Application of ALKBH9B gene in promoting the formation of plant callus

Abstract

The present invention discloses ALKBH9B the application of a gene in promoting the formation of plant callus, belonging to the technical field of genetic engineering, and provides ALKBH9B the application of a gene in promoting the formation of plant callus. It also provides the application of an expression cassette, vector or recombinant bacterium containing ALKBH9B the gene in promoting the formation of poplar leaf callus. By knocking out ALKBH9B the gene and reducing ALKBH9B the expression level of the gene, it is found that the gene-edited lines with gene knockout already start to form callus on the 5th day of culture on the callus induction medium, and the callus formation speed is relatively fast. The net weight of callus and the callus production rate are both significantly higher than those of the wild-type lines. It shows that ALKBH9B plays an inhibitory role in the process of inducing callus from leaves, and its deletion can promote the formation of callus.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to ALKBH9B the application of a gene in promoting the formation of plant callus. Background Art

[0002] Forests are the main components of terrestrial ecosystems and important natural resources that combine functions of reservoirs, granaries, treasure houses, and carbon sinks. As a part of forestry resources, wood plays an important role in fields such as construction, papermaking, and textiles, and has become an indispensable part of people's production and life. In recent years, per capita wood resources have been relatively scarce. Therefore, developing and protecting forestry resources and promoting the sustainable development of the ecological environment have become top priorities.

[0003] The formation of callus is an important self-repair mechanism when plants respond to environmental and mechanical injuries. It consists 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 techniques, the formation of callus has become an important link in research and application and affects the application of genetic engineering techniques. Especially in forest genetic improvement, it can effectively promote the transformation of foreign genes, thus quickly cultivating high-quality transgenic fast-growing forest trees, which is of great significance for meeting market demands, alleviating the contradiction between wood supply and demand, ensuring wood safety to a certain extent, and 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 widely present in eukaryotic mRNAs, referring to the addition of a methyl group to the 6th carbon atom of adenine. m 6 A modification is the most common and dynamically reversible RNA modification, and is involved in the regulation of gene expression during plant growth and development by m 6 A methyltransferases (Writers), demethylases (Erasers), and m 6 A-binding proteins (Readers) working together. The demethylase ALKBH in plants regulates gene expression related to plant flowering time, fruit ripening, signal transduction, and biotic and abiotic stresses by removing methylation modifications in mRNAs. Therefore, ALKBH plays a crucial role in the process of plant growth and development. If the methylation modifications in mRNAs cannot be removed, it may affect the normal growth and development of plants. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides ALKBH9B the application of the gene in promoting the formation of plant callus. By knocking down and / or knocking out ALKBH9B the gene, its expression level is reduced, promoting the rapid formation of plant callus.

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

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

[0008] Preferably, the plant is Populus.

[0009] More preferably, it promotes the formation of Populus leaf callus.

[0010] The present invention also provides the application of the expression cassette, vector or recombinant bacterium containing the ALKBH9B gene in promoting the formation of Populus leaf callus.

[0011] The present invention also provides a method for promoting the formation of Populus leaf callus by using the ALKBH9B gene. By means of transgenic method, the gene in Populus is knocked down and / or knocked out ALKBH9B to reduce its expression level, promoting the formation of Populus leaf callus.

[0012] Preferably, it further includes the step of knocking down and / or knocking out the gene in Populus, and the ALKBH9A nucleotide sequence of the gene is shown in SEQ ID NO.2. ALKBH9A

[0013] The present invention also provides the application of the ALKBH9A gene in promoting the formation of Populus leaf callus.

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

[0015] ALKBH9B The present invention also provides the application of the ALKBH9A gene and the ALKBH9B gene together in promoting the formation of Populus leaf callus. By knocking down and / or knocking out ALKBH9A the gene and ALKBH9B the gene, the expression levels of the gene and the ALKBH9A gene are reduced, promoting the rapid formation of plant callus.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] By knocking out ALKBH9B gene and / or ALKBH9A gene, the expression level of ALKBH9B gene and / or ALKBH9A gene is reduced, and it is found that ALKBH9B the gene editing strain with single gene knockout of ALKBH9B gene and the gene editing strain with double gene knockout of ALKBH9A gene both start to form callus on the 5th day of culturing on the callus induction medium, while no callus is formed in the wild strain at this time, and the wild strain starts to induce callus on the 8th day.

