Application of NtIPMD gene in regulating chlorophyll content in tobacco

Abstract

The present invention discloses the application of the NtIPMD gene in regulating the chlorophyll content of tobacco, and belongs to the field of plant genetic engineering technology. Based on the existing progress in tobacco genetic engineering and the observation and identification of related plant phenotypes, the present invention conducts in-depth research on the NtIPMD gene and finds that it is related to the development of tobacco chloroplasts. The nucleotide sequence of the NtIPMD gene is shown in SEQ ID NO.1. After silencing the gene, the chlorophyll content of the leaves decreases, the growth of the plants slows down, and transmission electron microscopy observation reveals abnormal chloroplast development. The present invention finds that the gene encoding tobacco isopropylmalate dehydrogenase, NtIPMD, plays a key role in regulating the chlorophyll content of tobacco leaves, laying the foundation for clarifying the molecular regulatory mechanism of chlorophyll biosynthesis in tobacco leaves and providing a new precise regulatory gene for regulating the chlorophyll content of tobacco leaves.

Description

Technical Field

[0001] The invention relates to application of NtIPMD gene in regulating tobacco chlorophyll content, and belongs to the technical field of plant gene engineering. Background Art

[0002] Tobacco is a dicotyledonous plant in the genus Nicotiana, Solanaceae, order Tubulales. The genus Nicotiana comprises over 60 species, of which two cultivated species, Nicotiana tabacum, dominates the cultivated area, while Nicotiana rustica (Nicotiana rustica) occupies a much smaller area. Cultivated tobacco can be divided into six types based on leaf quality, biological traits, and cultivation and curing methods: flue-cured tobacco, sun-cured tobacco, air-cured tobacco, burley tobacco, oriental tobacco, and rustica tobacco. Flue-cured tobacco is the most widely cultivated common tobacco, and my country ranks first in the world in both cultivated area and total production.

[0003] As a leaf-producing cash crop, flue-cured tobacco cultivation techniques differ from those of other field crops. Not only does it require a certain yield, but it also prioritizes leaf quality. Leaf quality determines its usability, directly impacting the color, aroma, flavor, and commercial value of cigarettes. It also impacts the economic benefits of tobacco farmers and is the lifeblood and starting point of the tobacco industry. To remain competitive in future domestic and international markets and meet the growing demand for high-quality tobacco from domestic and international cigarette companies, it is imperative to improve both leaf quality and safety.

[0004] Phytochromes are a key class of compounds in tobacco, primarily including chlorophyll and carotenoids. Chlorophyll is the most important pigment involved in photosynthesis. Chlorophyll degrades and disappears significantly during tobacco ripening and curing. This degradation occurs primarily through two pathways: First, chlorophyll degrades from the porphyrin ring to produce pyrrole compounds, which contribute to the aroma of aged tobacco leaves. Second, chlorophyll hydrolysis produces phytol, which can further degrade into neophytadiene and then furans, which are converted into the sweet, refreshing components of tobacco leaves. Generally speaking, sufficient chlorophyll degradation is beneficial to tobacco quality. However, incomplete degradation can transform chlorophyll into an unfavorable chemical component in dried tobacco leaves, imparting a distinct greenish odor. If chlorophyll is not fully degraded during the curing process before tobacco leaves are cured, varying degrees of "greenish-yellow" tobacco will result. This "greenish-yellow" tobacco not only exhibits poor appearance but also significantly impacts tobacco leaf quality. Therefore, sufficient chlorophyll degradation is a critically important indicator in tobacco grading.

[0005] Since the chlorophyll content in tobacco leaves is closely related to the quality of tobacco leaves and the growth of tobacco plants, sufficient and in-depth research on tobacco leaf chlorophyll-related genes, and thus targeted regulation of chlorophyll, has very important theoretical and practical significance for improving tobacco leaf quality and tobacco leaf variety improvement.

[0006] The Chinese invention patent with the announcement date of May 31, 2022 and the announcement number CN110205330B discloses tobacco heat shock protein HSP22 and its application. It specifically discloses that after reducing the expression of this protein in tobacco, the content of pigment substances in the leaves is significantly reduced. The pigment substances are: neoxanthin, violaxanthin, lutein, chlorophyll a / b, and β-carotene.

