Application of NtBCP gene in synthesis of cembranoids and improvement of tobacco germplasm resources

Abstract

The application discloses application of an NtBCP gene in synthesis of cembranoid diterpenoids in tobacco and improvement of tobacco germplasm resources and belongs to the technical field of tobacco genetic engineering. The application proves through experiments that the NtBCP gene is specifically expressed in tobacco trichome stalk cells and is highly related to metabolism of cembranoid diterpenoids in tobacco, and the nucleotide sequence of the NtBCP gene is shown as SEQ ID NO. 1. Further through overexpression technology, the application finds that after overexpression of the NtBCP gene in cultivated tobacco, the obtained transgenic tobacco has obviously increased cembranoid diterpenoids compared with normal control plants. The application can provide a new strategy and path for genetic engineering breeding of tobacco or cultivation of new varieties of other crops.

Description

Technical Field

[0001] This invention relates to the application of the NtBCP gene in the synthesis of diterpenoid compounds in tobacco and the improvement of tobacco germplasm resources, and belongs to the field of tobacco genetic engineering technology. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) belongs to the Solanaceae family and is an annual or short-lived perennial herb. In addition to being used to make cigarettes, dry tobacco, pipe tobacco, cigars, etc. for human consumption, it also has a variety of medicinal uses such as reducing swelling, detoxifying, and killing insects. It is an important economic crop and one of the plants with the largest biomass in nature, with great potential for exploitation and utilization.

[0003] As important secondary metabolites in plants, cembranes and diterpenoids act as phytoprotective agents to maintain normal plant growth and development, while also possessing favorable pharmacological properties. In tobacco (“tobacco cembranes and diterpenoids”), cembranes and diterpenoids exist primarily in two forms: cembrane-ol (CBT-ol) and cembrane-diol (CBD). They are synthesized in the glandular trichomes of tobacco and are the main components of the secretions from these trichomes, accounting for approximately 60% of the chemical composition of the leaves, and sometimes even exceeding 0.7% of the fresh leaf weight. Studies have reported that tobacco cembranes and diterpenoids exhibit good antifungal activity, and α-CBD, one isomer of tobacco CBD, possesses antitumor activity. Another isomer, β-CBD, exhibits significant neuroprotective activity and has been shown to be an excellent template for future drug development to treat Alzheimer's disease, Parkinson's disease, stroke, and other neurodegenerative diseases. Meanwhile, after the harvested tobacco leaves are roasted and processed, cepertrienol can be degraded into aroma substances such as solanone, which are closely related to human senses. These substances are an important source of tobacco aroma and give tobacco good economic value.

[0004] Therefore, cephaloditerpenoids have significant development and utilization value in plant-derived pesticide formulations and anticancer drugs. However, the low natural yield of cephaloditerpenoids limits their development in pesticides, food, and pharmaceuticals. With the rise of synthetic biology, targeted and precise regulation of plant terpenoid content has become an important need, and research on related metabolic pathways and regulatory mechanisms has gradually become a hot topic.

[0005] In tobacco, both the mevalonate pathway (MVA) and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway synthesize the central precursors of sisalpinoxans diterpenoids, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These two precursors undergo head-to-tail condensation under the catalysis of geranyl diphosphate synthase (GPPS) to form geranyl diphosphate (GPP) with a C10 skeleton. GPP then reacts with one molecule of IPP under the catalysis of farnesyl diphosphate synthase. Under the catalysis of geranylgeranyl diphosphate synthase (FPPS), farnesyl diphosphate (FPP) with one molecule of IPP forms a C15 backbone. FPP and one molecule of IPP then react with geranylgeranyl diphosphate synthase (GGPPS) to ultimately form geranylgeranyl diphosphate (GGPP) with a C20 backbone. GGPP, as an important precursor for the synthesis of cephalan diterpenoids, provides the cephalan skeleton structure for the formation of CBT-ol and CBD: it undergoes a cyclization reaction under the catalysis of cephalantrienol synthase (CBTS) to generate α- and β-CBT-ol. α- and β-CBT-ol then undergo hydroxylation reactions under the catalysis of cytochrome P450 hydroxylase (P450) to generate α- and β-CBD, respectively.

