Tobacco lipoxygenase NtLOX2 capable of regulating and controlling pigment synthesis and application of tobacco lipoxygenase NtLOX2

Abstract

The invention relates to tobacco lipoxygenase NtLOX2 capable of regulating and controlling pigment synthesis and application of the tobacco lipoxygenase NtLOX2. The amino acid sequence of the tobacco lipoxygenase NtLOX2 is shown as SEQ ID No.1. The invention provides tobacco lipoxygenase NtLOX2 which is found to be highly related to the content of tobacco pigment, and after the gene is silenced, the content of the pigment in tobacco is obviously increased.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco genetic engineering, and in particular to tobacco lipoxygenase NtLOX2 capable of regulating pigment synthesis and applications thereof. Background Art

[0002] In 1972, Roberts et al. discovered that the aroma of flue-cured tobacco leaves was inversely proportional to the carotenoid content, indicating that the decomposition of carotenoids during the curing and aging process is positively correlated with tobacco aroma. Weeks et al., studying the relationship between the aroma quality of American tobacco and its variety, found that while the aroma quality of flue-cured tobacco leaves significantly improved in the 1980s, the degradation products of carotenoids increased significantly, indicating that the degradation of pigment substances is a key process in the development of tobacco leaf quality.

[0003] Carotenoid degradation mainly involves two pathways: enzymatic degradation and oxidative degradation. Enzymatic degradation mainly involves carotenoid cleavage dioxygenase (CCD), which catalyzes the synthesis of two important plant hormones, strigolactones and abscisic acid.

[0004] The oxidative degradation pathway primarily involves the oxidation and degradation of carotenoids, catalyzed by lipoxygenase (LOX). Researchers have extensively studied the activity of LOX, a key enzyme in the degradation of lipids, particularly unsaturated higher fatty acids. LOX catalyzes the peroxidation of 1,4-diunsaturated fatty acids and carotenoids, producing volatile carbonyl compounds associated with aroma.

[0005] LOX catalyzes the dioxygenation of polyunsaturated fatty acids with cis-, cis-, and isoprene structures, such as linoleic acid and linolenic acid. During flour processing, it catalyzes the oxidation of prenyl double-bonded oils in flour with molecular oxygen. The resulting unstable hydroperoxides can induce polymerization of gluten protein molecules, thereby strengthening gluten and breaking down the double bonds of carotene, resulting in a whitening effect. Gayen et al. found that the lipoxygenase gene r9-LOX1 can reduce the degradation of carotenoids during rice storage.

[0006] However, there are still no reports on the regulation of carotenoids and chlorophyll by lipoxygenase in tobacco leaves. Therefore, the study of the regulation of pigments by lipoxygenase has become an urgent problem to be solved. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a tobacco lipoxygenase NtLOX2 capable of regulating pigment synthesis and its application. In tobacco, gene silencing technology is used to reduce the gene expression level of tobacco lipoxygenase NtLOX2, thereby regulating the pigment content in the leaves.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a tobacco lipoxygenase NtLOX2 capable of regulating pigment synthesis. The amino acid sequence of the tobacco lipoxygenase is shown in SEQ ID No. 1.

[0010] In the present invention, gene silencing technology is used to regulate the expression of the tobacco NtLOX2 protein, thereby significantly increasing the pigment content in tobacco leaves. This protein is composed of 553 amino acid residues, and its protein functional domain is similar to that of Lipoxygenase 2 in plants. Lipoxygenase 2 (LOX2) belongs to the non-heme iron dioxygenase family and is widely distributed in plants. It catalyzes the hydroperoxidation reaction of polyunsaturated fatty acids containing cis-cis-1,4-pentadiene structures (such as linoleic acid and α-linolenic acid) to produce oxylipins (such as jasmonic acid and green leaf volatiles). This protein is positively correlated with pigment degradation in plant leaves. When the expression of this protein is reduced, the amount of pigment degradation in the leaves is reduced and the content is increased.

