Method for constructing multi-gene expression vector or multi-target gene editing vector

Abstract

The invention discloses a method for constructing a multi-gene expression vector or a multi-target gene editing vector. The multi-gene expression vector or the multi-target gene editing vector comprises a transformation vector (pDF11) and two donor vectors (p103A and p103B), the transformation vector carries a CRI SPR-Act3.0 transcriptional activation system, a plant screening gene (hpt I I), two I-SceI restriction enzyme cutting sites in opposite directions, and a kanamycin (Km) resistance vector; the two donor vectors comprise a complete gRNA expression cassette and respective antibiotic selection marker genes, the two donor vectors respectively have spectinomycin (Spe) and ampicillin (Amp) resistance, and the two sides of the antibiotic selection marker genes also contain two I-Sce I restriction enzyme cutting sites in opposite directions; compared with the prior art, the invention has the advantages that the construction of the multi-gene and multi-target transcription activation vector is realized, the vector system constructs a series of single-target and multi-target transcription activation vectors, and one vector simultaneously contains four genes and 10 targets of two PtoWRKY41 targets, two PtoWRKY31 targets, two PtoWRKY70 targets and four PtoWRKY41a targets.

Description

Technical Field

[0001] The present invention relates to the field of gene editing vector technology, and specifically to a method for constructing a multi-gene expression vector or a multi-target gene editing vector. Background Art

[0002] By utilizing the principle that the original site disappears after ligation with the homologous endonuclease in the restriction endonuclease and combining it with different antibiotic screening genes, a vector system is constructed for constructing multi-gene expression vectors or multi-target gene editing vectors.

[0003] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above technical defects and provide a method for constructing a multi-gene expression vector or a multi-target gene editing vector.

[0005] In order to solve the above problems, the technical solution of the present invention is a method for constructing a multi-gene expression vector or a multi-target gene editing vector: comprising a transformation vector (pDF11) and two donor vectors (p103A, p103B);

[0006] The transformation vector carries the CRISPR-Act3.0 transcriptional activation system, a plant selection gene (hptII), two I-SceI restriction sites in opposite directions, and a kanamycin (Km) resistance vector; the two donor vectors contain complete gRNA expression cassettes and respective antibiotic selection marker genes, respectively confers spectinomycin (Spe) and ampicillin (Amp) resistance, and the antibiotic selection marker genes also contain two I-SceI restriction sites in opposite directions on both sides;

[0007] The method for constructing a multi-gene, multi-target gene editing vector includes the following steps:

[0008] Step 1: First, linearize the target gene with Esp3 I enzyme digestion and connect it with the linkers P1 and P2 designed according to the target sequence to construct their own complete gRNA expression cassettes. Then, use BstX I enzyme digestion to recover the gRNA expression cassettes carrying the P1 and P2 linkers.

[0009] Step 2: At the same time as step 1, the transformation vector pDF11 was digested with I-Sce I, and the target sequence P1 expression cassette containing the BstX I sticky end was ligated to it. The original BstX I and I-Sce I sites on the transformation vector disappeared, and the antibiotic marker gene, two I-Sce I sites, and the gRNA expression cassette containing the target sequence P1 on the donor vector were connected to the transformation vector. The ligation product was transformed into Escherichia coli competent cells and screened simultaneously with two antibiotics, Km and Spe, to obtain a transformation vector containing the target sequence P1;

[0010] Step three: Use I-Sce I to digest the vector, and connect the target sequence P2 expression cassette containing the BstX I sticky end to it. The ligation product is screened with Km and Amp to obtain the transformation vector containing the target sites P1 and P2. Different target gRNA expression cassettes are alternately connected in this order to realize the construction of multi-gene, multi-target transcription activation vector.

[0011] Furthermore, a BstX I site is contained on the outside of the antibiotic selection marker gene and the complete gRNA expression cassette, respectively, for connecting to the I-Sce I site on the transformation vector and making it disappear.

[0012] Furthermore, the vector system has a higher transcriptional activation ability of the CRISPR-Act3.0 system, in which one vector simultaneously contains a 4-gene 10-target transcriptional activation vector of 2 PtoWRKY41 targets, 2 PtoWRKY31 targets, 2 PtoWRKY70 targets and 4 PtoWRKY41 a targets.

[0013] Furthermore, the transformation vector is used for plant genetic transformation and has kanamycin (Km) resistance and two restriction enzyme sites, A and B. The donor vector carries four restriction enzyme sites, A, B, C, and D, and an ampicillin (Amp) or spectinomycin (Spe) resistance marker. Restriction sites A and C are homozygous, while restriction sites B and D are homozygous.

