit (Cat.EG101-01) recovers the target gene fragment and ligates it into a vector. -In Blunt cloning vector (Full Gold, Cat.CB501-01).

[0047] A single colony of *E. coli* DH5α was inoculated into 2 ml of LB medium and incubated overnight at 37°C and 200 rpm. The bacterial culture was then inoculated at a 1:50 ratio into 50 ml of LB medium and incubated at 20°C and 200 rpm until OD500 was reached. 600 =0.5, incubate on ice for 10 min, centrifuge at 5000 rpm for 10 min at 4℃, resuspend in 4 ml transformation buffer (10 mM PIPES, 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl), and aliquot into 100 μl. Add the ligation product to competent E. coli cells, mix well, incubate on ice for 30 min, heat shock in a 42℃ water bath for 90 s, place on ice for 2-3 min, add 1 ml LB medium, incubate at 37℃ for 1 h, centrifuge the bacterial suspension at 4000 rpm for 1 min, discard 0.8 ml of supernatant, resuspend the bacterial cells, spread on LB agar plates containing ampicillin (Amp, 100 mg / L), and incubate upside down in the dark at 37℃ for 12-16 h. Pick single colonies for PCR screening, extract plasmids from positive single clones and sequence them. The resulting recombinant plasmid was named pEASY-GRAS11.

[0048] Sequencing analysis revealed the GRAS11 gene of tomato, whose nucleotide sequence is shown in SEQ ID NO.1. It contains 1407 bases and encodes a protein of 468 amino acids, the specific amino acid sequence of which is shown in SEQ ID NO.2. Phylogenetic analysis showed that GRAS11 belongs to the Scarecrow-like subfamily and is most closely related to the sequence of Arabidopsis thaliana AtSCL3 (At1g50420). Figure 1A), and segment C contains leucine-rich region I (LRI), VHIID, leucine-rich region II (LRII), PFYRE, and SAW domains. Figure 1 B).

[0049] Example 2: Vector Construction

[0050] Using pEASY-GRAS11 as a template, the gene was amplified by PCR using the forward primer VIGS-GRAS11-F-BamHI (5'-GGATCCTTGATGTTGAGAAACTTCGC-3') and the reverse primer VIGS-GRAS11-R-XbaI (5'-TCTAGATGTCGAATCTTTGGAACCAC-3'). PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s; 30 cycles. The target gene fragment was recovered after agarose gel electrophoresis. The gene fragment and the vector pTRV2 were double-digested with BamHI and XbaI, respectively. The digestion products were recovered and mixed at a 1:1 molar ratio of vector to gene fragment, and ligated at 16℃ for 30 min (Takara, DNA Ligation Kit Ver. 2.1). The ligation product was transformed into *E. coli* DH5α and plated on LB agar plates containing 50 mg / L kanamycin (Kan). The plates were incubated in the dark at 37°C for 12–16 h. Single colonies were picked for PCR clone selection. Plasmids were extracted from positive single colonies and sequenced. The obtained plasmid was named pTRV2-GRAS11. This plasmid was used to silence GRAS11 expression.

[0051] Using pEASY-GRAS11 as a template, the gene was amplified by PCR using the forward primer GRAS11-F-BamHI (5'-GGATCCATGTTACAAGATGATGGTTCTTCA-3') and the reverse primer GRAS11-R-XbaI (5'-GAGCTCTCACTTCCTACATCGCCAAG-3'). PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 60 s; 35 cycles. After recovering the target gene fragment, it was double-digested with the vector pCAMBIA2301 using BamHI and SacI, respectively. The ligation product was transformed into E. coli DH5α, and selection was performed using 50 mg / L kanamycin (Kan). Plasmids were extracted from positive single colonies and sequenced. The obtained plasmid was named pCAMBIA-GRAS11. This plasmid was used for overexpression of GRAS11.

[0052] Example 3: Gene Expression Detection

[0053] Tomato leaves and roots were thoroughly ground in liquid nitrogen and transferred to a 2ml centrifuge tube. 1ml Trizol (Invitrogen, Cat. 15596-018) was added and mixed well, then incubated at room temperature for 5 minutes. The mixture was centrifuged at 12000 rpm for 10 minutes, and the precipitate was discarded. 0.2ml chloroform was added to the supernatant, mixed well, and centrifuged at 12000 rpm for 10 minutes. 0.5ml isopropanol was added to the supernatant to precipitate RNA. The mixture was centrifuged at 12000 rpm for 10 minutes, the precipitate was washed with 75% ethanol, vacuum dried, and dissolved in 50μl water.

