Application of NtFATB gene in regulation and control of development of tobacco glandular hair
By regulating tobacco glandular trichome development through the NtFATB gene and constructing an RNAi vector to silence the NtFATB gene, the problem of insufficient research on the molecular mechanism of tobacco glandular trichome development was solved, and tobacco quality and resistance were improved.
Patent Information
- Application Number
- CN202511140013.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-14
- Publication Date
- 2025-11-14
AI Technical Summary
Current research has limited understanding of the molecular mechanisms of tobacco glandular trichome development, lacking identification of key genes and regulatory mechanisms, which affects tobacco quality and resistance improvement.
The development of tobacco glandular trichomes was regulated by the NtFATB gene. The NtFATB gene was silenced by constructing an RNAi vector, and changes in glandular trichome density were observed to regulate tobacco glandular trichome density.
This research has enriched the understanding of the molecular regulatory mechanisms of tobacco glandular trichome development, improved the aroma quality and resistance of tobacco, and provided genetic resources for variety breeding.
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Abstract
Description
Technical Field
[0001] This invention relates to the application of the NtFATB gene in regulating the development of tobacco glandular trichomes, and belongs to the field of plant genetic engineering technology. Background Technology
[0002] Plant trichomes are specialized structures that develop from plant epidermal cells. Serving as a natural barrier between the plant and its environment, they play physiological roles such as preventing water evaporation, buffering direct sunlight, and resisting pathogens, insects, and extreme temperatures. Furthermore, the developmental model of trichomes provides a theoretical basis for cell differentiation research. Based on their type, trichomes can be classified as unicellular or multicellular, branched or unbranched, and glandular or non-glandular. The classification of glandular and non-glandular trichomes according to their biological functions has significant practical implications.
[0003] Glandular trichomes possess glands that secrete metabolically produced chemicals or toxic substances, such as alkaloids (nicotine, terpenoids, etc.), resins, and aromatic oils. They not only function to defend against biotic (e.g., repelling plant-like insects) and abiotic stresses, but also act as signaling molecules regulating plant growth and development. Non-glandular trichomes are typically epidermal appendages without glands and do not secrete metabolic substances. They play a role in resisting extreme heat and cold, inducing pollination, preventing UV radiation damage, resisting drought, adapting to high salinity, absorbing heavy metals, protecting plants from herbivores and pathogens, and resisting mechanical damage. Furthermore, some novel functions of epidermal trichomes have been studied. For example, the epidermal trichomes of cotton petals can maintain the shape of flower buds, ensuring seed production; after damage treatment, non-glandular trichomes in Asteraceae plants exhibit stronger stress resistance than glandular trichomes; and in pumpkins, non-glandular trichomes not only store water but also participate in petiole movement.
[0004] Tobacco (Nicotiana tabacum L.) is an important economic crop, and its yield and quality directly affect the development of the agricultural industry and farmers' economic income. The entire tobacco plant is densely covered with epidermal trichomes, exhibiting diverse morphologies and structures. Based on the presence or absence of secretory glands, they can be divided into protective trichomes and glandular trichomes. Tobacco glandular trichomes are formed by the differentiation of leaf primordia epidermal cells through division, and consist of three parts: basal cells, stalk cells, and head cells. Their secretions are an important component of the chemical substances on tobacco leaves. The density, type, and secretion accumulation of tobacco leaf glandular trichomes are closely related to tobacco plant resistance and tobacco leaf aroma quality. Therefore, the occurrence and metabolic regulation of tobacco glandular trichomes are of great significance for studying tobacco abiotic stress and aroma quality. Regulating glandular trichome development can improve tobacco resistance and enhance aroma quality.
