Application of ALKBH9B gene in promoting formation of plant callus

Abstract

The invention discloses an application of an ALKBH9B gene in promotion of plant callus formation, belongs to the technical field of gene engineering, and provides an application of the ALKBH9B gene in promotion of plant callus formation. The invention also provides an application of an expression cassette, a vector or a recombinant bacterium containing the ALKBH9B gene in promoting formation of poplar leaf calluses. The ALKBH9B gene is knocked out, the expression quantity of the ALKBH9B gene is reduced, it is found that when the gene editing strain with the knocked-out gene is cultured on a callus induction culture medium for 5 days, callus tissue starts to be formed, the callus forming speed is high, and the net weight of the callus and the callus generation rate are remarkably higher than those of a wild strain. The result shows that ALKBH9B has an inhibition effect in the process of inducing the callus by the leaf, and the deletion of ALKBH9B can promote the formation of the callus.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and particularly relates to ALKBH9B Application of genes in promoting plant callus formation. Background Art

[0002] Forests are the main component of terrestrial ecosystems and are important natural resources that combine water reservoirs, grain reservoirs, money reservoirs, and carbon reservoirs. As part of forest resources, wood plays an important role in the construction, papermaking, and textile industries, and has become an indispensable part of people's production and life. In recent years, per capita wood resources have been relatively scarce. Therefore, it has become an urgent task to develop and protect forest resources and promote the sustainable development of the ecological environment.

[0003] The formation of callus is an important self-repair mechanism for plants in response to environmental damage and mechanical damage. It is composed of amorphous cell clusters and provides a cell source for subsequent regeneration and reproduction. It is not only the key to plant wound healing, but also the basis for gene transformation, tissue culture and plant regeneration. With the development of plant breeding technology, the formation of callus has become an important link in research and application, and will affect the application of genetic engineering technology, especially in the genetic improvement of forest trees. It can effectively promote the transformation of exogenous genes, thereby quickly cultivating high-quality transgenic fast-growing forest trees. This is of great significance for meeting market demand and alleviating the contradiction between wood supply and demand, and can guarantee wood safety to a certain extent. It also has important application prospects for improving the biomass, wood quality and stress resistance of forest trees.

[0004] N 6 -methyladenosine (N 6 -Methyladenosine, m 6 A) is a post-transcriptional modification that is widely present in eukaryotic mRNA and refers to the addition of a methyl group to the sixth carbon atom of adenine. 6 A modification is the most common and dynamically reversible RNA modification. 6 A methyltransferase (Writer), demethylase (Eraser) and m 6 A binding protein (Reader) plays a role in the regulation of gene expression in plant growth and development. The demethylase ALKBH in plants regulates the expression of genes related to flowering time, fruit ripening, signal transduction, and biotic and abiotic stresses by removing methylation modifications in mRNA. Therefore, ALKBH plays a vital role in the growth and development of plants. If the methylation modifications in mRNA cannot be removed, it may affect the normal growth and development of plants. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes ALKBH9B Application of genes in promoting plant callus formation by knockdown and / or knockout ALKBH9B gene, reducing its expression level and promoting the rapid formation of plant callus.

[0006] To achieve the above object, the present invention provides ALKBH9B Application of gene in promoting plant callus formation, the ALKBH9B The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Preferably, by knocking down and / or knocking out ALKBH9B gene, reducing its expression level and promoting the rapid formation of plant callus.

[0008] Preferably, the plant is poplar.

[0009] Further preferably, the formation of poplar leaf callus is promoted.

[0010] The present invention also provides a ALKBH9B Application of gene expression cassette, vector or recombinant bacteria in promoting callus formation of poplar leaves.

[0011] The present invention also provides a method of using the ALKBH9B Method for genetically promoting callus formation in poplar leaves, knocking down and / or knocking out poplar by transgenic methods ALKBH9B gene, reducing its expression and promoting the formation of poplar leaf callus.

