Starting element, vector and method for carrying out gene editing on plant by using starting element and vector

Abstract

The invention relates to a promoter, a vector and a method for carrying out gene editing on a plant by using the promoter and the vector. The promoter comprises a core promoter, and the nucleotide sequence of the core promoter is shown as SEQ ID NO.1. The starting element and the vector can improve plant genetic transformation efficiency and expression level, and can enhance plant gene editing efficiency and transformation efficiency.

Description

Technical Field

[0001] The present disclosure relates to the field of genetic engineering, and in particular, to a promoter element and a vector, and a method for gene editing of plants using the same. Background Art

[0002] Plant transgenic technology refers to the use of genetic engineering and molecular biology techniques to introduce excellent target genes into recipient plants, thereby endowing plants with new excellent traits or enhancing the expression of endogenous genes, and has been widely applied in agricultural production and crop quality trait improvement. With technological progress, currently, on the basis of transgenic technology, genome editing technologies represented by CRISPR-Cas have been developed, which can modify the endogenous genes of recipient crops without introducing foreign genes, thereby enhancing excellent traits such as the yield and quality of crops. Agrobacterium-mediated genetic transformation is currently the most widely used biotechnological means for introducing foreign genes and gene editing vectors into recipient plants, and has currently been widely applied to major food crops such as rice, wheat, and corn, as well as important cash crops such as soybeans, cotton, vegetables, and tobacco. There are currently some studies that can improve the regeneration efficiency. For example, overexpression of the plant growth regulator GRF4-GIF1 can improve the regeneration efficiency of wheat. Although overexpression of plant growth factors can improve the regeneration frequency of plants to a certain extent, due to the continuous expression of growth factor genes, it causes adverse effects such as deformed regenerated seedlings and difficulty in passing on generations. Up to now, there are few reports on studies that can both improve the regeneration efficiency of plants and increase the genome editing efficiency of recipient plants.

[0003] Existing studies can use overexpression of plant growth factors to improve the regeneration efficiency, and the use of strong promoters such as the cauliflower mosaic virus CaMV35S of tobacco mosaic virus can improve the gene expression level. However, there are no reports on sequences and expression vectors that can simultaneously improve the regeneration efficiency, expression level, and editing efficiency. Using existing gene editing vectors to create an editing library covering the entire genome of plants is costly and laborious, and it is almost impossible to achieve for crops with complex genomes. Therefore, there is an urgent need for an expression vector system with high genome editing efficiency and high efficiency in obtaining regenerated seedlings. Summary of the Invention

[0004] The object of the present disclosure is to provide a promoter element and a vector, and a method for gene editing of plants using the same, which can improve the genetic transformation efficiency and expression level of plants, and can enhance the gene editing efficiency and transformation efficiency of plants.

[0005] To achieve the above object, in the first aspect of the present disclosure, a promoter element is provided, the promoter element includes a core promoter, and the nucleotide sequence of the core promoter is as shown in SEQ ID NO.1.

[0006] Optionally, the nucleotide sequence of the promoter element is as shown in SEQ ID NO.1.

[0007] Optionally, the promoter element further comprises an enhancer connected to the core promoter, and the nucleotide sequence of the enhancer is as shown in SEQ ID NO.2.

[0008] Optionally, the nucleotide sequence of the promoter element is as shown in SEQ ID NO.3.

[0009] The second aspect of the present disclosure provides a vector into which the promoter element described in the first aspect is inserted.

[0010] Optionally, the vector is an expression vector, and a target protein expression sequence insertion region, a terminator, and a hygromycin selection marker are further inserted downstream of the promoter element described in the first aspect in the vector.

[0011] Optionally, the vector is a gene editing vector, and a 2X35Sp promoter, a gRNA insertion region, and a eu terminator are further inserted upstream of the promoter element described in the first aspect in the vector; a spCas9 protein expression element, a ubp terminator, and a hygromycin selection marker are further inserted downstream of the promoter element described in the first aspect in the vector.