[0007] The Chinese invention patent with the announcement date of April 26, 2022 and the announcement number CN110862445B discloses the NtOEP1 gene that affects the pigment content of tobacco and its application. It specifically discloses that after silencing the gene, the content of pigment substances in transgenic tobacco plants is significantly reduced; the pigment substances include chlorophyll a and chlorophyll b.

[0008] The Chinese invention patent with announcement date of June 24, 2022 and announcement number CN113373166B discloses the application of tobacco NtAAP3 gene in tobacco. It specifically discloses that after silencing the gene, the chlorophyll a / b and total chlorophyll content in the tobacco leaves of transgenic tobacco plants are significantly increased.

[0009] In summary, the gene network regulating chlorophyll content in tobacco is very complex, and the specific regulatory mechanism is not clear. Therefore, discovering new chlorophyll content regulatory genes is crucial to clarifying the chlorophyll regulatory mechanism, and then regulating the chlorophyll content in tobacco through genetic engineering to improve tobacco leaf quality. Summary of the Invention

[0010] The purpose of the present invention is to provide an application of the NtIPMD gene in regulating the chlorophyll content of tobacco, thereby providing a new gene resource for regulating the chlorophyll content in tobacco in the prior art.

[0011] In order to achieve the above-mentioned object, the technical solution adopted by the present invention for the application of the NtIPMD gene in regulating the chlorophyll content of tobacco is:

[0012] The application of the NtIPMD gene in regulating tobacco chlorophyll content, the nucleotide sequence of the NtIPMD gene is shown in SEQ ID NO.1.

[0013] The beneficial effect of the above technical solution is that the application of the NtIPMD gene in regulating tobacco chlorophyll content is a groundbreaking invention. Using RNA interference silencing technology, the present invention interferes with NtIPMD gene expression in tobacco, generating NtIPMD gene-silenced transgenic plants. Testing revealed significantly reduced chlorophyll a / b and total chlorophyll content in tobacco leaves from NtIPMD gene-silenced plants, stunted chloroplast development, structural damage, uneven leaf green distribution, and slow growth, though plants can grow normally until harvest. Furthermore, long-term cultivation revealed that this condition is stably inherited, demonstrating that the NtIPMD gene plays a crucial role in regulating tobacco chlorophyll content.

[0014] As a further improvement, after inhibiting the expression of the NtIPMD gene, the chlorophyll content in tobacco leaves was significantly reduced.

[0015] As a further improvement, the inhibition is to construct an RNAi interference vector to silence the expression of the NtIPMD gene.

[0016] The beneficial effect of the above technical solution is that the interference strain obtained by RNAi has a better gene silencing effect than the VIGS strain, is more stable, and can be passed on to the next generation.

[0017] As a further improvement, the RNAi interference vector contains a specific nucleotide fragment of the NtIPMD gene as a guide sequence.

[0018] As a further improvement, the nucleotide sequence of the guide sequence is shown as SEQ ID NO.3.

[0019] As a further improvement, the preparation method of the RNAi interference vector includes the following steps: inserting the guide sequence into the PBWA(V)HS empty vector, screening, sequencing and identification.

[0020] The beneficial effect of the above technical solution is that: the present invention constructs an RNAi interference vector targeting the NtIPMD gene, uses the Agrobacterium transformation method to transfer the constructed RNAi interference vector into tobacco plants, and successfully constructs tobacco plants with silenced NtIPMD gene, providing a plant model for studying the function of the NtIPMD gene in tobacco.

[0021] As a further improvement, the tobacco is K326. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the relative expression level of the NtIPMD gene in Example 3 of the present invention (K326 is a normal control plant, R1, R2 and R3 are NtIPMD gene silenced plants, where ** represents P < 0.01);

[0023] Figure 2 The phenotypes of NtIPMD gene-silenced plants in Example 3 of the present invention (from left to right are K326 control plant, R1 strain, R2 strain, and R3 strain, of which R1, R2, and R3 are NtIPMD gene-silenced plants);

[0024] Figure 3 is the chlorophyll content of the NtIPMD gene-silenced plants in Example 4 of the present invention;

[0025] Figure 4 Transmission electron microscopy observation of leaves of NtIPMD gene-silenced plants in Example 4 of the present invention (the magnification of A and C is 500×, and the magnification of B and D is 5000×). DETAILED DESCRIPTION

[0026] Tobacco is an important model organism in biological research, and a large number of plant molecular biology studies and genetic engineering experiments are carried out on tobacco. Because of this situation, researchers have neglected the discovery, identification and utilization of tobacco's own functional genes.