[0011] SEQ ID No. 1:

[0012] .

[0013] Preferably, the nucleic acid sequence of the tobacco lipoxygenase is shown as SEQ ID No. 2.

[0014] Preferably, the specific nucleic acid fragment of the tobacco lipoxygenase nucleic acid sequence is bases 315-367, and this sequence is the conserved functional domain of the Lipoxygenase 2 gene.

[0015] SEQ ID No. 2:

[0016]

[0017] In a second aspect, the present invention provides a use of the tobacco lipoxygenase capable of regulating color synthesis and / or its expression regulating agent described in the first aspect in regulating pigment synthesis.

[0018] In a third aspect, the present invention provides a method for regulating pigment synthesis in leaves, the method comprising the following steps:

[0019] The gene silencing technology or gene overexpression technology is used to change the expression level of the tobacco lipoxygenase capable of regulating chromosynthesis in the leaves according to the first aspect.

[0020] In the present invention, gene silencing technology or gene overexpression method is used to regulate the expression level of tobacco NtLOX2 protein to regulate the content of pigments and their degradation products in tobacco leaves.

[0021] Preferably, the method comprises the following steps:

[0022] (1) amplifying the nucleotide sequence of tobacco lipoxygenase NtLOX2 as shown in SEQ ID No. 2;

[0023] (2) digesting the amplified product of step (1) and the vector separately and then ligating them to obtain a recombinant vector after identification;

[0024] (3) Transforming the recombinant vector obtained in step (2) into Agrobacterium to obtain a recombinant strain;

[0025] (4) amplifying and culturing the recombinant strain obtained in step (3), collecting the cells after centrifugation, resuspending them in a resuspension solution, and transforming them into plants after standing.

[0026] Preferably, in step (1), the amplification primers include SEQ ID No. 3 and SEQ ID No. 4.

[0027] SEQ ID No.3:

[0028] GGCGTTAAGAAATTAGAGG.

[0029] SEQ ID No.4:

[0030] ATTGACACCACAGGCACT.

[0031] Preferably, in step (1), the amplification procedure includes:

[0032] Pre-denaturation: 93-98°C, 1-5 min;

[0033] Denaturation: 93-98°C, 10-20s;

[0034] Annealing extension: 52-60°C, 10-20s; 65-80°C, 20-40s; cycle 30-38 times;

[0035] Extension: 65-80°C, 1-10 min. The 93-98°C may be, for example, 93°C, 94°C, 95°C, 96°C, 97°C, or 98°C. The 1-5 min may be, for example, 1 min, 2 min, 3 min, 4 min, or 5 min. The 52-60°C may be, for example, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C. The 10-20 s may be, for example, 10 s, 11 s, 12 s, 13 s, 14 s, 15 s, 16 s, 17 s, 18 s, 19 s, or 20 s. The 65-80°C may be, for example, 65°C, 66°C, 68°C, 70°C, 72°C, 74°C, 76°C, 78°C, or 80°C. The 20-40 s may be, for example, 20 s, 22 s, 24 s, 26 s, 28 s, 30 s, 32 s, 34 s, 36 s, 38 s, or 40 s. The 30-38 times may be, for example, 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, or 38 times. The 1-10 min may be, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0036] Preferably, in step (2), the vector comprises any one or a combination of at least two of a virus-induced silencing vector, an RNAi interference vector, an overexpression vector or a genome editing vector.

[0037] Preferably, the virus-induced silencing vector comprises any one of TRV2, PVX or CMV.

[0038] TRV2 has high silencing efficiency and a wide host range (including Solanaceae and Cruciferae, etc.), while also having mild symptoms and posing a low risk to plants. Other RNA viral vectors include the PVX viral vector, which has the disadvantages of a narrow host range (only applicable to Solanaceae crops), low efficiency, obvious symptoms, and high risk. The CMV viral vector has the advantages of a wide host range and high silencing efficiency, but its disadvantage is that the symptoms of the recipient host are too significant, which may directly cause disease and affect subsequent gene function analysis.