[0005] In recent years, research on the molecular mechanisms related to epidermal trichome development in the model plant Arabidopsis thaliana has been quite in-depth, revealing that epidermal cells form trichomes through processes such as initiation, branching, and elongation. Changes in the shape, size, and spatial structure of these trichomes are synergistically regulated by various plant hormones and numerous genes. Currently, there is considerable research on genes regulating epidermal trichome development, but research and descriptions of non-model plants are scarce. In contrast, tobacco glandular trichome secretions are closely related to the aroma and quality of tobacco leaves, and their development directly affects the content and composition of leaf secretions. Existing research mainly focuses on the morphological observation of tobacco glandular trichomes and the identification of secretions, with limited research on the molecular mechanisms of trichome development. Furthermore, the identification of genes related to trichome development in tobacco and the study of their molecular regulatory mechanisms are still in their early stages. Therefore, it is urgent to explore and utilize key genes for trichome development in tobacco, which is of significant practical importance for breeding new tobacco varieties that regulate trichome development and ensuring the quality and yield of tobacco. Summary of the Invention
[0006] The purpose of this invention is to provide the application of the NtFATB gene in regulating tobacco glandular trichome development, and to provide a gene resource that can participate in regulating tobacco glandular trichome development.
[0007] To achieve the above objectives, the technical solution adopted in this invention for the application of the NtFATB gene in regulating tobacco glandular trichome development is as follows:
[0008] The application of the NtFATB gene in regulating the development of tobacco glandular hairs, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0009] The beneficial effects of the above technical solution are as follows: The application of the NtFATB gene in regulating tobacco glandular trichome development is a pioneering invention. This invention provides a gene related to tobacco glandular trichome development, NtFATB. By constructing an RNAi vector for the NtFATB gene, cultivating tobacco plants with NtFATB gene silence, and observing the glandular trichome density in the leaves of the silent plants, analysis revealed a significant decrease in glandular trichome density in tobacco leaves. This proves that the NtFATB gene is a key gene regulating tobacco glandular trichome development. In the field of scientific research, this gene can be used to explore the molecular regulatory mechanism of glandular trichome development, identify genes related to glandular trichome development, and select key tobacco varieties that regulate glandular trichome development. The functional identification of the tobacco NtFATB gene not only enriches the gene resources for regulating tobacco glandular trichome development and quality breeding but also helps to elucidate the diverse functions of the tobacco NtFATB gene, possessing significant theoretical and applied value.
[0010] Specifically, the method of preparing transgenic tobacco germplasm using the NtFATB gene involves interfering with, silencing, knocking out, or overexpressing the NtFATB gene through transgenic technology, transient expression technology, or genome editing technology, thereby obtaining new tobacco varieties with changes in glandular trichome density.
[0011] As a further improvement, the density of tobacco glandular trichomes was regulated by controlling the expression level of the NtFATB gene.
[0012] As a further improvement, the regulation involves suppressing the expression of the NtFATB gene, which significantly reduces the density of glandular trichomes in tobacco leaves.
[0013] As a further improvement, the inhibition involves constructing an NtFATB gene RNAi vector, transforming it into plants, and inhibiting the expression of the NtFATB gene.
[0014] As a further improvement, the method for constructing the RNAi vector includes the following steps: using nucleotides 1-300 in the nucleotide sequence shown in SEQ ID NO.1 as the guide sequence for RNAi, the nucleic acid fragments are inserted into the empty backbone vector in the forward and reverse directions.
[0015] As a further improvement, the tobacco is K326. Attached Figure Description
[0016] Figure 1 This is an electrophoresis diagram of the PCR fragment product of the NtFATB gene in Example 1 of the present invention;
[0017] Figure 2 This is the RNAi map of the RNAi interference vector pBWA(V)HS-RNAi in Example 2 of the present invention;
[0018] Figure 3 This is the pBWA(V)HS-NtFATB-RNAi restriction map in Example 2 of the present invention;
[0019] Figure 4 Gene expression analysis of the NtFATB-RNAi transgenic line in Example 3 of this invention;
[0020] Figure 5 This study analyzes the morphology and density of glandular hairs in the NtFATB-RNAi transgenic line and the control K326 in Example 4 of this invention. Detailed Implementation
[0021] Currently, research on tobacco glandular trichomes in the industry mainly focuses on (1) the effects of trichome morphology, density, and secretion composition on tobacco resistance and quality, and (2) the regulation of tobacco glandular trichome development or trichome metabolites by a certain gene. Furthermore, reports on genes regulating glandular trichome development are very limited. Therefore, finding new targets for regulating glandular trichome development and using genetic engineering to improve existing major tobacco varieties is of significant theoretical and practical importance for breeding new varieties with different glandular trichome densities and types. Based on this, this invention provides the application of the NtFATB gene in regulating tobacco glandular trichome development.