[0006] Although the biosynthetic pathways of cephalotriene diterpenoids in plants are relatively clear, our understanding of the related regulatory mechanisms and networks is still limited, which has become a bottleneck restricting the precise regulation of cephalotriene diterpenoid production. Chinese invention patent CN111548399B, published on February 8, 2022, discloses a MYB transcription factor, its encoding gene, and its application for regulating cephalotriene diterpenoid accumulation in tobacco. Specifically, it discloses that overexpression of the NtMYB306a gene in cultivated tobacco significantly increased the expression levels of two key enzyme genes in the tobacco cephalotriene metabolic pathway, CBT and CYP71D16. The cephalotriene diterpenoid content in NtMYB306a overexpressing lines was significantly higher than that in the control group, indicating that NtMYB306a participates in the biosynthetic regulation of tobacco cephalotriene metabolism and simultaneously accumulates cephalotriene diterpenoids. However, there are still relatively few ways to precisely regulate the expression of cephalosporin diterpenoids in plants. Therefore, discovering more genes that can precisely regulate the expression of cephalosporin diterpenoids in plants is of great significance for elucidating the molecular regulatory mechanism of cephalosporin biosynthesis in tobacco and for the innovation of germplasm resources. Summary of the Invention

[0007] The first objective of this invention is to provide the application of the NtBCP gene in the synthesis of tobacco cephalosporin diterpenoids, providing a new and precise regulatory gene for the synthesis of tobacco cephalosporin diterpenoids in the prior art.

[0008] The second objective of this invention is to provide the application of the NtBCP gene in the improvement of tobacco germplasm resources, thereby providing new available gene resources for the improvement of tobacco aroma germplasm resources.

[0009] To achieve the above objectives, the technical solution adopted in this invention for the application of the NtBCP gene in the synthesis of tobacco cephalosporin diterpenoids is as follows:

[0010] The application of the NtBCP gene in the synthesis of diterpenoid compounds from tobacco, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] The beneficial effects of the above technical solution are as follows: The application of the NtBCP gene in the synthesis of cephalosporin diterpenoids in tobacco is a pioneering invention. Previous experimental studies have shown that the NtBCP gene is differentially expressed in tobacco glandular trichomes. To further study the function of this gene, this invention constructed an overexpression vector, transformed it into tobacco, and obtained transgenic tobacco lines overexpressing the NtBCP gene through identification. Observation of the transgenic tobacco lines revealed that the NtBCP gene is specifically expressed in the trichome stalk cells of tobacco. Moreover, detection showed that the content of cephalosporin diterpenoids in the transgenic tobacco lines was significantly increased compared to the normal control group. This invention lays the foundation for elucidating the molecular regulatory mechanism of cephalosporin biosynthesis in tobacco and provides a new, precise regulatory gene for the synthesis of cephalosporin diterpenoids in tobacco.

[0012] As a further improvement, the cephalosan diterpenoid compound is cephalotrienil diol.

[0013] The beneficial effects of the above technical solution are as follows: Cipterpenoid diol is the main form of cipterpenoid diterpenoids in tobacco. Finding the gene that precisely regulates cipterpenoid diol will help to elucidate the molecular regulatory mechanism of tobacco cipterpenoid biosynthesis.

[0014] As a further improvement, overexpression of the NtBCP gene increased the content of cephalosporin diterpenoids in tobacco.

[0015] As a further improvement, the overexpression involves constructing an NtBCP gene overexpression vector to overexpress the NtBCP gene.

[0016] As a further improvement, the tobacco is K326.

[0017] To achieve the above objectives, the technical solution adopted in the application of the NtBCP gene in tobacco germplasm resource improvement of this invention is as follows:

[0018] The application of the NtBCP gene in the improvement of tobacco germplasm resources, wherein the nucleotide sequence of the NtBCP gene is shown in SEQ ID NO.1; the germplasm resource improvement is to increase the concentration of diterpenoid compounds in tobacco.

