Application of NtHCH5 gene in regulation and control of plant glandular hair development

Abstract

The invention discloses application of an NtHCH5 gene in regulation and control of plant glandular hair development, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the gene for regulating and controlling plant glandular hair development provided by the invention is as shown in SEQ ID NO. 1. An RNAi carrier of the NtHCH5 gene is constructed through an RNAi technology, a tobacco plant silenced by the NtHCH5 gene is cultivated, and it is found that compared with a normal tobacco plant, the density of glandular hairs in tobacco leaves after the NtHCH5 gene is silenced is remarkably reduced, and the glandular hairs are shortened. An economic plant tobacco is used as a research object, and experiments prove that the NtHCH5 gene participates in regulation and control of plant glandular hair development, a new target gene is provided for regulation and control of plant glandular hair development, and a gene regulation and control network of plant in-vivo epidermal hair is enriched. The gene function of the plant NtHCH5 gene is also enriched, and the gene has important significance for analyzing the diversity of the plant NtHCH5 gene.

Description

Technical Field

[0001] This invention relates to the application of the NtHCH5 gene in regulating the development of plant glandular trichomes, and belongs to the field of plant genetic engineering technology. Background Technology

[0002] The appendages on the plant epidermis are collectively called epidermal trichomes. These are specialized hair-like appendages formed by plant epidermal cells. They have a simple structure, consisting of either a single cell or multiple cells; and exhibit diverse morphologies, commonly including hook-shaped, scale-like, branched, capitate, and stellate forms. Studies have found that some plants can simultaneously grow multiple types of epidermal trichomes, the type and size of which are determined by the plant organ or organ surface (adaxial and abaxial, leaf and stem) from which the trichomes are generated. There are many types of plant epidermal trichomes. Based on their secretory capacity, they are mainly divided into two categories: non-glandular trichomes (protective tissue) and glandular trichomes (secreting tissue). Based on cell composition, they can be divided into unicellular trichomes and multicellular trichomes, and can be further subdivided based on whether they are branched.

[0003] Plant epidermal hairs have a wide range of applications, helping to resist external biological invasions and various stresses. Studies have found that epidermal hairs, with their superior physical structure—dense, long, and curly—defend against pests. The terpenes, alkaloids, resins, and volatile oils secreted by these hairs can also poison some bacteria or insects, thus resisting their invasion. Furthermore, epidermal hairs exhibit significant resistance to external stressors such as drought, cold, and strong light radiation. Epidermal hairs also have economic value. For example, cotton fiber, derived from the glandular hairs of cotton seeds, is a single-celled fiber formed from the differentiation and development of epidermal cells of the cotton coxoderm, and is a major raw material for textiles. Epidermal hairs also have medicinal value, such as artemisinin and menthol. Artemisinin, a sesquiterpene component secreted by the capitulum of Artemisia annua, is an important raw material for treating malaria; while menthol, a fragrance derived from the glandular hairs of peppermint, possesses various biological activities including analgesia, anti-inflammation, hepatoprotection, choleretic effects, penetration enhancement, and antimicrobial activity. Therefore, molecular-level research on the developmental patterns of plant epidermal hairs is particularly important, as it not only involves the theoretical basis of cell differentiation but also has potential guiding significance for agricultural production.

[0004] Tobacco (Nicotiana tabacum L.) is an important economic crop with significant economic value globally. 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 from 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 accumulation of secretions in tobacco leaves are closely related to tobacco plant resistance and aroma quality. Therefore, the occurrence and metabolic regulation of tobacco glandular trichomes are of great significance for studying abiotic stress and aroma quality in tobacco.

[0005] However, current research on tobacco glandular trichomes mainly focuses on: (1) the effects of trichome morphology, density, and secretion composition on tobacco resistance and quality; and (2) the regulation of glandular trichome metabolites by a certain gene. 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 great theoretical and practical significance for breeding new varieties with different glandular trichome densities and types. Summary of the Invention

[0006] The purpose of this invention is to apply the NtHCH5 gene to the regulation of plant glandular trichome development, providing a new regulatory target for plant glandular trichome development.

