Nicotiana tabacum isopropyl malate dehydratase NtIPDS, encoding gene, RNAi interference vector and application

Abstract

The application discloses tobacco isopropyl malate dehydrase NtIPDS, a coding gene, an RNAi interference carrier and application. The application successfully clones a coding gene of isopropyl malate dehydrase in tobacco, and the nucleotide sequence is as follows: (1) the nucleotide sequence shown in SEQ ID NO. 1; (2) the nucleotide sequence shown in SEQ ID NO. 1 is substituted and / or deleted and / or added with one or more nucleotides and the nucleotide sequence of the same functional protein. Based on the existing tobacco genetic engineering progress and related plant phenotype observation and identification, the NtIPDS gene is deeply researched, and it is found that the NtIPDS gene is related to tobacco chloroplast development and acyl sugar metabolism. After the gene is silenced, the chlorosis plant growth is slow, the chloroplast development is found to be abnormal through transmission electron microscope observation, and meanwhile, it is found that the acyl sugar content of the silenced plant is significantly increased through metabolic detection analysis.

Description

Technical Field

[0001] This invention relates to tobacco isopropyl malate dehydratase NtIPDS, its encoding gene, RNAi interference vector, and its applications, belonging to the field of plant genetic engineering technology. Background Technology

[0002] Acyl sugars (glycoesters, mainly in the form of sucrose esters) are a class of secondary metabolites detected to date in the glandular hairs of plants in the Solanaceae, Caryophyllaceae, Pedaliaceae, Rosaceae, and Geraniaceae families, as well as in the roots of Brassicaceae plants. They possess various biological activities, including insect resistance and antibacterial activity. Increasing the content of glycoesters has long been an important goal in the breeding of Solanaceae plants such as tomatoes and potatoes. Tobacco (Nicotiana tabacum) is an important economic crop, and glycoesters are important aroma precursors in tobacco, contributing significantly to the aroma quality of tobacco leaves. They are also important components in the flavoring and additive production of cigarettes. With the development of Chinese-style cigarettes and the development of new tobacco products, the cigarette industry has a huge demand for high-end cigarette raw materials with good aroma quality, sufficient aroma volume, and distinctive aroma characteristics. Therefore, increasing the glycoester content of tobacco leaves through various means, especially cultivating new tobacco varieties with high glycoester content, is of great significance.

[0003] Isopropyl malate dehydratase (IPDS) is primarily involved in leucine metabolism in plants, hence its abbreviation LeuC. In Arabidopsis thaliana, this protein also participates in the methionine chain elongation process in the glucosinolate biosynthesis pathway. There are three IPDS subtypes (AtIPDS1-3) in Arabidopsis. Silencing AtIPDS2 or AtIPDS3 genes alone does not produce a significant phenotype, but silencing both AtIPDS2 and AtIPDS3 together can affect pollen and embryo sac development. However, research on IPDS is currently limited, and its function in tobacco has not been reported. Therefore, investigating the function of IPDS in tobacco is an urgent problem to be solved. Summary of the Invention

[0004] To address the aforementioned problems, the first objective of this invention is to provide the NtIPDS ​​gene, which encodes the tobacco isopropyl malate dehydratase. This invention has successfully cloned the NtIPDS ​​gene, which encodes the tobacco isopropyl malate dehydratase NtIPDS.

[0005] The second objective of this invention is to provide tobacco isopropyl malate dehydratase NtIPDS, which is closely related to tobacco chloroplast development and acyl sugar content.

[0006] The third objective of this invention is to provide the application of the tobacco isopropyl malate dehydratase encoding gene NtIPDS ​​in regulating tobacco chloroplast development and / or acyl sugar content, in order to solve the problem that the function of isopropyl malate dehydratase in tobacco is unknown in the prior art.

[0007] The fourth objective of this invention is to provide an RNAi interference vector that can efficiently silence the NtIPDS ​​gene, successfully obtaining NtIPDS ​​gene-silenced tobacco plants, thus laying the foundation for studying the function of the NtIPDS ​​gene in tobacco.

