Novel gene editing element self-cleaning recombinant vector suitable for tobacco and application of novel gene editing element self-cleaning recombinant vector

Abstract

The invention discloses a novel gene editing element self-cleaning recombinant vector suitable for tobacco and application thereof.The recombinant vector comprises an expression cassette of a germ cell specific expression Barnase gene, the expression cassette comprises a germ cell specific expression promoter DLL, a lethal gene Barnase and an NOS terminator, the recombinant vector is obtained by integrating the expression cassette into a tobacco editing vector, and the expression cassette is a recombinant vector with a specific expression function. A linked recombinant vector is formed with other gene editing elements, and the sequence of an expression cassette is as shown in SEQ ID No.1. After a plant cell is transformed by the gene editing recombinant vector, the gene editing element of a T0-generation plant is not influenced to play a role, Barnase protein capable of killing female and male gametes is expressed during sexual propagation, most of T0-generation seeds do not contain CRISPR editing elements, filial generation plants do not contain CRISPR editing elements any more, the efficiency of obtaining the CRISPR-free editing element is improved, and the method is suitable for large-scale popularization and application. And manpower, material resources and financial resources are greatly saved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a novel gene editing element self-clearing recombinant vector suitable for tobacco and applications thereof. Background Art

[0002] Gene editing is a disruptive biotechnology in the current life sciences. The emergence and application of gene editing tools has opened up the possibility of genetic modification in plant breeding. In recent years, crop breeding has made great strides towards the era of intelligent design breeding, and genome editing technology is an indispensable key biotechnology. Conventional plant gene editing generally achieves targeted gene editing through stable transformation, followed by selfing or backcrossing to remove the CRISPR editing elements. This prevents residual CRISPR elements in the host, which can cause off-target effects, trait instability, and regulatory issues. Furthermore, conventional removal of transgenic elements is typically achieved through selfing or backcrossing. Because these elements must be simultaneously tested and verified to identify the correct edited plants and whether they contain the transgenic elements, large-scale propagation and testing of progeny plants is required, a process that consumes considerable time, material, and financial resources. Currently, methods to improve the efficiency of screening for non-transgenic plants primarily rely on fluorescent screening and resistance markers. Non-transgenic methods can also be used to obtain transgenic-free plants, such as ribonucleoprotein (RNP) transfection, transient expression techniques without DNA integration, nanobiotechnology, and plant virus-mediated editing, but these methods are relatively inefficient. To further reduce the labor and time costs of screening, researchers developed the TKC (Transgene Killer CRISPR) system in rice, utilizing it to achieve self-elimination of transgenes. This method combines gene-editing elements with pollen- and embryo-specific suicide elements into a linked system. After gene editing, the suicide elements specifically kill transgenic pollen and seeds, allowing for the autonomous selection of DNA-free, stably inherited gene-edited plants without the need for additional time and labor for identification.

[0003] However, there are currently no reports on TKC editing systems in dicotyledons. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new type of gene editing element self-clearing recombinant vector suitable for tobacco and its application, which effectively reduces the manpower, material resources and financial resources required to obtain edited plant offspring without CRISPR editing elements.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] A novel self-clearing recombinant vector for gene editing elements suitable for tobacco. The recombinant vector contains an expression cassette for germ cell-specific expression of the Barnase gene. The expression cassette includes the germ cell-specific expression promoter DLL, the lethal gene Barnase, and the NOS terminator. The recombinant vector integrates the expression cassette into the tobacco editing vector to form a linked recombinant vector with other gene editing elements. The sequence of the expression cassette is shown in SEQ ID No. 1.

[0007] Preferably, in the above technical solution, the germ cell-specific expression promoter DLL gene is derived from Arabidopsis thaliana and is used to drive the specific expression of Barnase in germ cells.

[0008] Preferably, in the above technical solution, the coding sequence of the lethal gene Barnase is codon-optimized for plant preference, and a plant-derived intron is added for cloning of the gene in prokaryotes.

[0009] Preferably, in the above technical solution, the recombinant vector uses pOREU3TR as its skeleton.