[0007] To achieve the above objectives, the technical solution adopted in this invention for the application of the NtHCH5 gene in regulating plant glandular trichome development is as follows:

[0008] The application of the NtHCH5 gene in regulating the development of plant glandular hairs, the nucleotide sequence of the NtHCH5 gene is shown in SEQ ID NO.1.

[0009] The beneficial effects of the above technical solution are as follows: The application of the NtHCH5 gene in regulating plant glandular trichome development is a pioneering invention. The nucleotide sequence of the gene regulating plant glandular trichome development provided by this invention is shown in SEQ ID NO.1. This invention constructs an RNAi vector for the NtHCH5 gene using RNAi technology, cultivates tobacco plants with NtHCH5 gene silence, and finds that compared with normal tobacco plants, the density of glandular trichomes in tobacco leaves with NtHCH5 gene silence is significantly reduced, and the glandular trichomes are shorter. This invention uses the economically important plant tobacco as the research object, and experimentally demonstrates that the NtHCH5 gene participates in regulating plant glandular trichome development, providing a new target gene for the regulation of plant glandular trichome development, enriching the gene regulatory network of plant epidermal trichomes; moreover, this invention also enriches the gene function of the plant NtHCH5 gene, which is of great significance for analyzing the diversity of the plant NtHCH5 gene.

[0010] Specifically, the nucleotide sequence of the plant glandular trichome development-related gene (encoding gene for 3-hydroxyisobutyryl-CoA hydrolase-like protein 5) NtHCH5 provided by this invention is shown in SEQ ID NO.1. Alternatively, it may be a nucleotide sequence that can hybridize with the DNA sequence defined in SEQ ID NO.1 under highly stringent conditions; or a DNA sequence that has more than 90% homology with the DNA sequence defined in SEQ ID NO.1 and encodes a protein with the same function.

[0011] Tobacco NtHCH5 encodes a protein associated with plant 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 derivative polypeptides that affect changes in plant glandular trichome density and length.

[0012] 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.

[0013] As a further improvement, the regulation involves suppressing the expression of the NtHCH5 gene, which significantly reduces the density of glandular hairs in plant leaves and shortens their length.

[0014] As a further improvement, the inhibition was achieved by constructing an RNAi vector of the NtHCH5 gene, transforming plants, and screening and identifying plants with significantly reduced glandular hair density.

[0015] As a further improvement, the RNAi vector of the NtHCH5 gene is prepared by the following steps: using a specific sequence in the NtHCH5 gene as a guide sequence, it is inserted into the starting vector in the forward and reverse directions.

[0016] As a further improvement, the nucleotide sequence of the guide sequence is shown in SEQ ID NO.3.

[0017] As a further improvement, the launch carrier is PBWA(V)HS.

[0018] As a further improvement, the plant is a member of the Solanaceae family.

[0019] As a further improvement, the Solanaceae plant is tobacco. Attached Figure Description

[0020] Figure 1 This is an electrophoresis diagram of the PCR fragment product of the NtHCH5 gene in Example 1 of the present invention;

[0021] Figure 2This is the RNAi map of the RNAi interference vector pBWA(V)HS-RNAi in Example 2 of the present invention;

[0022] Figure 3 This is the pBWA(V)HS-NtHCH5-RNAi restriction enzyme digestion pattern in Example 2 of the present invention;

[0023] Figure 4 This is an analysis of gene expression in the NtHCH5-RNAi transgenic line in Example 3 of the present invention;

[0024] Figure 5 This study analyzes the morphology and density of glandular hairs in the NtHCH5-RNAi transgenic line and the control K326 in Example 4 of this invention. Detailed Implementation

[0025] The development of plant epidermal trichomes is influenced by multiple regulatory factors. In recent years, significant progress has been made in studying the developmental patterns of Arabidopsis thaliana using molecular genetics methods, establishing gene regulatory networks. However, the regulation of epidermal trichome development in other plants differs greatly from that in Arabidopsis, requiring further research to elucidate the molecular mechanisms of epidermal trichome development in these plants. Based on this, this invention provides the application of the NtHCH5 gene in regulating plant glandular trichome development.