[0008] The fifth objective of this invention is to provide the application of the tobacco isopropyl malate dehydrase encoding gene NtIPDS ​​or the RNAi interference vector in the breeding of new tobacco varieties. By silencing the NtIPDS ​​gene in tobacco plants using RNAi interference technology, it was found that the acyl sugar content in the silenced lines was significantly increased, thus obtaining new tobacco varieties with increased acyl sugar content.

[0009] To achieve the above objectives, the technical solution adopted by the tobacco isopropyl malate dehydrase encoding gene NtIPDS ​​in this invention is as follows:

[0010] The nucleotide sequence of the NtIPDS ​​gene, which encodes tobacco isopropyl malate dehydratase, is as follows:

[0011] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0012] (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.1 that has been substituted and / or deleted and / or added with one or more nucleotides and expresses the same functional protein.

[0013] The beneficial effects of the above technical solution are as follows: The present invention successfully cloned the NtIPDS ​​gene, which encodes tobacco isopropyl malate dehydratase, using specific primers, laying the foundation for studying the function of tobacco isopropyl malate dehydratase in tobacco.

[0014] To achieve the above objectives, the technical solution adopted by the tobacco isopropyl malate dehydratase NtIPDS ​​of the present invention is as follows:

[0015] The amino acid sequence of tobacco isopropyl malate dehydratase (NtIPDS) is as follows:

[0016] (1) The amino acid sequence shown in SEQ ID NO.2;

[0017] (2) Derivative proteins with the same function, but with the amino acid sequence shown in SEQ ID NO.2 replaced and / or deleted and / or added to one or more amino acid residues.

[0018] The beneficial effects of the above technical solution are as follows: This invention demonstrates through experiments that tobacco isopropyl malate dehydratase NtIPDS ​​regulates chloroplast development and acyl sugar content in tobacco plants during tobacco growth, thus revealing the function of isopropyl malate dehydratase in tobacco and providing a new direction for studying the function of isopropyl malate dehydratase in plants.

[0019] To achieve the above objectives, the technical solution adopted by the present invention for the application of the tobacco isopropyl malate dehydrase encoding gene NtIPDS ​​in regulating tobacco chloroplast development and / or acyl sugar content is as follows:

[0020] Application of the NtIPDS ​​gene, which encodes tobacco isopropyl malate dehydratase, in regulating tobacco chloroplast development and / or acyl sugar content.

[0021] The beneficial effects of the above technical solution are as follows: This invention demonstrates through experiments that the NtIPDS ​​gene, which encodes tobacco isopropyl malate dehydratase, is significantly correlated with chloroplast development and acyl sugar content in tobacco.

[0022] As a further improvement, after inhibiting the expression of the NtIPDS ​​gene, chloroplasts in tobacco plants showed poor development, structural damage, and / or a significant increase in acyl sugar content.

[0023] The beneficial effects of the above technical solution are as follows: This invention constructs an RNAi interference vector targeting the NtIPDS ​​gene, and transforms the constructed RNAi interference vector into tobacco plants using Agrobacterium-mediated transformation. Detection revealed that inhibiting NtIPDS ​​gene expression resulted in poor chloroplast development and structural damage in tobacco plants, and a significant increase in acyl sugar content. This provides new gene resources for the regulation of chloroplast development in tobacco and enriches the gene network regulating acyl sugar in tobacco.

[0024] To achieve the above objectives, the technical solution adopted by the RNAi interference vector of the present invention is as follows:

[0025] The RNAi interference vector contains a specific nucleotide fragment of the NtIPDS ​​gene as a guide sequence.

[0026] The beneficial effects of the above technical solution are that the interference lines obtained by RNAi have better and more stable gene silencing effects than the VIGS lines, and can be passed on to the next generation.

[0027] As a further improvement, the nucleotide sequence of the guide sequence is shown in SEQ ID NO.3.

[0028] As a further improvement, the guide sequence was inserted into the PBWA(V)HS empty vector for screening and sequencing identification.

[0029] To achieve the above objectives, the technical solution adopted in the application of the tobacco isopropyl malate dehydratase encoding gene NtIPDS ​​or the RNAi interference vector in the breeding of new tobacco varieties is as follows:

[0030] Application of the tobacco isopropyl malate dehydrase encoding gene NtIPDS ​​or RNAi interference vector in the breeding of new tobacco varieties.