[0019] The beneficial effects of the above technical solution are as follows: the application of the NtBCP gene in tobacco germplasm resource improvement is a pioneering invention. This invention cloned the tobacco NtBCP gene and, after overexpressing the NtBCP gene in tobacco using Agrobacterium-mediated transformation, found a significant increase in the content of cephalothrix trienol in tobacco. Cephalothrix trienol can be degraded into solanone and other aroma compounds closely related to human sensory perception during tobacco processing. These aroma compounds are important sources of tobacco aroma; therefore, by regulating the content of cephalothrix trienol in tobacco, the aroma of tobacco can be controlled, providing new gene resources for tobacco germplasm resource improvement.

[0020] As a further improvement, the cephalosan diterpenoid compound is cephalotrienil diol.

[0021] As a further improvement, the content of cephalosporin diterpenoids in tobacco was increased by overexpressing the NtBCP gene.

[0022] As a further improvement, the tobacco is K326. Attached Figure Description

[0023] Figure 1 This is a gel electrophoresis image of the NtBCP gene clone in Example 1 of the present invention (where M represents DNA marker, and 1 and 2 represent PCR reaction products).

[0024] Figure 2 The expression level of NtBCP gene in the NtBCP overexpression lines in Example 3 of the present invention was analyzed (where CK represents control K326, OE-1 / OE-2 / OE-3 represent NtBCP overexpression lines, and ** represents P<0.01).

[0025] Figure 3 This is a confocal phenotypic observation of NtBCP overexpression lines in Example 4 of the present invention (TG:GFP represents the control line, TG:NtBCP-GFP represents the NtBCP overexpression line, green fluorescence represents the expressed protein, and red fluorescence represents chloroplasts).

[0026] Figure 4 This is an analysis of the content of ceperane diterpenoids in the NtBCP overexpression strain in Example 5 of the present invention (CK represents control K326, OE-1 / OE-2 / OE-3 represent NtBCP overexpression strains, α-CBD represents α-ceperanetrienediol, β-CBD represents β-ceperanetrienediol, * represents P<0.05, ** represents P<0.01). Detailed Implementation

[0027] Previous experimental studies in this invention have demonstrated the differential expression of the NtBCP gene in tobacco glandular trichomes. To further investigate the function of this gene, an overexpression vector was constructed and transformed into tobacco. Transgenic lines overexpressing the NtBCP gene were obtained through identification. Observation of the transgenic lines revealed that the NtBCP gene is specifically expressed in the trichome stalk cells of tobacco. Furthermore, detection showed that the content of diterpenoids in tobacco was significantly increased in the transgenic lines compared to the normal control group. This invention lays the foundation for elucidating the molecular regulatory mechanism of diterpenoid biosynthesis in tobacco and provides a new, precise regulatory gene for the synthesis of diterpenoids in tobacco.

[0028] Furthermore, cepertrienol can be degraded into aroma compounds such as solanone during tobacco processing, which are closely related to human sensory experience. These aroma compounds are an important source of tobacco aroma. Therefore, by adjusting the content of cepertrienol in tobacco, the aroma of tobacco can be controlled, providing new genetic resources for the improvement of tobacco germplasm resources.

[0029] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment are commercially available.

[0030] Biomaterials:

[0031] Tobacco material: Cultivated tobacco (Nicotiana tabacum) variety K326, purchased from Yuxi China Tobacco Seed Co., Ltd.;

[0032] The vector plasmid pCAMBIA1300 used for overexpressing the tobacco gene was purchased from the China Plasmid Vector Strains Cell Line Gene Preservation Center.

