Application of tobacco NtIDD9 gene in leaf included angle regulation and control

Abstract

The invention provides an application of a tobacco NtIDD9 gene in leaf included angle regulation and control, and belongs to the technical field of tobacco gene engineering. By inhibiting expression of the tobacco NtIDD9 gene, regulating and controlling increase of a plant leaf included angle, in-situ hybridization detection shows that the tobacco NtIDD9 gene is expressed in an endoderm of a petiole base, the NtIDD9 gene is silenced through a gene engineering method, and the tobacco NtIDD9 gene is expressed in an endoderm of the petiole base. The tobacco transgenic line with the increased tobacco leaf included angle is obtained through screening, and a technical support is provided for improving the tobacco yield and the tobacco quality.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology and specifically relates to a tobacco NtIDD9 Application of genes in leaf angle regulation. Background Art

[0002] As a key economic crop, tobacco has a wide consumer market around the world. With the increasing market demand for tobacco, improving the yield and quality of tobacco leaves has become an important topic in tobacco research.

[0003] The growth process of tobacco is affected by many factors, among which the angle of the leaves is an important physiological characteristic. The angle of the leaves affects the incident angle of light, which has a direct impact on the efficiency of photosynthesis. Reasonable leaf arrangement can reduce the shadow effect and increase the light capture rate, thereby promoting the growth and development of plants. The optimal angle of the leaves can maximize the use of sunlight, enhance the efficiency of photosynthesis, and thus accelerate the growth rate of tobacco and increase yield.

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

[0005] In recent years, advances in genomics and molecular biology techniques have gradually revealed the genetic factors that affect the angle of plant leaves. Studies have found some genes that are closely related to leaf growth angles. The functions and regulatory mechanisms of these genes provide a theoretical basis for genetic improvement and optimization of leaf angles. In-depth exploration of tobacco leaf angle regulatory genes not only helps to understand the basic mechanism of plant growth, but also provides new ideas and methods for breeding higher-yielding and more stress-resistant tobacco varieties. Summary of the invention

[0006] To solve the above problems, the present invention aims to provide a tobacco NtIDD9 The application of genes in the regulation of leaf angle is proved by experiments NtIDD9 The expression of the gene can affect the angle of tobacco leaves, which helps the leaves to maximize the use of sunlight and enhance the efficiency of tobacco photosynthesis.

[0007] To achieve the above object, tobacco of the present invention NtIDD9 The technical solution adopted for the application of genes in leaf angle regulation is: tobacco NtIDD9 The application of genes in leaf angle regulation by inhibiting tobacco NtIDD9 Gene expression regulates the increase in leaf angle in tobacco plants NtIDD9 The base sequence of the gene is shown in SEQ ID NO.1.

[0008] The beneficial effect of the above technical solution is that: in tobacco, by inhibiting NtIDD9 Gene expression can regulate the increase of tobacco leaf angle, which indicates NtIDD9 Genes play an important role in regulating the angle of tobacco leaves. The optimal angle of leaves can maximize the use of sunlight, enhance the efficiency of photosynthesis, regulate tobacco growth and development, and lay the foundation for improving tobacco yield and quality.

[0009] Preferably, the tobacco NtIDD9 Gene expression methods include constructing NtIDD9 Gene RNAi vector, NtIDD9 Inhibition of gene expression.

[0010] Further preferably, the target sequence of the RNAi vector is shown as SEQ ID NO.3.

[0011] Further preferably, the method for constructing the RNAi vector comprises obtaining the target sequence fragment for constructing the RNAi vector by amplifying with forward and reverse primers, and the forward and reverse primers are shown as SEQ ID NO.6-SEQ ID NO.9.

[0012] Further preferably, by genetic engineering NtIDD9 Gene silencing was used to screen and obtain transgenic tobacco lines with increased leaf angles. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 In Example 2 of the present invention NtIDD9 The results of in situ hybridization detection of the gene in the base tissue of tobacco petiole; Figure 2 In Example 5 of the present invention NtIDD9 Gene silencing in plants NtIDD9 Gene expression detection; Figure 3 In Example 5 of the present invention NtIDD9 Leaf angle statistics of gene silenced plants; Figure 4 In Example 5 of the present invention NtIDD9 Comparison of phenotypes of gene-silenced plants (left: wild type WT, right: silenced plants RNAi). DETAILED DESCRIPTION

[0014] The present invention is constructed by NtIDD9Gene RNAi vector, NtIDD9 Gene expression is inhibited, the angle of the plant leaves is regulated to increase, and transgenic tobacco strains with increased leaf angles are screened.