[0054] cDNA was synthesized using an RNA PCR system (TaKaRa, Cat.DRR019A). The reaction solution consisted of: 10×RT buffer: 1 μl; dNTP: 1 μl; MgCl2: 2 μl; Oligo dT: 0.5 μl; RNA: 1 μl; RNase inhibitor: 0.5 μl; AMV reverse transcriptase: 0.5 μl; H2O: 4 μl. The reaction conditions were: 42℃ for 30 min, followed by 85℃ for 5 min.

[0055] Quantitative real-time PCR (qRT-PCR) was performed using the CFX Connect Real-Time System (Bio-Rad Laboratories, Inc.). The forward primer was qRT-GRAS11-F (5'-TTGTCTTGAATCGACGCTGC-3'), and the reverse primer was qRT-GRAS11-R (5'-GCATATCACCACGCAACCAT-3'). Tomato actin was used as an internal control gene, with primers qRT-Actin-F (5'-CAGCAGATGTGGATCTCAAA-3') and qRT-Actin-R (5'-CTGTGGACAATGGAAGGAC-3'). PCR conditions were: 95℃ for 15 min; 95℃ for 15 s, 56℃ for 30 s, 72℃ for 30 s; 40 cycles. Data analysis was performed using CFX Manager Software.

[0056] GRAS11 is expressed in all tissues of tomato, with higher expression levels in leaves, roots, and young fruits, such as... Figure 2 As shown in Figure A; the expression level in glandular trichomes was significantly higher than that in leaves, such as Figure 2 As shown in B. Methyljasmonic acid can induce high expression of GRAS11, such as... Figure 2 C; Gibberellin can inhibit GRAS11 expression, such as Figure 2 As shown in D.

[0057] Example 4: Agrobacterium-mediated transformation

[0058] Agrobacterium (GV3101) monoclonal inoculation was carried out in 2 ml LB medium (containing 25 mg / L rifampicin and 50 mg / L gentamicin) and incubated overnight at 28°C and 200 rpm. The inoculation was then carried out at a ratio of 1:50 in 100 ml LB medium (containing 25 mg / L rifampicin and 50 mg / L gentamicin) and incubated for 4 h. The medium was then placed on ice for 10 min, centrifuged at 8000 rpm for 10 min, resuspended in 2 ml 20 mM CaCl2, and aliquoted into 100 μl containers.

[0059] The vector plasmid described in Example 2 was added to different competent cells, flash-frozen in liquid nitrogen for 1 min, incubated at 37°C for 10 min, then 1 ml of LB medium was added and incubated at 28°C for 3 h. The culture was then spread on LB agar plates (containing 25 mg / L rifampin, 50 mg / L gentamicin, and 50 mg / L kanamycin) and incubated upside down at 28°C for 3-4 days. Single colonies were picked for PCR clone screening, and positive single colonies were selected for subsequent experiments.

[0060] Example 5: Analysis of Terpenoid Compounds

[0061] Tomato leaves were flash-frozen in liquid nitrogen, ground, and transferred to 2 ml centrifuge tubes. 1 ml of n-hexane was added, and the mixture was shaken for 1 min. The mixture was centrifuged at 13000 rpm for 2 min, and the supernatant was collected. Terpenoid compounds were detected using gas chromatography-mass spectrometry (GC-MS, Thermo Scientific). Gas chromatography separation was performed using a Trace1300 system and an HP-5ms capillary column (30 m × 0.32 mm) with the following parameters: helium carrier gas 1 ml / min, injection port 250℃, 50℃ for 5 min, 10℃ / min to 300℃, and hold for 5 min. Mass spectrometry was performed using an ITQ900 with the following parameters: 70 eV full scan, m / z range 50-650. Data analysis was performed using Xcalibur software (Thermo Scientific).