[0012] The third aspect of the present disclosure provides a method for gene editing of plants, the method comprising the following steps:

[0013] S1. Insert a gRNA coding sequence targeting the gene to be edited into the gRNA insertion region of the gene editing vector described in the second aspect to obtain a gene editing plasmid;

[0014] S2. Transform Agrobacterium with the gene editing plasmid to obtain a transformant;

[0015] S3. Infect the plant culture with the transformant to obtain an infected plant culture;

[0016] S4. Culture the infected plant culture in a medium containing hygromycin to obtain a gene-edited plant.

[0017] Optionally, the nucleotide sequence of the gRNA targeting the gene to be edited is as shown in SEQ ID NO.4.

[0018] Optionally, the plant is selected from at least one of tobacco, tomato, soybean, cotton, pepper, rape, eggplant, Chinese cabbage, lettuce, cabbage, potato, peanut, cucumber, watermelon, sunflower, strawberry, radish, citrus, alfalfa, poplar, Arabidopsis thaliana, sesame, quinoa, sesbania, grape, apple, pear, kiwifruit, chrysanthemum, dandelion, rabdosia rubescens, rice, wheat, corn, sorghum, wild rice, millet, sugarcane, bamboo, onion, leek, ginger, banana, and lily; preferably, the plant is tobacco.

[0019] Through the above technical solutions, the present disclosure provides a starting element, a vector, and a method for gene editing of plants; the starting element and the vector containing the starting element can improve the plant genetic transformation efficiency and expression level, and can enhance the plant gene editing efficiency and transformation efficiency.

[0020] Other features and advantages of the present disclosure will be described in detail in the following specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings are used to provide a further understanding of the present disclosure, and constitute a part of the specification. Together with the following specific implementation, they are used to explain the present disclosure, but do not constitute a limitation to the present disclosure. In the drawings:

[0022] Figure 1 is a schematic structural diagram of the expression vector of the present disclosure.

[0023] Figure 2 is the regeneration and expression of the expression vector of the present disclosure in tobacco T0 generation transformed seedlings.

[0024] Figure 3 is a schematic structural diagram of the gene editing vector of the present disclosure.

[0025] Figure 4 is a comparative analysis diagram of the editing efficiency of different editing vectors in tobacco T0 generation stable transformation lines.

[0026] Figure 5 is the expression of betacyanin in the transient expression system of Nicotiana benthamiana leaves by different expression vectors. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The following will describe in detail the specific implementation of the present disclosure with reference to the drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0028] The first aspect of the present disclosure provides a starting element, and the starting element includes a core promoter, and the nucleotide sequence of the core promoter is shown in SEQ ID NO.1.

[0029] In the present disclosure, the inventors of the present disclosure found and screened two genes, mRNA_60132 and mRNA_24715, which were highly expressed in callus during the tobacco regeneration process, while identifying genes highly expressed in callus. These two genes are derived from the ancestral species of allotetraploid tobacco, Nicotiana sylvestris and Nicotiana tomentosiformis, respectively, and encode two histone H2A proteins. Since the process of dedifferentiation and redifferentiation experienced during tobacco tissue culture involves changes in histone genes, the inventors of the present disclosure speculated that given that histone promoters can be applied to drive high expression of genes in callus and may cause changes in tobacco regeneration, therefore, according to the information of Arabidopsis thaliana HTA6, the core promoter (i.e., the HTA6p promoter element) was synthesized.

[0030] In one embodiment of the present disclosure, the promoter element may be a core promoter, and the nucleotide sequence of the promoter element is shown in SEQ ID NO.1.

[0031] In another embodiment of the present disclosure, the promoter element may further include an enhancer connected to the core promoter, wherein the nucleotide sequence of the enhancer is shown in SEQ ID NO.2.

[0032] In the present disclosure, the inventors designed to remove the conventional restriction enzyme sites in the octopine synthase (OCS) and mannopine synthase (MAS) activation sequences, artificially synthesized the nucleotide sequences of OCS and MAS, and used linker1 (tcgag) as the linking sequence to connect OCS and MAS to form an enhancer (OCS_MAS, SEQ ID NO.2).