[0012] Preferably, it also includes knocking down and / or knocking out poplar ALKBH9A The genetic steps ALKBH9A The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0013] The present invention also provides the ALKBH9A Application of genes in promoting callus formation in poplar leaves.

[0014] Preferably, by knocking down and / or knocking out ALKBH9A gene, reducing its expression level and promoting the rapid formation of plant callus.

[0015] The present invention also provides the ALKBH9B Genes and ALKBH9A Gene co-expression in promoting leaf callus formation in poplar trees by knocking down and / or knocking out ALKBH9B Genes and ALKBH9A Gene, lower ALKBH9B The expression levels of the ALKBH9A gene and ALKBH9A gene promote the rapid formation of plant callus.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention knocks out ALKBH9B Genes and / or ALKBH9A Gene, lower ALKBH9B Genes and / or ALKBH9A The expression of genes was found ALKBH9B Gene editing lines with single gene knockout and ALKBH9B Genes and ALKBH9A The gene-edited strains with double gene knockout began to form callus tissue on the 5th day of culture on the callus induction medium, while the wild strain did not form callus tissue at this time, and the wild strain only began to induce callus on the 8th day.

[0017] The callus tissues of the wild strain were observed at 11, 14, and 20 days. ALKBH9B Gene editing lines with single gene knockout and ALKBH9B Genes and ALKBH9A The callus formation rate was faster in the gene-edited strain with double gene knockout.

[0018] ALKBH9B Gene editing lines with single gene knockout and ALKBH9B Genes and ALKBH9A The net callus weight and callus production rate of the gene-edited strain with double gene knockout were significantly higher than those of the wild-type strain. These results indicate that ALKBH9B It plays an inhibitory role in the process of leaf callus induction, while its absence can promote the formation of callus. ALKBH9A As a paralogous gene, it also plays a certain regulatory role in this process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Map of the gene editing pYLCRISPR / Cas9-DH vector; Figure 2 The genetic transformation process of poplar, wherein A is callus infection and then placed on co-culture medium, B is callus formation adventitious buds on differentiation medium, and C is adventitious buds screening in rooting medium with hygromycin added; Figure 3 for PagALKBH9BThe editing status of the edited plants. The “*” in the figure indicates that the translation is terminated prematurely; Figure 4 The process of leaf induction callus formation in different strains, where A is the state of the wild type 84K on the 0th day, B is the state of the gene-edited strain #8-3-KO on the 0th day, C is the state of the gene-edited strain 7-2-KO on the 0th day, D is the state of the wild type 84K on the 5th day, E is the state of the gene-edited strain #8-3-KO on the 5th day, F is the state of the gene-edited strain 7-2-KO on the 5th day, G is the state of the wild type 84K on the 8th day, H is the state of the gene-edited strain #8-3-KO on the 8th day, I is the state of the gene-edited strain 7-2-KO on the 8th day, J is the state of the wild type 84K on the 11th day, and K is the state of the gene-edited strain #8-3-KO on the 1st day. The status of the gene-edited strain 7-2-KO was 1d, L was the status of the gene-edited strain 7-2-KO on the 11th day, M was the status of the wild-type 84K on the 14th day, N was the status of the gene-edited strain #8-3-KO on the 14th day, O was the status of the gene-edited strain 7-2-KO on the 14th day, P was the front status of the wild-type 84K on the 20th day, Q was the front status of the gene-edited strain #8-3-KO on the 20th day, R was the front status of the gene-edited strain 7-2-KO on the 20th day, S was the back status of the wild-type 84K on the 20th day, T was the back status of the gene-edited strain #8-3-KO on the 20th day, and U was the back status of the gene-edited strain 7-2-KO on the 20th day. The scale bar is 1 cm. Figure 5 Comparison of callus net weight and callus production rate in different strains, where A is callus net weight, B is callus production rate, and *** represents P < 0.05. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Example 1 1. Experimental Materials and Methods Plant material and growth conditions: The plant material is the silver gland poplar '84K' tissue culture seedlings from the research group of Professor Lu Mengzhu / Zhang Jin of Zhejiang Agricultural and Forestry University. The gene-edited tissue culture seedlings of ALKBH9B are derived from the genetic transformation of silver gland poplar '84K' tissue culture seedlings. Its growth conditions are a temperature range of 24±2°C and a light intensity of 16h / 8h (light / dark).