[0022] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.
[0023] Unless otherwise specified, the following examples were conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or as recommended by the manufacturer's instructions.
[0024] Biomaterials:
[0025] All plant tissue materials in the following embodiments of the present invention were obtained from flue-cured tobacco (Nicotiania tabacum L.) variety K326 and K326 transgenic plants with RNAi interference NtFATB. The tobacco materials were grown in an artificial climate chamber at a growth temperature of 25°C and a photoperiod of 12 hours of light / 12 hours of darkness.
[0026] Tobacco seedlings were harvested, and leaves were flash-frozen in liquid nitrogen for subsequent molecular experiments; leaves were also collected, freeze-dried, and used for subsequent metabolic assays. The nucleotide sequence of the tobacco glandular trichome development-related gene (fatty acyl-ACP thioesterase B) NtFATB is shown in SEQ ID NO. 1. Alternatively, it could be a nucleotide sequence that hybridizes to the DNA sequence defined in SEQ ID NO. 1 under highly stringent conditions; or a DNA sequence that shares more than 90% homology with the DNA sequence defined in SEQ ID NO. 1 and encodes a protein with the same function.
[0027] Tobacco NtFATB encodes a protein associated with tobacco glandular trichome development, the amino acid sequence of which is shown in SEQ ID NO.2. The protein may also be formed by substitution and / or deletion and / or addition of one or more amino acid residues from the amino acid sequence shown in SEQ ID NO.2, and may contain a derivative polypeptide that affects changes in tobacco glandular trichome density. The substitution and / or deletion and / or addition of one or more amino acid residues refers to the substitution and / or deletion and / or addition of no more than 10 amino acid residues.
[0028] Specific embodiments of the application of the NtFATB gene in regulating tobacco glandular trichome development:
[0029] Example 1: Tobacco RNA Extraction and cDNA Synthesis
[0030] In this embodiment, young leaves of cultivated tobacco K326 seedlings were used as samples. RNA was extracted and reverse-engineered into cDNA. Using the cDNA as a template, the NtFATB gene was amplified by PCR to obtain the PCR amplification product. The specific implementation steps are as follows:
[0031] RNA extraction: Young leaves of cultivated tobacco K326 seedlings were used as samples. After being thoroughly ground into powder with liquid nitrogen, approximately 100 mg of the powder was placed in a 1.5 mL centrifuge tube containing 1.0 mL of TRIZOL reagent. 200 μL of chloroform was added, the mixture was shaken and centrifuged, and the supernatant was carefully removed and transferred to another centrifuge tube. 500 μL of isopropanol was added, the precipitate was collected, and the RNA was separated by centrifugation. The RNA was then washed with 75% alcohol, allowed to dry slightly at room temperature, and then dissolved in an appropriate volume of RNase-free water. The extracted total RNA was then treated with DNase I. The digestion reaction system was: 1 μg RNA, 1 μL 10×reaction buffer with MgCl2, 1 μL (1 U) DNase I, RNase-free, and 10 μL DEPC-treated water. The mixture was incubated in a 37°C water bath for 30 min.
[0032] cDNA first-strand synthesis: Prepare the template RNA / primer mixture shown in Table 1 in a sterile 0.2 mL centrifuge tube, incubate at 70 °C for 10 min, then rapidly cool on ice for at least 2 min, and centrifuge for a few seconds to allow the denatured template RNA / primer solution to accumulate at the bottom of the centrifuge tube.