[0027] Existing research suggests that isopropylmalate dehydrogenase (IPMD) is primarily involved in leucine metabolism in plants, hence its abbreviation, LeuB. In Arabidopsis thaliana, studies on IPMD suggest that this protein also participates in methionine chain elongation in the glucosinolate biosynthesis pathway. Studies have shown that there are three IPMD isoforms (AtIPMD1-3) in Arabidopsis thaliana. Silencing either AtIPMD2 or AtIPMD3 alone does not produce noticeable phenotypes in the plants. However, silencing both AtIPMD2 and AtIPMD3 together affects pollen and embryo sac development (Functional characterization of Arabidopsis thaliana isopropylmalate dehydrogenases reveals their important roles in gametophyte development, New Research Phytologist, 2010). In Nicotiana benthamiana, it has been reported that IPMD is involved in the acyl sugar metabolism pathway (Transcriptomic and Reverse Genetic Analyses of Branched-Chain Fatty Acid and Acyl Sugar Production in Solanum pennellii and Nicotiana benthamiana, Plant Physiology, 2008), but the specific function of this gene in tobacco has not been reported.

[0028] Based on previous research, the present invention used the cDNA of tobacco K326 leaves as a template and PCR technology to clone the gene encoding tobacco isopropylmalate dehydrogenase, NtIPMD. Based on the existing progress in tobacco genetic engineering and the observation and identification of related plant phenotypes, the NtIPMD gene was deeply studied and found to be related to tobacco chloroplast development. After silencing the gene, the chlorophyll content of the leaves decreased, the growth of the plants slowed down, and transmission electron microscopy revealed abnormal chloroplast development. The present invention found that the gene encoding tobacco isopropylmalate dehydrogenase, NtIPMD, plays a key role in regulating the chlorophyll content of tobacco leaves, laying the foundation for clarifying the molecular regulatory mechanism of chlorophyll biosynthesis in tobacco leaves and providing a new precise regulatory gene for regulating the chlorophyll content of tobacco leaves.

[0029] The present invention is further described in detail below with reference to specific examples. Unless otherwise specified, the equipment and reagents used in each embodiment, experimental example and comparative example can be obtained from commercial sources.

[0030] All plant tissue materials in the following examples were taken from the flue-cured tobacco (Nicotiania tabacum L.) variety K326 and K326 transgenic plants expressing RNAi interference with NtIPMD. The tobacco materials were grown in an artificial climate chamber at a growth temperature of 25°C and a photoperiod of 12 hours light / 12 hours dark.

[0031] The chlorophyll content in the following examples was determined using a plant chlorophyll content detection kit with the product number A04782-S, purchased from Shanghai Jingkang Bioengineering Co., Ltd.

[0032] Specific examples of the application of the NtIPMD gene of the present invention in regulating tobacco chlorophyll content:

[0033] Example 1 Cloning of NtIPMD gene fragment

[0034] In this example, total RNA was extracted from young leaves of tobacco K326, reverse transcribed into cDNA, and the NtIPMD gene was successfully cloned using the cDNA as a template. The specific implementation steps are as follows:

[0035] 1. Extraction of total RNA from tobacco

[0036] Young leaves of tobacco K326, approximately 4 weeks old, were used as samples. After being thoroughly ground into a powder using 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 then added, the mixture was shaken and centrifuged, and the upper aqueous phase was carefully transferred to another centrifuge tube. 500 μL of isopropanol was added to precipitate the RNA, which was then separated by centrifugation and washed with 75% ethanol. After being slightly dried at room temperature, an appropriate volume of RNase-free water was added to fully dissolve the extracted total RNA. Finally, the extracted total RNA was treated with DNase I for subsequent cDNA preparation. The DNase I digestion system for a 10 μL reaction was as follows:

[0037] Tobacco total RNA, 1 μg; 10× reaction buffer with MgCl2, 1 μL; DNase I (RNase-free), 1 μL (1 U); DEPC-treated water added to 10 μL.

