CN115851756A - Plant gene editing recombinant vector capable of screening out false positive callus and application - Google Patents

Abstract

The invention discloses a plant gene editing recombinant vector capable of screening out false positive callus and application thereof. The recombinant vector can screen out false positive callus and transformant, contains NtAN2 gene element, adds an NtU4M promoter-driven 35S-terminated expression frame containing NtAN2 gene element into T-DNA, and forms a linked recombinant vector with other gene editing elements; the whole expression frame sequence is SEQ ID No.1. When the scheme of the invention is adopted to carry out plant gene editing technology to create the mutant, whether the callus is false positive of the transgene can be judged by the red color which can be distinguished by naked eyes at the stage of the resistant callus. The screening of the false positive callus reduces the appearance of false positive plants, reduces the investment of the steps of cultivation, detection and the like of the false positive plants in the later period, and further improves the probability of the occurrence of the plants with effectively generated gene editing.

Description

Plant gene editing recombinant vector capable of screening out false positive callus and application thereof

Technical Field

The invention relates to the technical field of plant gene engineering, in particular to a plant gene editing recombinant vector capable of screening out false positive callus and application thereof.

Background

The CRISPR/Cas9 technology has been developed to date since 2012 and has been widely used in the field of gene editing. It consists of two parts, an artificially engineered guide RNA (sgRNA) and a Cas9 nuclease. The gene editing principle is that a Nuclear Localization Signal (NLS) is added into a Cas9 nuclease expression frame, cas9 is guided into a cell nucleus by the nuclear localization signal after being translated in cytoplasm to be combined with sgRNA, a 20nt sequence-specific guide sequence is added into the sgRNA to form a complex, a DNA site which is complementary to the 20nt specific sequence in the sgRNA is identified, and a DNA double-strand break is formed by cutting. In general, DNA repair is dominated by non-homologous end joining (NHEJ), which results in the formation of short indels (base insertions or deletions) that result in frame shift mutations in the target gene.

In the implementation process of plant gene editing, tissue culture is generally required to form resistant callus, and the resistant callus is differentiated to form a resistant plant. False positive resistant callus exists in the tissue culture process, the false positive resistant callus can be further differentiated into false positive plants, the appearance of the false positive callus in large-scale mutant creation increases the screening range and difficulty of editing plants, and the detection cost is greatly improved.

At present, the conventional genes such as fluorescent protein, GUS, luciferase and the like can also be used for eliminating and screening false positive callus. The detection of these genes either requires expensive instruments or requires specific substrates for catalytic reactions, which has great limitations in large-scale mutant creation applications.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a plant gene editing recombinant vector capable of screening out false positive callus and application thereof, which overcome the defects of the prior art, solve the problem that false positive callus exists in the tissue culture stage and promote the large-scale mutant creation by utilizing the gene editing technology.

The technical problem to be solved by the invention is realized by the following technical scheme:

a recombinant vector in plant gene editing can screen out false positive callus, the recombinant vector contains NtAN2 gene elements, an expression frame which is driven by NtU4M and terminated by 35S and contains the NtAN2 gene elements is added into T-DNA to form a linked recombinant vector with other elements in gene editing; the whole expression frame sequence is SEQ ID No.1.

Preferably, the genetic element is capable of performing a gene editing function, and the NtAN2 gene is a MYB transcription factor derived from tobacco, for promoting the synthesis of plant anthocyanins.

Preferably, the UNS expression cassette is capable of expressing proteins that regulate anthocyanin synthesis, resulting in the accumulation of large amounts of anthocyanin in plant tissues, producing a visible red color, and rendering the entire callus red.

Application of a recombinant vector capable of screening out false positive callus in screening out false positive callus.