[0015] The technical solution of the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment can be obtained from commercial sources.

[0016] In the following examples, unless otherwise specified, the examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions.

[0017] Biomaterials: Tobacco material: Cultivated tobacco ( Nicotiana tabacum ) variety K326, available from public channels; Tobacco of the present invention NtIDD9 Specific examples of the application of genes in leaf angle regulation: Example 1 This embodiment is the angle of tobacco leaves NtIDD9 The specific implementation process of gene cloning is as follows: (1) Total RNA extraction Tobacco (K326) leaves were taken as samples and ground into powder in liquid nitrogen; 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, shake to mix, centrifuge, and carefully transfer the upper aqueous phase to another centrifuge tube; Add 500 μl of isopropanol to precipitate and separate RNA by centrifugation, then wash with 75% alcohol, dry slightly at room temperature, and add an appropriate volume of RNase-free water to fully dissolve; Finally, the extracted total RNA was treated with DNase I for subsequent cDNA preparation. During DNase I digestion, the 10 μl reaction system was as follows: The extracted total RNA, 1 μg; 10×reaction buffer with MgCl2, 1 μl; DNase I (RNase-free), 1 μl (1U); Add DEPC-treated water to 10 μl; Place in a 37°C water bath for 30 min.

[0018] (2) cDNA synthesis Prepare the template RNA / primer mixture in a sterile 0.2 ml centrifuge tube, keep it at 70℃ for 10 min, then quickly cool it on ice for more than 2 min, and centrifuge it for a few seconds to allow the denatured solution of the template RNA / primer to accumulate at the bottom of the centrifuge tube; The template RNA / primer mixture (7 μl system) is specifically: 1 μl of RNA (100 ng / μl) digested with DNase I in step (1); Oligo (dT) Primer (50 μM), 1 μl; RNase-free dH2O, 5 μl; After the reverse transcription reaction solution is prepared in the above centrifuge tube, it is kept at 42°C for 1 hour; kept at 70°C for 15 minutes and then cooled on ice, and the obtained result is cDNA; The specific reverse transcription reaction solution system (10 μl) is: The above template RNA / primer denaturation solution, 7 μl; 5× M-MLV buffer, 2 μl; dNTP Mixture (10 mM each), 0.5 μl; RNase Inhibitor (40 U / μl), 0.25 μl; RTase M-MLV (RNase H-) (200 U / μl), 0.25 μl.

[0019] (3) PCR amplification First, the primer sequences for PCR amplification were designed as shown in SEQ ID NO.4 - SEQ ID NO.5: Amplification NtIDD9 Primers for gene fragments: NtIDD9 -F: 5 '-ATGGCAACAAACAGATTCTTATGTG-3 ', NtIDD9 -R: 5 '-TCACTGAAAATTTCCAGCACTTGCA-3 '.

[0020] Subsequently, PCR amplification was performed using the cDNA prepared in step (2) as a template. The 50 μl amplification system was designed as follows: cDNA template, 1 μl; GXL polymerase, 1 μl; 5×GXL buffer, 10 μl; dNTP Mixture (10 mM), 4 μl; NtIDD9 -Primer-F / R, 8 μl; ddH2O, 26 μl; The PCR reaction program was as follows: 98°C, 10 sec; 55°C, 15 sec, 68°C, 30 sec, 35 cycles, and extension at 72°C for 5 min.

[0021] The PCR product obtained by amplification was subjected to 1% agarose gel electrophoresis, and then the PCR amplification product was recovered and purified, and then sequenced to obtain a 1227 bp nucleotide sequence as shown in SEQ ID NO. NtIDD9 Gene coding sequence, and obtain the sequence shown in SEQ ID NO.2 NtIDD9 The amino acid sequence of the protein.

[0022] Example 2 This example uses in situ hybridization technology to NtIDD9 The cellular expression pattern of the gene is detected, and the specific implementation process is as follows: Tissue fixation: Rinse the tissue with PBS, then immediately place it in in situ hybridization fixative (plant) for more than 12 hours, evacuate with a vacuum pump, and store and transport at 4°C.

[0023] Dehydration, wax immersion and embedding: After tissue fixation, cut the tissue block of the target area in the fume hood, about 3mm thick, dehydrate through low to high gradient alcohol, make it transparent through xylene, and then embed it in wax.

[0024] Paraffin sectioning: The tissue paraffin block was sliced ​​into 6 μm thick slices using a microtome, and the slices were scooped out using a slide spreader and baked in a 62°C oven for 2 h.