[0027] Agrobacterium resuspension formula: Dissolve 2.14 g / L WPM449 (Phyto Technology, USA, catalog number: #L449) and 20 g / L sucrose in deionized water, adjust the pH to 5.6, and sterilize by high temperature and high pressure. Before use, add 100 μM acetosyringone (AS) on the clean bench.

[0028] Acetosyringone (AS) formulation: Dissolve 392.4 mg of acetosyringone powder in 20 mL of dimethyl sulfoxide (DMSO) and filter through a 0.22 μm filter into a sterile centrifuge tube to obtain a 100 mM stock solution.

[0029] Co-culture medium formula: Dissolve 2.4g / L WPM449, 0.5g / L morpholineethanesulfonic acid (MES), and 20g / L sucrose in deionized water, adjust the pH to 5.9, add 3.2g / L plant gel and sterilize. After cooling to below 60°C, add 100μM acetosyringone on a clean bench.

[0030] Differentiation screening medium formula: 2.4g / L WPM449, 0.5g / L MES, 0.1mg / L naphthaleneacetic acid (NAA), 0.5mg / L 6-benzyladenine (6-BA), 20g / L sucrose, pH adjusted to 5.9, add 3.2g / L plant gel and sterilize. After cooling to below 60°C, add 200mg / L timentin and 1.5mg / L hygromycin.

[0031] Formula of callus induction medium: dissolve 2.4 g / L WPM449, 0.5 g / L MES, 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg / L NAA, 0.1 mg / L furanylaminopurine (KT) and 20 g / L sucrose in deionized water, adjust the pH to 5.9, add 3.2 g / L plant gel and sterilize.

[0032] Rooting medium formula: Dissolve 2.2g / L MS519 (Phyto Technology, USA, catalog number: #M519), 0.5g / L MES, 0.05mg / L NAA, 0.02mg / L indolebutyric acid (IBA), and 20g / L sucrose in deionized water, adjust the pH to 5.9, add 7.8g / L agar and sterilize. After cooling to below 60°C, add 200mg / L timentin.

[0033] 2. Construction of ALKBH9B gene-edited poplar S1. Construction of PagALKBH9B gene editing vector: The gene editing vector CRISPR / Cas9 system used was developed by the team of Academician Liu Yaoguang from the School of Life Sciences of South China Agricultural University, and the target was designed through its online tool MMEJ-KO.

[0034] 1) Target design: The gene editing pYLCRISPR / Cas9-DH vector map used is as follows Figure 1 As shown, in the early stage of vector construction, sequences with a GC content greater than 50%, a low self-folding score, and high specificity are preferred as targets. The target sequence needs to be specifically verified in the silver gland poplar '84K' genome database. According to the target sequence, the adapter primer is designed and the gene editing vector is constructed.

[0035] Considering PagALKBH9A yes PagALKBH9B In order to prevent the phenotypic complementation caused by functional redundancy, a paralogous gene targeting PagALKBH9A and PagALKBH9B Double gene knockout vector.

[0036] Nucleotide sequence of ALKBH9B (SEQ ID NO.1):

[0037] Nucleotide sequence of ALKBH9A (SEQ ID NO.2):

[0038] The target site sequences of gRNA are shown in Table 1 below.

[0039] Table 1 Dual gene editing targets ;

[0040] 2) Preparation of adapter primers Mix and dilute 1 μL of each upstream and downstream primer of the target adapter with 8 μL of water, set the PCR instrument at 90°C for 30 seconds, and then cool it at room temperature to complete the preparation of the adapter primer.