[0039] Preferably, in step (2), the vector includes a promoter and a terminator.

[0040] Preferably, in step (2), the enzyme cleavage site includes any one or a combination of at least two of BamH I, Sac I, EcoR I, Nde I, Nco I, Pac I, Pst I, Sca I, Spe I, Xba I or Xho I;

[0041] Preferably, in step (2), the identification method includes any one or a combination of at least two of resistance screening, PCR identification or whole-genome sequencing.

[0042] Preferably, in step (3), the transfer method includes electroporation.

[0043] Preferably, in step (3), the Agrobacterium comprises one or a combination of at least two of GV3101, EHA101, EHA105 or LBA4404.

[0044] Preferably, in step (4), the resuspension comprises 5-15 mM MgCl2, 0.1-0.3 mM acetosyringone, and 5-15 mM 2-(N-morpholino)ethanesulfonic acid. The 5-15 mM concentration may be, for example, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM. The 0.1-0.3 mM concentration may be, for example, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, or 0.3 mM.

[0045] Preferably, in step (4), the standing time is 1-5 hours and the temperature is 20-30° C. The 1-5 hours can be, for example, 1 hour, 2 hours, 3 hours, 4 hours or 5 hours. The 20-30° C. can be, for example, 20° C., 22° C., 24° C., 26° C., 28° C. or 30° C.

[0046] Preferably, in step (4), the transformation method includes a transient transformation method.

[0047] Compared with the prior art, the present invention has at least the following beneficial effects:

[0048] The present invention provides a tobacco lipoxygenase gene, NtLOX2, which has been found to be highly correlated with tobacco pigment content. Silencing this gene significantly increases pigment content in tobacco. This property provides a foundation and reference for the development of new tobacco leaf varieties with high pigment content. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a phenotypic comparison of the TRV2-PDS, TRV2-GFP and TRV2-NtLOX2 vector transformation groups.

[0050] Figure 2 The graph shows the relative expression levels of the NtLOX2 gene in the TRV2-GFP group and the TRV2-NtLOX2 group plants.

[0051] Figure 3 The results show the pigment content in the plants of TRV2-GFP group and TRV2-NtLOX2 group. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0053] Sources of reagents and materials used in the following examples:

[0054] Tobacco K326: Zhengzhou Tobacco Institute Planting Base;

[0055] Ben's tobacco: Zhengzhou Tobacco Institute planting base;

[0056] TRV2 vector: Zhengzhou Tobacco Research Institute Gene Center.

[0057] Example 1

[0058] This example clones the tobacco NtLOX2 gene.

[0059] The tobacco K326 used in this example was planted at the Zhengzhou Tobacco Institute's planting base. Seedlings were raised in seedling pots and transplanted two weeks after germination. The seedlings were planted in plastic pots (10 cm × 10 cm) and maintained at 22°C under 16 h light / 8 h dark conditions with daily fertilization and watering. PCR amplification of the NtLOX2 gene was performed using cDNA from tobacco K326 leaves as a template. The primer sequences are SEQ ID No. 3 and SEQ ID No. 4. The PCR amplification procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s. After 34 cycles, the final extension was performed at 72°C for 5 min. The PCR amplification products were analyzed by agarose gel electrophoresis and recovered for later use.

[0060] By sequencing the PCR amplified product, the tobacco NtLOX2 gene comprises 1662 bases, the base sequence of which is shown in SEQ ID NO. 1. The tobacco lipoxygenase NtLOX2 comprises 553 amino acids, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0061] Example 2

[0062] This example constructs a recombinant vector

[0063] The PCR amplification product in Example 1 and the empty vector TRV2 were double-digested with EcoRI and BamHI, respectively. The digestion products were recovered and ligated using T4 DNA ligase.