[0010] Preferably, in the above technical solution, after the expression cassette of the Barnase gene is transformed into plant cells by the leaf disc method, it does not affect the function of the gene editing element, but during sexual reproduction, the Barnase toxin protein is expressed through the specific regulation of the DNA element in the recombinant vector, which can kill the female and male gametes of the T0 generation plants containing T-DNA elements, and only the female and male gametes of the T0 generation plants without T-DNA elements survive.

[0011] A method for preparing an edited plant without CRISPR editing elements using a recombinant vector comprises the following steps:

[0012] (1) Preparation of Barnase gene expression cassette specifically expressed in germ cells:

[0013] The Barnase gene expression cassette containing two introns is driven by the DLL promoter and terminated by the NOS terminator. The expression cassette sequence is shown in SEQ ID No. 1. PCR amplification is performed using primers containing 15 bp of homologous sequences at both ends of the backbone vector. The expression cassette is subjected to agarose gel electrophoresis and gel purification to obtain a linearized DNA fragment.

[0014] (2) Construction of recombinant vector: The backbone vector pOREU3TR was double-digested with restriction endonucleases KpnI and BsrGI, and the purified linearized backbone DNA was recovered by agarose gel electrophoresis. The expression cassette linearized DNA was recombined with the purified linearized backbone DNA; the recombinant product was transformed into Escherichia coli, and the gene editing recombinant vector PEB138 was obtained after screening and sequencing;

[0015] (3) Construction of gene editing vector: Design 23nt specific sequence and upstream and downstream primers according to the sequence specificity of the target gene, anneal and connect by PCR, and transform Agrobacterium;

[0016] (4) Creation and testing of T0 generation edited plants: The gene editing vector is transformed into explants, and T0 generation plants are obtained through callus induction, differentiation and rooting; T0 generation plants are sampled for sgRNA and target mutation detection, and their transformation efficiency and editing efficiency are tested, and T0 generation seeds are harvested;

[0017] (5) Detection of CRISPR editing elements and editing efficiency in T1 generation plants: T0 generation seeds were sown, and plants were randomly selected for detection of CRISPR editing elements and target editing. The proportion of T1 generation plants without CRISPR editing elements and the proportion of edited plants were statistically analyzed.

[0018] Preferably, in the above technical solution, the linearized DNA of the Barnase gene expression cassette is subjected to a recombination reaction with the purified linearized backbone DNA, and the reaction system is as follows:

[0019]

[0020] Preferably, in the above technical solution, the recombination reaction conditions are: 37° C. for 30 min; then cooled to 4° C. or immediately placed on ice for cooling.

[0021] Preferably, in the above technical solution, in step (2), the annealing program of the PCR instrument is: 95°C for 5 min, -0.1°C / 8 s, and annealing to 25°C.

[0022] Preferably, in the above technical solution, the PEB138 backbone is cleaved by enzyme, and the enzyme cleavage system is:

[0023]

[0024] Incubate overnight at 37°C for 4 h, perform electrophoresis on a 1.5% agarose gel, and use a gel recovery kit to recover the backbone fragments.

[0025] The connection system is:

[0026]

[0027] The ligation was carried out at 16°C for 2 hours. The ligation product was purified using a gel recovery kit and transformed into Escherichia coli by electroporation to form a gene knockout vector. A large amount of plasmid was extracted by shaking for later use.

[0028] Preferably, in the above technical solution, the Agrobacterium is LBA4404, and the transformed Agrobacterium is evenly spread on YEB solid culture medium, and transformed Agrobacterium is obtained after overnight culture.

[0029] Preferably, in the above technical solution, the NOS primer sequence is as follows:

[0030] NOS-F: 5'-GATTGAATCCTGTTGCCGGT-3';

[0031] NOS-R:5'-GTAACATAGATGACACCGCG-3'.

[0032] The invention discloses an application of a novel self-clearing recombinant vector for gene editing elements suitable for tobacco in the preparation of T-DNA element-free edited plants.

[0033] Preferably, in the above technical solution, the Barnase gene expression cassette expresses a Barnase protein that can kill female and male gametes during sexual reproduction. The Barnase protein has strong cytotoxicity and can kill female and male gametes containing CRISPR editing elements. In this way, most of the T0 generation seeds do not contain CRISPR editing elements, and their offspring plants no longer contain CRISPR editing elements.