[0025] Dewaxing of paraffin sections: put the sections into dewaxing transparent liquid I for 15 min - dewaxing 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 soak in DEPC water.

[0026] Repair and digestion: Repair with repair solution, draw circles with a gene pen after natural cooling, add proteinase K (20ug / ml) at 40℃ for digestion, rinse with pure water and then wash 3 times with PBS, each time for 5 minutes.

[0027] Prehybridization: add prehybridization solution and incubate at 65℃ for 25min.

[0028] Hybridization: Pour off the pre-hybridization solution, add probe-containing hybridization solution, and hybridize overnight in a constant temperature incubator.

[0029] Washing after hybridization: wash away the hybridization solution, wash with 2×SSC at 40℃ for 10min, wash with 1×SSC at 40℃ for 2×5min, and wash with 0.5×SSC at room temperature for 10min.

[0030] Add branched probe for hybridization: Gently shake dry the slices, add preheated branched probe hybridization solution (60 μl), place horizontally in a wet box and hybridize at 40°C for 45 minutes. During this process, add 50 ml of 2×SSC to the bottom of the wet box to prevent the slices from drying out.

[0031] Washing after hybridization: Pour off the hybridization solution and rinse the sections in 2×SSC, 1×SSC, 0.5×SSC, and 0.1×SSC preheated at 40°C for 5 minutes.

[0032] Add the corresponding signal probe: add the hybridization solution containing the signal probe, dilute it at a ratio of 1:400, incubate at 40°C for 3 hours, and then wash with the following SSC in sequence: 2×SSC, wash at 40°C for 10 minutes, 1×SSC, wash at 40°C for 2×5 minutes, and 0.5×SSC at 40°C for 10 minutes.

[0033] Add blocking solution: add blocking serum (normal rabbit serum) and incubate at room temperature for 30 minutes.

[0034] Add AP-labeled mouse anti-digoxigenin antibody (anti-DIG-AP): Pour off the serum blocking solution, add anti-DIG-AP, incubate at 40°C for 50 min, and wash with TBS 4 times for 5 min.

[0035] BCIP / NBT color development: add BCIP / NBT color development solution, observe the positive result under microscope, and rinse with pure water.

[0036] Sealing: Glycerol gelatin sealing.

[0037] Microscopy, image acquisition and analysis.

[0038] Specific results such as Figure 1 As shown in the figure, it can be seen that tobacco NtIDD9 The gene is expressed in the endoderm at the base of the petiole.

[0039] Example 3 This embodiment is to construct NtIDD9 Gene RNAi vector (pBWA(V)HS-NtIDD9-RNAi vector), the specific implementation process is as follows: (1) Obtain the target sequence First, according to the sequencing results in Example 1 NtIDD9 Gene coding sequence, and based on the characteristics of RNAi interference technology, selection NtIDD9The 360bp sequence in the gene coding region was used as the target site, and two pairs of specific primers were designed, one pair was a forward fragment primer, and the other pair was a reverse fragment primer. The specific designed PCR amplification primer sequences are shown in SEQ ID NO.6-SEQ ID NO.9: Forward Primer: IDD F(+):5'-cagtGGTCTCacaacGGCAATACTAATCACAACCTCATTG-3', IDD F(-):5'-cgatGGTCTCacaggATTACTTGTAGTAGATCCAATTTGA-3'; Reverse Primer: IDD R(+):5'-cagtGGTCTCagggcATTACTTGTAGTAGATCCAATTTGA-3', IDD R(-):5'-cagtGGTCTCatacaGGCAATACTAATCACAACCTCATTG-3'; PCR amplification was performed using tobacco cDNA as a template.

[0040] The PCR amplification system was designed as follows: cDNA template, 1 μl; GXL polymerase, 1 μl; 5×GXL buffer, 10 μl; dNTP Mixture (10 mM), 4 μl; IDD-Primer-F / R, 8 μl; ddH2O, 26 μl; The PCR reaction program was as follows: 98°C, 10 sec; 55°C, 15 sec, 68°C, 30 sec, 35 cycles, and extension at 72°C for 5 min.

[0041] The target sequence fragment for constructing the RNAi vector is obtained, and the target sequence is shown as SEQ ID NO.3.

[0042] (2) Enzyme digestion and ligation Plasmid pBWA(V)HS-RNAi was used as a vector. The pBWA(V)HS vector was double-digested with BsaI and Eco31I, and the digestion products were recovered. The target sequence product obtained in step (1) was ligated with the digested pBWA(V)HS vector using Infusion ligase.