[0033] Experimental reagents:

[0034] Restriction endonucleases, dATP, dTTP, PrimerSTAR GXL DNA polymerase, MiniBEST Agarose Gel DNA Extraction Kit, and MiniBEST Plasmid Purification Kit were all purchased from TAKARA Biotechnology Co., Ltd.; T4 DNA polymerase and RNA extraction TRIZOL reagent were purchased from Invitrogen; reverse transcription kit was purchased from Roche; infusion enzyme was purchased from Clontech; DNase enzyme was purchased from Fermentas; and antibiotics such as kanamycin and rifampin were purchased from Shanghai Sangon Biotech Co., Ltd.

[0035] Experimental apparatus:

[0036] The target gene fragment was cloned using a gradient PCR instrument manufactured by Eppendorf, model: Mastercycler epgradient, made in Germany.

[0037] Specific examples of the application of the NtBCP gene of this invention in the synthesis of tobacco cephalosporin diterpenoids and the improvement of tobacco germplasm resources:

[0038] Example 1 Cloning of the NtBCP gene

[0039] In this embodiment, RNA was extracted from K326, and cDNA was synthesized using the extracted RNA as a template. After PCR reaction, the NtBCP gene was obtained. The specific implementation steps are as follows:

[0040] 1. RNA extraction

[0041] Young leaves of cultivated tobacco K326, aged 4 weeks, were used as samples. After flash freezing in liquid nitrogen, surface glandular hairs were collected, ground, and approximately 100 mg of powdered material 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 vortexed, and centrifuged. The supernatant was carefully removed and transferred to another centrifuge tube. 500 μL of isopropanol was added, and the precipitate was separated by centrifugation. The RNA was then washed with 75% ethanol, allowed to dry slightly at room temperature, and then dissolved in an appropriate volume of RNase-free water. The extracted total RNA was treated with DNase I. The digestion reaction system is shown in Table 1, and the treatment conditions were: incubation in a 37°C water bath for 30 min.

[0042] Table 1. DNase I-treated systems

[0043] reagents volume RNA 1μg <![CDATA[10×reaction buffer with MgCl2]]> 1μL DNase I, RNase-free 1 μL (1 U) DEPC-treated water to 10μL

[0044] 2. cDNA synthesis

[0045] Prepare the template RNA / primer mixture in Table 2 below 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.

[0046] Table 2 Template RNA / primer mixture preparation system

[0047] reagents volume RNA (100 ng / μL) 1μL Oligo(dT) Primer (50μM) 1μL <![CDATA[RNase free dH2O]]> 5μL Total Volume 7μL

[0048] Note: The RNA in Table 2 is the RNA that has been treated with DNase I in step 1.

[0049] After preparing the reverse transcription reaction solution in Table 3 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.

[0050] Table 3 cDNA reaction system

[0051] reagents volume The above template RNA / primer denaturing solution 7μL 5×M-MLV buffer 2μL dNTP Mixture (10mM each) 0.5μL RNase Inhibitor (40 U / μL) 0.25μL RTase M-MLV(RNase H-)(200U / μL) 0.25μL Total Volume 10μL

[0052] 3. Obtaining the NtBCP gene

[0053] The primer sequences used to amplify the full-length NtBCP gene are as follows:

[0054] NtBCP-F:5'-ATGGTTACAAGGATGAATATTGCA-3' (shown in SEQ ID NO.3);

[0055] NtBCP-R: 5'-CTACCACATTATAACTAATGGCAAT-3' (shown in SEQ ID NO. 4).

[0056] The PCR amplification system is shown in Table 4, and the reaction conditions are shown in Table 5.

[0057] Table 4 PCR amplification system

[0058] reagents volume GXL polymerase 1μL 5×GXL buffer 10μL dNTP Mixture (10 mM) 4μL Primer-F / R 8μL <![CDATA[ddH2O]]> 26μL cDNA 1μL Total Volume 20μL

[0059] Table 5 PCR reaction procedure

[0060]

[0061] The PCR products obtained from the amplification were subjected to 1% agarose gel electrophoresis (the gel electrophoresis results are shown in the figure). Figure 1 (As shown in the figure), the PCR amplification product was then recovered, purified, and sequenced.