[0033] Table 1 Template RNA / Primer Mixture System
[0034]
[0035] After preparing the reverse transcription reaction solution shown in Table 2 in the centrifuge tubes, incubate at 42°C for 1 hour; incubate at 70°C for 15 minutes and then cool on ice. The resulting cDNA is used for PCR amplification.
[0036] Table 2 Reverse Transcription Reaction Solution
[0037]
[0038]
[0039] Using K326 cDNA as a template, primers were designed based on information from the tobacco genome database to perform PCR amplification of the NtFATB gene, and the PCR amplification product was obtained (the fragment amplified in this embodiment is a specific sequence of the NtFATB gene, which is used as the guide sequence for the subsequent construction of the RNAi vector; specifically, nucleotides 1-300 in the nucleotide sequence shown in SEQ ID NO.1 are used as the guide sequence for RNAi).
[0040] Primers for amplifying the forward NtFATB gene fragment:
[0041] NtFATB-F: 5'-CAGTGGTCTCATGATTGTAACTGCTACT-3' (shown in SEQ ID NO.3);
[0042] NtFATB-R: 5'-CAGTGGTCTCTTCATATGACCTGATGCT-3' (shown in SEQ ID NO.4).
[0043] LOOP primer:
[0044] F: 5'-CAGTGGTCTCACAGGTCTAGTTTTTCTCCTT-3' (shown in SEQ ID NO.5);
[0045] R: 5'-CAGTGGTCTCAGCCCGGGCTCTGTAACTATC-3' (shown in SEQ ID NO. 6).
[0046] Primers for amplifying the reverse NtFATB gene fragment:
[0047] NtFATB-F: 5'-CAGTGGTCTCTTGATGCTGACGATATC-3' (shown in SEQ ID NO.7);
[0048] NtFATB-R: 5'-CAGTGGTCTCGCCTTGAGAAACCCCAT-3' (shown in SEQ ID NO.8).
[0049] The PCR amplification system is shown in Table 3 below, and the PCR reaction procedure is shown in Table 4 below.
[0050] Table 3 PCR amplification system
[0051]
[0052] Table 4 PCR reaction procedures
[0053]
[0054]
[0055] The amplified PCR products were subjected to 1% agarose gel electrophoresis. The gel electrophoresis results are as follows: Figure 1 As shown in the figure (M: marker (DL5000); 1: forward sequence PCR product; 2: reverse sequence PCR product). After electrophoresis, the PCR products were purified using the Takara PCR product purification kit according to the product instructions and sent to Shanghai Sangon Biotech for sequencing to verify the sequence results.
[0056] Example 2: Construction of gene silencing vector
[0057] Based on the NtFATB gene fragment obtained in Example 1, this invention further constructs an RNAi vector to silence the NtFATB gene. The specific implementation steps are as follows:
[0058] After purification, the PCR product containing the Infusion adapter sequence from Example 1 was used to ligate the target fragment to the following method using Infusion ligase: Figure 2 The pBWA(V)HS-RNAi vector is shown. The infusion ligation system is shown in Table 5 below.
[0059] Table 5 Infusion Connection System
[0060]
[0061] The fragment mixture was reacted at 50°C for 15 minutes, then placed on ice for 2-3 minutes.
[0062] Transformation of E. coli competent cells using the ligation product (heat shock method): Under aseptic conditions, add 10 μL of the ligation product to competent cells, mix gently, and incubate on ice for 30 min. Heat shock at 42℃ for 90 s, then quickly transfer the centrifuge tube to an ice bath for 2-3 min. Add 800 μL of antibiotic-free LB medium and incubate at 37℃ with gentle shaking (100-160 rpm) for about 1 h. Spread 200 μL of the culture solution onto LB solid medium containing 50 μg / mL antibiotics. Before spreading the culture, add X-Gal and IPTG and spread evenly. Incubate upside down at 37℃ for 12-16 h.