[0014] Furthermore, the method for constructing a multi-gene transformation vector or a multi-target gene editing vector comprises the following steps:

[0015] An exogenous gene or target gene gRNA expression cassette is constructed on the donor vector, and the target fragment and antibiotic screening fragment are cut using endonucleases C and D. At the same time, the transformation vector is linearized by cutting with endonucleases A and B, and the target fragment is cut by C and D respectively. New fragments can be continuously connected based on the principle that the restriction site disappears after homotypic enzyme ligation. Escherichia coli is transformed and screened with kanamycin (Km) plus the antibiotic corresponding to the screening marker on the donor. The double-resistant single colony obtained by culture is the constructed multi-gene or multi-target gene editing transformation vector.

[0016] The advantages of the present invention compared with the existing technology are:

[0017] 1. The present invention realizes the construction of multi-gene, multi-target transcriptional activation vectors. This vector system not only has the high transcriptional activation ability of the CRISPR-Act3.0 system, but also breaks through the limitation of the original vector system that can only contain up to 6 targets. Currently, using this vector system, a series of single- and multi-target transcriptional activation vectors have been constructed, including one vector containing 4 genes and 10 targets, 2 PtoWRKY41 targets, 2 PtoWRKY31 targets, 2 PtoWRKY70 targets, and 4 PtoWRKY41a targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the transformation vector in Example 1 of the present invention.

[0019] Figure 2 This is a diagram of the donor carrier in Example 1 of the present invention.

[0020] Figure 3 This is a process diagram of the vector construction process in Example 1 of the present invention.

[0021] Figure 4 This is a second process diagram of the vector construction process in Example 1 of the present invention.

[0022] Figure 5 3 is a process diagram of the vector construction process in Example 1 of the present invention.

[0023] Figure 6 This is a diagram of the gene editing vector system in Example 2 of the present invention, A: transformation vector; B: donor vector.

[0024] Figure 7 This is a process diagram of the multi-target gene editing vector construction process in Example 2 of the present invention.

[0025] Figure 8 This is the second process diagram of the multi-target gene editing vector construction process in Example 2 of the present invention.

[0026] Figure 9 This is the third process diagram of the multi-target gene editing vector construction process in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the contents of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0028] Example 1

[0029] A method for constructing a multi-gene expression vector or a multi-target gene editing vector, wherein the entire vector system includes a transformation vector and a donor vector:

[0030] The transformation vector is used for plant genetic transformation and inherently confers kanamycin (Km) resistance and two restriction enzyme sites, A and B. The donor vector carries four restriction enzyme sites, A, B, C, and D, and an ampicillin (Amp) or spectinomycin (Spe) resistance marker. Restriction sites A and C are homozygous, while restriction sites B and D are homozygous.

[0031] When constructing a multi-gene transformation vector or a multi-target gene editing vector, first construct the exogenous gene or target gene gRNA expression cassette on the donor vector, and use endonucleases C and D to cut out the target fragment and antibiotic screening fragment; at the same time, use endonucleases A and B to cut the transformation vector to linearize it, and then continuously connect it to the donor vector in sequence to cut out the target fragment by C and D. Utilizing the principle that the restriction site disappears after homotypic enzyme ligation, new fragments can be continuously connected. Escherichia coli is transformed and screened with kanamycin (Km) plus the antibiotic corresponding to the selection marker on the donor. The double-resistant single colony obtained by culture is the constructed multi-gene or multi-target gene editing transformation vector.

[0032] Example 2

[0033] A method for constructing a multi-gene expression vector or a multi-target gene editing vector, the vector system comprising a transformation vector (pDF11) and two donor vectors (p103A and p103B). The transformation vector carries the CRISPR-Act3.0 transcriptional activation system, a plant selection gene (hptII), two I-SceI restriction sites in opposite directions, and a kanamycin (Km) resistance vector; the two donor vectors contain a complete gRNA expression cassette and respective antibiotic selection marker genes, conferring resistance to spectinomycin (Spe) and ampicillin (Amp), respectively. The antibiotic selection marker genes are also flanked by two I-SceI restriction sites in opposite directions; a BstXI site is contained on the outside of each of the antibiotic selection marker genes and the complete gRNA expression cassette, which is used to ligate to and eliminate the I-SceI site on the transformation vector.