[0033] According to the present disclosure, linker2 (cctcag) was used as the linking sequence to connect the core promoter and the enhancer to form a promoter element (i.e., the OMH6p promoter element), and the nucleotide sequence of the promoter element is shown in SEQ ID NO.3.

[0034] The second aspect of the present disclosure provides a vector into which the promoter element described in the first aspect is inserted.

[0035] In one embodiment of the present disclosure, the vector is an expression vector, and a target protein expression sequence insertion region, a terminator, and a hygromycin selection marker are further inserted downstream of the promoter element described in the first aspect in the vector.

[0036] In the present disclosure, the target protein expression sequence insertion region may be the modified RUBY sequence constructed by the inventors of the present disclosure. Figure 1As shown, when the starting element is the core promoter, a RUBY sequence, a ubp terminator, and a hygromycin selection marker are inserted downstream of the starting element to construct an HTA6p::RUBY expression vector. When the starting element is a starting element with an enhancer connected to the core promoter, a RUBY sequence, a ubp terminator, and a hygromycin selection marker are inserted downstream of the starting element to construct an OMH6p::RUBY expression vector.

[0037] In another embodiment of the present disclosure, the vector is a gene editing vector, and a 2X35Sp promoter, a gRNA insertion region, and a eu terminator are further inserted upstream of the starting element described in the first aspect; a spCas9 protein expression element, a ubp terminator, and a hygromycin selection marker are further inserted downstream of the starting element described in the first aspect.

[0038] In the above embodiment, the starting element can be a starting element with an enhancer connected to the core promoter, such as Figure 3 As shown, a 2X35Sp promoter, a gRNA insertion region, and a eu terminator are inserted upstream of the starting element, and a spCas9 protein expression element, a ubp terminator, and a hygromycin selection marker are further inserted downstream of the starting element to construct an OMH6p::Cas9 gene editing vector.

[0039] The third aspect of the present disclosure provides a method for gene editing of plants, which includes the following steps:

[0040] S1. Insert a gRNA coding sequence targeting the gene to be edited into the gRNA insertion region of the gene editing vector described in the second aspect to obtain a gene editing plasmid;

[0041] S2. Transform Agrobacterium with the gene editing plasmid to obtain a transformant;

[0042] S3. Infect the plant culture with the transformant to obtain an infected plant culture;

[0043] S4. Culture the infected plant culture in a medium containing hygromycin to obtain a gene-edited plant.

[0044] According to the present disclosure, the nucleotide sequence of the gRNA targeting the gene to be edited is as shown in SEQ ID NO.4.

[0045] In the present disclosure, the plant may be selected from one of dicotyledonous plants and monocotyledonous plants. Preferably, the dicotyledonous plant may be selected from at least one of tobacco, tomato, soybean, cotton, pepper, rapeseed, eggplant, Chinese cabbage, lettuce, cabbage, potato, peanut, cucumber, watermelon, sunflower, strawberry, radish, citrus, alfalfa, poplar, Arabidopsis thaliana, sesame, quinoa, sesbania, grape, apple, pear, kiwifruit, chrysanthemum, dandelion, and isodon rubescens. The monocotyledonous plant may be selected from at least one of rice, wheat, corn, sorghum, wild rice, millet, sugarcane, bamboo, onion, leek, ginger, banana, and lily. More preferably, the plant may be tobacco.

[0046] The present invention will be further described in detail below in conjunction with embodiments, but the scope of the present invention is not limited to the following embodiments.

[0047] Unless otherwise specified, the materials used in this embodiment are all commercially available products.

[0048] Example 1

[0049] This example is used to illustrate the construction of an expression vector.

[0050] The expression tracer system uses betacyanin RUBY, and the nucleotide sequence encoding RUBY is modified. Specifically, the methionine (Met) at the eighth position of RUBY is modified to leucine (Leu) to construct a modified RUBY sequence.