[0063] Screening and identification of positive clones: Numerous blue and white bacterial colonies grew in the culture medium. Once the colonies reached a suitable size, several white colonies were picked using a sterilized pipette tip and cultured with shaking in LB broth containing 50 μg / mL kanamycin for 12-16 hours. Plasmids were extracted, and the vector construction was identified by enzyme digestion. Results are as follows: Figure 3 As shown in the figure (M: marker; 1: restriction enzyme band), the RNAi vector for silencing the NtFATB gene (pBWA(V)HS-NtFATB-RNAi vector) was successfully constructed.
[0064] Example 3: Agrobacterium-mediated tobacco transformation and identification of transgenic plants
[0065] In this embodiment, the RNAi vector (pBWA(V)HS-NtFATB-RNAi vector) for silencing the NtFATB gene constructed in Example 2 was transformed into Agrobacterium, and then tobacco was infected. The transgenic plants were then identified. The specific implementation steps are as follows:
[0066] 1. Freeze-thaw transformation of Agrobacterium
[0067] Add 1 μg pBWA(V)HS-NtFATB-RNAi vector to 100 μL of EHA105 Agrobacterium L competent cells, mix well, and incubate on ice for 30 min. Then freeze in liquid nitrogen for 5 min, remove from liquid nitrogen, and incubate in a 37°C water bath for 5 min. After incubating on ice for 5 min, add 500 μl LB solution and incubate at 28°C with full shaking for 4 h. Finally, spread the bacterial culture evenly on selective agar plates and incubate at 28°C for 24-48 h.
[0068] 2. Leaf disc method for converting tobacco variety K326
[0069] (a) Under sterile conditions, tobacco K326 seeds were placed in EP tubes and rinsed 2-3 times with sterile water; then soaked in 75% alcohol for 30-60 seconds, treated with 0.1% mercuric chloride for 5 minutes, rinsed 5 times with sterile water, and sown on MS medium. The culture bottles were placed in an artificial climate chamber to ensure normal germination and growth of tobacco seedlings.
[0070] (b) When the tobacco seedlings reach 3-5cm in length (approximately 20-30 days), take the terminal bud and place it on MS+BA (6-benzylaminopurine) 0.2mg / L medium (to promote bud growth and accelerate development) for subculture. After 14 days of subculture (until small leaves appear), take leaves measuring 1cm × 1cm, remove the petiole, and make incisions on the leaf surface and edges. Place the leaves on MS+BA 1.0mg / L pH 6.0-6.5 pre-culture medium, with the upper surface facing down and in close contact with the medium, and pre-culture in the dark for 2-3 days.
[0071] The MS medium consisted of the following components: NH4NO3 1650 mg / L, KNO3 1900 mg / L, KH2PO4 170 mg / L, MgSO4·7H2O 370 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, Na2EDTA 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, myo-Inositol 100 mg / L, Glycine 2 mg / L, and Nicotinic acid. 0.5 mg / L, Pyridoxine·HCl 0.5 mg / L, Thiamine·HCl 0.1 mg / L, sucrose 30 g / L, agar 8 g / L.
[0072] (c) Remove the pre-cultured leaves or stem segments and infect them with Agrobacterium infection solution. The night before infection, shake two bottles of Agrobacterium. Fill 2 mL centrifuge tubes with bacterial suspension, centrifuge at 4000 rpm for 5 min, and wash twice with bacterial suspension. Add 1.5 mL of bacterial cells to the bacterial suspension at a 1:10 ratio (10 mL of bacterial suspension to 1 tube of acetylsuccinyl ethylsuccinate (As) 25 mg / L), and continuously shake the infection solution to ensure full contact with the cut surfaces of the leaves and stem segments. After 15 min, remove the tubes and blot the bacterial suspension dry on sterilized, dry filter paper.