[0031] The beneficial effects of the above technical solution are as follows: This invention uses RNA interference silencing technology to interfere with the expression of the NtIPDS ​​gene in tobacco, obtains transgenic plants with NtIPDS ​​gene silence, and detects that the acyl sugar content in the tobacco leaves of NtIPDS ​​gene-silenced plants is significantly increased. This provides a new strategy and path for molecular breeding to regulate the acyl sugar content of tobacco and other plants, and also lays a theoretical foundation for improving tobacco quality and enhancing tobacco smoke.

[0032] As a further improvement, silencing the NtIPDS ​​gene significantly increased the acyl sugar content in tobacco, resulting in a new tobacco variety with increased acyl sugar content.

[0033] As a further improvement, the new tobacco variety is obtained by the following method: transforming Agrobacterium tumefaciens with RNAi vector as an infection solution, then transforming tobacco, and obtaining a new tobacco variety with significantly increased leaf acyl sugar content through screening and identification.

[0034] The beneficial effects of the above technical solution are as follows: This invention constructs an RNAi interference vector targeting the NtIPDS ​​gene, and uses Agrobacterium-mediated transformation to transfer the constructed RNAi interference vector into tobacco plants, successfully constructing tobacco plants with silenced NtIPDS ​​genes, providing a plant model for studying the function of NtIPDS ​​genes in tobacco.

[0035] Preliminary research on the NtIPDS ​​gene in this invention shows that the gene is related to chloroplast development. Silencing the gene can increase the acyl sugar content in leaves. Utilizing this characteristic, the gene can be silenced using gene silencing technology, which is of great significance for the breeding of new tobacco varieties with high acyl sugar content and provides a technical means for high-quality tobacco breeding. Therefore, it has significant practical value. Attached Figure Description

[0036] Figure 1 The relative expression level of the NtIPDS ​​gene in Experiment Example 3 of this invention (K326 is a normal control plant, and R1, R2 and R3 are NtIPDS ​​gene-silenced plants);

[0037] Figure 2The phenotypes of NtIPDS ​​gene-silenced plants in Experiment Example 3 of this invention are shown (from left to right: K326 control plant, R1 plant, R2 plant and R3 plant, where R1, R2 and R3 are NtIPDS ​​gene-silenced plants, all of which are seedlings).

[0038] Figure 3 The chlorophyll content of NtIPDS ​​gene-silenced plants in Experiment Example 4 of this invention (K326 is a normal control plant, and R1, R2 and R3 are NtIPDS ​​gene-silenced plants);

[0039] Figure 4 The chlorophyll fluorescence parameter Fv / Fm in Experiment Example 4 of this invention (K326 is a normal control plant, and R1 is a NtIPDS ​​gene-silenced plant);

[0040] Figure 5 The transmission electron microscopy observation of the leaves of NtIPDS ​​gene-silenced plants in Experiment Example 4 of this invention (AB, CD and EF are each a group, all of which are leaves in the vigorous growth stage, and BDF are excerpts from ACE, where AB is the K326 control leaf (relatively green), CD and EF are leaves of RNAi gene-silenced plants, CD leaves are wrinkled and unevenly chlorotic, EF leaves are wrinkle-free and uniformly chlorotic).

[0041] Figure 6 The content of acyl sugar in the NtIPDS ​​gene-silenced plant in Experimental Example 5 of this invention. Detailed Implementation

[0042] 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.

[0043] All plant tissue materials in the following examples were obtained from the flue-cured tobacco (Nicotiania tabacum L.) variety K326 and K326 transgenic plants with RNAi interference NtIPDS. 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.

[0044] Example 1: Tobacco isopropyl malate dehydratase encoding gene NtIPDS ​​gene

[0045] The nucleotide sequence of the tobacco isopropyl malate dehydrase encoding gene NtIPDS ​​in this embodiment is as follows:

[0046] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0047] (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.1 that has been substituted and / or deleted and / or added with one or more nucleotides and expresses the same functional protein.