[0041] 3) Construction of gRNA expression cassette The target linker was combined with the gRNA by the cutting and ligation method, and the reaction system was shown in Table 2, followed by PCR amplification. The amplification conditions were 37°C for 5 min, 20°C for 5 min, and 5 cycles.

[0042] Table 2 gRNA expression cassette reaction system ;

[0043] After the first round of PCR amplification, each product of the gRNA-target connection in the previous round was subjected to two reactions respectively, and the PCR amplification program was set as follows: pre-denaturation at 95°C for 5 min, followed by denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, 29 cycles, and finally full extension at 72°C for 5 min (as shown in Table 3 below).

[0044] Table 3 Target connection system ;

[0045] In the second round of nested PCR amplification, the PCR products of the previous round were diluted 10 times, and 1 μL of each was taken as a template to mix the reaction system (as shown in Table 4 below), and the PCR amplification conditions were set as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, and finally 72°C extension for 5 min. 5 μL of the amplified product was tested by 1% agarose gel electrophoresis, and the next step was carried out after confirming the size of the gRNA.

[0046] Table 4 Second round nested PCR system ;

[0047] 4) Ligation of pYLCRISPR / Cas9-DH plasmid The products of target one and target two were mixed and purified, and then connected to the pYLCRISPR / Cas9-DH plasmid using the Golden Gate method. The connection system is shown in Table 5 below.

[0048] Table 5 pYLCRISPR / Cas9-DH terminal connection system ;

[0049] Set the PCR program: incubate at 37°C for 10 min, incubate at 37°C for 5 min, incubate at 10°C for 5 min, incubate at 20°C for 5 min, cycle the first two to four steps 15 times, and finally incubate at 37°C for 2 min.

[0050] 5) E. coli transformation and sequencing The ligation product was transformed into Escherichia coli, cultured at 37°C overnight (14 h), and single clones were picked from the screening culture plate for PCR detection and sequencing verification.

[0051] S2. Transfection: The PagALKBH9B-DH gene editing vector was transferred into the Agrobacterium strain GV3101 by the heat shock method, and then introduced into 84K poplar by the Agrobacterium-mediated method.

[0052] 1) Select 84K tissue culture seedlings about one month old, select the 3rd to 6th healthy leaves from the top of the seedling, use a scalpel to lightly scratch the surface of the leaf and place it in callus induction medium for one month, and use it after it grows callus.

[0053] 2) When the optical density of Agrobacterium containing the PagALKBH9B-DH gene editing vector reaches OD 600 =0.6, centrifuge at 3500rpm for 15min, remove the supernatant and resuspend to OD 600 =0.4.

[0054] 3) Divide the callus tissue grown from the leaves into small pieces and soak them in the infection solution for 15 minutes, shaking gently to ensure uniform contact. After infection, place the callus tissue on sterile filter paper, wait for the excess liquid in the callus tissue to drain, and then transfer it to the co-culture medium and culture it in the dark for 3 days. During this period, observe whether there are colonies growing around the callus tissue to ensure the co-culture effect.

[0055] 4) After three days, callus tissue was transferred to differentiation medium and resistant adventitious buds were selected at 24±2℃ and 16 / 8h day and night photoperiod. The medium was changed every three weeks for 3 months until adventitious buds were observed to grow in callus tissue.

[0056] 5) Transfer the adventitious buds to the rooting screening medium.

[0057] 6) Extract DNA from the leaves of the rooted plants and perform PCR verification. Amplify the DNA expressing Cas9 protein in the verified plants by PCR and perform high-throughput sequencing. Select the gene-edited plants as transgenic plants for subsequent experiments.

[0058] The main steps of cultivating PagALKBH9B gene-edited transgenic plants are as follows: Figure 2 As shown, callus infection with Agrobacterium tumefaciens (e.g. Figure 2 A), bud differentiation of callus (such as Figure 2 B) and the cultivation of transgenic plants through resistance screening (e.g. Figure 2 C). Figure 3 The figure shows the editing status of gene-edited plants. PagALKBH9B Single gene knockout resulted in gene-edited strain #8-3-KO. PagALKBH9A and PagALKBH9B Double gene knockout resulted in the gene-edited strain #7-2-KO.