[0038] The reaction conditions were 37°C in a water bath for 30 min.

[0039] 2. cDNA Synthesis

[0040] Prepare the template RNA / primer mixture listed in Table 1 in a sterile 0.2 mL centrifuge tube. Incubate at 70°C for 10 min, then quickly cool on ice for at least 2 min. Centrifuge for a few seconds to allow the denatured template RNA / primer solution to accumulate at the bottom of the tube.

[0041] Table 1 RNA / primer mixture preparation

[0042] name Dosage RNA (100 ng / μL) 1 μL Oligo(dT)Primer (50μM) 1 μL <![CDATA[RNase free dH2O]]> 5μL Total Volume 7μL

[0043] After preparing the reverse transcription reaction solution in Table 2 in the above centrifuge tube, incubate at 42°C for 1 hour; incubate at 70°C for 15 minutes, and then cool on ice. The obtained cDNA was used for PCR amplification.

[0044] Table 2 Reverse transcription reaction solution preparation

[0045] Reagent name Dosage The above template RNA / primer denaturation solution 7μL 5×M-MLV buffer 2μL dNTP Mixture (10 mM each) 0.5μL RNase Inhibitor (40U / μL) 0.25 μL RTase M-MLV(RNase H-)(200U / μL) 0.25 μL Total Volume 10 μL

[0046] 3. PCR amplification

[0047] The primer sequences for PCR amplification are as follows:

[0048] Primers for amplifying the NtIPMD gene fragment (the underlined portion is the vector linker sequence):

[0049] NtIPMD-F:5'- TTTCAGGGCCAT ATGGCGGCTTCCTTACAATT-3' (shown in SEQ ID NO.4);

[0050] NtIPMD-R:5'- CTCGAATTCGGATCC TTAAACAGCAGCGGGAGTT-3' (shown in SEQ ID NO. 5).

[0051] 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.

[0052] Table 3 PCR reaction system

[0053] Reagent name Dosage cDNA template 1 μL GXL polymerase 1 μL 5×GXL buffer 10 μL dNTP Mixture (10mM) 4μL Primer-F / R 8μL <![CDATA[ddH2O]]> 26μL

[0054] The PCR reaction program is shown in Table 4.

[0055] Table 4 PCR reaction program

[0056]

[0057] The amplified PCR products were subjected to 1% agarose gel electrophoresis, and then the PCR amplified products were recovered and purified before sequencing.

[0058] Example 2 Construction of RNAi vector

[0059] Based on the cloning of the NtIPMD gene in Example 1, this example further verifies the function of the gene by constructing an RNAi interference vector targeting the NtIPMD gene. The specific implementation steps are as follows:

[0060] 1. Enzyme digestion and ligation

[0061] Using nucleotides 67-344 of the NtIPMD gene as the RNAi guide sequence (as shown in SEQ ID NO. 3), PCR amplification was performed in both forward and reverse directions. Using the pBWA(V)HS-RNAi plasmid as the vector, the pBWA(V)HS vector was digested with Pst I, and the digestion products were recovered. The RNAi product obtained by PCR amplification was ligated with the digested pBWA(V)HS vector using Infusion ligase. The 10 μL ligation system is shown in Table 5.

[0062] Table 5 Infusion connection system

[0063] name Dosage 5×Infusion Ligase 2μL pBWA(V)HS-RNAi after enzyme digestion (Pst I) 2μL PCR amplification products 6μL Total Volume 10 μL

[0064] After reacting at 50°C for 15 minutes, place on ice for 2-3 minutes and set aside.

[0065] After the forward fragment was ligated to pBWA(V)HS, the pBWA(V)HS ligated with the forward fragment was digested with Sma I, and then the reverse sequence was ligated to pBWA(V)HS using the same ligation method as above.