[0051] (1) Construction of the 35Sp::RUBY expression vector:

[0052] The RUBY-NOS sequence in the DR5::RUBY vector was amplified using a kit (2×Phanta Max Master Mix, Novoprotein, P525-03), and the amplified sequence was replaced with the sequence in the SalI to EcoRI region of the pDC 45 vector (the sequence of this vector is the same as that in ZL202110786304.5) using a homologous recombination kit (ClonExpress II One Step Cloning Kit, Novoprotein, C112-01) to construct 35Sp::RUBY as a positive control.

[0053] (2) Construction of the HTA6p::RUBY expression vector:

[0054] The HTA6 promoter element sequence shown in SEQ ID NO.1 was amplified and inserted into the SpeI to NcoI sites of the 35Sp::RUBY vector using homologous recombination to construct the HTA6p::RUBY expression vector.

[0055] (3) Construction of the OMH6p::RUBY expression vector:

[0056] The conventional restriction enzyme sites in the activation sequences of octopine synthase (OCS) and mannopine synthase (MAS) were designed to be removed. The OCS nucleotide sequence and MAS nucleotide sequence were artificially synthesized. Using linker1 as the linking sequence, OCS and MAS were linked to synthesize the OCS_MAS combined sequence shown in SEQ ID NO.2. Then, the HTA6 promoter element and the OCS_MAS combined sequence were linked through linker2 to synthesize the OMH6p promoter element shown in SEQ ID NO.3.

[0057] The OMH6p promoter element was inserted into the SpeI to NcoI sites of the 35Sp::RUBY vector to construct the OMH6p::RUBY expression vector.

[0058] The structures of the 35Sp::RUBY expression vector, HTA6p::RUBY expression vector, and OMH6p::RUBY expression vector are as Figure 1 shown.

[0059] Example 2

[0060] This example is used to illustrate the application of the expression vector in improving the efficiency and expression level of plant genetic transformation.

[0061] The three expression vectors, 35Sp::RUBY, HTA6p::RUBY, and OMH6p::RUBY, were introduced into competent Agrobacterium tumefaciens EH105. The three binary vectors with correct sequencing were transformed into Agrobacterium EH105 and stored at -80°C for later use. The leaves of tobacco (wild Honghua Dajinyuan) were infected using the optimized leaf disc transformation method. The specific steps are as follows:

[0062] (1) Pre-culture: Take the leaves of sterile seedlings that have grown to 4 - 6 true leaves, are robust, have dark green leaves, and clear leaf veins. Cut off the edges of the sterile leaves, and cut the leaves into 1.0×1.0 cm leaf discs along the main vein. Place them in MS medium, at 28°C, with a light time of 16 h / d and a light intensity of 2000 LX, and pre-culture for 2 days.

[0063] (2) Agrobacterium activation and infection: Using the identified and preserved original Agrobacterium liquid, after it completely melts, take 100 μL and add it to 45 ml of LB liquid medium containing 50 mg / L kanamycin and 10 mg / L rifampicin. Incubate with shaking at 28°C and 220 rpm overnight until OD600 is about 1.0. Transfer the bacterial liquid to a pre-sterilized and pre-cooled 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells with pre-cooled MS liquid medium to OD600 of about 0.6. Then add acetosyringone with a final concentration of 20 mg / L, and let it stand for 30 minutes to 2 hours, preparing for infection.

[0064] (3) Infection: Place the pre-cultured leaf discs into the Agrobacterium infection solution for infection for 5 minutes, with intermittent gentle shaking.

[0065] (4) Co-cultivation: Fish out the infected leaves, blot dry the Agrobacterium liquid on sterile filter paper, lay them face down on the MS medium supplemented with 1 mg / L 6-BA and 0.1 mg / L IBA, and place them in an artificial climate chamber (temperature 26°C, humidity 40%), and culture in the dark for 3 days.

