Application of tobacco NtIDD9 gene in leaf angle regulation

Abstract

The application provides tobacco NtIDD9 The application belongs to the technical field of tobacco genetic engineering, and provides a method for regulating leaf angle by inhibiting the expression of a tobacco NtIDD9 The expression of the tobacco NtIDD9 Gene in the inner germ layer of the base of the petiole is detected by in situ hybridization, the tobacco NtIDD9 Gene silencing is achieved by a genetic engineering method, and a tobacco transgenic line with a larger leaf angle is screened, thereby providing technical support for improving tobacco yield and quality.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and specifically relates to a tobacco NtIDD9 gene in the regulation of leaf angle. BACKGROUND

[0002] Tobacco is a key economic crop with a wide range of consumer markets worldwide. As market demand for tobacco continues to increase, improving the yield and quality of tobacco leaves has become an important research topic in the tobacco industry.

[0003] The growth of tobacco is influenced by various factors, among which the leaf angle is an important physiological characteristic. The leaf angle affects the angle of light incidence, directly impacting the efficiency of photosynthesis. Reasonable leaf arrangement can reduce the shadow effect, increase light capture, and promote plant growth and development. The optimal leaf angle can maximize the use of sunlight, enhance the efficiency of photosynthesis, and thus accelerate the growth rate and increase the yield of tobacco.

[0004] In addition, different growth environments (such as light, temperature, humidity, etc.) also have a significant impact on the growth characteristics of plants. Related studies have shown that by adjusting the leaf angle, plants can better adapt to different environmental conditions. For example, in environments with insufficient light, properly adjusting the leaf angle can enable plants to more effectively capture light energy, thereby improving their growth potential. For tobacco, regulating leaf angle not only helps to improve photosynthetic efficiency, but also enhances its resistance and adaptability, allowing it to maintain good growth under different climate and soil conditions.

[0005] In recent years, advances in genomics and molecular biology techniques have gradually revealed the genetic factors affecting plant leaf angle. Studies have found some genes closely related to leaf growth angle, and the functions and regulatory mechanisms of these genes provide a theoretical basis for genetic improvement and optimization of leaf angle. Further exploration of tobacco leaf angle regulation genes not only helps to understand the basic mechanisms of plant growth, but also provides new ideas and methods for cultivating higher-yielding, more resistant tobacco varieties. SUMMARY

[0006] To solve the above problems, the purpose of the present application is to provide a tobacco NtIDD9 gene in the regulation of leaf angle, which has been proven through experiments that the expression of the regulation NtIDD9 gene can affect the size of the tobacco leaf angle, allowing the leaf to maximize the use of sunlight and enhance the efficiency of photosynthesis in tobacco.

[0007] To achieve the above purpose, the technical solution adopted by the tobacco NtIDD9 gene in the regulation of leaf angle is as follows:

[0008] tobacco NtIDD9 The application of genes in the regulation of leaf angle, through the inhibition of tobacco NtIDD9 Gene expression regulates the increase of leaf angle in tobacco plants. NtIDD9 The base sequence of the gene is shown in SEQ ID NO.1.

[0009] The beneficial effect of the above technical solution is that it inhibits... NtIDD9 Gene expression can regulate the increase of the leaf angle in tobacco, which indicates that... NtIDD9 Genes play an important role in regulating the leaf angle of tobacco. The optimal leaf angle can maximize the use of sunlight, enhance the efficiency of photosynthesis, regulate the growth and development of tobacco, and lay the foundation for improving tobacco yield and quality.

[0010] Preferably, suppressing tobacco NtIDD9 Methods of gene expression include constructing NtIDD9 Gene RNAi vectors, for NtIDD9 Gene expression is suppressed.

[0011] More preferably, the target sequence of the RNAi vector is shown in SEQ ID NO.3.

[0012] More preferably, the method for constructing the RNAi vector includes obtaining the target sequence fragment for constructing the RNAi vector by amplification using forward and reverse primers, as shown in SEQ ID NO.6-SEQ ID NO.9.