[0066] 2. Heat shock transformation

[0067] Under sterile conditions, add 10 μL of the ligation product to the competent E. coli cells, mix gently and place on ice for 30 minutes; heat shock at 42°C for 90 seconds, then quickly transfer the centrifuge tube to an ice bath for 2-3 minutes; add 800 μL of LB medium without antibiotics and shake on a shaker at 37°C and 120 rpm for about 1 hour; take 200 μL of the culture solution and apply it to LB solid medium containing 50 μg / mL of antibiotics (add X-Gal and IPTG before applying the bacterial solution) and culture it upside down at 37°C for 12 hours.

[0068] 3. Screening and identification

[0069] During the culture process, bacterial plaques in the culture medium will separate into two types: blue and white. When the plaques reach the appropriate size, pick a few white spots with a sterile pipette tip and culture them separately in LB liquid medium containing 50 μg / mL kanamycin for 12 hours with shaking. Then, the plasmids are extracted and digested with enzymes to ensure that the recombinant vector is correctly constructed. The correctly constructed plasmids (or strains containing the plasmids) are stored for future use. The correctly constructed recombinant plasmid is named: pBWA(V)HS-NtIPMD-RNAi.

[0070] Example 3 Agrobacterium-mediated tobacco transformation and identification of transgenic plants

[0071] In this example, the RNAi interference vector constructed in Example 2 was transformed into tobacco using the Agrobacterium-mediated tobacco transformation method to obtain tobacco plants with NtIPMD gene silenced. The specific implementation steps are as follows:

[0072] 1. Freeze-thaw transformation of Agrobacterium

[0073] 1 μg of pBWA(V)HS-NtIPMD-RNAi vector was added to 100 μL of EHA105 Agrobacterium competent medium, mixed well and allowed to stand on ice for 30 minutes; then placed in liquid nitrogen for freezing for 5 minutes, taken out of liquid nitrogen and immediately placed in a 37°C water bath for 5 minutes, and then allowed to stand on ice for 5 minutes; 500 μL of LB culture medium was added and cultured at 28°C with shaking for 4 hours; finally, the bacterial liquid was evenly spread on a solid culture medium supplemented with antibiotics (50 mg / L kanamycin and 50 mg / L rifampicin) and cultured at 28°C for about 24 hours.

[0074] 2. Preparation of bacterial solution for transfection

[0075] Pick a single plaque and place it in 5 ml LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin. Culture it overnight at 28°C and 200 rpm until the bacterial concentration reaches OD 600 = about 1.5; take 2 mL of bacterial solution and add it to a centrifuge tube, centrifuge at 4000 r / min for 5 min; remove the supernatant, aspirate 1 mL of new MS liquid culture medium, resuspend Agrobacterium, centrifuge at 4000 r / min for 5 min, repeat this operation once; add the resuspended bacterial solution of 1 mL of MS liquid culture medium to 40 mL of MS liquid culture medium (containing 40 μL, 25 mg / L acetosyringone), which is the infection solution, and let it stand for 2 hours before use for infection.

[0076] 3. Leaf disc method transformation

[0077] (a) Sterile tobacco K326 seeds were sown on MS medium for culture. When the tobacco seedlings grew to 3-5 cm (approximately 20-30 days), the terminal buds were placed on MS medium supplemented with 0.2 mg / L BA (to strengthen the buds and promote rapid growth) for subculture.

[0078] (b) After 14 days of subculture (small leaves are sufficient), take a leaf of approximately 1 cm × 1 cm in size, remove the petiole, scratch the leaf surface and leaf edge, and place it on a pre-culture medium containing MS + 1.0 mg / L BA (pH 6.0-6.5), facing down and close to the medium, and pre-culture in the dark for 2 days.

[0079] (c) removing the leaves pre-cultured in step (b), placing them in an Agrobacterium infection solution for 15 minutes, and then absorbing the bacterial solution onto sterilized dry filter paper;

[0080] (d) Place the infected leaves back on the pre-culture medium and incubate at 28°C in the dark for 2-3 days until microplaques form around the leaf cuts.