[0073] The preparation method of Agrobacterium infection solution is as follows: Take the transformed Agrobacterium stored at -80℃, streak it onto agar plates, and add 50 mg / L Kan and 50 mg / L Rif to the LB solid plate; pick a single colony and transfer it to 5 mL of LB liquid medium containing 50 mg / L Kan and 50 mg / L Rif, and incubate overnight (12-16 h) at 28℃ and 200 rpm in a shaker; when the bacterial concentration reaches OD 600When the concentration reaches approximately 1.5, add 2 mL of bacterial culture to a centrifuge tube and centrifuge at 4000 rpm for 5 min. Remove the supernatant, aspirate 1 mL of fresh MS liquid medium, resuspend the Agrobacterium, and centrifuge at 4000 rpm for 5 min. Repeat the above steps once. After resuspending the bacteria in 1 mL of MS liquid medium, add it to 40 mL of MS liquid medium (containing 40 μL of 25 mg / L As) to prepare the infection solution. Infect the bacteria after standing for 2 hours.
[0074] (d) Place the leaves and stem segments back onto the pre-culture medium and co-culture at 28°C in the dark for 2-3 days until micro-bacterial spots form around the leaf cuts; remove the co-cultured tobacco leaves and stem segments and rinse them 6 times with sterile water containing 500 mg / L Cef. The first rinse is placed on a shaker and shaken for 30 minutes, followed by 5 minutes each time, to wash away Agrobacterium on the surface of the explants.
[0075] (e) Blot dry with filter paper and transfer to tobacco budding medium. The budding medium is MS + BA 1.0 mg / L + Hyg 25 mg / L + Cef 500 mg / L pH 5.8. Observe after 2 weeks. If no bacteria are found, reduce the Cef concentration. If bacteria are found, continue to maintain the Cef concentration.
[0076] (f) Change the culture medium every 2 weeks until adventitious buds appear. Cut off the regenerated seedlings (about 1 cm long) and transfer them to subculture medium MS + BA 0.2-0.1 mg / L + Hyg 25 mg / L + Cef 500 mg / L pH 5.8. When the seedlings grow to 2 cm in length (with small buds), transfer them to rooting medium MS + NAA 0.2 mg / L and culture at 25℃ with 12h light for about 3 weeks until robust roots develop.
[0077] (g) When the roots grow to 2-3cm and the seedlings are about 7-10cm tall, remove them from the Erlenmeyer flasks, wash off the culture medium from the roots, and transplant them into flower pots for greenhouse cultivation.
[0078] 3. Identification of NtFATB gene silencing lines
[0079] Genomic DNA was extracted from transgenic tobacco seedlings using a DNA extraction kit from Takara. Primers for the Kan resistance gene were designed for PCR amplification, and positive plants were screened. Ten positive plants were detected.
[0080] The primers for the Kan resistance gene are:
[0081] Kan-F: 5'-TCTGGACGAAGAGCATCAGG-3' (shown in SEQ ID NO. 9);
[0082] Kan-R: 5'-ATGAATCCAGAAAAGCGGCC-3' (shown in SEQ ID NO. 10).
[0083] RNA was extracted from the K326 control and three NtFATB-RNAi transgenic lines as described in Example 1, and cDNA was synthesized. Quantitative PCR was used to detect the expression of NtFATB in different transgenic lines. The detection primers and internal control primers are shown below:
[0084] The qRT-PCR primers are:
[0085] q NtFATB-F: 5'-ATCGCTATCCTACTTGGG-3' (shown in SEQ ID NO.11);
[0086] q NtFATB-R: 5'-AACCTGACCTGGTATTTT-3' (shown in SEQ ID NO.12).
[0087] The primers for the internal reference gene are:
[0088] 26s-F: 5'-GAAGAAGGTCCCAAGGGTTC-3' (shown in SEQ ID NO. 13);
[0089] 26s-R: 5'-TCTCCCTTTAACACCAACGG-3' (shown in SEQ ID NO.14).
