Tetrastigma hemsleyanum ThHCT gene as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of genes, and discloses a Tetrastigma hemsleyanum ThHCT gene and application of a coding gene thereof, and the Tetrastigma hemsleyanum ThHCT gene has a base sequence as shown in SEQ ID NO.1 in a sequence table. The invention relates to application of a Tetrastigma hemsleyanum ThHCT gene and a coding gene thereof in improving plant lignin and o-methoxyphenyl (G subunit) monomers. The gene is over-expressed in tobacco leaves, so that the content of lignin in the tobacco leaves can be increased. If the gene is used for transforming plants such as rice, pepper, eggplant, strawberry, tomato and the like, the content of lignin in stalks can be increased, and the lodging resistance and disease resistance of the plants can be enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of functional genomics, and in particular to a Tripterygium wilfordii Hook.f. ThHCT gene, its coding gene and application. BACKGROUND

[0002] Lignin is a kind of phenolic polymer polymerized by cinnamyl alcohol monomers, is a main natural polymer compound in plants, and widely exists in vascular plants, and plays an important role in mechanical support, water transport and defense of plant body. It is usually deposited on the wall of vessel molecular secondary thickening of xylem and thick-walled tissue. Lignin, cellulose and chitin are one of the most abundant natural polymers in the world. Lignin is a complex polymer polymerized by three monomers, which are usually coniferyl alcohol, coniferyl alcohol and sinapyl alcohol, represented by symbols G, S and H respectively. Coniferyl alcohol (G) is the most common monomer in lignin, which belongs to aromatic alcohol compounds. In plants, it usually combines with other monomers to form lignin to enhance the plant cell wall. Due to its special chemical structure, coniferyl alcohol also has wide application in industry, such as used for manufacturing spices, medicines and dyes, etc. Coniferyl alcohol (S) and sinapyl alcohol (H) are mainly involved in the synthesis of lignin, and provide good durability and stability for lignin. In the wood industry, it has important value, and can be used for manufacturing paper, plywood and wood products, etc. In dicotyledonous plants, S subunit and G subunit are mainly used, and there are various types of lignin and various lignin monomers in plants, which fully shows that plants have different adaptability or functional characteristics in evolution and development.

[0003] Lignin biosynthesis is a process of gradually converting into lignin monomers and finally polymerizing into lignin under the catalysis of a series of enzymes. The content, composition of lignin monomers and total lignin content vary with plant species. During the process of lignin synthesis, caffeoyl-CoA is an essential intermediate for the synthesis of three lignin monomers (G-type, S-type and H-type). In recent years, the research of lignin biosynthesis enzyme genes has become one of the hotspots in plant molecular biology. Among them, phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD) and caffeoyl-CoA O-methyltransferase (CCoAOMT) have been reported to affect lignin synthesis. However, the lignin synthesis pathway is complex, and the function of hydroxycinnamoyl-CoA: shikimate transferase (HCT) in lignin synthesis has been reported less. Current research has found that the addition of caffeoyl-CoA in cotton can promote the expression of HCT13 and promote the growth of cotton fiber [Teng L, Zheng K, Qu Y Y, et al. Cloning and Expression Analysis of Gossypium barbadense GbHCT13 Gene [J]. Chinese Journal of Agricultural Biotechnology, 2021, 29(6): 11. DOI:10.3969 / j.issn.1674-7968.2021.06.003.]. In tobacco, through RNAi interference technology, the NtHCT gene was inhibited, and it was found that the lignin content was partially reduced, and the cleavable p-hydroxyphenyl (H) monomer increased slightly [RNAi Modulation of Chlorogenic Acid and Lignin Deposition in Nicotiana tabacum and Insufficient Compensatory Metabolic Cross-Talk. Claudia L. Cardenas, Michael A. Costa, Dhrubojyoti D. Laskar, Syed G. A. Moinuddin, Choonseok Lee, Laurence B. Davin, and Norman G. Lewis. Journal of Natural Products 2021 84(3), 694-706. DOI:10.1021 / acs.jnatprod.1c00054]. It has also been reported that HCT / C3H can catalyze coumaroyl-CoA to form caffeoyl-CoA, and the inhibition of HCT expression in Arabidopsis and tobacco was found to cause a decrease in S monomers and an increase in H monomers. Therefore, HCT is considered to be involved in lignin biosynthesis. However, whether overexpression of HCT affects lignin has not been reported.Tetrastigma hemsleyanum Diels et Gilg is a rare medicinal plant in China. The biological function of HCT in Tetrastigma hemsleyanum Diels et Gilg has not been reported.