[0018] And subsequent observation of the callus on the 11th, 14th, and 20th days shows that the formation rate of the callus in the wild strain is slow, while ALKBH9B the gene editing strain with single gene knockout of ALKBH9B gene and the gene editing strain with double gene knockout of ALKBH9A gene have a faster callus formation rate.

[0019] ALKBH9B The gene editing strain with single gene knockout of ALKBH9B gene and the gene editing strain with double gene knockout of ALKBH9A gene have significantly higher net callus weight and callus production rate than the wild type strain. These results indicate that ALKBH9B plays an inhibitory role in the process of inducing callus in leaves, and its deletion can promote the formation of callus. At the same time, ALKBH9A as a paralogous gene, also plays a certain regulatory role in this process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 is the gene editing pYLCRISPR / Cas9-DH vector map;

[0022] Figure 2 is the genetic transformation process of poplar, where A is the callus after infection placed on the co-culture medium, B is the formation of adventitious buds from the callus on the differentiation medium, and C is the screening of adventitious buds in the rooting medium containing hygromycin;

[0023] Figure 3 For PagALKBH9B The editing status of the edited plants, where "*" in the figure represents the premature termination of translation;

[0024] Figure 4 It is the process of callus induction from leaves in different plant lines. Among them, A is the state of wild-type 84K at 0 d, B is the state of gene-edited line #8-3-KO at 0 d, C is the state of gene-edited line 7-2-KO at 0 d, D is the state of wild-type 84K at 5 d, E is the state of gene-edited line #8-3-KO at 5 d, F is the state of gene-edited line 7-2-KO at 5 d, G is the state of wild-type 84K at 8 d, H is the state of gene-edited line #8-3-KO at 8 d, I is the state of gene-edited line 7-2-KO at 8 d, J is the state of wild-type 84K at 11 d, K is the state of gene-edited line #8-3-KO at 11 d, L is the state of gene-edited line 7-2-KO at 11 d, M is the state of wild-type 84K at 14 d, N is the state of gene-edited line #8-3-KO at 14 d, O is the state of gene-edited line 7-2-KO at 14 d, P is the front state of wild-type 84K at 20 d, Q is the front state of gene-edited line #8-3-KO at 20 d, R is the front state of gene-edited line 7-2-KO at 20 d, S is the back state of wild-type 84K at 20 d, T is the back state of gene-edited line #8-3-KO at 20 d, U is the back state of gene-edited line 7-2-KO at 20 d, and the scale bar is 1 cm;

[0025] Figure 5 It is the comparison of the net callus weight and the production rate in different plant lines. Among them, A is the net callus weight, B is the callus production rate, and *** represents P < 0.05. Detailed implementation mode

[0026] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] I. Experimental Materials and Methods

[0033] Plant Materials and Growth Conditions: The plant material is tissue-cultured seedlings of Populus alba×P. glandulosa‘84K’ preserved by the research group of Professor Lu Mengzhu / Zhang Jin from Zhejiang A&F University. The gene-edited tissue-cultured seedlings of ALKBH9B are obtained from the genetic transformation of tissue-cultured seedlings of Populus alba×P. glandulosa‘84K’. Its growth conditions are within the temperature range of 24±2°C and the light is 16h / 8h (light / dark).

[0034] Formula of Agrobacterium resuspension solution: 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 perform autoclaving. Before use, add 100 μM acetosyringone (AS) on the ultra-clean workbench.

[0035] Formula of acetosyringone (AS): Dissolve 392.4 mg of acetosyringone powder in 20 mL of dimethyl sulfoxide (DMSO), filter it through a 0.22 μm filter membrane into a sterile centrifuge tube to obtain a 100 mM stock solution.

[0036] Formula of co-culture medium: Dissolve 2.4 g / L WPM449, 0.5 g / L morpholineethanesulfonic acid (MES), and 20 g / L sucrose in deionized water, adjust the pH to 5.9, add 3.2 g / L of phytagel and sterilize. After cooling to below 60°C, add 100 μM acetosyringone on the ultra-clean workbench.

[0037] Differentiation screening medium formula: 2.4 g / L WPM449, 0.5 g / L MES, 0.1 mg / L naphthaleneacetic acid (NAA), 0.5 mg / L 6-benzyladenine (6-BA), 20 g / L sucrose, adjust the pH to 5.9, add 3.2 g / L phytagel and sterilize. After cooling to below 60 °C, add 200 mg / L ticarcillin and 1.5 mg / L hygromycin.