[0043] (3) Conversion Under sterile conditions, add 10 μl of the ligation product from step (2) to the competent cells, mix gently, and place on ice for 30 minutes; Heat shock at 42°C for 90 seconds, then quickly transfer the tube to an ice bath for 2-3 minutes; Add 800 μl of LB medium without antibiotics and shake at 37°C and 120 rpm for about 1 h; Take 200 μl of culture solution and apply it on LB solid medium containing 50 μg / ml antibiotics (add X-Gal and IPTG before applying the culture solution) and culture it upside down at 37℃ for 12 hours.

[0044] (4) Screening and identification During the culture process, the plaques in the culture medium will be divided into two categories: blue and white. When the plaques grow to a suitable size, pick a few white spots with a sterilized pipette tip and culture them in LB liquid culture medium containing 50μg / ml kanamycin for 12 hours. Then extract the plasmid and perform enzyme digestion identification to ensure that the recombinant vector is constructed correctly. The correctly constructed plasmid (or strain containing the plasmid) is saved for future use. Finally, the correctly constructed recombinant plasmid is named: pBWA(V)HS- NtIDD9- RNAi.

[0045] Example 4 This example is a method for constructing tobacco transformation mediated by Agrobacterium tumefaciens NtIDD9 Gene silencing plants, the specific implementation process is as follows: (1) Agrobacterium transformation and preparation of bacterial solution for transfection 1 μg of pBWA(V)HS- NtIDD9 -RNAi recombinant vector was added to 100 μl of EHA105 Agrobacterium competent cells, mixed and placed on ice for 30 min; Then, freeze it in liquid nitrogen for 5 min, take it out of liquid nitrogen and immediately put it in a 37°C water bath for 5 min, and then let it stand on ice for 5 min; Add 500 μl of LB culture medium and culture at 28°C with shaking for 4 h. Finally, spread the bacterial solution evenly on a solid culture medium supplemented with antibiotics (50 mg / L kanamycin and 50 mg / L rifampicin) and culture at 28°C for about 24 h. Pick a single plaque and place it in 5 ml LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and culture it overnight at 28°C and 200 rpm until the bacterial solution 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 medium, resuspend the Agrobacterium, centrifuge at 4000 r / min for 5 min, and repeat this operation once; Finally, resuspend the bacterial solution in 1 ml of MS liquid culture medium and add it to 40 ml of MS liquid culture medium (containing 40 μL, 25 mg / L acetosyringone), which is the infection solution. Leave it for 2 hours before use for infection.

[0046] (2) Leaf disk method transformation (a) Sow sterile tobacco K326 seeds on MS medium for cultivation. When the tobacco seedlings grow to 3-5 cm (about 20-30 days), take the top buds and place them on MS+BA (6-benzyladenine) 0.2 mg / L (to strengthen the buds and make them grow quickly) medium for subculture; (b) After 14 days of subculture (small leaves are sufficient), take leaves of about 1 cm × 1 cm in size, cut off the petiole, scratch the leaf surface and leaf edge, and place them on the pre-culture medium of MS + BA 1.0 mg / L (pH 6.0-6.5), facing down and close to the culture medium, and pre-culture in the dark for 2 days; (c) taking out the leaves pre-cultured in step (b), placing them in the Agrobacterium infection solution for infection for 15 min, and then taking them out and absorbing the bacterial solution on sterilized dry filter paper; (d) Place the infected leaves back on the pre-culture medium and co-culture at 28°C in the dark for 2-3 days until micro-bacteria spots form around the cut edges of the leaves. Take out the co-cultured tobacco leaves again and rinse them with sterile water supplemented with 500 mg / L Cef (cephalosporin) to wash off the Agrobacterium on the surface of the explants. (e) After drying the surface liquid in step (d) with filter paper, transfer the culture medium to tobacco budding medium (MS + BA1.0mg / L + Hyg (hygromycin) 25mg / L + Cef 500mg / L pH 5.8). During the culture process, change the culture medium every 2 weeks until adventitious buds grow (usually 2 weeks), cut the regenerated seedlings (about 1 cm), transfer to subculture medium (MS + BA 0.2mg / L + Hyg 25mg / L + Cef 500mg / L, pH 5.8) for culture, and when the seedlings grow to 2 cm in length (small buds are sufficient), transfer to rooting medium (MS + NAA (naphthaleneacetic acid) 0.2mg / L) and culture at 25°C with 12h light for about 3 weeks to ensure that a strong root system grows; (g) When the roots grow to 2-3 cm and the seedlings are about 7-10 cm tall, remove the flask, wash off the root culture medium, transplant it into a flowerpot, and culture it in the greenhouse.