[0081] The co-cultivated tobacco leaves were removed again and rinsed with sterile water supplemented with 500 mg / L Cef to wash away the Agrobacterium on the explant surface;

[0082] (e) After absorbing the surface liquid in step (d) with filter paper, the cells were transferred to tobacco budding medium (MS + BA 1.0 mg / L + Hyg 25 mg / L + Cef 500 mg / L pH 5.8);

[0083] During the culture process, the culture medium was changed every 2 weeks until adventitious buds grew (usually 2 weeks);

[0084] The regenerated seedlings (about 1 cm) were cut off and transferred to subculture medium (MS + BA 0.2 mg / L + Hyg 25 mg / L + Cef 500 mg / L, pH 5.8) for culture;

[0085] When the seedlings grow to 2 cm in length (small buds are sufficient), transfer them to rooting medium (MS + NAA 0.2 mg / L) and culture them at 25°C with 12 h light for about 3 weeks to ensure that strong roots grow.

[0086] (f) When the roots grow to 2-3 cm and the seedlings are about 7-10 cm tall, remove the flasks, wash off the root culture medium, and transplant them into pots for greenhouse cultivation.

[0087] 4. Identification of transgenic lines and plant phenotype observation

[0088] Extract genomic DNA from tobacco seedlings in step 3, design primers, and use PCR to identify Kan resistance. The PCR system and reaction conditions are as described in Experimental Example 1. The specific primer sequences are designed as follows:

[0089] Kan-F: 5'-TCTGGACGAAGAGCATCAGG-3' (shown in SEQ ID NO. 6),

[0090] Kan-R: 5'-ATGAATCCAGAAAAGCGGCC-3' (shown in SEQ ID NO. 7).

[0091] The identification results showed that a total of 20 Kan-positive resistant plants were obtained.

[0092] Furthermore, real-time quantitative PCR was used to analyze the expression of the NtIPMD gene in the transgenic plants. For qRT-PCR analysis, the reaction system consisted of 10 μL 2×SYBR I Master, 0.5 μL each of upstream and downstream primers, 50 ng of cDNA, and ddH2O to a volume of 20 μL. The PCR procedure was as follows: 94°C pre-denaturation for 30 seconds, followed by 45 cycles of denaturation at 94°C for 5 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds. Primer sequences were designed as follows:

[0093] qNtIPMD-F: 5'-TGAAATATGGCCTAGGTG-3' (shown in SEQ ID NO. 8);

[0094] qNtIPMD-R: 5'-CTGAATGAATGTCACCAG-3' (shown in SEQ ID NO. 9);

[0095] During the analysis, the internal reference gene primers were designed as follows:

[0096] 26s-F: 5'-GAAGAAGGTCCCAAGGGTTC-3' (shown in SEQ ID NO. 10);

[0097] 26s-R: 5'-TCTCCCTTTAACACCAACGG-3' (shown in SEQ ID NO. 11).

[0098] Some test results such as Figure 1 As shown. Figure 1 It can be seen that the expression levels of the NtIPMD gene in different transgenic lines were reduced to varying degrees, indicating that the constructed RNAi interference vector can effectively interfere with the expression of the NtIPMD gene and obtain tobacco plants with NtIPMD gene silenced.

[0099] The phenotypes of tobacco plants with NtIPMD gene silenced were observed at the T1 seedling stage. Figure 2 As shown in Figure 3, after NtIPMD gene silencing, the plants grew slowly and the leaves lost their green color.

[0100] Example 4: Verification of NtIPMD gene function

[0101] The NtIPMD gene-silenced tobacco plants obtained in Example 3 were used as research subjects. The chlorophyll content of tobacco leaves was measured, and the leaf cell structure was observed using a transmission electron microscope to further study the function of the NtIPMD gene. The specific implementation procedures were as follows:

[0102] 1. Chlorophyll content determination

[0103] Weigh about 0.1g of tobacco leaves or other green tissues, remove the midrib, cut into pieces, and wash with distilled water. Preparation of the extract: Take 200mL of anhydrous ethanol and 400mL of acetone, mix thoroughly and set aside. Add 1mL of distilled water and a small amount of reagent 1 (about 10mg), grind thoroughly in the dark or under weak light conditions, and transfer to a 10mL glass test tube. Rinse the mortar with the extract, transfer all the rinse liquid into the glass test tube, and make up to 10mL with the extract. Place the glass test tube in the dark or wrap it with tin foil and soak for 3h. Observe that the tissue residue at the bottom of the test tube turns completely white, which means the extraction is complete. If the tissue residue does not turn completely white, continue to extract until it turns completely white. Take 1mL of the extract in a 1mL glass cuvette, adjust the extract to zero, measure the absorbance at 663nm and 645nm, record them as A663 and A645 respectively, and calculate the chlorophyll a, chlorophyll b and total chlorophyll content by the formula. The results are as follows Figure 3 shown.