[0034] Preferably, in the above technical solution, the recombinant vector is used as a backbone vector to develop a variety of editing vectors, including but not limited to single-target gene editing, multi-target gene editing, guide editing, base editors, etc.

[0035] The above technical solution of the present invention has the following beneficial effects:

[0036] The gene editing recombinant vector of the present invention contains a Barnase gene expression cassette specifically expressed in germ cells. After transforming plant cells, it does not affect the function of the gene editing elements in the T0 generation plants. However, during sexual reproduction, it expresses a Barnase protein that can kill both female and male gametes. Most T0 generation seeds do not contain CRISPR editing elements, and their daughter plants no longer contain CRISPR editing elements. This can greatly improve the efficiency of obtaining plants without CRISPR editing elements and save a lot of manpower, material resources and financial resources required for the separation and removal of CRISPR editing elements. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0038] Figure 1 Schematic diagram of the PEB138 recombinant vector structure. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention.

[0040] Unless otherwise specified, all experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained through commercial channels.

[0041] The present invention integrates an expression cassette containing the germ cell-specific promoter DLL, the lethal gene Barnase, and the NOS terminator into a tobacco editing vector, forming a linked recombinant vector with other gene editing elements. When using the present invention to create gene-edited mutants, the gene editing elements can perform gene editing during vegetative growth; however, during sexual reproduction, they express the Barnase protein, which is lethal to both male and female gametes. As a result, the majority of pollen and female gametes containing the CRISPR editing elements in T0 plants abort. The resulting T0 seeds have a significantly higher proportion of CRISPR-free T0 seeds than those containing conventional editing vectors, allowing efficient generation of CRISPR-free edited plant progeny.

[0042] Example 1 Preparation of Barnase Gene Expression Cassette Specific to Germ Cells

[0043] The backbone vector pOREU3TR was used for the construction of the recombinant vector in Example 1.

[0044] The Barnase gene expression cassette is driven by the DLL promoter and terminated by the NOS terminator. The entire expression cassette sequence is shown in SEQ ID No. 1 and was synthesized by Nanjing GenScript. PCR amplification was performed using primers containing 15 bp of homologous sequence at both ends of the backbone vector. A linearized DNA fragment of the expression cassette was obtained by agarose gel electrophoresis and gel purification.

[0045] Example 2 Construction of recombinant vector

[0046] (1) The backbone vector pOREU3TR was double-digested with restriction endonucleases KpnI and BsrGI, and the purified linearized backbone DNA was recovered by agarose gel electrophoresis.

[0047] (2) The linearized DNA of the Barnase gene expression cassette in Example 1 was subjected to a recombination reaction with the purified linearized backbone DNA. The reaction system was as follows:

[0048]

[0049]

[0050] (3) Recombination reaction conditions: 37°C for 30 min; then cool to 4°C or immediately place on ice.

[0051] (4) The recombinant product was transformed into Escherichia coli, and the gene editing recombinant vector PEB138 was obtained through screening and sequencing.

[0052] Specifically:

[0053] PEB138 backbone enzyme digestion, the enzyme digestion system is:

[0054]

[0055] Incubate overnight at 37°C for 4 h, perform electrophoresis on a 1.5% agarose gel, and use a gel recovery kit to recover the backbone fragments.

[0056] The connection system is:

[0057]

[0058] The ligation was carried out at 16°C for 2 hours. The ligation product was purified using a gel recovery kit and transformed into Escherichia coli by electroporation to form a gene knockout vector. A large amount of plasmid was extracted by shaking for later use.

[0059] The Agrobacterium is LBA4404. The transformed Agrobacterium is evenly spread on a YEB solid culture medium and cultured overnight to obtain the transformed Agrobacterium.

[0060] Preferably, the NOS primer sequence is as follows:

[0061] NOS-F: 5'-GATTGAATCCTGTTGCCGGT-3';

[0062] NOS-R:5'-GTAACATAGATGACACCGCG-3'.