[0062] Sequencing results show that the NtBCP gene cloned in this embodiment includes 789 bp, and its base sequence is shown in SEQ ID NO.1. The amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0063] Example 2 Construction of NtBCP gene overexpression vector

[0064] Based on the NtBCP gene cloned in Example 1, this embodiment constructs an overexpression vector for the NtBCP gene to further verify its function. The specific implementation steps are as follows:

[0065] 1. Enzyme digestion and ligation

[0066] Based on the promoter sequence of the NtBCP gene and the information of the vector pCAMBIA1300-GFP, SacI (5' end) and BamHI (3' end) restriction sites were designed at both ends of the target fragment. The fragment was sent to a synthesis company for full-sequence synthesis and assembled into the pCAMBIA1300-GFP vector to obtain the TG-p1300-GFP expression vector.

[0067] Using plasmid TG-p1300-GFP as a vector, the TG-p1300-GFP vector was double-digested with SacI and BamHI, and the digestion products were recovered. The NtBCP fragment obtained by PCR amplification was ligated into the digested TG-p1300-GFP vector using Infusion ligase. The ligation system (10 μL) was designed as shown in Table 6. The reaction conditions were 50℃ for 15 minutes, followed by incubation on ice for 2-3 minutes.

[0068] Table 6 Connection System

[0069] reagents volume 5×Infusion ligase 2μL TG-p1300-GFP after enzyme digestion 2μL PCR amplification products 6μL

[0070] 2. Conversion (Heat Shock Method)

[0071] Under aseptic conditions, add 10 μL of the ligation product from step 1 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 shake on a shaker at 37℃ and 120 rpm for about 1 h; take 200 μL of culture medium and spread it on LB solid medium containing the antibiotic kanamycin (50 μg / mL), and incubate upside down at 37℃ for 12 h.

[0072] 3. Screening and identification

[0073] Once the plaques reached a suitable size, several white spots were picked up with a sterilized pipette tip and cultured in LB liquid medium containing 50 μg / mL kanamycin for 12 hours with shaking. The plasmid was then extracted and subjected to enzyme digestion to confirm correct recombinant vector construction. Correctly constructed plasmids (or strains containing the plasmid) were preserved for later use. Finally, the correctly constructed recombinant plasmid was named TG-p1300-NtCBP-GFP.

[0074] Example 3: Construction of transgenic tobacco lines overexpressing the NtBCP gene

[0075] Based on the overexpression vector constructed in Example 2, this embodiment utilizes Agrobacterium-mediated tobacco transformation to transform the correctly constructed plasmid into tobacco, obtaining a transgenic tobacco line overexpressing the NtBCP gene. The specific implementation steps are as follows:

[0076] 1. Preparation before transformation (freeze-thaw transformation of Agrobacterium and preparation of bacterial solution for transfection)

[0077] 1 μg of the TG-p1300-GFP and TG-p1300-NtCBP-GFP vectors prepared in Example 2 were added to 100 μL of EHA105 Agrobacterium competent cells, mixed well, and incubated on ice for 30 min. Then, the cells were frozen in liquid nitrogen for 5 min, removed from the liquid nitrogen, and immediately placed in a 37°C water bath for 5 min, and then incubated on ice for 5 min. 500 μL of LB medium was added, and the cells were incubated at 28°C with shaking for 4 h. Finally, the bacterial culture was evenly spread on solid culture medium containing antibiotics (50 mg / L kanamycin and 50 mg / L rifampin) and incubated at 28°C for about 24 h.

[0078] Pick a single bacterial plaque and place it in 5 mL of LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampin. Incubate overnight at 28°C and 200 rpm until the bacterial concentration reaches OD500. 600 = Approximately 1.5; Add 2 mL of bacterial culture to a centrifuge tube and centrifuge at 4000 r / min for 5 min; Remove the supernatant, aspirate 1 mL of fresh MS liquid medium, resuspend Agrobacterium, centrifuge at 4000 r / min for 5 min, and repeat this operation once; Finally, add 1 mL of the resuspended bacterial culture to 40 mL of MS liquid medium (containing 40 μL, 25 mg / L acetylsyl syringone), which is the infection solution. After standing for 2 h, it can be used for infection.