[0062] Example 6: Virus-Induced Gene Silencing (VIGS)

[0063] Agrobacterium carrying pTRV1 and pTRV2-GRAS11 plasmids were inoculated at a 1:100 ratio into LB medium (containing 25 mg / L rifampin, 50 mg / L gentamicin, and 50 mg / L kanamycin) and incubated overnight at 28°C. The pellets were centrifuged at 8000 rpm for 10 min, and resuspended in transfection buffer (10 mM MES, 10 mM MgCl2, 0.1 mM acetylsyleugenone) to OD200. 600=2.0, incubate at room temperature for 4-6 hours. Mix Agrobacterium carrying pTRV1 and pTRV2-GRAS11 plasmids at a 1:1 ratio and inject into tomato leaves using a syringe. Inoculated tomatoes are then cultured at 25℃ for 4 weeks for gene expression and compound content analysis.

[0064] In tomato variety LA4024, GC-MS analysis showed that the contents of β-phellandrene, β-caryophyllene, and α-humulene were reduced by 63%, 71%, and 57%, respectively. Figure 3 As shown in AE; qRT-PCR analysis showed that the expression level of GRAS11 in virus-infected tomato leaves decreased by 85%; TPS20 and TPS12 decreased by 73% and 65%, respectively. Figure 3 As shown in FH.

[0065] In the 'Micro-Tom' variety, the plant cannot synthesize β-phellandrene due to the lack of expression of the TPS20 gene. In virus-infected tomato leaves, the contents of β-ocimene, (-)-β-pinene, β-(E)-caryophyllene, and α-humulene were reduced by 48%, 29%, 63%, and 62%, respectively. Figure 4 As shown in AF, the expression levels of GRAS11, TPS5, and TPS12 decreased by 67%, 28%, and 52%, respectively. Figure 4 As shown in GI.

[0066] The results above indicate that reducing or silencing GRAS11 expression can significantly decrease the content of tomato terpenoids.

[0067] Example 7: Tomato Conversion

[0068] Tomato (variety 'Micro-Tom') seeds were soaked in 70% ethanol for 2 min, then in 2% sodium hypochlorite solution for 10 min, washed four times with sterile water, and germinated on MS agar plates (25℃, 16h light). Agrobacterium bacteria cultured overnight were resuspended in MS liquid medium to OD. 600=0.6. Cotyledons were excised from seedlings 10 days after germination and soaked in Agrobacterium resuspension for 20 min, then cultured in the dark at 25℃ for 2 days. The explants were then transferred to callus induction medium (MS medium containing 2 mg / L zeatin, 100 mg / L kanamycin, and 200 mg / L cephalosporin) and cultured, with the medium changed every 2 weeks until shoots appeared. The shoots were then cut off and cultured on rooting medium (MS medium containing 100 mg / L kanamycin) for 4-6 weeks. When the test-tube seedlings had two true leaves, they were transplanted into flowerpots and placed in a greenhouse for further growth (16 hours light, 8 hours dark, 250 μmol / L saturated oxygen). -2 s -1 Light intensity, 60% humidity, 25℃).

[0069] After screening, three transgenic tomato lines overexpressing GRAS11 were obtained (OE-GRAS11-8 / 9 / 10). GC-MS analysis showed that compared with control plants, the contents of β-ocimene, (-)-β-pinene, β-(E)-caryophyllene, and α-humulene in the leaves of transgenic plants overexpressing GRAS11 were increased by 2-3 times, respectively. Figure 5 As shown in AD. The expression levels of TPS5 and TPS12 in these lines were also increased by 3.1-5.5 and 2.7-3.5 times, respectively. Figure 5 As shown in EF.

[0070] qRT-PCR analysis revealed significantly elevated expression levels of several genes in the upstream precursor synthesis pathways MEP and MVA, including 1-deoxy-D-xylulose-5-phosphate synthase 2 (DXS2), 4-(cytidine5'-diphospho)-2-C-methyl-D-erythritol kinase (CMK), and isopentenyl-diphosphate delta-isomerase 1 (IDI1) in the MEP pathway; and acetoacetyl-CoA thiolase 2 / 3 (AACT2 / 3), 3-hydroxy-3-methylglutaryl-CoA synthase 2 / 3 (HMGS2 / 3), 3-hydroxy-3-methylglutaryl-CoA reductase 4 (HMGR4), and mevalonate kinase (MK) in the MVA pathway. Figure 6 As shown.

[0071] The above results indicate that GRAS11 overexpression can not only significantly increase TPS expression levels, but also promote the expression of upstream precursor synthesis pathway genes, thereby increasing the yield of terpenoids.