[0013] More preferably, genetic engineering methods are used to make NtIDD9 Gene silencing was used to screen for transgenic tobacco lines with increased leaf angle. Attached Figure Description

[0014] Figure 1 In Embodiment 2 of the present invention NtIDD9 Results of in situ hybridization detection of genes in the base tissue of tobacco petioles;

[0015] Figure 2 In Embodiment 5 of the present invention NtIDD9 In gene-silenced plants NtIDD9 Gene expression level detection;

[0016] Figure 3 In Embodiment 5 of the present invention NtIDD9 Statistics on leaf angle in gene-silenced plants;

[0017] Figure 4 In Embodiment 5 of the present invention NtIDD9 Phenotypic comparison of gene-silenced plants (left: wild-type WT, right: RNAi of silenced plants). DETAILED DESCRIPTION

[0018] The present application constructs a gene RNAi vector to inhibit gene expression, regulate leaf angle, and screen tobacco transgenic lines with large leaf angle. NtIDD9 NtIDD9 The present application constructs a gene RNAi vector to inhibit gene expression, regulate leaf angle, and screen tobacco transgenic lines with large leaf angle.

[0019] The technical solutions of the present application are further described below in combination with specific examples. Unless otherwise specified, the equipment and reagents used in each example can be obtained from commercial channels.

[0020] In the following examples, unless otherwise specified, the examples are carried out according to the conventional experimental conditions or the conditions suggested by the manufacturer's instructions.

[0021] Biological material:

[0022] Tobacco material: tobacco (Nicotiana tabacum) cultivar K326, which can be obtained from public channels; Nicotiana tabacum

[0023] The tobacco of the present application NtIDD9 Specific examples of the application of the gene in the regulation of leaf angle:

[0024] Example 1

[0025] This example is the cloning of the gene regulating the leaf angle of tobacco, and the specific implementation process is as follows: NtIDD9

[0026] (1) Total RNA extraction

[0027] Take tobacco (K326) leaves as samples, grind them into powder in liquid nitrogen;

[0028] Take about 100 mg of powdered material and place it in a 1.5 ml centrifuge tube containing 1.0 ml of TRIZOL reagent, then add 200 μl of chloroform, mix well by shaking, and centrifuge. Carefully transfer the upper aqueous phase to another centrifuge tube;

[0029] Add 500 μl of isopropanol, precipitate and centrifuge to separate the RNA, then wash with 75% alcohol, and after drying at room temperature, add an appropriate volume of RNase free water and dissolve thoroughly;

[0030] Finally, the extracted total RNA is treated with DNase I for subsequent cDNA preparation. The 10 μl reaction system for DNase I digestion is as follows:

[0031] 1 μg of extracted total RNA;

[0032] 1 μl of 10×reaction buffer with MgCl2;​​​

[0033] DNase I (RNase-free), 1 μl (1 U);

[0034] DEPC-treated water to 10 μl;

[0035] Put in 37°C water bath for 30 min.

[0036] (2) cDNA synthesis

[0037] Prepare the template RNA / primer mixture in a sterile 0.2 ml centrifuge tube, incubate at 70°C for 10 min, then quickly cool on ice for 2 min or more, and centrifuge for a few seconds to make the denatured template RNA / primer solution gather at the bottom of the centrifuge tube;

[0038] The template RNA / primer mixture (7 μl system) is specifically:

[0039] RNA (100 ng / μl) after DNase I digestion in step (1), 1 μl;

[0040] Oligo (dT) Primer (50 μM), 1 μl;

[0041] RNase free dH2O, 5 μl;

[0042] After preparing the reverse transcription reaction solution in the above centrifuge tube, incubate at 42°C for 1 h; cool on ice after incubating at 70°C for 15 min, and the obtained is cDNA;

[0043] The specific reverse transcription reaction solution system (10 μl) is:

[0044] The above template RNA / primer denatured solution, 7 μl;

[0045] 5×M-MLV buffer, 2 μl;

[0046] dNTP Mixture (each 10 mM), 0.5 μl;

[0047] RNase Inhibitor (40 U / μl), 0.25 μl;

[0048] RTase M-MLV (RNase H-) (200 U / μl), 0.25 μl.

[0049] (3) PCR amplification

[0050] First, the primer sequence for PCR amplification is shown as SEQ ID NO. 4 - SEQ ID NO. 5:

[0051] Amplification NtIDD9 Primers of the gene fragment:

[0052] NtIDD9 -F: 5 '-ATGGCAACAAACAGATTCTTATGTG-3 ',

[0053] NtIDD9 -R: 5 '-TCACTGAAAATTTCCAGCACTTGCA-3 '.