[0079] 2. Leaf disc method conversion

[0080] (a) Sterile tobacco K326 seeds were sown on MS medium and cultured. When the tobacco seedlings grew to 3-5 cm (about 20-30 days), the apical buds were taken and placed on MS+BA (0.2 mg / L) medium (to strengthen the buds and promote rapid growth) for subculture.

[0081] (b) After 14 days of subculture (as long as small leaves are present), take leaves about 1cm×1cm in size, cut off the petiole, and make scratches on the surface and edges of the leaves. Place them on MS+BA (1.0mg / L) pre-culture medium (pH 6.0-6.5), with the upper side facing down and in close contact with the medium. Pre-culture in the dark for 2 days.

[0082] (c) Take out the leaves that were pre-cultured in step (b), immerse them in Agrobacterium infection solution for 15 minutes, and then remove them and blot dry the bacterial solution on sterilized dry filter paper.

[0083] (d) Place the infected leaves back onto the pre-culture medium and co-culture them 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 again and rinse them with sterile water containing 500 mg / L cephalosporin (Cef) to wash away Agrobacterium on the surface of the explants.

[0084] (e) After blotting off the surface liquid from step (d) with filter paper, transfer the sample to tobacco budding medium (MS + 1.0 mg / L BA + 25 mg / L Hyg + 500 mg / L Cef, pH 5.8);

[0085] During the cultivation process, the culture medium should be changed every 2 weeks until adventitious buds grow (usually 2 weeks).

[0086] Cut off the regenerated seedlings (about 1 cm long) and transfer them to subculture medium (MS + 0.2 mg / L BA + 25 mg / L Hyg + 500 mg / L Cef, pH 5.8) for culture;

[0087] When the seedlings grow to 2cm in length, they are transferred to rooting medium (MS + 0.2mg / L NAA) and cultured at 25℃ with 12h light for about 3 weeks to ensure the growth of robust roots.

[0088] (f) When the roots grow to 2-3cm and the seedlings are about 7-10cm tall, remove the Erlenmeyer flasks, wash off the culture medium from the roots, and transplant them into flower pots for greenhouse cultivation.

[0089] 3. Identification of transgenic lines

[0090] Genomic DNA was extracted from tobacco seedlings in step 2, primers were designed, and PCR was used to identify Kan resistance. The specific primer sequences are as follows:

[0091] Kan-F: 5'-TCTGGACGAAGAGCATCAGG-3' (shown in SEQ ID NO.5);

[0092] Kan-R: 5'-ATGAATCCAGAAAAGCGGCC-3' (shown in SEQ ID NO. 6).

[0093] The identification results showed that a total of 15 Kan-positive resistant plants were obtained.

[0094] Furthermore, the expression level of the NtBCP gene in transgenic plants was analyzed using real-time quantitative PCR. The primer sequences for the specific qRT-PCR analysis were designed as follows:

[0095] qNtBCP-F: 5'-GCCAAAGTCACAAAGAGTG-3' (shown in SEQ ID NO.7);

[0096] qNtBCP-R: 5'-GGCTGGTCCAACAGTTAT-3' (shown in SEQ ID NO. 8).

[0097] In the analysis process, the primers for the internal reference gene were designed as follows:

[0098] q26s-F: 5'-GAAGAAGGTCCCAAGGGTTC-3' (shown in SEQ ID NO. 9);

[0099] q26s-R: 5'-TCTCCCTTTAACACCAACGG-3' (shown in SEQ ID NO.10).

[0100] Some test results are as follows Figure 2 As shown. From Figure 2 It can be seen that the expression level of the NtBCP gene was increased to varying degrees in different transgenic lines. For ease of observation and research, the three transgenic lines with the highest expression levels (OE-1, OE-2, and OE-3) were selected as the subjects for subsequent confocal observation and NtBCP gene function research.