[0066] (5) S1 Subculture: Transfer the leaves after 3 days of co-cultivation face up to the S1 differentiation medium. The S1 medium is the MS medium supplemented with 1 mg / L 6-BA, 0.1 mg / L NAA, 8 mg / L hygromycin, and 150 mg / L ticarcillin. Place 6 - 8 leaves in each petri dish, culture in the dark in the artificial climate chamber for about 2 days and then continue to culture under light until adventitious buds grow from the leaf margins and the buds grow to about 0.5 cm.

[0067] (6) S2 Subculture: Use forceps to transfer the adventitious buds grown on S1 to the S2 differentiation medium. The S2 medium is the MS medium supplemented with 1 mg / L 6-BA, 0.1 mg / L NAA, 10 mg / L hygromycin, and 150 mg / L ticarcillin. Culture under light conditions for 1 - 2 weeks, and the adventitious buds grow into young seedlings.

[0068] Take pictures of the transformed materials at S2, and conduct statistical analysis on the number of regenerated seedlings and the color depth. The results are as Figure 2 shown in the figure and Table 1.

[0069] Table 1

[0070]

[0071] Note: a: The transformation efficiency is the ratio of the number of purple regenerated seedlings to the number of tobacco explants; b: The improvement multiple is the multiple of improvement compared with the conversion rate of 35Sp::RUBY; c: The high-expression regenerated seedlings refer to the T0 generation regenerated seedlings of tobacco with leaves showing dark purple; d: The increase multiple is the multiple of increase in the ratio of high-expression regenerated seedlings compared with 35Sp::RUBY; e: The first resistant bud refers to the number of days when the first purple regenerated bud can be seen with the naked eye. The transfer of tobacco co-cultured leaf discs to the S1 medium is defined as the first day.

[0072] From Figure 2As can be seen from Table 1, the overall transformation efficiency of purple regenerated seedlings increased by 3.34 times, and that of regenerated seedlings with high expression levels increased by 5.67 times. Moreover, the time to the emergence of the first resistant bud was advanced by 4 - 5 days. This shows that the HTA6p::RUBY expression vector containing the HTA6 promoter element and the OMH6p::RUBY expression vector containing the OMH6p promoter element can enhance the genetic transformation efficiency and expression level of tobacco.

[0073] Example 3

[0074] This example is used to illustrate the construction of a gene editing vector.

[0075] Amplify the OMH6p promoter element sequence shown in SEQ ID NO.3. Use SbfI and NcoI double digestion on the pDC45 vector (the sequence of this vector is the same as that in ZL202110786304.5). After gel extraction of the upper band, use a seamless ligation kit (ClonExpress II One Step Cloning Kit, Novoprotein, C112 - 01) to insert OMH6p into the linearized pDC45 vector to construct the OMH6p::Cas9 gene editing vector. The structure of the OMH6p::Cas9 gene editing vector is as Figure 3 shown. Synthesize the U6 - sgRNA - 2X35S promoter sequence (as shown in SEQ ID NO.5) at BGI. Use the seamless ligation kit to insert U6 - sgRNA and the 2X35S promoter into the pDC45 vector linearized by HindIII and NcoI double digestion to construct the 35Sp::Cas9 gene editing vector control group.

[0076] Example 4

[0077] This example is used to illustrate the method for gene editing of plants.

[0078] Construction of an editing vector targeting the PDS gene: Use the BsaI restriction endonuclease to cut OMH6p::Cas9 and 35Sp::Cas9 (control group) to obtain linearized expression vectors. Insert the targeting sequence of the tobacco PDS gene into the linearized OMH6p::Cas9 and 35Sp::Cas9 expression vectors using T4 ligase (M0202S, NEB ENGLAND BioLabs) to obtain the OMH6p::Cas9 - PDS_sg4 binary vector and the 35Sp::Cas9 - PDS_sg4 binary vector (control group).