[0026] The application of the plant glandular trichome development-related gene NtHCH5 in this invention involves inhibiting its expression in plants, which can alter the density and length of glandular trichomes in seedling leaves. NtHCH5 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 NtHCH5 gene expression is acceptable.

[0027] When the NtHCH5 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 used can be processed, 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 NtHCH5 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.

[0028] 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.

[0029] 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.

[0030] Biomaterials:

[0031] 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 NtHCH5. 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.

[0032] Tobacco seedlings were harvested, and their leaves were flash-frozen in liquid nitrogen for subsequent molecular experiments; other leaves were collected, freeze-dried, and used for subsequent metabolic assays.

[0033] Specific embodiments of the application of the NtHCH5 gene in regulating plant glandular trichome development:

[0034] Example 1: Tobacco RNA Extraction and cDNA Synthesis

[0035] 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, PCR amplification of the NtHCH5 gene was performed to obtain the PCR amplification product. The specific implementation steps are as follows:

[0036] 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.

[0037] 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.

[0038] Table 1 Template RNA / Primer Mixture System

[0039]

[0040]

[0041] 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.

[0042] Table 2 Reverse Transcription Reaction Solution

[0043]

[0044] Using K326 cDNA as a template, primers were designed based on information from the tobacco genome database to perform PCR amplification of the NtHCH5 gene, and the PCR amplification product was obtained (the fragment amplified in this embodiment is the specific sequence of the NtHCH5 gene, which is used as the guide sequence for the subsequent construction of the RNAi vector, and the nucleotide sequence is shown in SEQ ID NO3).

[0045] Primers for amplifying the forward NtHCH5 gene fragment:

[0046] NtHCH5-F: 5'-CAGTGGTCTCTGCCAATGAAGTTCTCTG-3' (shown in SEQ ID NO.4);

[0047] NtHCH5-R: 5'-CGATGGTCTCGAAGCCACAGTCTGGATG-3' (shown in SEQ ID NO. 5).

[0048] LOOP primer:

[0049] F: 5'-CGATGGTCTCACAGGTCTAGTTTTTCTCCTT-3' (shown in SEQ ID NO. 6);

[0050] R: 5'-CGATGGTCTCAGCCCGGGCTCTGTAACTATC-3' (shown in SEQ ID NO.7).

[0051] Primers for amplifying the reverse NtHCH5 gene fragment:

[0052] NtHCH5-F: 5'-CAGTGGTCTCTCCAGACTGTGGCTTC-3' (shown in SEQ ID NO.8);

[0053] NtHCH5-R: 5'-CAGTGGTCTAGAGAACTTCATTGGCA-3' (as shown in SEQ ID NO.9).

[0054] The PCR amplification system is shown in Table 3 below, and the PCR reaction procedure is shown in Table 4 below.

[0055] Table 3 PCR amplification system

[0056]

[0057]

[0058] Table 4 PCR reaction procedures

[0059]

[0060] 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.

[0061] Example 2: Construction of gene silencing vector

[0062] Based on the NtHCH5 gene fragment obtained in Example 1, this invention further constructs an RNAi vector to silence the NtHCH5 gene. The specific implementation steps are as follows:

[0063] 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.

[0064] Table 5 Infusion Connection System

[0065]

[0066] The fragment mixture was reacted at 50°C for 15 minutes, then placed on ice for 2-3 minutes.

[0067] 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.

[0068] 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 NtHCH5 gene (pBWA(V)HS-NtHCH5-RNAi vector) was successfully constructed.

[0069] Example 3: Agrobacterium-mediated tobacco transformation and identification of transgenic plants

[0070] In this embodiment, the RNAi vector (pBWA(V)HS-NtHCH5-RNAi vector) for silencing the NtHCH5 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:

[0071] 1. Freeze-thaw transformation of Agrobacterium

[0072] Add 1 μg pBWA(V)HS-NtHCH5-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.

[0073] 2. Leaf disc method for converting tobacco variety K326

[0074] (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.

[0075] (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.

[0076] 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.

[0077] (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 acetylsuccinone (As, 25 mg / L) to the bacterial suspension at a 1:10 ratio (10 mL of bacterial suspension to 1.5 mL of bacterial cells in one tube), 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 dry the bacterial suspension on sterilized, dry filter paper.