[0004] The chromosomal level genome is assembled by using the genome sequencing technology. Based on the assembled high-quality Tetrastigma hemsleyanum Diels et Gilg genome, the candidate gene HCT family is screened out by using the reported tobacco HCT gene as a reference and using the evolutionary relationship. The final candidate gene HCT is determined by using the transcriptome, metabolite detection different tissues, combined analysis of lignin and HCT family differential genes. The full-length of the gene is cloned by using the RT-PCR technology. The function of the gene is determined by detecting the change of lignin and lignin monomer substances of the transgenic tobacco, and the application of the gene is explored. SUMMARY

[0005] The application provides a Tetrastigma hemsleyanum Diels et Gilg ThHCT gene, a coding gene and application thereof. The Tetrastigma hemsleyanum Diels et Gilg ThHCT gene has a base sequence as shown in SEQ ID NO. 1 in the sequence listing. The open reading frame (ORF) of the gene is 1296 bp, that is, the CDS sequence; the length of the full-length transcript of the gene, that is, the mRNA, is 1616 bp, and the length of the translated protein is 431 amino acids. The gene can be applied to improve the content of plant lignin, especially the content of lignin monomer G.

[0006] The technical scheme of the application is as follows:

[0007] A Tetrastigma hemsleyanum Diels et Gilg ThHCT gene has a gene coding nucleotide sequence as shown in SEQ ID NO. 1 in the sequence listing.

[0008] A Tetrastigma hemsleyanum Diels et Gilg ThHCT gene has a gene coding nucleotide sequence as shown in SEQ ID NO. 1 in the sequence listing.

[0009] The protein encoded by the Tetrastigma hemsleyanum Diels et Gilg ThHCT gene has an amino acid sequence as shown in SEQ ID NO. 3.

[0010] A recombinant plant expression vector comprising the Tetrastigma hemsleyanum Diels et Gilg ThHCT gene.

[0011] A method for improving the content of plant lignin and the proportion of o-methoxyphenyl monomer, the method comprising introducing the Tetrastigma hemsleyanum Diels et Gilg ThHCT gene into a plant cell and allowing it to express.

[0012] A method for improving the content of plant lignin and the proportion of o-methoxyphenyl monomer, the method comprising the following steps:

[0013] The *Trifolium repens* ThHCT gene, or the recombinant plant expression vector, is introduced into a plant or plant cell, and the gene is expressed in the plant or plant cell.

[0014] A method for increasing the lignin content and the proportion of o-methoxyphenyl monomers in plants, the method comprising applying to the plant a protein encoded by the ThHCT gene of *Trifolium repens*, or a composition comprising the protein.

[0015] The plants mentioned are rice, corn, strawberries, peppers, eggplants, and tomatoes.

[0016] The application of the *Trifolium repens* ThHCT gene in increasing lignin and o-methoxyphenyl (G subunit) monomers in plants, thereby increasing the content of lignin, especially the content of lignin monomer G, is described in the following methods:

[0017] a. The ThHCT gene was ligated into the 35S-driven pCAMBIA1300 vector;

[0018] b. Introduce the recombinant pCAMBIA1300 vector into plants via Agrobacterium-mediated transformation, including but not limited to rice, corn, strawberry, pepper, eggplant and tomato;

[0019] c. Positive plants were obtained by screening with hygromycin B as a resistance marker.

[0020] The plants mentioned are rice, corn, strawberries, peppers, eggplants, and tomatoes.

[0021] The *Trifolium repens* gene ThHCT of this invention can increase the content of lignin and lignin monomer G. If this gene is introduced into other plants, such as strawberries, peppers, eggplants, and tomatoes, it may also increase the content of lignin and monomers in these plants. Attached Figure Description

[0022] Figure 1 Evolutionary analysis of HCT in *Trifolium repens* and NtHCT in tobacco

[0023] Figure 2 Phenotypic analysis of transgenic tobacco plants and quantitative expression detection of the HCT gene.