[0063] Example 3 Construction of gene editing vector

[0064] (1) Targeting primer design

[0065] Design a 23-nt specific sequence (sgRNA) based on the sequence specificity of the target gene. The design method can be found in the following instructions: http: / / crispor.tefor.net / crispor.py. Synthesize the upstream primer (5'-TGACNNNNNNNNNNNNNNNNNNNNNN-3') and the downstream primer (5'-AAACNNNNNNNNNNNNNNNNNNNNNN-3'). Specifically, the sgRNA is an arbitrary 20-base sequence, the 5'-end adapter sequence of the upstream primer is TGAC, and the 5'-end adapter sequence of the downstream primer is AAAC.

[0066] (2) Annealing

[0067] After diluting the synthesized primers to a concentration of 100 ng / μL, take 5 μL of each upstream and downstream primers and mix them in a PCR tube. Anneal the PCR instrument using the following program: 95°C for 5 min, -0.1°C for 8 s, and then anneal to 25°C. Dilute the annealed product 10-fold with water and store in a refrigerator at -20°C until ready for use.

[0068] pOREU3TR NtDFR backbone cleavage

[0069] Enzyme digestion system (50 μL):

[0070]

[0071] The fragments were digested overnight at 37°C, and electrophoresed on 1.5% agarose gel. The backbone fragments were recovered using an EZNA Gel Extraction Kit.

[0072] (3) Connection

[0073] The annealed and diluted products were mixed one by one to form a mixed annealed product, which was then ligated with the enzyme-digested pOREU3TR / NtDFR backbone vector. The ligation system (10 μL) was as follows:

[0074]

[0075] The ligation was carried out at 16°C for 2 hours. The ligation product was purified using an EZNAGel Extraction Kit and transformed into E. coli by electroporation to form a gene knockout vector. The plasmid was extracted in large quantities by shaking for later use.

[0076] (4) Preparation of transformed Agrobacterium

[0077] The gene knockout vector library plasmid was transformed into Agrobacterium LBA4404 by electroporation. The transformed Agrobacterium was evenly spread on YEB solid medium and cultured overnight to obtain transformed Agrobacterium.

[0078] Example 4 Creation and Detection of T0 Generation Edited Plants

[0079] The leaf disc method was used to transform the base plate of red flower Dajinyuan tobacco. After co-cultivation, callus induction, differentiation culture and rooting culture, T0 generation rooted seedlings were obtained. The rooted seedlings were transplanted into pots, and samples were taken to test the editing efficiency and editing type. The test results are shown in Table 1 below:

[0080] Table 1

[0081]

[0082]

[0083] As can be seen from the table, after PEB138 was transformed into tobacco leaf discs, it was able to normally produce T0 generation plants. The T0 generation plants included various types of mutations, including biallelic mutations, homozygous mutations, and heterozygous mutations. The overall editing efficiency reached 82.19%, indicating that this recombinant vector can be used to create gene editing materials.

[0084] Example 5 Screening and identification of edited plants without CRISPR editing elements in T1 generation plants

[0085] Three T0 generation seeds with biallelic mutations, homozygous mutations, and heterozygous mutations were selected for sowing, and 24 plants were randomly selected to detect CRISPR editing elements and target editing. The proportion of T1 generation plants without CRISPR editing elements and the proportion of edited plants were statistically analyzed.

[0086] After sowing three T0 generation seeds, thinning and transplanting were performed, and 24 plants were randomly selected. Individual samples were taken at the 4-6 leaf stage and sent to Huazhi Biotechnology for CRISPR editing element and target site testing to determine whether they contained CRISPR editing elements and target site editing, and to analyze whether the target site mutations were consistent with those of the T0 generation. The test results are shown in Table 2 below:

[0087] Table 2

[0088]

[0089] The test results of three seeds showed that the proportion of recombinant editing vector offspring plants without CRISPR editing elements was 100%, which was 4.8 times the control's 20.83%, close to the theoretical value, indicating that the recombinant vector can efficiently obtain edited plant offspring without CRISPR editing elements; in addition, the editing efficiency of the T1 generation plants was not much different from that of the control, both at around 70%, and the mutation type was also consistent with the T0 generation, indicating that the mutations produced by the recombinant vector can be stably inherited.