[0054] Subsequently, the cDNA prepared in step (2) is used as a template to perform PCR amplification, and the 50 μl amplification system is designed as follows:

[0055] cDNA template, 1 μl;

[0056] GXL polymerase, 1 μl;

[0057] 5×GXL buffer, 10 μl;

[0058] dNTP Mixture (10 mM), 4 μl;

[0059] NtIDD9 -Primer-F / R, 8 μl;

[0060] ddH2O, 26 μl;

[0061] The PCR reaction program is shown as follows: 98℃, 10 sec; 55℃, 15 sec, 68℃, 30 sec, 35 cycles, 72℃ extension 5 min.

[0062] The PCR product obtained by amplification is subjected to 1% agarose gel electrophoresis, and then the PCR amplification product is recovered and purified, and sequenced to obtain a nucleotide sequence containing 1227 bp of the gene coding sequence shown as SEQ ID NO. 1, and obtain the amino acid sequence of the protein shown as SEQ ID NO. 2. NtIDD9 NtIDD9

[0063] Example 2

[0064] This example is to detect the cell expression pattern of the tobacco NtIDD9 gene by using in situ hybridization technology, and the specific implementation process is as follows:

[0065] ​​Tissue fixation: The tissue was rinsed with PBS and then immediately immersed in the in situ hybridization fixation solution (plant) for more than 12 h, vacuum pumping, and stored and transported at 4°C.

[0066] Dehydration and wax immersion embedding: After the completion of tissue fixation, the tissue block of the target region was cut in a fume hood, about 3 mm thick, dehydrated by alcohol from low to high gradient, and then transparentized by xylene and embedded by wax immersion.

[0067] Paraffin sectioning: The paraffin block was sectioned by a microtome to a thickness of 6 μm, and the section was taken out by a section lifter and baked in a 62°C oven for 2 h.

[0068] Paraffin section deparaffination: The section was sequentially placed in deparaffination transparent liquid I for 15 min, deparaffination transparent liquid II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and DEPC water for immersion.

[0069] Repair and digestion: Repair was performed by using a repair solution, and after natural cooling, the section was circled by a gene pen, and proteinase K (20 ug / ml) was added for digestion at 40°C, and then the section was washed with pure water and PBS for 3 times, each for 5 min.

[0070] Pre-hybridization: Pre-hybridization solution was added, and incubation was performed at 65°C for 25 min.

[0071] Hybridization: The pre-hybridization solution was poured off, and hybridization solution containing a probe was added, and hybridization was performed overnight in a thermostat.

[0072] Post-hybridization washing: The hybridization solution was washed off, and the section was sequentially washed with 2×SSC at 40°C for 10 min, 1×SSC at 40°C for 2×5 min, and 0.5×SSC at room temperature for 10 min.

[0073] Dropwise addition of branch probe hybridization: The section was gently shaken and dried, and preheated branch probe hybridization solution (60 μl) was added, and the section was horizontally placed in a wet box for hybridization at 40°C for 45 min, and 50 ml of 2×SSC was added to the bottom of the wet box to prevent the section from drying.

[0074] Post-hybridization washing: The hybridization solution was poured off, and the section was sequentially washed with 2×SSC, 1×SSC, 0.5×SSC, and 0.1×SSC preheated at 40°C for 5 min.

[0075] Dropwise addition of corresponding signal probe: Signal probe hybridization solution was added, and the dilution ratio was 1:400, and incubation was performed at 40°C for 3 h, and then the section was sequentially washed with 2×SSC at 40°C for 10 min, 1×SSC at 40°C for 2×5 min, and 0.5×SSC at 40°C for 10 min.

[0076] Dropwise addition of blocking solution: Normal rabbit serum was added as a blocking solution, and incubation was performed at room temperature for 30 min.

[0077] Add anti-DIG-AP: pour off the serum blocking solution, add anti-DIG-AP, incubate at 40°C for 50 min, and then wash with TBS 4 times for 5 min each time.

[0078] Coloring with BCIP / NBT color developing agent: add BCIP / NBT color developing solution, observe the positive under a microscope, and then rinse with pure water.