[0024] Figure 3 Detection of lignin content in tobacco overexpression plants

[0025] Figure 4 Detection of lignin monomer content in tobacco overexpression plants Detailed Implementation

[0026] A *Trifolium repens* ThHCT gene having the gene-encoding nucleotide sequence described in SEQ ID NO.1 of the sequence listing.

[0027] A gene encoding *Trifolium repens* ThHCT has the full-length transcript nucleotide sequence described in SEQ ID NO.2 of the sequence listing.

[0028] The protein encoded by the *Trifolium repens* ThHCT gene is characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.3.

[0029] A recombinant plant expression vector containing the aforementioned *Trifolium repens* ThHCT gene.

[0030] A method for increasing the lignin content and the proportion of o-methoxyphenyl monomers in plants, the method comprising introducing the *Trifolium repens* ThHCT gene into plant cells and expressing it.

[0031] A method for increasing the lignin content and the proportion of o-methoxyphenyl monomers in plants, the method comprising the following steps:

[0032] The *Trifolium repens* ThHCT gene, or the recombinant plant expression vector, is introduced into a plant or plant cell, and the gene is expressed in the plant or plant cell.

[0033] A method for increasing the lignin content and the proportion of o-methoxyphenyl monomers in plants, the method comprising applying to the plant a protein encoded by the ThHCT gene of *Trifolium repens*, or a composition comprising the protein.

[0034] The application of the *Trifolium repens* ThHCT gene in increasing lignin and o-methoxyphenyl (G subunit) monomers in plants, thereby increasing the content of lignin, especially the content of lignin monomer G, is described in the following methods:

[0035] a. The ThHCT gene was ligated into the 35S-driven pCAMBIA1300 vector;

[0036] b. Introduce the recombinant pCAMBIA1300 vector into plants via Agrobacterium-mediated transformation, including but not limited to rice, corn, strawberry, pepper, eggplant and tomato;

[0037] c. Positive plants were obtained by screening with hygromycin B as a resistance marker.

[0038] The plants include rice, corn, strawberries, peppers, eggplants, and tomatoes.

[0039] Gene acquisition and transformation

[0040] Target gene acquisition

[0041] Phylogenetic analysis was performed, and the sequence of the BAHD acyltransferase NtHCT (gene ID: AJ507825) was obtained from the UniProt protein database. The protein sequence was arranged in ClustalW and analyzed using MEGA-X software. A Poisson model was employed, and the Neighbor-Joining (NJ) bootstrap test was run 1000 times. Reliability was assessed by a bootstrap support value ≥50%. The closest related gene to NtHCT, *Trifolium repens* HCT (The05G013290), was obtained.

[0042] (I) Total RNA Extraction

[0043] 1) Weigh 0.1g of fresh leaves of *Trifolium repens* and place them in a nitrogen-pre-cooled mortar. Grind the tissue with a pestle while continuously adding liquid nitrogen until it is ground into powder. Then quickly transfer the powder to a 10mL centrifuge tube and add an appropriate amount of RNAiso Plus that matches the sample homogenate volume. Homogenize thoroughly.

[0044] 2) Transfer the homogenate to centrifuge tubes and let it stand at room temperature (15-30℃) for 5 minutes.

[0045] 3) Centrifuge at 12,000×g at 4℃ for 5 minutes.

[0046] 4) Carefully aspirate the supernatant and transfer it to a new centrifuge tube (do not aspirate the precipitate).

[0047] 5) Add chloroform (1 / 5 volume of RNAiso Plus) to the supernatant, tighten the centrifuge tube cap, and mix until the solution emulsifies and turns milky white.

[0048] 6) Let stand at room temperature for 5 minutes, then centrifuge at 12000 rpm at 4℃ for 15 minutes.

[0049] 7) Transfer the supernatant to another new centrifuge tube.

[0050] 8) Add 1 volume of isopropanol to the supernatant, invert the centrifuge tube to mix thoroughly, and let stand at room temperature for 10 minutes.

[0051] 9) Centrifuge at 12000 rpm at 4℃ for 10 minutes, carefully discard the supernatant, add an equal amount of 75% ethanol to RNAiso Plus, gently invert the centrifuge tube to wash the wall, centrifuge at 7500 rpm at 4℃ for 5 minutes, and carefully discard the supernatant.