[0104] The calculation formula is as follows:

[0105] Chlorophyll a content (mg / g) = (12.7 × A 663 -2.69×A 645 )×V 提 ×D÷m÷1000

[0106] =0.01×(12.7×A 663 -2.69×A 645 )×D÷m

[0107] Chlorophyll b content (mg / g) = (22.9 × A 645 -4.68×A 663 )×V 提 ×D÷m÷1000

[0108] =0.01×(22.9×A 645 -4.68×A 663 )×D÷m

[0109] Total chlorophyll content (mg / g) = (20.21 × A 645 +8.02×A 663 )×V 提 ×D÷m÷1000

[0110] =0.01×(20.21×A 645 +8.02×A 663 )×D÷m

[0111] Where V 提 : extraction volume, 10 mL; D: dilution factor; m: sample mass, g.

[0112] 2. Observation of leaf ultrastructure

[0113] The leaves of T1 seedlings with large differences in chlorophyll content (control, partially chlorotic, and completely yellow) were selected for chloroplast ultrastructure observation. The same part of the leaves was selected for sampling, avoiding the veins. The fresh tissue was cut into 1 mm pieces with a scalpel. 2 The small pieces were immediately placed in a headspace bottle filled with electron microscopy fixative and evacuated with a vacuum pump until they sank to the bottom. After post-fixation, room temperature dehydration, infiltration embedding, polymerization, ultrathin sectioning, staining and other treatments, the chloroplast ultrastructure was observed under a transmission electron microscope, and images were collected for analysis.

[0114] The results are as follows Figure 4 As shown, in the mesophyll cells of K326 (control), chloroplasts were well-developed, elongated, and regularly distributed close to the cell membrane. The chloroplast structure was clear, with intact inner and outer membranes, and the starch particles within the chloroplasts were large. Compared to the control, in NtIPMD gene-silenced plants, leaf cell volume increased, the number of chloroplasts decreased significantly, the leaf green color was unevenly distributed, and the starch granules within the chloroplasts were significantly smaller. Abnormal chloroplast development in NtIPMD gene-silenced plants may be one of the causes of chlorosis in silenced plants.

[0115] In summary, the present invention uses RNA interference silencing technology to interfere with NtIPMD gene expression in tobacco, generating NtIPMD gene-silenced transgenic plants. Testing revealed significantly reduced chlorophyll a / b and total chlorophyll content in the leaves of NtIPMD gene-silenced plants, stunted chloroplast development and structural damage, uneven leaf green distribution, and slow growth, though they continued to grow normally until harvest. This demonstrates that the NtIPMD gene plays a crucial role in regulating tobacco chlorophyll content. Furthermore, long-term cultivation revealed that this effect is stably inherited, and cultivating multiple generations of transgenic tobacco revealed no significant effects on other tobacco phenotypes besides the aforementioned phenotypes.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. NtIPMD The application of the gene in regulating tobacco chlorophyll content is characterized by: described NtIPMD The nucleotide sequence of the gene is shown in SEQ ID NO.1; the tobacco is the flue-cured tobacco variety K326; NtIPMD After the gene was expressed, the chlorophyll content in tobacco leaves was significantly reduced.

2. according to claim 1 NtIPMD The application of the gene in regulating tobacco chlorophyll content is characterized by: The inhibition is to construct an RNAi interference vector, NtIPMD Silencing gene expression.

3. according to claim 2 NtIPMD The application of the gene in regulating tobacco chlorophyll content is characterized by: The RNAi interference vector comprises NtIPMD The specific nucleotide fragment of the gene serves as a guide sequence; the nucleotide sequence of the guide sequence is shown in SEQ ID NO.

3.

4. according to claim 3 NtIPMD The application of the gene in regulating tobacco chlorophyll content is characterized by: The preparation method of the RNAi interference vector comprises the following steps: inserting the guide sequence into the PBWA (V) HS empty vector, screening, sequencing and identification.

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