[0079] Mounting: mount with glycerol gelatin.

[0080] Microscopic examination, image acquisition and analysis.

[0081] Specific results are shown in Figure 1 the figures, from which it can be seen that the tobacco NtIDD9 gene is expressed in the endoderm of the leaf petiole base.

[0082] Example 3

[0083] This example is directed to constructing NtIDD9 a gene RNAi vector (pBWA(V)HS-NtIDD9-RNAi vector), and the specific implementation process is as follows:

[0084] (1) Obtain the target sequence

[0085] First, according to the gene coding sequence obtained by sequencing in Example 1, and based on the characteristics of the RNAi interference technology, a 360bp length sequence in the coding region of the gene is selected as the target site, and two pairs of specific primers are designed, one pair being forward fragment primers and the other pair being reverse fragment primers. The specific design of the PCR amplification primer sequences is shown in SEQ ID NO. 6-SEQ ID NO. 9: NtIDD9 NtIDD9 Forward primer:

[0086] IDD F(+): 5'-cagtGGTCTCacaacGGCAATACTAATCACAACCTCATTG-3',

[0087] IDD F(-): 5'-cgatGGTCTCacaggATTACTTGTAGTAGATCCAATTTGA-3';

[0088] Reverse primer:

[0089] IDD R(+): 5'-cagtGGTCTCagggcATTACTTGTAGTAGATCCAATTTGA-3',

[0090] IDD R(-): 5'-cgatGGTCTCacaggATTACTTGTAGTAGATCCAATTTGA-3'.

[0091] ​IDD R(-): 5'-cagtGGTCTCatacaGGCAATACTAATCACAACCTCATTG-3';

[0092] PCR amplification was performed using tobacco cDNA as template.

[0093] The PCR amplification system was designed as follows:

[0094] cDNA template, 1 μl;

[0095] GXL polymerase, 1 μl;

[0096] 5×GXL buffer, 10 μl;

[0097] dNTP Mixture (10 mM), 4 μl;

[0098] IDD-Primer-F / R, 8 μl;

[0099] ddH2O, 26 μl;

[0100] The PCR reaction program was as follows: 98℃, 10 sec; 55℃, 15 sec, 68℃, 30 sec, 35 cycles, 72℃ extension for 5 min.

[0101] The target sequence fragment for constructing the RNAi vector was obtained, and the target sequence was shown as SEQ ID NO. 3.

[0102] (2) Enzymatic digestion and ligation

[0103] The plasmid pBWA(V)HS-RNAi was used as a vector, and the pBWA(V)HS vector was subjected to double digestion with BsaI and Eco31I, and the digestion products were recovered, respectively. The target sequence product obtained in step (1) and the digested pBWA(V)HS vector were ligated using Infusion ligase.

[0104] (3) Transformation

[0105] Under sterile conditions, 10 μl of the ligation product in step (2) was added to the competent cells, and then mixed gently, followed by ice bath for 30 min;

[0106] 42℃ heat shock for 90 s, and then the centrifuge tube was quickly transferred to the ice bath for 2-3 min;

[0107] 800 μl of LB medium without antibiotics was added, and then the mixture was shaken at 37℃ and 120 rpm for about 1 h;

[0108] Take 200 μl culture liquid to be coated on (firstly add X-Gal and IPTG before coating the bacterial liquid) LB solid culture medium containing antibiotic 50 μg / ml, 37°C inverted culture for 12 h.

[0109] (4) Screening and identification

[0110] During the culture process, the bacterial plaque in the culture medium will be divided into blue and white two types, when the plaque grows to the appropriate size, use sterilized gun head to pick up several white plaques, respectively in LB liquid medium containing 50 μg / ml kanamycin, shake culture for 12 h, then extract plasmid, perform enzyme digestion identification, ensure that the recombinant vector construction is correct, for the plasmid (or bacterial strain containing plasmid) construction correct, finally, the correct recombinant plasmid is named: pBWA(V)HS- NtIDD9- RNAi.