[0048] Example 2 Tobacco Isopropyl Malate Dehydrase NtIPDS

[0049] The amino acid sequence of the tobacco isopropyl malate dehydrase NtIPDS ​​in this embodiment is as follows:

[0050] (1) The amino acid sequence shown in SEQ ID NO.2;

[0051] (2) Derivative proteins with the same function, but with the amino acid sequence shown in SEQ ID NO.2 replaced and / or deleted and / or added to one or more amino acid residues.

[0052] Example 3: RNAi interference vector

[0053] The RNAi interference vector of this embodiment contains a specific nucleotide fragment of the NtIPDS ​​gene as a guide sequence, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0054] Experimental Example 1: Cloning of the NtIPDS ​​gene fragment

[0055] In this experiment, total RNA was extracted from young leaves of tobacco K326, reverse transcribed into cDNA, and the NtIPDS ​​gene was successfully cloned using the cDNA as a template. The specific experimental procedures are as follows:

[0056] 1. Extraction of total RNA from tobacco

[0057] Young leaves of tobacco K326, approximately 4 weeks old, were used as samples. After thorough grinding into powder using liquid nitrogen, about 100 mg of the powder was placed in a 1.5 mL centrifuge tube containing 1.0 mL of TRIZOL reagent. Then, 200 μL of chloroform was added, the mixture was vortexed, centrifuged, and the upper aqueous phase was carefully 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% ethanol, allowed to dry slightly at room temperature, and then dissolved in an appropriate volume of RNase-free water. Finally, the extracted total RNA was treated with DNase I for subsequent cDNA preparation. The DNase I digestion system was as follows: 10 μL.

[0058] Total RNA from tobacco, 1 μg; 10× reaction buffer with MgCl2, 1 μL; DNase I (RNase-free), 1 μL (1 U); DEPC-treated water added to 10 μL.

[0059] The reaction conditions were: place in a 37°C water bath for 30 minutes.

[0060] 2. cDNA synthesis

[0061] Prepare the template RNA / primer mixture 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.

[0062] Table 1. Preparation of RNA / primer mixture

[0063] name Dosage RNA (100 ng / μL) 1μL Oligo(dT) Primer (50μM) 1μL <![CDATA[RNase free dH2O]]> 5μL Total Volume 7μL

[0064] After preparing the reverse transcription reaction solution 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.

[0065] Table 2 Preparation of Reverse Transcription Reaction Solution

[0066]

[0067]

[0068] 3. PCR amplification

[0069] The primer sequences for PCR amplification are as follows:

[0070] Primers for amplifying the NtIPDS ​​gene fragment (underlined parts are vector adapter sequences):

[0071] NtIPDS-F: 5'- TTTCAGGGCCAT ATGGCATCTTCTGCCATC-3' (shown as SEQ ID NO.4);

[0072] NtIPDS-R: 5'- CTCGAATTCGGATCC TCACTGCAAAAACTCTCTG-3' (shown in SEQ ID NO.5).

[0073] Using the cDNA prepared in step 2 as a template, PCR amplification was performed. The design of the 50 μL amplification system is shown in Table 3.

[0074] Table 3 PCR reaction system

[0075] Reagent Name Dosage cDNA template 1μL GXL polymerase 1μL 5×GXL buffer 10μL dNTP Mixture (10mM) 4μL Primer-F / R 8μL <![CDATA[ddH2O]]> 26μL

[0076] The PCR reaction procedure is shown in Table 4.

[0077] Table 4 PCR reaction procedure

[0078]

[0079] The PCR products obtained from the amplification were subjected to 1% agarose gel electrophoresis, and then the PCR amplification products were recovered, purified, and sequenced.

[0080] Experiment Example 2: Construction of RNAi Vectors

[0081] Based on the NtIPDS ​​gene cloned in Experiment 1, this experiment further validates the function of this gene by constructing an RNAi interference vector targeting the NtIPDS ​​gene. The specific experimental procedure is as follows:

[0082] 1. Enzyme digestion and ligation

[0083] Nucleotides 204-404 of the NtIPDS ​​gene were used as the guide sequence for RNAi, as shown in SEQ ID NO.3, for PCR amplification in both directions. The plasmid pBWA(V)HS-RNAi was used as the vector; the pBWA(V)HS vector was digested with Pst I, and the digestion products were recovered separately. The RNAi product obtained from PCR amplification was ligated to the digested pBWA(V)HS vector using Infusion ligase. The 10 μL ligation system is shown in Table 5.