[0072] Two homozygous mutant lines, gras11-1, were obtained by introducing a mutation at the GRAS11 gene locus using CRISPR-Cas9 genome editing technology. - / - The mutant has a 40bp deletion, gras11-2 - / - The mutant has a 7bp deletion and an 11bp insertion sequence, such as Figure 7 As shown in Figure A, both mutants result in frameshift mutations and fail to produce functional GRAS11 protein. GC-MS analysis indicates that gras11-1 - / - and gras11-2 - / - The contents of β-ocimene, (-)-β-pinene, β-(E)-caryophyllene, and α-humulene in the mutant leaves were reduced by 30-50%, respectively. Figure 7 As shown in BE, the expression levels of TPS5 and TPS12 also decreased by 20-40%, as... Figure 7 As shown in FG. Furthermore, the expression levels of precursor synthesis pathway genes, including DXS2 and IDI1 in the MEP pathway and AACT2 / 3 and HMGR4 in the MVA pathway, were also significantly reduced, such as... Figure 8 As shown.

[0073] Example 8: Glandular hair analysis

[0074] The density and diameter of glandular trichomes on the surface of young leaves were analyzed using a digital microscope (Digital Microscope VHX-6000, Keyence). The density and diameter of the glandular trichomes were quantitatively analyzed using ImageJ software (https: / / imagej.nih.gov / ij).

[0075] Compared with the control material, the diameter of glandular trichomes in the leaves of the GRAS11-overexpressing transgenic plants OE-GRAS8 / 9 / 10 increased by 1.4-1.5 times, equivalent to a 2.7-3.4-fold increase in volume, while the density of glandular trichomes did not change significantly. Figure 9 As shown.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. sequence list <110> Tobacco Research Institute, Chinese Academy of Agricultural Sciences <120> Application of GRAS11 in regulating the synthesis of plant terpenoids and / or glandular trichome development <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1407 <212> DNA <213> Artificial sequence() <400> 1 atgttacaag atgatggttc ttcatctgtg acctcatcat cacctattca agtatttcca 60 atgatgtctg tatcacctag ctttggttca tcaaatcagt ggcttaagga gctgaaatct 120 gaagaaagag ggttgtattt gatacatctt ttgcttgctt gtgctaatca tgttgcttct 180 ggtagccttg agaatgctaa tatagcactt gaccatattt cccaacttgc atctcctagt 240 ggagatacca tgcaaaggat tgcttcatattttactgagg ctttagctga taggattcta 300 aggagttggc ctggtcttta taaggcgttg cgttcgacta agttatcggt tgtctcagaa 360 gaaattcttg ttaggaagat gtttttcgag atctttcctt tcttgaaggt ggcgtttgtg 420 gtcacaaatc aagctataat tgaagctatg gaaggtgaaa agatggttca tattgtggat 480 cttaatgctg ctgaacccct gcaatggcgt gcgttgcttc aggacttgag cgcgcgtcct 540 gaaggaccgc cccatctgcg cattactggg gttcatcagc aaaagaggt gttagatcaa 600 atggcacatg tgcttactca agaagcagaa aactggata tccctttca gttcaatcaa 660 gtagttagca gattgaaaa tctgatgtt gagaaacttc gcgtgaaaac gggggaggct 720 cttgcgatta gttcaattat gcaattgcac acccttctag cccatgataa tgacagaag 780 tcccctttgc cttttaagca ttcaatggt gttaacttaa acagggcact agtcaccaa 840 atactttag gggaatttct tgaaaagat atggctaatg gttgcagtcc aagcaatgac 900 accgctctt catccccgct atgttcact ggttcacaa agatgatag tttcctcaat 960 gctttgtggg gtttatcacc aaagttatg gtggtacag aaaagatgc taaccataat 1020 gggacaactc ttatggagag gctatcagag tcgttacatt tttagctgc attatttgat 1080 tgtcttgaat cgacgctgcc aagacatca ttagagagat taaagtgga aagatgtta 1140 taggtgaag agattagaaa cattatagca tgtgagga tcgaacgaa gagaggcat 1200 