[0038] Callus induction medium formula: 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 kinetin (KT) and 20 g / L sucrose in deionized water, adjust the pH to 5.9, add 3.2 g / L phytagel and sterilize.

[0039] Rooting medium formula: Dissolve 2.2 g / L MS519 (Phyto Technology, USA, catalog number: #M519), 0.5 g / L MES, 0.05 mg / L NAA, 0.02 mg / L indolebutyric acid (IBA), 20 g / L sucrose in deionized water, adjust the pH to 5.9, add 7.8 g / L agar and sterilize. After cooling to below 60 °C, add 200 mg / L ticarcillin.

[0040] II. Construction of ALKBH9B gene-edited poplar

[0041] 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 College of Life Sciences, South China Agricultural University, and the target was designed through its online tool MMEJ-KO.

[0042] 1) Target design: The gene-editing pYLCRISPR / Cas9-DH vector map used is as Figure 1 shown. At the initial stage of vector construction, sequences with a GC content greater than 50%, a lower self-folding score, and high specificity were preferentially selected as targets. The target sequence needs to be specifically verified in the Populus alba × Populus glandulosa '84K' genome database. According to the target sequence, adapter primers were designed and the gene-editing vector was constructed.

[0043] Considering that PagALKBH9A is PagALKBH9B a paralogous gene of PagALKBH9A and PagALKBH9B To prevent phenotypic rescue caused by functional redundancy, a double-gene knockout vector targeting

[0044] Nucleotide sequence of ALKBH9B (SEQ ID NO.1):

[0045]

[0046] Nucleotide sequence of ALKBH9A (SEQ ID NO.2):

[0047]

[0048] The target site sequences of the gRNAs are shown in Table 1 below.

[0049] Table 1 Dual-gene editing targets

[0050] ;

[0051] 2) Preparation of adapter primers

[0052] Mix 1 μL of each of the upstream and downstream primers of the target adapter with 8 μL of water for dilution, set at 90 °C for 30 s in a PCR instrument, and then cool to room temperature to complete the preparation of the adapter primers.

[0053] 3) Construction of gRNA expression cassette

[0054] By the method of cutting and ligating simultaneously, bind the target adapter to the gRNA. The reaction system is shown in Table 2 below, and then perform PCR amplification. The amplification conditions are incubation at 37 °C for 5 min, incubation at 20 °C for 5 min, with 5 cycles.

[0055] Table 2 Reaction system for gRNA expression cassette

[0056] ;

[0057] After the first-round PCR amplification, perform two reactions respectively on the products of each gRNA ligated to the target in the previous round. Set the PCR amplification program: pre-denaturation at 95 °C for 5 min, then denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, with 29 cycles, and finally complete extension at 72 °C for 5 min (as shown in Table 3 below).

[0058] Table 3 Target ligation system

[0059] ;

[0060] In the second-round nested PCR amplification, dilute the PCR products of the previous round by 10 times, take 1 μL of each as templates, mix the reaction systems (as shown in Table 4 below), and set the PCR amplification conditions: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, with 35 cycles, and finally extension at 72 °C for 5 min. Take 5 μL of the amplification products for detection by 1% agarose gel electrophoresis. After confirming the size of the gRNA, proceed to the next step.

[0061] Table 4 Second-round nested PCR system

[0062] ;

[0063] 4) Ligate the pYLCRISPR / Cas9-DH plasmid

[0064] Mix the products of target one and target two and then purify them. Use the Golden Gate method to ligate the pYLCRISPR / Cas9-DH plasmid. The ligation system is shown in Table 5 below.

[0065] Table 5 Final vector ligation system of pYLCRISPR / Cas9-DH

[0066] ;

[0067] 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.

[0068] 5) Escherichia coli transformation and sequencing

[0069] Transform the ligation product into Escherichia coli. After culturing overnight (14 h) at 37 °C, pick monoclonal colonies from the screening culture plate for PCR detection and sequencing verification.

[0070] S2. Transfection: Transfer the PagALKBH9B-DH gene editing vector into the Agrobacterium strain GV3101 by heat shock method, and then introduce it into Populus alba×Populus glandulosa by Agrobacterium-mediated method.