[0064] The ligation product was transformed into competent E. coli DH5α. After the transformation, the product was plated on LB solid medium containing 50 mg / L Kan and incubated at 37°C for 12 hours. Positive single colonies were selected for amplification and further identified by PCR. Verification was also performed by sequencing to ensure that the recombinant vector TRV2-NtLOX2 was constructed correctly.

[0065] Example 3

[0066] This example is to prepare the recombinant Agrobacterium liquid

[0067] (1) Transformation of Agrobacterium

[0068] In this example, TRV2-GFP (blank control group) and TRV2-PDS (VIGS efficiency control group) were prepared simultaneously using the methods of Examples 1-2. GFP (green fluorescent protein) is a commonly used reporter gene. By observing whether GFP can be successfully expressed in plants, the infection efficiency and expression capacity of the VIGS system can be verified; TRV2-PDS carries the plant's endogenous phytoene dehydrogenase (PDS) gene fragment, the silencing of which blocks carotenoid synthesis, leading to photooxidative damage to chloroplasts, manifested as leaf bleaching (photobleaching). This significant phenotype makes it a positive control for the VIGS system.

[0069] The TRV2-GFP, TRV2-PDS and TRV2-NtLOX2 recombinant plasmids were transformed into Agrobacterium GV3101 competent cells by electroporation, and cultured and screened on YEB plates containing 50 mg / L Kan and 50 mg / L R-Ref. After inverted culture at 28°C for 2 days, colony PCR was used to screen Agrobacterium carrying the target gene.

[0070] (2) Preparation of bacterial solution for transfection

[0071] The positive Agrobacterium clones obtained by screening in step (1) were cultured in 5 mL of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) at 28° C. and 250 rpm for 12 h.

[0072] Take 50uL of culture and inoculate it into 50mL of YEB liquid medium (containing 50mg / L Kan), cultivate to OD600=1.2, then centrifuge at 4000g for 5min, collect the bacteria, and resuspend them in resuspension solution (10mM magnesium chloride, 10mM 2-(N-morpholino)ethanesulfonic acid and 0.15mM acetosyringone), adjust OD600=1.0, and place at 25℃ for 3h.

[0073] Example 4

[0074] In this example, plants were transformed

[0075] Three-week-old Nicotiana benthamiana leaves were used as experimental materials. The transfection bacterial solution prepared in Example 3 was injected into the tobacco leaves using a 1 mL syringe. The injection volume per leaf was approximately 1500 μL. The injected tobacco was continued to be cultured at a temperature of approximately 25°C, a humidity of 60-70%, and a photoperiod of 16 hours light / 8 hours dark. Phenotypical changes were observed. Ten Nicotiana benthamiana plants were tested in each group.

[0076] Phenotypic changes of tobacco leaves three weeks after injection Figure 1 As shown in the results, it can be seen that the new leaves of the plants infected with Agrobacterium containing TRV2-PDS showed bleaching, indicating that the infection was successful; while there was no obvious change in the TRV2-GFP group, and the corresponding tobacco plants in the TRV2-NtLOX2 group showed no significant changes, indicating that the NtLOX2 gene had no obvious effect on other basic physiological states of tobacco.

[0077] The expression of NtLOX2 gene was detected by qRT-PCR. Figure 2 As shown, it can be seen that the expression level of NtLOX2 was significantly reduced in the plants infected with TRV2-NtLOX2.

[0078] The pigment content in the plants of TRV2-NtLOX2 group and TRV2-GFP group was detected. Figure 3 As shown in the results, the expression of this gene in the TRV2-NtLOX2 group was reduced by approximately 73%, while the pigment content in gene-silenced plants increased by approximately 25%. This result indicates that the NtLOX2 gene is a negative pigment regulator, capable of pigment degradation, effectively regulating the content of pigment substances in tobacco leaves without affecting the tobacco plant phenotype. Overexpression of the gene in tobacco leaves can effectively degrade pigments. This has broad prospects for increasing the content of tobacco leaf aroma compounds and cultivating new tobacco varieties with high tolerance and high aroma.