[0052] 10) RNA dissolution. Open the centrifuge tube cap and allow the precipitate to dry at room temperature for 3-5 minutes. After the precipitate has dried, add 30-50 μL of RNase-free water to dissolve it.

[0053] (II) Gene Cloning and Transformation

[0054] Total RNA was extracted from fresh leaves of *Trifolium repens* using the above method. Then, using *Trifolium repens* mRNA as a template, the coding sequence of the ThHCT gene was amplified by RT-PCR.

[0055] The specific steps are as follows: First, mRNA is reverse transcribed into first-strand cDNA. The reverse transcription kit used is PrimeScript from TaKaRa. TM II. Using the 1st Strand cDNA Synthesis Kit, in a 20 μl reaction volume, add 1 μl of 50 μM random primers (Random 6mers), 1 μl of 10 mM dNTPs, 2 μl of total RNA (2 μg), and DEPC water to a final volume of 10 μl. Denature at 65°C for 5 minutes, then rapidly cool on ice for 2 minutes. Centrifuge briefly, then add 4 μl of 5×PrimerScript II buffer, 0.5 μl of RNase inhibitor, 0.5 μl of PrimerScript RTase, and 5 μl of DEPC water. Mix gently, incubate at 30°C for 10 minutes, then at 42°C for 60 minutes, and finally at 95°C for 5 minutes. Then, amplify the target gene ThHCT using the first-strand cDNA as a template. The amplification primers used are: ThHCT-F, 5'-CCGGAATTCCGGATGATCATAAACGTGAGGGA-3', and ThHCT-R, 5'-CCCAAGCTTGGGTTAAATGTCGTATAAGTACT-3'.

[0056] The PCR reaction volume was 50 μL. The following were added sequentially: 25 μL of 2×PCR buffer, 4 μL of 2.5 mM dNTPs, 2 μL of reverse transcription product, 2 μL of 10 μM forward primer (ThHCT-F), 2 μL of 10 μM reverse primer (ThHCT-R), and 0.5 μL of 2.5 U / μL Tag DNA polymerase. Water was then added to a final volume of 50 μL. PCR reaction conditions were: pre-denaturation at 94℃ for 3 min, denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, for 30 cycles, followed by a final extension at 72℃ for 10 min. The mixture was then stored at 4℃.

[0057] After amplification, the ThHCT gene was inserted into the pMD19-T vector (produced by TakaRa). The purified ThHCT gene DNA was ligated into the pMD19-T vector in a 10 μL ligation system, consisting of 0.5 μL pMD19-T vector, 4.5 μL purified ThHCT gene DNA, and 5 μL Solution I. Ligation was carried out at 14-16°C for 8-12 hours, and the ligation product was then transformed into *E. coli* DH5α competent cells. After successful sequencing, the ThHCT gene was digested with EcoRI and HindIII (produced by TakaRa) as follows: a 40 μL digestion system was prepared, including 4 μL 10× buffer, 8 μL pMD19-T vector with the ThHCT gene inserted, 1 μL EcoRI, 1 μL HindIII, and 26 μL water, and incubated at 37°C for 6 hours.

[0058] Gene fragments were recovered using the Gel Extraction Kit produced by Kangwei Century Company. The procedure was as follows: After gel electrophoresis of the above-mentioned DNA product, the single target DNA band was excised from the agarose gel and placed in a clean centrifuge tube. The weight of the gel was calculated, and 1 volume of BufferPG was added to the gel block. The centrifuge tube was incubated at 50°C, with the centrifuge tube gently inverted every 2-3 minutes until the gel solution turned yellow, ensuring that the gel block was fully dissolved. At the same time, 200 μL of BufferPS was added to the adsorption column (DM) already loaded into the collection tube, and the column was centrifuged at 13000 rpm for 1 minute. The waste liquid in the collection tube was discarded, and the adsorption column was returned to the collection tube. After the gel block was completely dissolved, the gel solution was cooled to room temperature and added to the adsorption column already loaded into the collection tube. The column was incubated at room temperature for 2 minutes, centrifuged at 13000 rpm for 1 minute, and the waste liquid in the collection tube was discarded. The adsorption column was returned to the collection tube. Add 450 μL of Buffer PW to the adsorption column, centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Repeat the above steps once. Centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube. Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 μL of Buffer EB dropwise to the center of the adsorption membrane, and incubate at room temperature for 2 minutes. Centrifuge at 13000 rpm for 1 minute. The resulting solution is the purified ThHCT gene and should be stored at -20°C.