[0090] Quantitative PCR test results as follows Figure 4 As shown, the transgenic line with the lowest expression level (NtFATB-RNAi5) was selected as the research object to observe the morphology and density of leaf glandular hairs.
[0091] Example 4: Observation and Counting Analysis of Glandular Trinus Morphology in Tobacco Leaves
[0092] In this embodiment, the transgenic line with the lowest yield in Example 3 (NtFATB-RNAi5) was selected as the research object to observe the morphology and density of leaf glandular hairs. The specific real-time operation is as follows:
[0093] When the seeds of the transgenic line NtFATB-RNAi5 germinated to the 4-6 leaf stage, three plants were randomly selected, and the first true leaf was taken for histochemical staining, glandular trichome morphology observation, and density statistics. Surface observation was performed using a super depth-of-field microscope. Newly sprouted leaves less than 5 cm in diameter were immersed in a 0.2% (w / v) Rhodamine B aqueous solution for 30 min, followed by rinsing three times with distilled water to remove unbound dye. After drying the surface, the leaf surface was observed using a super depth-of-field microscope, and three fields of view were randomly selected in the middle of the upper epidermis for glandular trichome density statistical analysis.
[0094] Results of observation of glandular hair quantity as follows Figure 5 As shown, after the NtFATB gene was silenced, the density of glandular hairs in the leaves of NtFATB-RNAi transgenic tobacco plants decreased significantly by 50%, indicating that the NtFATB gene plays an important role in regulating the development of glandular hairs in tobacco leaves.
[0095] The application of the tobacco glandular trichome development-related gene NtFATB in this invention involves inhibiting the expression of the NtFATB gene in tobacco plants, which can alter the density of glandular trichomes on tobacco leaves during the seedling stage. NtFATB gene expression can be inhibited through various RNA-mediated methods, such as: gene silencing mediated by plant virus vectors, Agrobacterium-mediated transformation into RNAi interference vectors, optimization of gene coding frames, and optimization of gene promoters. The methods for inhibiting gene expression described in this invention are not limited to the above-mentioned methods; any method that can inhibit NtFATB expression is acceptable.
[0096] When the NtFATB gene of this invention is constructed into a plant expression vector, any enhancing or inducible promoter can be added before its transcription initiation nucleotide. To facilitate the identification and screening of transgenic plant cells or plants, the vector can be modified, such as by adding plant-selective markers (GUS gene, luciferase gene, etc.) or antibiotic resistance markers (gentamicin, kanamycin, etc.). The transformed plant host can be either a monocotyledonous or dicotyledonous plant, such as tobacco, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, or alfalfa. The expression vector carrying the NtFATB gene of this invention can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plants can be cultured into plants.
[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. The application of the NtFATB gene in regulating tobacco glandular trichome development, characterized by: The nucleotide sequence of the NtFATB gene is shown in SEQ ID NO.
1.
2. The application of the NtFATB gene in regulating tobacco glandular trichome development according to claim 1, characterized in that: The density of tobacco glandular trichomes can be regulated by controlling the expression level of the NtFATB gene.
3. The application of the NtFATB gene according to claim 1 or 2 in regulating tobacco glandular trichome development, characterized in that: The regulation involved suppressing the expression of the NtFATB gene, which significantly reduced the density of glandular trichomes in tobacco leaves.
4. The application of the NtFATB gene according to claim 3 in regulating tobacco glandular trichome development, characterized in that: The inhibition was achieved by constructing an NtFATB gene RNAi vector, transforming it into plants, and inhibiting the expression of the NtFATB gene.
5. The application of the NtFATB gene according to claim 4 in regulating tobacco glandular trichome development, characterized in that: The method for constructing the RNAi vector includes the following steps: using nucleotides 1-300 in the nucleotide sequence shown in SEQ ID NO.1 as the guide sequence for RNAi, and inserting the nucleic acid fragments into the empty backbone vector in the forward and reverse directions.
6. The application of the NtFATB gene according to claim 5 in regulating tobacco glandular trichome development, characterized in that: The tobacco is K326.