[0084] Table 5 Infusion Connection System

[0085] name Dosage 5×Infusion ligase 2μL pBWA(V)HS-RNAi after enzyme digestion (Pst I) 2μL PCR amplification products 6μL Total Volume 10μL

[0086] React at 50℃ for 15 minutes, then place on ice for 2-3 minutes before use.

[0087] After the forward sequence is ligated to pBWA(V)HS, the pBWA(V)HS containing the forward sequence is digested with Sma I enzyme, and then the reverse sequence is ligated to pBWA(V)HS. The ligation method is the same as described above.

[0088] 2. Thermal shock conversion

[0089] Under aseptic conditions, add 10 μL of the ligation product to competent E. coli cells, mix gently, and incubate on ice for 30 min. Heat shock at 42°C for 90 s, then quickly transfer the centrifuge tube to ice for 2-3 min. Add 800 μL of antibiotic-free LB medium and shake on a shaker at 37°C and 120 rpm for approximately 1 h. Spread 200 μL of the culture solution onto LB solid medium containing 50 μg / mL antibiotics (add X-Gal and IPTG before spreading and mix well), and incubate upside down at 37°C for 12 h.

[0090] 3. Screening and identification

[0091] During the culture process, bacterial plaques in the culture medium will separate into blue and white categories. Once the plaques reach a suitable size, several white plaques are picked using a sterilized pipette tip and cultured separately in LB liquid medium containing 50 μg / mL kanamycin for 12 hours with shaking. Then, plasmids are extracted and subjected to enzyme digestion identification to ensure correct recombinant vector construction. Correctly constructed plasmids (or strains containing the plasmids) are preserved for later use. The correctly constructed recombinant plasmid is finally named: pBWA(V)HS-NtIPDS-RNAi.

[0092] Experimental Example 3: Agrobacterium-mediated tobacco transformation and identification of transgenic plants

[0093] This experiment utilizes Agrobacterium-mediated tobacco transformation to transform the RNAi interference vector constructed in Example 2 into tobacco plants, obtaining tobacco plants with the NtIPDS ​​gene silenced. The specific experimental procedure is as follows:

[0094] 1. Freeze-thaw transformation of Agrobacterium

[0095] Add 1 μg pBWA(V)HS-NtIPDS-RNAi vector to 100 μL of EHA105 Agrobacterium competent cells, mix well, and incubate on ice for 30 min; then freeze in liquid nitrogen for 5 min, remove from liquid nitrogen and immediately place in a 37℃ water bath for 5 min, then incubate on ice for 5 min; add 500 μL LB medium, incubate at 28℃ with shaking for 4 h, and finally spread the bacterial culture evenly on solid culture medium supplemented with antibiotics (50 mg / L kanamycin and 50 mg / L rifampin), and incubate at 28℃ for about 24 h.

[0096] 2. Preparation of bacterial suspension for transfection

[0097] 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; Add the resuspended bacterial culture to 1 mL of MS liquid medium and add it to 40 mL of MS liquid medium (containing 40 μL, 25 mg / L acetylsuccinone), which is the infection solution. After standing for 2 h, it can be used for infection.

[0098] 3. Leaf disc method conversion

[0099] (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+BA0.2 mg / L (to strengthen the buds and promote rapid growth) medium for subculture.

[0100] (b) After subculture for 14 days (as long as small leaves are available), take leaves about 1cm×1cm in size, cut off the petiole, scratch the surface and edge of the leaf, and place them on MS+BA1.0mg / L (pH6.0-6.5) pre-culture medium, with the upper side facing down and close to the medium, and pre-culture in the dark for 2 days.

[0101] (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 the bacterial solution with sterilized dry filter paper.

[0102] (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-microbes form around the leaf cuts;

[0103] The co-cultured tobacco leaves were removed again and rinsed with sterile water containing 500 mg / L Cef to wash away Agrobacterium on the surface of the explants.