[0071] 1) Select Populus alba×Populus glandulosa tissue culture seedlings about one month old. Select the 3rd to 6th healthy leaves from the top of the seedlings. After gently scratching the leaf surface with a scalpel, place them in the callus induction medium and culture for one month. Use them after callus grows.

[0072] 2) When the optical density of the Agrobacterium containing the PagALKBH9B-DH gene editing vector reaches OD 600 = 0.6, centrifuge at 3500 rpm for 15 min, remove the supernatant and resuspend to OD 600 = 0.4.

[0073] 3) Divide the callus grown from the above leaves into small pieces and soak them in the infection bacterial solution for 15 min, and gently shake to ensure uniform contact. After infection, place the callus on the sterilized filter paper. Wait for the excess liquid in the callus to drain, and then transfer it to the co-culture medium and culture in the dark for 3 d. During this period, observe whether there is colony growth around the callus to ensure the co-culture effect.

[0074] 4) After three days, transfer the callus to the differentiation medium and screen for resistant adventitious buds under a day / night photoperiod of 24 ± 2 °C and 16 / 8 h. Replace the medium every three weeks for 3 months until adventitious buds grow from the callus are observed.

[0075] 5) Transfer the adventitious buds to the rooting screening medium.

[0076] 6) Extract DNA from the leaves of the rooted plants for PCR verification. After PCR amplification of the DNA with Cas9 protein expression in the verified plants, perform high-throughput sequencing, and select the plants with gene editing as transgenic plants for subsequent experiments.

[0077] The main steps for cultivating PagALKBH9B gene-edited transgenic plants are as Figure 2 shown, including the infection of callus with Agrobacterium (such as Figure 2 A in Figure 2 ), the bud differentiation of callus (such as Figure 2 B in Figure 3 ), and the cultivation of transgenic plants through resistance screening (such as PagALKBH9B C in PagALKBH9A ). As PagALKBH9B shown, for the editing situation of gene-edited plants,

[0078] a single-gene knockout yielded the gene-edited line #8-3-KO,

[0079] and a double-gene knockout yielded the gene-edited line #7-2-KO.

[0080] III. Results

[0081] 1. Knockout PagALKBH9B Promotes the induction of callus formation from poplar leaves

[0082] Select one-month-old, vigorously growing and identically growing gene-edited lines #7-2-KO, #8-3-KO and wild-type 84K (WT) lines, and select more than 8 tissue culture seedlings from each line for the experiment of inducing callus from leaves. As Figure 4 shown, when the gene-edited lines were cultured on the callus induction medium for 5 days, callus had already started to form, while no callus formation was observed in the WT lines at this time (such as Figure 4 D in Figure 4 E in Figure 4 F in Figure 4 G in Figure 4 H in Figure 4As shown in I), during subsequent observations of the calli of the 11th, 14th, and 20d calli, the calli of the WT line formed slowly, while the calli of the gene-edited lines formed faster (as Figure 4 in J, Figure 4 in K, Figure 4 in L, Figure 4 in M, Figure 4 in N, Figure 4 in O, Figure 4 in P, Figure 4 in Q, Figure 4 in R, Figure 4 in S, Figure 4 in T and Figure 4 in U). To exclude the influence of initial leaf differences, the average value of the callus production rate was also calculated (callus production rate = net callus weight / leaf weight). As Figure 5 shown, both the net callus weight and the callus production rate of the gene-edited lines were significantly higher than those of the wild-type lines. In addition, the net callus weight and the callus production rate of the double-gene-edited line #7-2-KO were also significantly higher than those of the single-gene-edited line #8-3-KO. As Figure 5 in A and Figure 5 in B, these results indicate that PagALKBH9B plays an inhibitory role during the process of inducing callus from leaves, and its deletion can promote the formation of callus. At the same time, PagALKBH9A as a paralogous gene, also plays a certain regulatory role in this process.

[0083] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention 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; By knockdown and / or knockout ALKBH9B Gene, reduce its expression, and promote the rapid formation of plant callus; The plant is poplar.

2. The application according to claim 1, characterized in that: Promote the formation of callus tissue of poplar leaves.

3. A kind of utilization 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 and promoting the formation of poplar leaf callus; Said ALKBH9B The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The method for promoting callus formation in poplar leaves according to claim 3, 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.

5. ALKBH9B Genes and ALKBH9A The invention relates to 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; 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; The plant is poplar.

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