[0059] The recovered gene fragment was ligated into the pCAMBIA1300 vector as follows: The ligation system consisted of 10 μL, including 2 μL of pCAMBIA1300 vector, 6 μL of purified gene DNA, 1 μL of 10×T4 ligase buffer, and 1 μL of T4 ligase. Ligation was carried out at 4-10℃ for 12 h. The ligation product was then transformed into E. coli DH5α competent cells, and plasmids were extracted for identification.

[0060] The gene fragment was ligated into the pCAMBIA1300 vector and then transformed into AGL1 Agrobacterium. The procedure was as follows: 200 μL of competent Agrobacterium cells were taken, and 5-10 μL of the constructed plasmid DNA was added. The cells were incubated at 30°C on ice for 30 min, flash-frozen in liquid nitrogen for 1 min, and then incubated at 37°C for 5 min. Then, 1 mL of LYEB medium (product number CM2010S-02, Weidi Biotechnology) was added. The cells were incubated at 28°C for 4 h. After centrifugation at 10000g for 30 s, the supernatant was discarded, and the cells were resuspended in 0.1 mL of LYEB medium. The cells were then plated on YEB plates containing 100 μg / mL kanamycin and 125 μg / mL rifampin and incubated at 28°C for approximately 48 h.

[0061] After successful identification (positive clones were selected as templates and identified using colony PCR), the model plant tobacco was transformed using Agrobacterium-mediated transformation. The procedure was as follows: Agrobacterium colonies containing the target plasmid were inoculated into 10 ml of YEB medium (containing 100 μg / mL kanamycin and 125 μg / mL rifampin) and cultured overnight at 28°C with shaking at 200 rpm. The day before transformation, the colonies were inoculated at a 1:50 ratio into 200 mL of YEB medium containing the same antibiotics and cultured until OD was reached. 600 The OD value was 0.6-0.8. Take the bacterial suspension and transfer it to freshly prepared antibiotic-free YEB liquid medium at a ratio of 1%-2%. After 6 hours, the OD value of the bacterial suspension was... 600 A value of 0.4 to 0.6 is sufficient for conversion.

[0062] The transformation of the model plant tobacco using the Agrobacterium-mediated leaf disc method was performed as follows: A single colony of Agrobacterium containing the target gene plasmid was inoculated into 10 mL of YEB medium (containing 100 μg / mL kanamycin and 125 μg / mL rifampin) and cultured overnight at 28°C with shaking at 200 rpm. The day before transformation, the colony was inoculated 1:50 into 200 mL of YEB medium containing the same antibiotics and cultured until the OD600 reached 1.2–1.6. After approximately 6 hours, the colony was collected by centrifugation at 5000g for 15 min and resuspended in osmotic buffer to achieve an OD600 of 0.8. The 200 mL resuspension could be reused three times. Healthy leaves were cut from the sterile tobacco culture using scissors and forceps and placed in a glass dish. The leaves were then cut into pieces approximately 0.5–1 cm in length using a scalpel. 2Cut small pieces of leaf by quickly slicing them with a blade, ensuring a clean cut. Use tweezers to place the cut leaves into Agrobacterium resuspension for 8-10 minutes. Pour out the bacterial suspension and use tweezers to transfer the leaves to filter paper in a glass dish to absorb the bacterial suspension. Place the leaves on a clean bench for a period of time to allow the bacterial suspension to dry. Place the dried leaves on a co-culture medium (MS medium + NAA 0.1 mg / L + 6-BA 1.5 mg / L) and incubate in the dark at 25°C for 3 days. After 3 days of co-culture, transfer the tobacco leaves to differentiation and selection medium (MS + 6-BA 1.5 mg / L + NAA 0.1 mg / L + hygromycin 30 mg / L + cephalosporin 100 mg / L). Ensure the leaves are in contact with and adhere to the medium surface, leaving some space between the leaves, and then incubate at 25°C under light. Tobacco seedlings sprouted in a light-cured culture chamber after two to three weeks. These seedlings were then cut and transferred to a seedling growth medium (MS + 6-BA 0.1 mg / L + NAA 0.01 mg / L + hygromycin 30 mg / L + cephalosporin 100 mg / L). After about a week, rooted seedlings were selected and transferred to a rooting medium (MS + hygromycin 30 mg / L + cephalosporin 100 mg / L). One week after transplanting, large buds showed rooting. After another week of growth, the rooted seedlings were removed from the culture flasks, the solid culture medium was removed from the roots, and they were transplanted into flowerpots. A light-transmitting cover was used to maintain humidity. After 2-3 days of growth, the cover was removed. Seeds of the T2 generation were harvested from individual plants, propagated, and identified up to the T3 generation, yielding eight homozygous transgenic lines.