[0104] (e) After blotting off the surface liquid from step (d) with filter paper, transfer the sample to tobacco budding medium (MS + BA 1.0 mg / L + Hyg 25 mg / L + Cef 500 mg / L pH 5.8);

[0105] During the cultivation process, the culture medium should be changed every 2 weeks until adventitious buds grow (usually 2 weeks).

[0106] Cut off the regenerated seedlings (about 1 cm long) and transfer them to subculture medium (MS + BA 0.2 mg / L + Hyg 25 mg / L + Cef 500 mg / L, pH 5.8) for culture;

[0107] When the seedlings grow to 2cm in length (with small buds), they are transferred to rooting medium (MS + NAA 0.2mg / L), cultured at 25℃ under 12h light for about 3 weeks to ensure the growth of robust roots.

[0108] (g) 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.

[0109] 4. Identification of transgenic lines and observation of plant phenotypes

[0110] Genomic DNA was extracted from tobacco seedlings in step 3. Primers were designed, and PCR was used to identify Kan resistance. The PCR system and reaction conditions were as described in Experiment 1. The specific primer sequences are designed as follows:

[0111] Kan-F: 5'-TCTGGACGAAGAGCATCAGG-3' (shown in SEQ ID NO. 6),

[0112] Kan-R: 5'-ATGAATCCAGAAAAGCGGCC-3' (shown in SEQ ID NO. 7).

[0113] The identification results showed that a total of 20 Kan-positive resistant plants were obtained.

[0114] Furthermore, the expression level of the NtIPDS ​​gene in transgenic plants was analyzed using real-time quantitative PCR (qRT-PCR). Specifically, the qRT-PCR reaction system was as follows: 10 μL 2×SYBR I Master, 0.5 μL each of forward and reverse primers, 50 ng cDNA, and ddH2O added to a final volume of 20 μL. The PCR program was: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 60℃ annealing for 10 s, 72℃ extension for 10 s, for 45 cycles. The primer sequences were designed as follows:

[0115] qNtIPDS-F: 5'-GGGTGTTTGCCACGTTGCTCT-3' (shown in SEQ ID NO. 8);

[0116] qNtIPDS-R: 5'-TGCCCGAAAGCTCCAGCAGT-3' (shown in SEQ ID NO. 9);

[0117] In the analysis process, the primers for the internal reference gene were designed as follows:

[0118] 26s-F: 5'-GAAGAAGGTCCCAAGGGTTC-3' (shown in SEQ ID NO. 10);

[0119] 26s-R: 5'-TCTCCCTTTAACACCAACGG-3' (shown in SEQ ID NO.11).

[0120] Some test results are as follows Figure 1 As shown. From Figure 1 It can be seen that the expression level of NtIPDS ​​gene was reduced to varying degrees in different transgenic lines, indicating that the constructed RNAi interference vector can effectively interfere with the expression of NtIPDS ​​gene, and tobacco plants with NtIPDS ​​gene silence were obtained.

[0121] The phenotype of tobacco plants with silenced NtIPDS ​​gene was observed during the seedling stage, and the results were as follows: Figure 2 As shown, after the NtIPDS ​​gene is silenced, the plant grows slowly and the leaves become chlorotic and mottled.

[0122] Example 4: Application of the NtIPDS ​​gene, which encodes tobacco isopropyl malate dehydrase, in regulating tobacco chloroplast development.

[0123] Using tobacco plants with silenced NtIPDS ​​gene obtained in Experiment 3 as the research object, the chlorophyll content of tobacco leaves was measured, chlorophyll fluorescence parameters were measured, and the leaf cell structure was observed using transmission electron microscopy to further study the function of NtIPDS ​​gene. The specific experimental procedure is as follows:

[0124] 1. Chlorophyll content determination

[0125] Accurately weigh 0.2 g (accurate to 0.1 mg) of freeze-dried fresh tobacco leaves (seedling stage) into a 50 mL Erlenmeyer flask, accurately add 25 mL of extraction solvent (90% acetone solution), and ultrasonically extract for 20 min under ice bath conditions. Filter an appropriate amount of the extract through a 0.45 μm micromembrane filter. Transfer the filtrate to a 2 mL amber chromatographic bottle for HPLC analysis.