gaaaagctcg aaagtggtt ccaagattc gandacatctg gttttgggaa tgtgcctttg 1260 agttattatg ctatgttgca ggcaagaagg ttgttgcaga gttacagttg tgaaggatac 1320 aagatcaaag aagataatgg ttgcgtggtg atatgctggc aggatcgccc acttttctca 1380 gtgtcgtctt ggcgatgtag gaagtga 1407 <210> 2 <211> 468 <212> PRT <213> Artificial Sequence()​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Thr Lys Leu Ser Val Val Ser Glu Glu Ile Leu Val Arg Lys Met Phe 115 120 125 Phe Glu Ile Phe Pro Phe Leu Lys Val Ala Phe Val Val Thr Asn Gln 130 135 140 Ala Ile Ile Glu Ala Met Glu Gly Glu Lys Met Val His Ile Val Asp 145 150 155 160 Leu Asn Ala Ala Glu Pro Leu Gln Trp Arg Ala Leu Leu Gln Asp Leu 165 170 175 Ser Ala Arg Pro Glu Gly Pro Pro His Leu Arg Ile Thr Gly Val His 180 185 190 Gln Gln Lys Glu Val Leu Asp Gln Met Ala His Val Leu Thr Gln Glu 195 200 205 Ala Glu Lys Leu Asp Ile Pro Phe Gln Phe Asn Gln Val Val Ser Arg 210 215 220 Leu Glu Asn Leu Asp Val Glu Lys Leu Arg Val Lys Thr Gly Glu Ala 225 230 235 240 Leu Ala Ile Ser Ser Ile Met Gln Leu His Thr Leu Leu Ala His Asp 245 250 255 Asn Asp Lys Lys Ser Pro Leu Pro Phe Lys His Ser Asn Gly Val Asn 260 265 270 Leu Asn Arg Ala Leu Val Asn Gln Asn Thr Leu Gly Glu Phe Leu Glu 275 280 285 Lys Asp Met Ala Asn Gly Cys Ser Pro Ser Asn Asp Thr Ala Ser Ser 290 295 300 Ser Pro Leu Cys Ser Thr Gly Ser Thr Lys Met Asp Ser Phe Leu Asn 305 310 315 320 Ala Leu Trp Gly Leu Ser Pro Lys Val Met Val Val Thr Glu Gln Asp 325 330 335 Ala Asn His Asn Gly Thr Thr Leu Met Glu Arg Leu Ser Glu Ser Leu 340 345 350 His Phe Tyr Ala Ala Leu Phe Asp Cys Leu Glu Ser Thr Leu Pro Arg 355 360 365 Thr Ser Leu Glu Arg Leu Lys Val Glu Lys Met Leu Leu Gly Glu Glu 370 375 380 Ile Arg Asn Ile Ile Ala Cys Glu Gly Ile Glu Arg Lys Glu Arg His 385 390 395 400 Glu Lys Leu Glu Lys Trp Phe Gln Arg Phe Asp Thr Ser Gly Phe Gly 405 410 415 Asn Val Pro Leu Ser Tyr Tyr Ala Met Leu Gln Ala Arg Arg Leu Leu 420 425 430 Gln Ser Tyr Ser Cys Glu Gly Tyr Lys Ile Lys Glu Asp Asn Gly Cys 435 440 445 Val Val Ile Cys Trp Gln Asp Arg Pro Leu Phe Ser Val Ser Ser Trp 450 455 460 Arg Cys Arg Lys 465

Claims

1. Application of the GRAS11 gene in regulating the synthesis of plant terpenoids and / or increasing the volume of glandular trichomes in plant leaves; wherein the plant is tomato, and the terpenoids are β-ocimene, (-)-β-pinene, β-(E)-caryophyllene and α-humulene, and the nucleotide sequence of the GRAS11 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the GRAS11 gene is shown in SEQ ID NO.2; overexpression of the GRAS11 gene can increase the content of tomato terpenoids and / or increase the volume of tomato leaf glandular trichomes, and silencing the GRAS11 gene can inhibit the synthesis of tomato terpenoids.

2. Application of GRAS11 gene overexpression in at least one of the following (a)-(b): (a) Application in increasing the content of terpenoid compounds in tomatoes; (b) Application in increasing the volume of glandular hairs in tomato leaves; The terpenoids are β-ocimene, (-)-β-pinene, β-(E)-caryophyllene and α-humulene, and the GRAS11 gene sequence is shown in SEQ ID NO.

1.

3. Application of silencing the GRAS11 gene in inhibiting the synthesis of tomato terpenoids; The terpenoids are β-ocimene, (-)-β-pinene, β-(E)-caryophyllene and α-humulene, and the GRAS11 gene sequence is shown in SEQ ID NO.1.

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