[0111] Example 4

[0112] This embodiment is to construct NtIDD9 gene silencing plant by using Agrobacterium-mediated tobacco transformation method, the specific implementation process is as follows:

[0113] (1) Agrobacterium transformation, preparation of transfection bacterial liquid

[0114] 1 μg of pBWA(V)HS- NtIDD9 -RNAi recombinant vector prepared in example 2 is added to 100 μl of EHA105 Agrobacterium competent cells, mixed and placed on ice for 30 min;

[0115] Then freeze in liquid nitrogen for 5 min, immediately put into 37°C water bath after taking out from liquid nitrogen, water bath for 5 min, and then place on ice for 5 min;

[0116] Add 500 μl of LB culture solution, recover and culture at 28°C under shaking condition for 4 h, and finally evenly smear the bacterial liquid on the solid culture medium added with antibiotics (50 mg / L kanamycin and 50 mg / L rifampicin), and culture at 28°C for 24 h or so;

[0117] Pick single plaque in 5 ml LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and culture at 28°C, 200 r / min overnight, and culture until the bacterial liquid concentration reaches OD 600 =1.5 or so;

[0118] Take 2 ml bacterial liquid and add to centrifuge tube, centrifuge at 4000 r / min for 5 min;

[0119] Remove the supernatant, resuspend the Agrobacterium in 1 ml of fresh MS liquid medium, centrifuge at 4000 r / min for 5 min, and repeat this operation once;

[0120] Finally, resuspend the bacterial solution in 1 ml of MS liquid medium and add it to 40 ml of MS liquid medium containing 40 μL of 25 mg / L acetosyringone. The resulting solution is the infection solution, which is used to infect after 2 h.

[0121] (2) Leaf disc transformation

[0122] (a) Sterilized tobacco K326 seeds are sown on MS medium for culture. When the tobacco seedlings grow to 3-5 cm (about 20-30 days), the apical buds are placed on MS + BA (6-benzyladenine) 0.2 mg / L medium for subculture to make them grow rapidly;

[0123] (b) After 14 days of subculture (when small leaves appear), leaf pieces of about 1 cm x 1 cm are taken, the petioles are cut off, and the leaf surface and edges are scratched. Then, the leaf pieces are placed on the pre-culture medium of MS + BA 1.0 mg / L (pH 6.0-6.5) with the front surface down and close to the medium, and pre-cultured in the dark for 2 days;

[0124] (c) The pre-cultured leaf pieces in step (b) are taken out and infected in the Agrobacterium infection solution for 15 min. Then, the bacterial solution is absorbed with sterilized dry filter paper;

[0125] (d) The infected leaf pieces are placed back on the pre-culture medium and cultured at 28°C in the dark for 2-3 days until microcalli form around the cut edges. Then, the cultured tobacco leaf pieces are taken out and washed with sterile water containing 500 mg / L Cef (cephalosporin) to wash off the Agrobacterium on the surface of the explants;

[0126] (e) After the surface liquid in step (d) is absorbed with filter paper, the leaf pieces are transferred to the tobacco bud-inducing medium (MS + BA 1.0 mg / L + Hyg (hygromycin) 25 mg / L + Cef 500 mg / L, pH 5.8). During the culture, the medium is replaced every 2 weeks until adventitious buds grow (usually 2 weeks). The regenerated seedlings (about 1 cm) are cut and transferred to the subculture medium (MS + BA 0.2 mg / L + Hyg 25 mg / L + Cef 500 mg / L, pH 5.8). When the seedlings grow to 2 cm long (with small buds), they are transferred to the rooting medium (MS + NAA (naphthalene acetic acid) 0.2 mg / L) and cultured at 25°C with 12 h light for about 3 weeks to ensure the growth of strong root systems.

[0127] (g) When the roots grow to 2-3 cm and the seedlings grow to 7-10 cm, the triangular bottle is removed, the root culture medium is washed away, and the seedlings are transplanted into a flowerpot and cultured in a greenhouse.

[0128] (3) Transgenic strain identification

[0129] After the tobacco seedlings in the above (2) grow for about a month, a small amount of leaf is taken as a sample, DNA is extracted, and positive transgenic strains are detected by PCR amplification, cloning and sequencing. The specific identification method is as follows:

[0130] A pair of PCR detection primers is designed as shown in SEQ ID NO. 10 and SEQ ID NO. 11 for identifying positive transformed plants by PCR. Specifically:

[0131] NtIDD9 J-F: 5 '-TCACTGAAAATTTCCAGCACTTGCA-3 ',

[0132] NtIDD9 J-R: 5 '-ATGGCAACAAACAGATTCTTATGTG-3 '.