[0101] Example 4: NtBCP gene expression localization

[0102] Once the control plants and the three overexpressing transgenic lines (OE-1, OE-2 and OE-3) reached the seedling stage (about 4 weeks), the tobacco leaves were subjected to laser confocal analysis and photographed for record-keeping.

[0103] The results are as follows Figure 3 As shown, in the control line transformed with TG-p1300-GFP (TG:GFP), GFP signal was present and expressed throughout the entire glandular trichome; however, after overexpression of the NtBCP gene (TG:NtCBP-GFP), the fusion protein NtBCP-GFP was expressed only in the stalk cells of the glandular trichome (the stalk cells adjacent to the head cells). Compared with the control, NtBCP-GFP was expressed only in the stalk cells, indicating that the NtBCP gene can serve as a stalk cell marker gene for the classification of glandular trichome cell subtypes.

[0104] Example 5 Functional verification of the NtBCP gene

[0105] This embodiment selects the NtBCP overexpression lines (OE-1, OE-2, and OE-3) from Example 3 as the research object, and detects the content of cephalosporin diterpenoids in the leaves to verify the function of the NtBCP gene. The specific implementation operation is as follows:

[0106] Transgenic tobacco seeds from control plants and NtBCP overexpression lines were evenly sown in small pots containing nutrient soil and cultured in a light-controlled incubator. During the seedling stage, leaves were rapidly frozen in liquid nitrogen and then lyophilized. 20 mg of lyophilized fresh leaf powder was weighed, and dichloromethane (containing eicosatrienoic acid as an internal standard) was added. Extraction was performed by sonication for 60 min, followed by derivatization with BSTFA / DMF (1:1 v / v) at 70 °C for 60 min. After concentration and drying, 500 μL of the supernatant was loaded onto GC-MS for analysis.

[0107] Test results as follows Figure 4 As shown in the figure, compared with normal control plants, the content of α-cephalantrienide diterpenoids (α-CBD) and β-cephalantrienide diterpenoids (β-CBD) in the leaves of NtBCP-overexpressing tobacco plants was significantly increased. This result indicates that the NtBCP gene is closely related to the metabolism of cephalantrienide diterpenoids in tobacco, and based on this result, a certain technical and theoretical foundation can be laid for the breeding of new tobacco varieties.

[0108] 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. NtBCP The application of genes in the synthesis of tobacco cephalosporin diterpenoids is characterized by: The NtBCP The nucleotide sequence of the gene is shown in SEQ ID NO.1; the cephalosan diterpenoid compound is cephalosandiol.

2. As described in claim 1 NtBCP The application of genes in the synthesis of tobacco cephalosporin diterpenoids is characterized by: overexpression NtBCP Genes, the content of cephalosporin diterpenoids in tobacco increases.

3. As described in claim 2 NtBCP The application of genes in the synthesis of tobacco cephalosporin diterpenoids is characterized by: The overexpression is for constructing NtBCP Gene overexpression vectors, for NtBCP The gene is overexpressed.

4. As described in claim 1 NtBCP The application of genes in the synthesis of tobacco cephalosporin diterpenoids is characterized by: The tobacco is K326.

5. NtBCP The application of genes in the improvement of tobacco germplasm resources is characterized by: The NtBCP The nucleotide sequence of the gene is shown in SEQ ID NO.1; the germplasm resource improvement is to increase the content of tobacco cephalosporin diterpenoids; the cephalosporin diterpenoids are cephalosporin trienyl alcohol.

6. The method according to claim 5 NtBCP The application of genes in the improvement of tobacco germplasm resources is characterized by: Through overexpression NtBCP Genes that increase the content of cephalosporin diterpenoids in tobacco.

7. The method according to claim 5 NtBCP The application of genes in the improvement of tobacco germplasm resources is characterized by: The tobacco is K326.

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