[0079] The correctly sequenced binary vector (OMH6p::Cas9-PDS_sg4) and the control group (35Sp::Cas9-PDS_sg4) were transformed into Agrobacterium tumefaciens EH105 and stored at -80°C for later use. The leaves of tobacco (wild Honghua Dajinyuan) were infected using the optimized leaf disc transformation method, and the specific steps are as follows:

[0080] (1) Pre-culture: Take the leaves of aseptic seedlings that have grown to 4 - 6 true leaves, are robust, have dark green leaves, and clear leaf veins. Cut off the edges of the aseptic leaves, and cut the leaves into 1.0×1.0 cm leaf discs along the main vein. Place them in MS medium, at 28°C, with a light duration of 16 h / d and a light intensity of 2000 LX, and pre-culture for 2 days.

[0081] (2) Agrobacterium activation and infection: Using the preserved original Agrobacterium liquid after identification, after it completely melts, take 100 μL and add it to 45 ml of LB liquid medium containing 50 mg / L kanamycin and 10 mg / L rifampicin. Shake and culture overnight at 28°C and 220 rpm until OD600 is about 1.0. Transfer the bacterial liquid to a pre-sterilized and pre-cooled 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells with pre-cooled MS liquid medium to OD600 of about 0.6. Then add acetosyringone with a final concentration of 20 mg / L, and let it stand for 30 minutes to 2 hours, preparing for infection.

[0082] (3) Infection: Put the pre-cultured leaf discs into the Agrobacterium infection solution for infection. The infection time is 5 min, with intermittent gentle shaking.

[0083] (4) Co-culture: Take out the infected leaves, blot dry the Agrobacterium liquid on sterile filter paper, lay them face down on MS medium supplemented with 1 mg / L 6-BA and 0.1 mg / L IBA, put them into an artificial climate chamber (temperature 26°C, humidity 40%), and culture in the dark for 3 days.

[0084] (5) S1 subculture: Transfer the leaves after 3 days of co-culture face up to the S1 differentiation medium. The S1 medium is MS medium supplemented with 1 mg / L 6-BA, 0.1 mg / L NAA, 8 mg / L hygromycin, and 150 mg / L ticarcillin. Place 6 - 8 leaves in each petri dish, culture in the dark in an artificial climate chamber for about 2 days and then continue to culture under light until adventitious buds grow at the leaf edges and the bud length reaches about 0.5 cm.

[0085] (6) S2 subculture: Use forceps to transfer the adventitious buds grown on S1 to the S2 differentiation medium. The S2 medium is MS medium supplemented with 1 mg / L 6-BA, 0.1 mg / L NAA, 10 mg / L hygromycin, and 150 mg / L ticarcillin. Culture under light conditions for 1 - 2 weeks, and the adventitious buds grow into young seedlings.

[0086] (7) Transfer the regenerated buds growing on S2 to the same medium and continue culturing for 2 weeks. Respectively count the number of regenerated tobacco plants with albino phenotypes in 35Sp::Cas9-PDS_sg4 and OMH6p::Cas9-PDS_sg4 (the albino phenotype is due to the simultaneous knockout of two gene copies in allopolyploid tobacco).

[0087] The results are as Figure 4 shown. Under the same explant conditions, the number of albino PDS-edited seedlings obtained with the OMH6p::Cas9-PDS_sg4 editing vector is 5.3 times that of the control group, significantly increasing the total number of gene-edited seedlings.

[0088] Example 5

[0089] This example is used to illustrate the improvement of the expression level of transient transformation in tobacco.

[0090] Pick monoclonal colonies of Agrobacterium tumefaciens EH105 containing the negative control pDC45, positive control 35Sp::RUBY, HTA6p::RUBY, and OMH6p::RUBY and culture them overnight in kanamycin antibiotic. Take 1 ml of the small culture solution and inoculate it into 50 ml of LB medium containing kanamycin antibiotic and culture it overnight with shaking. Use a centrifugation speed of 5000 revolutions per minute to precipitate Agrobacterium tumefaciens and use a resuspension solution (100 mmol / L magnesium chloride, 10 mmol / L morpholineethanesulfonic acid, 0.1 mmol / L acetosyringone) to adjust the OD value of the enriched Agrobacterium tumefaciens to 0.5. Inject the back leaves of Nicotiana benthamiana using a 1 ml syringe. After 2 - 3 days, remove the leaves and immediately soak them in absolute ethanol. Take pictures of the leaves after 2 days (as Figure 5 shown). For the decolorized leaves, after rinsing the surface ethanol with sterile water, use a hole punch to take the purple areas of the injection holes of the same bacterial solution at 3 different positions and place them in 0.5 ml of sterile water overnight. Take 200 ul of the betacyanin aqueous solution and place it in a 96-well microplate. Use a multifunctional microplate reader (TECAN, infinite M200PRO) to detect the absorbance value (OD value) of the betacyanin aqueous solution at 538 nm. The results are shown in Table 2.