[0059] Callus formation from leaves of S3, gene-edited lines and wild-type 84K One-month-old robust and growing wild-type plants were selected, and the fourth leaf was uniformly selected from the top of the seedling. The leaf surface was lightly scratched with a scalpel, and the leaf was placed in callus induction medium for light-proof culture. During the culture process, the growth of transgenic and wild-type callus was regularly observed, and photographed and recorded in time.

[0060] 3. Results 1. Knockout PagALKBH9B Promoting the induction of callus formation in poplar leaves One-month-old gene-edited lines #7-2-KO, #8-3-KO and wild-type 84K (WT) were selected. More than 8 tissue culture seedlings of each line were selected for leaf callus induction experiments. Figure 4 As shown in Figure 2, callus formation began to form in the gene-edited strain on the callus induction medium on the 5th day, while no callus formation was observed in the WT strain at this time (e.g. Figure 4 Middle D, Figure 4 ZhongE and Figure 4 F); on the 8th day, the WT line began to induce callus, while the callus formation of the gene-edited line further expanded (as shown in Figure 4 Medium G, Figure 4 Medium H and Figure 4 In the subsequent observation of callus tissues at 11, 14, and 20 days, the callus formation rate of the WT strain was slow, while the callus formation rate of the gene-edited strain was fast (as shown in Figure 1). Figure 4 Middle J, Figure 4 Middle K, Figure 4 Medium L, Figure 4 Medium M, Figure 4 Medium N, Figure 4 Middle O, Figure 4 Medium P, Figure 4 Middle Q, Figure 4 Middle R, Figure 4 Medium S, Figure 4 Medium T and Figure 4 In order to exclude the influence of initial leaf differences, the average callus production rate was also calculated (callus production rate = callus net weight / leaf weight). Figure 5 As shown in the figure, the net callus weight and callus production rate of the gene-edited strains were significantly higher than those of the wild-type strains. In addition, the net callus weight and callus production rate of the double-gene-edited strain #7-2-KO were also significantly higher than those of the single-gene-edited strain #8-3-KO. Figure 5 Middle A and Figure 5 In B, these results show that PagALKBH9B It plays an inhibitory role in the process of leaf callus induction, while its absence can promote the formation of callus. PagALKBH9A As a paralogous gene, it also plays a certain regulatory role in this process.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. ALKBH9B The use of a gene in promoting plant callus formation is characterized in that: Said ALKBH9B The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: By knockdown and / or knockout ALKBH9B gene, reducing its expression level and promoting the rapid formation of plant callus.

3. The application according to claim 1, characterized in that: The plant is poplar.

4. The use according to claim 3, characterized in that: Promote the formation of callus tissue of poplar leaves.

5. Containing as claimed in claim 1 ALKBH9B Application of gene expression cassette, vector or recombinant bacteria in promoting callus formation of poplar leaves.

6. A kind of use ALKBH9B A method for genetically promoting callus formation in poplar leaves, characterized in that: Knockdown and / or knockout of poplar trees by transgenic methods ALKBH9B Gene, reducing its expression, promoting the formation of poplar leaf callus; Said ALKBH9B The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The method for promoting callus formation in poplar leaves according to claim 6, characterized in that: Also included are knockdown and / or knockout poplar ALKBH9A The genetic steps ALKBH9A The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

8. ALKBH9B Genes and ALKBH9A The invention discloses an application of genes in promoting the formation of callus tissue of poplar leaves, characterized in that: Said ALKBH9B The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ALKBH9A The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

9. The use according to claim 8, characterized in that: By knockdown and / or knockout ALKBH9B Genes and ALKBH9A Gene, lower ALKBH9B Genes and ALKBH9A The expression level of the gene promotes the rapid formation of plant callus.

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