[0047] (3) Identification of transgenic lines After the tobacco seedlings in (2) grow for about one month, take a small amount of leaves as samples, extract DNA, and detect positive transgenic strains through PCR amplification, cloning, and sequencing. The specific identification method is as follows: A pair of PCR detection primers as shown in SEQ ID NO.10 and SEQ ID NO.11 were designed to identify positive transformed plants by PCR: specifically: NtIDD9 -JF: 5 '-TCACTGAAAATTTCCAGCACTTGCA-3 ', NtIDD9 -JR: 5'-ATGGCAACAAACAGATTCTTATGTG-3'.

[0048] PCR amplification was performed using DNA templates of T0 transgenic strains; PCR conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 40 s, for a total of 25 cycles; and final extension at 72°C for 10 min. The PCR amplification was performed on agarose gel electrophoresis to identify the positive transformed plants.

[0049] Example 5 This example is to determine NtIDD9 Gene expression, leaf angle measurement and phenotypic changes were observed. The specific implementation process is as follows: (1) NtIDD9 Expression quantification Wild-type WT and RNAi plant samples were collected, RNA was extracted, and cDNA was synthesized as a template using a reverse transcription kit (refer to the kit instructions for operation). NtL25 The gene was used as an internal reference for fluorescence quantitative PCR detection.

[0050] Detection NtIDD9 The fluorescent quantitative primers of the gene, the primer sequences are shown in SEQ ID NO.12 and SEQ ID NO.13: NtIDD9 -qF: 5'-GAGCAGCAACAGCACCAATT-3', NtIDD9 -qR: 5'- TGCTGTTGTTGCTGTTGCTC-3'; Testing tobacco NtL25 When the gene is expressed, the primer sequences are shown in SEQ ID NO.14 and SEQ ID NO.15: NtL25 -F: 5'-CAAAAGTTACATTCCACCG-3', NtL25 -R: 5'-TTTCTTCGTCCCCATCAGGC-3'.

[0051] The conditions of fluorescence quantitative PCR were as follows: the first step was pre-denaturation at 95°C for 10 s; the second step was PCR reaction at 95°C for 5 s, 60°C for 30 s, and 39 cycles; the third step was melting curve.

[0052] Each sample was biologically replicated three times. -△△CT Methods Relative gene expression was analyzed.

[0053] The results are as follows Figure 2 As shown, compared with the wild type WT, in RNAi plants, tobacco NtIDD9 The gene expression level was significantly decreased.

[0054] (2) Phenotypic observation and measurement The rooted seedlings of the wild-type WT and RNAi-silenced T0 plants were transplanted into pots and cultured in the greenhouse for about 6 weeks. The leaf angles were observed and measured. Figure 3 and Figure 4 (Left: wild type WT, right: RNAi silenced plant) shows that compared with wild-type tobacco, the leaf angle of gene-silenced positive plants is significantly larger.

[0055] From the above results, it can be seen that NtIDD9 The expression level of the gene can control the angle of tobacco leaves. Based on this result, it can lay the foundation for tobacco growth and development as well as improving tobacco yield and quality.

[0056] Finally, it is 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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. Tobacco NtIDD9 The application of genes in regulating leaf angle is characterized in that: By suppressing tobacco NtIDD9 The expression of the gene regulates the increase of the leaf angle of the plant, and the tobacco NtIDD9 The base sequence of the gene is shown in SEQ ID NO.

1.

2. Tobacco according to claim 1 NtIDD9 The application of genes in regulating leaf angle is characterized in that: The inhibition of tobacco NtIDD9 Gene expression methods include constructing NtIDD9 Gene RNAi vector, NtIDD9 Inhibition of gene expression.

3. Tobacco according to claim 2 NtIDD9 The application of genes in regulating leaf angle is characterized in that: The target sequence of the RNAi vector is shown in SEQ ID NO.

3.

4. Tobacco according to claim 3 NtIDD9 The application of genes in regulating leaf angle is characterized in that: The method for constructing the RNAi vector includes obtaining a target sequence fragment for constructing the RNAi vector by amplifying with forward and reverse primers, wherein the forward and reverse primers are shown in SEQ ID NO.6 to SEQ ID NO.

9.

5. The tobacco according to any one of claims 1 to 4 NtIDD9 The application of genes in regulating leaf angle is characterized in that: Through genetic engineering NtIDD9 Gene silencing was used to screen and obtain transgenic tobacco lines with increased leaf angles.

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