[0133] PCR amplification is performed using the DNA template of the T0 generation transgenic strain. The PCR conditions are as follows: 94°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 40 s, a total of 25 cycles; and 72°C final extension for 10 min.

[0134] PCR amplification is detected by agarose gel electrophoresis to identify positive transformed plants.

[0135] Example 5

[0136] In this example, the expression amount of the gene is determined, the leaf angle is measured, and the phenotype change is observed. The specific implementation process is as follows: NtIDD9

[0137] (1) NtIDD9 Expression quantification

[0138] Wild type WT and RNAi plant samples are collected, RNA is extracted, and cDNA is synthesized as a template using a reverse transcription kit (operation according to the kit instruction manual). Tobacco NtL25 gene is used as an internal reference for fluorescence quantitative PCR detection.

[0139] Detection NtIDD9 The fluorescence quantitative primers of the gene are as follows: the primer sequences are shown in SEQ ID NO. 12 and SEQ ID NO. 13:

[0140] NtIDD9 ​q-F: 5'-GAGCAGCAACAGCACCAATT-3',

[0141] NtIDD9 q-R: 5'-TGCTGTTGTTGCTGTTGCTC-3';

[0142] Detection of tobacco NtL25 gene, the primer sequences are shown in SEQ ID NO. 14 and SEQ ID NO. 15:

[0143] NtL25 F: 5'-CAAAAGTTACATTCCACCG-3',

[0144] NtL25 R: 5'-TTTCTTCGTCCCATCAGGC-3'.

[0145] The conditions of the fluorescent quantitative PCR are as follows: the first step is pre-denaturation at 95℃ for 10 s; the second step is PCR reaction at 95℃ for 5 s and at 60℃ for 30 s, 39 cycles; and the third step is melting curve.

[0146] Each sample was biologically repeated for 3 times, and the relative gene expression amount was analyzed by 2 -△△CT method.

[0147] The results are shown in Figure 2 compared with the wild type WT, the expression level of tobacco NtIDD9 gene in the RNAi plant was significantly reduced.

[0148] (2) Phenotype observation and measurement

[0149] The T0 generation plant rooting seedlings of wild type WT and silencing line RNAi were transplanted into pots respectively, and cultured in the greenhouse for about 6 weeks. The leaf angle was observed and measured, and the results are shown in Figure 3 and Figure 4 (Left: wild type WT, right: silencing plant RNAi), compared with the wild type tobacco, the leaf angle of the positive plant of gene silencing was significantly larger.

[0150] From the above results, it can be seen that NtIDD9 the expression level of the gene can control the size of the tobacco leaf angle, and based on this result, it can lay a foundation for the growth and development of tobacco, and the improvement of tobacco yield and quality.

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Tobacco NtIDD9 application of the gene in leaf angle regulation, characterized in that, By inhibiting the expression of tobacco NtIDD9 gene, the angle between the leaves of tobacco plants is regulated to be larger, and the tobacco NtIDD9 The base sequence of the gene is shown in SEQ ID NO.

1.

2. The tobacco of claim 1 NtIDD9 application of the gene in leaf angle regulation, characterized in that, The inhibition of tobacco NtIDD9 Methods of gene expression include constructing NtIDD9 Gene RNAi vectors, to NtIDD9 Inhibit gene expression.

3. The tobacco of claim 2 NtIDD9 application of the gene in leaf angle regulation, characterized in that, The target sequence of the RNAi carrier is shown as SEQ ID NO.

3.

4. The tobacco of claim 3 NtIDD9 application of the gene in leaf angle regulation, characterized in that, The construction method of the RNAi carrier comprises obtaining a target sequence fragment for constructing the RNAi carrier by amplification of forward and reverse primers, and the forward and reverse primers are shown as SEQ ID NO. 6 - SEQ ID NO.

9.

5. The tobacco of any one of claims 1-4 NtIDD9 application of the gene in the regulation of leaf angle, characterized in that, by genetic engineering methods to NtIDD9 gene silencing, screening for tobacco transgenic lines with increased leaf angle in tobacco.

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