[0079] In summary, the present invention utilizes gene silencing technology in tobacco to reduce the expression level of the NtLOX2 gene, thereby achieving regulation of the pigment content in leaves.

[0080] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A tobacco lipoxygenase NtLOX2 capable of regulating pigment synthesis, characterized in that: The amino acid sequence of the tobacco lipoxygenase is shown in SEQ ID No.

1.

2. The tobacco lipoxygenase NtLOX2 capable of regulating chromosynthesis according to claim 1, characterized in that The nucleic acid sequence of the tobacco lipoxygenase is shown in SEQ ID No.

2.

3. Use of the tobacco lipoxygenase capable of regulating color synthesis and / or its expression regulating agent according to claim 1 or 2 in regulating pigment synthesis.

4. A method for regulating pigment synthesis in leaves, characterized in that: The method comprises the following steps: The gene silencing technology or gene overexpression technology is used to change the expression level of the tobacco lipoxygenase capable of regulating color synthesis according to claim 1 or 2 in the leaves.

5. The method for regulating pigment synthesis in leaves according to claim 4, characterized in that: The method comprises the following steps: (1) amplifying the nucleotide sequence of tobacco lipoxygenase NtLOX2 as shown in SEQ ID No. 2; (2) digesting the amplified product of step (1) and the vector separately and then ligating them to obtain a recombinant vector after identification; (3) Transforming the recombinant vector obtained in step (2) into Agrobacterium to obtain a recombinant strain; (4) amplifying and culturing the recombinant strain obtained in step (3), collecting the cells after centrifugation, resuspending them in a resuspension solution, and transforming them into plants after standing.

6. The method for regulating pigment synthesis in leaves according to claim 5, characterized in that: In step (1), the amplification primers include SEQ ID No. 3 and SEQ ID No. 4; Preferably, in step (1), the amplification procedure includes: Pre-denaturation: 93-98°C, 1-5 min; Denaturation: 93-98°C, 10-20s; Annealing extension: 52-60°C, 10-20s; 65-80°C, 20-40s; cycle 30-38 times; Extension: 65-80℃, 1-10min.

7. The method for regulating pigment synthesis in leaves according to claim 5 or 6, characterized in that: In step (2), the vector comprises any one or a combination of at least two of a virus-induced silencing vector, an RNAi interference vector, an overexpression vector, or a genome editing vector; Preferably, the virus-induced silencing vector comprises any one of TRV2, PVX or CMV; Preferably, in step (2), the vector includes a promoter and a terminator.

8. The method for regulating pigment synthesis in leaves according to any one of claims 5 to 7, characterized in that: In step (2), the enzyme cleavage site includes any one of BamH I, Sac I, EcoR I, Nde I, Nco I, Pac I, Pst I, Sca I, Spe I, Xba I or Xho I, or a combination of at least two thereof; Preferably, in step (2), the identification method includes any one or a combination of at least two of resistance screening, PCR identification or whole-genome sequencing.

9. The method for regulating pigment synthesis in leaves according to any one of claims 5 to 7, characterized in that: In step (3), the method of transformation includes electroporation; Preferably, in step (3), the Agrobacterium comprises one or a combination of at least two of GV3101, EHA101, EHA105 or LBA4404.

10. The method for regulating pigment synthesis in leaves according to any one of claims 5 to 8, characterized in that: In step (4), the resuspension solution comprises 5-15 mM MgCl2, 0.1-0.3 mM acetosyringone and 5-15 mM 2-(N-morpholino)ethanesulfonic acid; Preferably, in step (4), the standing time is 1-5 hours and the temperature is 20-30°C; Preferably, in step (4), the transformation method includes a transient transformation method.

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