[0126] Analytical conditions: Column: Waters Nova-Pak-C18 (3.9×150mm, 4μm); Column temperature: 30℃; Column flow rate: 0.5mL / min; Injection volume: 10μL; Mobile phase A: isopropanol; Mobile phase B: 80% acetonitrile aqueous solution (V / V); Equilibrium time: 6min; Gradient elution, conditions are shown in Table 6.

[0127] Table 6 Eluting gradient of the mobile phase

[0128]

[0129] Detection wavelengths: chlorophyll a was detected at 448 nm, and chlorophyll b at 428 nm. Qualitative analysis was performed using retention time combined with the absorption spectra of the standard, and quantification was performed using the external standard method.

[0130] The results are as follows Figure 3 As shown, the contents of chlorophyll a and chlorophyll b were significantly reduced in plants with the NtIPDS ​​gene silenced.

[0131] 2. Measurement of chlorophyll fluorescence parameters

[0132] Chlorophyll fluorescence parameters Fv / Fm were measured using a DUAL-PAM-100 dual-channel modulated chlorophyll fluorometer. Samples were selected from K326 and R1 (one plant from a NtIPDS ​​gene-silenced tobacco plant, both in the seedling stage). Before measurement, the plants were allowed to dark adapt for 30 minutes, followed by chlorophyll fluorescence parameter analysis. Three leaves from each plant were measured.

[0133] The results are as follows Figure 4 As shown, compared with the control, the chlorophyll fluorescence parameter Fv / Fm of NtIPDS ​​gene-silenced plants was reduced, indicating that the reduction in NtIPDS ​​expression affected the photosynthesis of plant leaves.

[0134] 3. Observation of the ultrastructure of the blade

[0135] Leaves with significant differences in chlorophyll content (control, partially chlorotic, and completely yellowed, taken during the vigorous growth period) were selected for chloroplast ultrastructure observation. Sampling was performed on the same parts of the leaves, avoiding the veins. The fresh tissue was cut into 1mm pieces using a scalpel. 2 Small pieces were then immediately placed in headspace vials containing electron microscopy fixative, and vacuum pumped until they sank to the bottom. After post-fixation, room temperature dehydration, infiltration embedding, polymerization, ultrathin sectioning, and staining, the ultrastructure of chloroplasts was observed under a transmission electron microscope, and images were collected for analysis.

[0136] The results are as follows Figure 5 As shown, by Figure 5 A and Figure 5 B indicates that in the K326 (control) mesophyll cells, the chloroplasts are fully developed, elongated oval in shape, regularly distributed close to the inner cell membrane, with a clear chloroplast structure and intact inner and outer membranes. The amylopectin within the chloroplasts is relatively large. Figure 5 C and Figure 5 As shown in D, compared to the control, in NtIPDS ​​gene-silenced plants with unevenly distributed green leaves, chloroplast content was significantly reduced, chloroplast distribution was irregular and boundaries were unclear, chloroplast traits were irregular, chloroplast structure was abnormal, some chloroplast membranes were damaged, and the number and size of starch granules within chloroplasts were reduced; Figure 5 E and Figure 5 F indicates that in NtIPDS ​​gene-silenced plants with uniform yellowing of leaves, chloroplasts are relatively evenly distributed, but the chloroplast boundaries are unclear, the chloroplast membranes are damaged, and the number of starch grains is reduced or even disappears.

[0137] In summary, the poor development and structural damage of chloroplasts in NtIPDS ​​gene-silenced plants may be one of the reasons for chlorosis in these plants.

[0138] Experimental Example 5: Application of the tobacco isopropyl malate dehydrase encoding gene NtIPDS ​​or RNAi interference vector in the breeding of new tobacco varieties.