[0078] 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 600 When 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.

[0079] (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.

[0080] (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.

[0081] (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.

[0082] (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.

[0083] 3. Identification of NtHCH5 gene silencing lines

[0084] 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. Five positive plants were detected.

[0085] The primers for the Kan resistance gene are:

[0086] Kan-F: 5'-TCTGGACGAAGAGCATCAGG-3' (shown in SEQ ID NO. 10);

[0087] Kan-R: 5'-ATGAATCCAGAAAAGCGGCC-3' (shown in SEQ ID NO. 11).

[0088] RNA was extracted from the K326 control and three NtHCH5-RNAi transgenic lines as described in Example 1, and cDNA was synthesized. Quantitative PCR was used to detect the expression of NtHCH5 in different transgenic lines. The detection primers and internal control primers are shown below:

[0089] The qRT-PCR primers are:

[0090] q NtHCH5-F: 5'-GTTGCTGCTGGACTTGCC-3' (shown in SEQ ID NO.12);

[0091] q NtHCH5-R: 5'-TCCCACCTGAACTCTTCTTTGT-3' (shown in SEQ ID NO.13).

[0092] The primers for the internal reference gene are:

[0093] 26s-F: 5'-GAAGAAGGTCCCAAGGGTTC-3' (shown in SEQ ID NO. 14);

[0094] 26s-R: 5'-TCTCCCTTTAACACCAACGG-3' (shown in SEQ ID NO.15).

[0095] Quantitative PCR test results as follows Figure 4As shown, the transgenic line with the lowest expression level (NtHCH5-RNAi4) was selected as the research object to observe the morphology and density of glandular hairs on the leaves.

[0096] Example 4: Observation and Counting Analysis of Glandular Trinus Morphology in Tobacco Leaves

[0097] In this embodiment, the transgenic line with the lowest yield in Example 3 (NtHCH5-RNAi4) was selected as the research object to observe the morphology and density of leaf glandular hairs. The specific real-time operation is as follows:

[0098] When the seeds of the transgenic line NtHCH5-RNAi4 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.

[0099] Results of observation of glandular hair quantity as follows Figure 5 As shown, after the NtHCH5 gene was silenced, the density of glandular hairs in the leaves of NtHCH5-RNAi transgenic tobacco plants decreased significantly by 66.7%, and the length of the glandular hairs was significantly shortened, indicating that the NtHCH5 gene plays an important role in regulating the development of glandular hairs in tobacco leaves.

[0100] 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 NtHCH5 gene in regulating plant glandular trichome development, characterized by: The nucleotide sequence of the NtHCH5 gene is shown in SEQ ID NO.

1.

2. The application of the NtHCH5 gene in regulating plant glandular trichome development according to claim 1, characterized in that: The regulation involved suppressing the expression of the NtHCH5 gene, resulting in a significant decrease in glandular hair density and a shortening of glandular hair length in plant leaves.

3. The application of the NtHCH5 gene in regulating plant glandular trichome development according to claim 2, characterized in that: The inhibition was achieved by constructing an RNAi vector for the NtHCH5 gene, transforming plants, and screening and identifying plants with significantly reduced glandular hair density.

4. The application of the NtHCH5 gene in regulating plant glandular trichome development according to claim 3, characterized in that: The RNAi vector for the NtHCH5 gene is prepared by the following steps: using a specific sequence from the NtHCH5 gene as a guide sequence, inserting it sequentially into the starting vector in both forward and reverse directions.

5. The application of the NtHCH5 gene in regulating plant glandular trichome development according to claim 4, characterized in that: The nucleotide sequence of the guide sequence is shown in SEQ ID NO.

3.

6. The application of the NtHCH5 gene in regulating plant glandular trichome development according to claim 4, characterized in that: The launch vehicle is PBWA(V)HS.

7. The application of the NtHCH5 gene according to any one of claims 1 to 6 in regulating plant glandular trichome development, characterized in that: The plant in question belongs to the Solanaceae family.

8. The application of the NtHCH5 gene according to claim 8 in regulating plant glandular trichome development, characterized in that: The plant in question is tobacco, belonging to the Solanaceae family.

Images