[0091] Table 2

[0092] pDC45 35Sp::RUBY HTA6p::RUBY OMH6p::RUBY OD value 0.08 0.41 0.58 0.89

[0093] From Table 2 and Figure 5It can be seen that, compared with the negative control pDC45 and the positive control 35Sp::RUBY, the absorbance values of HTA6p::RUBY and OMH6p::RUBY of the present disclosure are larger, especially for OMH6p::RUBY, indicating that in the transient transformation system of tobacco, HTA6p::RUBY and OMH6p::RUBY can produce more betacyanin, especially OMH6p::RUBY, which produces the most betacyanin.

[0094] The preferred embodiments of the present disclosure have been described in detail above in conjunction with the accompanying drawings. However, the present disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0095] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0096] Furthermore, any combination can be made among various different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, and it should also be regarded as the content disclosed by the present disclosure.

Claims

1. A starting element, characterized in that, The starting element includes a core promoter, and the nucleotide sequence of the core promoter is as shown in SEQ ID NO.

1.

2. The starting element according to claim 1, wherein, The nucleotide sequence of the starting element is as shown in SEQ ID NO.

1.

3. The starting element according to claim 1, wherein, The starting element further includes an enhancer connected to the core promoter, and the nucleotide sequence of the enhancer is as shown in SEQ ID NO.

2.

4. The starting element according to claim 3, wherein, The nucleotide sequence of the starting element is as shown in SEQ ID NO.

3.

5. A carrier, characterized in that, The vector is inserted with the starting element described in any one of claims 1-4.

6. The carrier according to claim 5, wherein, The vector is an expression vector, and a target protein expression sequence insertion region, a terminator, and a hygromycin selection marker are further inserted downstream of the starting element described in any one of claims 1-4 in the vector.

7. The carrier according to claim 5, wherein, The vector is a gene editing vector, and a 2X35Sp promoter, a gRNA insertion region, and a eu terminator are further inserted upstream of the starting element described in any one of claims 1-4 in the vector; a spCas9 protein expression element, a ubp terminator, and a hygromycin selection marker are further inserted downstream of the starting element described in any one of claims 1-4 in the vector.

8. A method for gene editing of plants, characterized in that, The method includes the following steps: S1. Insert a gRNA coding sequence targeting the gene to be edited into the gRNA insertion region of the gene editing vector described in claim 7 to obtain a gene editing plasmid; S2. Transform Agrobacterium with the gene editing plasmid to obtain a transformant; S3. Infect a plant culture with the transformant to obtain an infected plant culture; S4. Culture the infected plant culture in a medium containing hygromycin to obtain a gene-edited plant.

9. The method according to claim 8, wherein, The nucleotide sequence of the gRNA targeting the gene to be edited is as shown in SEQ ID NO.

4.

10. The method according to claim 8, wherein The plant is selected from at least one of tobacco, tomato, soybean, cotton, pepper, rape, eggplant, Chinese cabbage, lettuce, cabbage, potato, peanut, cucumber, watermelon, sunflower, strawberry, radish, citrus, alfalfa, poplar, Arabidopsis, sesame, quinoa, sesbania, grape, apple, pear, kiwifruit, chrysanthemum, dandelion, rabdosia rubescens, rice, wheat, corn, sorghum, wild rice, millet, sugarcane, bamboo, onion, leek, ginger, banana, and lily; Preferably, the plant is tobacco.

Images