[0139] Using tobacco plants with silenced NtIPDS ​​gene obtained in Experiment 3 as the research object, the acyl sugar content in leaves was measured to further explore the function of NtIPDS ​​gene. The specific experimental procedure is as follows:

[0140] 1. Extraction of secretions from tobacco leaf surface

[0141] During the vigorous growth period, select T1 generation NtIPDS ​​gene-silenced plants with growth consistent with the control K326. Take 5g of tobacco leaves from the 3rd and 4th leaf positions (from the top downwards), freeze-dry them, and take 50mg. Place the 50mg of the leaf in a 5mL centrifuge tube, add 2.5mL of CH2Cl2 solution (chromatographic grade, the same below), and shake at 37℃ for 20min to ensure sufficient contact between the leaves and the CH2Cl2 solution and dissolve the secretions on the surface of the tobacco leaves. Then centrifuge and pour the CH2Cl2 extract (supernatant) into a new 5mL centrifuge tube. Add another 2.5mL of CH2Cl2 solution to the tube containing the leaves, shake at 37℃ for 20min, centrifuge again, and pour the CH2Cl2 extract (supernatant) into the same new 5mL centrifuge tube containing the CH2Cl2 extract. Obtain a total of 5mL of extract from the two extractions. Add 50μL of internal standard solution (sucrose octaacetate, dissolved in dichloromethane, concentration 200μg / mL), and concentrate and dry under low temperature vacuum.

[0142] Add 100 μL of a silanizing reagent mixture of BSTFA:DMF = 1:1 (volume ratio) to each sample, heat in a water bath at 75°C for 30 min, cool to room temperature, then add 100 μL of a BSA:pyridine mixture of 1:1 (volume ratio) to the sample, mix thoroughly, and filter through a 0.25 μm organic phase filter into a sample vial.

[0143] 2. Determination of acyl sugar (sucrose ester) content

[0144] Sucrose ester content was determined using GC-MS. The chromatographic column was a DB-5MS (30m × 0.25μm × 0.25μm). Injector temperature: 265℃; ion source temperature: 280℃; interface temperature: 280℃; column oven temperature: 165℃; transfer line temperature: 280℃. Temperature program: Starting at 165℃, increased to 280℃ at 4℃ / min, held for 25 min. Injection volume: 1 μL; split ratio: 15:1; column head pressure: 100 kPa. Qualitative analysis of acyl sugars was performed according to published literature (Characterization of trichohome-specific BAHD acyltransferases involved in acylsugar biosynthesis in Nicotiana tabacum. 2022. J Exp Bot.). Results are as follows: Figure 6 As shown, compared with the control, the acyl sugar content in NtIPDS ​​gene-silenced plants of different lines was significantly increased.

[0145] 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. A gene encoding tobacco isopropyl malate dehydratase NtIPDS The application of genes in regulating tobacco acyl sugar content is characterized by: inhibition NtIPDS Following gene expression, the acyl sugar content in tobacco plants significantly increased; NtIPDS The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A gene encoding a tobacco isopropyl malate dehydratase NtIPDS The application of gene or RNAi interference vectors in the breeding of new tobacco varieties is characterized by: The NtIPDS The nucleotide sequence of the gene is shown in SEQ ID NO.1; the RNAi interference vector contains the... NtIPDS A specific nucleotide fragment of the gene is used as a guide sequence; NtIPDS After gene silencing, the acyl sugar content in tobacco increases significantly, resulting in new tobacco varieties with increased acyl sugar content.

3. The tobacco isopropyl malate dehydratase encoding gene according to claim 2 NtIPDS The application of gene or RNAi interference vectors in the breeding of new tobacco varieties is characterized by: The nucleotide sequence of the guide sequence is shown in SEQ ID NO.

3.

4. The tobacco isopropyl malate dehydratase encoding gene according to claim 3. NtIPDS The application of gene or RNAi interference vectors in the breeding of new tobacco varieties is characterized by: The method for preparing the RNAi interference vector includes the following steps: inserting the guide sequence into the PBWA(V)HS empty vector, and then screening and sequencing for identification.

5. The tobacco isopropyl malate dehydratase encoding gene according to claim 4. NtIPDS The application of gene or RNAi interference vectors in the breeding of new tobacco varieties is characterized by: The new tobacco varieties were obtained by the following method: RNAi vector was transformed into Agrobacterium as an infection solution, and then transformed into tobacco. Through screening and identification, new tobacco varieties with significantly increased leaf acyl sugar content were obtained.

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