[0090] Overall, this recombinant vector can significantly improve the efficiency of obtaining edited plants without CRISPR editing elements, which will greatly reduce the manpower, material and financial resources required for the self-pollination process to remove T-DNA.

[0091] Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A novel gene editing element self-clearing recombinant vector suitable for tobacco, It is characterized in that The recombinant vector contains an expression frame for the Barnase gene specifically expressed in germ cells, and the expression frame contains the promoter DLL, the lethal gene Barnase and the NOS terminator specifically expressed in germ cells. The recombinant vector integrates the expression frame into the editing vector of tobacco to form a linked recombinant vector with other gene editing elements. The sequence of the expression frame is shown in SEQ ID No.

1.

2. The novel gene editing element self-clearing recombinant vector suitable for tobacco according to claim 1, It is characterized in that The promoter DLL gene specifically expressed in reproductive cells is derived from Arabidopsis thaliana and is used to drive Barnase to be specifically expressed in reproductive cells.

3. The novel gene editing element self-clearing recombinant vector suitable for tobacco according to claim 1, It is characterized in that The coding sequence of the lethal gene Barnase is codon-optimized according to plant preference, and an intron from plant source is added for cloning the gene in prokaryotes.

4. The novel gene editing element self-clearing recombinant vector suitable for tobacco according to claim 1, It is characterized in that The recombinant vector uses pOREU3TR as a backbone.

5. A method for preparing an edited plant without CRISPR editing elements using the recombinant vector according to any one of claims 1 to 4, specifically The following steps are involved: (1) Preparation of Barnase gene expression cassette specifically expressed in germ cells: The Barnase gene expression cassette containing two introns is driven by the DLL promoter and terminated by the NOS terminator. The expression cassette sequence is shown in SEQ ID No.

1. The expression cassette is amplified by PCR using primers containing 15 bp homologous sequences at both ends of the backbone vector at the upstream and downstream ends, and the expression cassette is purified by agarose gel electrophoresis and gel cutting to obtain a linearized DNA fragment; (2) Construction of recombinant vector: The backbone vector pOREU3TR was double-digested with restriction endonucleases KpnI and BsrGI, and the purified linearized backbone DNA was recovered by agarose gel electrophoresis. The expression cassette linearized DNA and the purified linearized backbone DNA were subjected to a recombination reaction; the recombinant product was transformed into Escherichia coli, and the gene editing recombinant vector PEB138 was obtained after screening and sequencing; (3) Construction of gene editing vector: Design 23 nt specific sequence and upstream and downstream primers according to the sequence specificity of the target gene, PCR annealing and transformation of Agrobacterium; (4) Creation and testing of T0-generation edited plants: The gene editing vector is transformed into explants, and T0-generation plants are obtained through callus induction, differentiation and rooting. T0-generation plants are sampled for sgRNA and target mutation detection, transformation efficiency and editing efficiency are tested, and T0-generation seeds are harvested. (5) Detection of CRISPR editing elements and editing efficiency in T1 generation plants: T0 generation seeds were sown, and plants were randomly selected for detection of CRISPR editing elements and target editing. The proportion of T1 generation plants without CRISPR editing elements and the proportion of edited plants were statistically analyzed.

6. The use according to claim 5, It is characterized in that The linearized DNA of the Barnase gene expression cassette was recombined with the purified linearized backbone DNA. The reaction system was as follows:

7. The use according to claim 5, It is characterized in that The enzyme digestion system is: The connection system is:

8. The use according to claim 5, It is characterized in that The Agrobacterium is LBA4404. The transformed Agrobacterium is evenly spread on a YEB solid culture medium, and the transformed Agrobacterium is obtained after overnight culture.

9. Use of the novel gene editing element self-clearing recombinant vector suitable for tobacco according to any one of claims 1 to 4 in preparing edited plants without CRISPR editing elements.

10. The use according to claim 9, It is characterized in that During sexual reproduction, the Barnase gene expression frame expresses Barnase protein that can kill female and male gametes. Barnase protein can kill female and male gametes containing CRISPR editing elements, so that the T0 generation seeds do not contain CRISPR editing elements, and their offspring plants no longer contain CRISPR editing elements.

Images