[0063] Gene screening and functional identification

[0064] Using reported tobacco HCT gene sequences, and through evolutionary analysis by comparing them with the *Trifolium repens* genome, it was found that NtHCT is most closely related to a gene on chromosome 5 of *Trifolium repens*, namely The05G013290. The *Trifolium repens* HCT gene number (e.g., ...) Figure 1 (As shown).

[0065] When the HCT gene of the Trifoliales order was transferred into Nicotiana benthamiana, the transgenic spiders were found to be more robust (e.g., Figure 2 As shown above, qPCR verification revealed that the expression level of HCT in the HCT-OE#1 and HCT-OE#2 lines was significantly higher than that in the wild-type plants (see above). Figure 2 Down).

[0066] Lignin content was measured in HCT gene transgenic lines and control plants. The results are shown below. In both overexpression lines, lignin content was significantly higher than in the control group. Figure 3 As shown.

[0067] Lignin polymers are composed of varying amounts of p-hydroxyphenyl (H subunit), o-methoxyphenyl (G subunit), and sinapyl (S subunit) monomers. Analysis of monomer content in leaves of tobacco overexpressing the OE#2 mutant revealed a significant increase primarily in the G subunit, while the contents of the other two monomers showed no significant difference. This indicates that HCT overexpression promotes G subunit formation, thereby leading to lignin accumulation. Figure 4 As shown.

[0068]

[0069]

[0070]

[0071]

[0072]

Claims

1. A type of three-leaf clover ThHCT A gene, characterized in that it has the gene-encoding nucleotide sequence described in SEQ ID NO.1 of the sequence listing.

2. A coding scheme for the *Trifolium repens* as described in claim 1 ThHCT The gene is characterized by having the full-length transcript nucleotide sequence described in SEQ ID NO.2 of the sequence listing.

3. A type of *Trifolium repens* as described in claim 1 ThHCT Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

3.

4. A recombinant plant expression vector, characterized in that, It contains the *Trifolium repens* ThHCT gene as described in claim 1.

5. A method for increasing the lignin content and the proportion of o-methoxyphenyl monomers in plants, characterized in that, The method includes introducing the *Trifolium repens* ThHCT gene of claim 1 into plant cells and expressing it.

6. A method for increasing the lignin content and the proportion of o-methoxyphenyl monomers in plants, characterized in that, The method includes the following steps: Introduce the *Trifolium repens* ThHCT gene as described in claim 1, or the recombinant plant expression vector as described in claim 4, into a plant or plant cell, and express the gene in the plant or plant cell.

7. A method for increasing the lignin content and the proportion of o-methoxyphenyl monomers in plants, characterized in that, The method comprises applying to the plant the protein encoded by the *Trifolium repens* ThHCT gene as described in claim 3, or a composition comprising said protein.

8. The application according to claim 5, characterized in that: The plants mentioned are rice, corn, strawberries, peppers, eggplants, and tomatoes.

9. The application according to claim 5, characterized in that: The following methods can be used to increase the lignin content in plants, especially the content of lignin monomer G: a. Genes ThHCT Connect to the 35S driven pCAMBIA1300 carrier or other types of carriers such as pCAMBIA1390; b. Introduce the recombinant pCAMBIA1300 vector into plants, including but not limited to rice, corn, strawberry, pepper, eggplant and tomato, via Agrobacterium-mediated transformation; c. Positive plants were obtained by screening with hygromycin B as a resistance marker.

10. The application according to claim 6, characterized in that: The plants mentioned are rice, corn, strawberries, peppers, eggplants, and tomatoes.

Images