[0034] In the process of constructing a multi-gene, multi-target gene editing vector, first, Esp3 I enzyme digestion and linearization are performed, and then they are connected with the linkers P1 and P2 designed according to the target sequence to construct their own complete gRNA expression cassettes, and then BstX I enzyme digestion is used to recover the gRNA expression cassettes carrying the P1 and P2 linkers; at the same time, the transformation vector pDF11 is digested with I-Sce I enzyme, and the target sequence P1 expression cassette containing the BstX I sticky end is connected to it, the original BstX I and I-Sce I sites on the transformation vector disappear, and the antibiotic marker gene, two I-Sce I sites and the gRNA expression cassette containing the target sequence P1 on the donor vector are connected to the transformation vector, the ligation product is transformed into Escherichia coli competent cells and screened with two antibiotics, Km and Spe, to obtain the transformation vector containing the target P1; similarly, I-Sce I enzyme digestion is used to digest this vector, and the target sequence P1 containing the BstX I sticky end is connected to it. The target sequence P2 expression cassette at the I sticky end is ligated to it. The ligation product is screened with Km and Amp to obtain a transformation vector containing the target sites P1 and P2. Different target gRNA expression cassettes are then alternately connected in this order to achieve the construction of a multi-gene, multi-target transcription activation vector. This vector system not only has the high transcription activation ability of the CRISPR-Act3.0 system, but also overcomes the limitation of the original vector system that can only contain a maximum of six targets. Currently, a series of single- and multi-target transcription activation vectors have been constructed using this vector system, including a transcription activation vector for four genes and ten targets simultaneously containing two PtoWRKY41 targets, two PtoWRKY31 targets, two PtoWRKY70 targets, and four PtoWRKY41a targets.

[0035] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A method for constructing a multi-gene expression vector or a multi-target gene editing vector, characterized by: Includes a transformation vector (pDF11) and two donor vectors (p103A, p103B); The transformation vector carries the CRISPR-Act3.0 transcriptional activation system, a plant selection gene (hptII), two I-SceI restriction sites in opposite directions, and a kanamycin (Km) resistance vector; the two donor vectors contain complete gRNA expression cassettes and respective antibiotic selection marker genes, respectively confers spectinomycin (Spe) and ampicillin (Amp) resistance, and the antibiotic selection marker genes also contain two I-SceI restriction sites in opposite directions on both sides; The method for constructing a multi-gene, multi-target gene editing vector includes the following steps: Step 1: First, linearize the target gene with Esp3 I enzyme digestion and connect it with the linkers P1 and P2 designed according to the target sequence to construct their own complete gRNA expression cassettes. Then, use BstX I enzyme digestion to recover the gRNA expression cassettes carrying the P1 and P2 linkers. Step 2: At the same time as step 1, the transformation vector pDF11 was digested with I-Sce I, and the target sequence P1 expression cassette containing the BstX I sticky end was ligated to it. The original BstX I and I-Sce I sites on the transformation vector disappeared, and the antibiotic marker gene, two I-Sce I sites, and the gRNA expression cassette containing the target sequence P1 on the donor vector were connected to the transformation vector. The ligation product was transformed into Escherichia coli competent cells and screened simultaneously with two antibiotics, Km and Spe, to obtain a transformation vector containing the target sequence P1; Step three: Use I-Sce I to digest the vector, and connect the target sequence P2 expression cassette containing the BstX I sticky end to it. The ligation product is screened with Km and Amp to obtain the transformation vector containing the target sites P1 and P2. Different target gRNA expression cassettes are alternately connected in this order to realize the construction of multi-gene, multi-target transcription activation vector.

2. A method for constructing a multi-gene expression vector or a multi-target gene editing vector according to claim 1, characterized in that: There is a BstX I site on the outside of the antibiotic selection marker gene and the complete gRNA expression cassette, which is used to connect to the I-Sce I site on the transformation vector and eliminate it.

3. The method for constructing a multi-gene expression vector or a multi-target gene editing vector according to claim 1, characterized in that: The vector system has a high transcriptional activation ability of the CRISPR-Act3.0 system, in which one vector simultaneously contains a 4-gene 10-target transcriptional activation vector with 2 PtoWRKY41 targets, 2 PtoWRKY31 targets, 2 PtoWRKY70 targets and 4 PtoWRKY41 a targets.

4. The method for constructing a multi-gene expression vector or a multi-target gene editing vector according to claim 1, characterized in that: The transformation vector is used for plant genetic transformation and has kanamycin (Km) resistance and two restriction enzyme sites, A and B. The donor vector carries four restriction enzyme sites, A, B, C, and D, and an ampicillin (Amp) or spectinomycin (Spe) resistance marker. Restriction sites A and C are homozygous, while restriction sites B and D are homozygous.

5. The method for constructing a multi-gene expression vector or a multi-target gene editing vector according to claim 4, characterized in that: The method for constructing a multi-gene transformation vector or a multi-target gene editing vector comprises the following steps: S1: Construct the exogenous gene or target gene gRNA expression cassette on the donor vector, and use endonucleases C and D to cut out the target fragment and antibiotic screening fragment; S2: Use endonucleases A and B to cut the transformation vector to linearize it, and then continuously connect it into the donor vector in sequence. Cut the target fragments through C and D respectively. Utilizing the principle that the restriction site disappears after ligation with the same tail enzyme, new fragments can be continuously connected. Transform Escherichia coli and screen with kanamycin (Km) plus the antibiotic corresponding to the selection marker on the donor. The double-resistant single colony obtained by culture is the constructed multi-gene or multi-target gene editing transformation vector.

Images