The application of a recombinant vector capable of screening out false positive callus in screening out false positive callus comprises the following steps:

(1) Preparation of NtAN2 gene elements and expression cassettes: the NtAN2 gene is driven by an NtU4M promoter, the termination is carried out by a 35S terminator, the sequence of the whole expression frame is SEQ ID No.1, primers with 15bp homologous sequences at two ends of a skeleton vector are used for PCR amplification at upstream and downstream, and a linearized DNA fragment of the expression frame is obtained through agarose gel electrophoresis and gel cutting purification;

(2) Construction of recombinant vectors: utilizing restriction enzymes KasI and HindIII to double-enzyme-cut a skeleton vector pORE-Cas9, cutting gel by agarose gel electrophoresis to recover purified linearized skeleton DNA, and carrying out recombination reaction on the linearized DNA of the NtAN2 expression frame and the purified linearized skeleton DNA; transforming the recombinant product into escherichia coli, and obtaining a gene editing recombinant vector pORE-Cas9/NtAN2 through screening and sequencing;

(3) Construction of gene knockout vector libraries: designing a 23nt specific sequence and upstream and downstream primers according to the sequence specificity of the target gene, carrying out PCR annealing connection and transforming agrobacterium;

(4) Transforming plant explants and resistant callus by using the gene knockout vector library to obtain: infecting wild tobacco by using the prepared agrobacterium, and inducing to form resistant callus;

(5) T-DNA tag selection of resistant calli: extracting the genome DNA of the resistant callus, and performing PCR amplification by using a specific primer of the NOS terminator to identify the existence of the T-DNA.

The technical scheme of the invention has the following beneficial effects:

(1) The gene editing recombinant vector comprises the NtAN2 gene element, and when the mutant is created by adopting the scheme of the invention to carry out plant gene editing technology, whether the callus is false positive can be judged by visually distinguishing red at the resistant callus stage, namely the red callus is a positive callus containing T-DNA transfer, and the green callus is a false positive callus without T-DNA transfer. The screening of the false positive callus reduces the occurrence of false positive plants, reduces the investment of steps of cultivation, detection and the like of the false positive plants in the later period, and further improves the probability of effectively generating gene editing plants.

Drawings

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.

FIG. 1 is a diagram of a callus cultured with an editing vector of the NtAN2 gene of the present invention, wherein A is a callus cultured with an editing vector containing the NtAN2 gene, and positive color shows red color, and false positive color shows green color; b is the callus cultured by the control group, and the control group can only grow green callus and can not distinguish positive from false positive.

Detailed Description

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

All experimental procedures used in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention adds an NtU4M driven, 35S terminated expression frame (named UNS) containing NtAN2 gene elements into T-DNA to form a linkage recombination vector with other gene editing elements. The gene editing element can exert a gene editing function, the NtAN2 gene is a MYB transcription factor derived from tobacco and can promote the synthesis of plant anthocyanin, so that the whole plant body can be red due to the existence of the UNS expression frame, and a system which is tightly linked with other elements of T-DNA is formed by the UNS expression frame, the transfer or false positive of the T-DNA can be indirectly represented by the existence and nonexistence of red callus, and the problem of false positive callus existing in the tissue culture stage in the large-scale mutant creation process of the plant can be solved by the application of the UNS.

Example 1 preparation of NtAN2 Gene elements and expression cassettes

The backbone vector pORE-Cas9 (from university of southwest) used for the recombinant vector construction of this example 1.

Preparation of the NtAN2 gene element and expression cassette:

the NtAN2 gene is driven by an NtU4M promoter, and is terminated by a 35S terminator, and the whole expression frame sequence is SEQ ID No.1 and is synthesized by Nanjing Jinsrui. And performing PCR amplification by using primers respectively containing 15bp homologous sequences at two ends of the skeleton vector at the upstream and the downstream, performing agarose gel electrophoresis, cutting gel and purifying to obtain the linearized DNA fragment of the expression frame.

EXAMPLE 2 construction of recombinant vectors

(1) The restriction enzyme KasI and HindIII are used for double digestion of the skeleton vector pORE-Cas9, and agarose gel electrophoresis is used for cutting gel to recover purified linearized skeleton DNA.

(2) The NtAN2 expression cassette linearized DNA from example 1 and the linearized backbone DNA purified in (1) were subjected to recombination reaction as follows:

(3) The recombination reaction condition is as follows: reacting at 37 ℃ for 30min; cooled to 4 ℃ or immediately placed on ice to cool.

(4) And transforming the recombinant product into escherichia coli, and screening and sequencing to obtain a gene editing recombinant vector pORE-Cas9/NtAN2.

Example 3 construction of Gene knock-out vector library

(1) Targeted primer design

A23 nt specific sequence (sgRNA) was designed based on the sequence specificity of the target gene, and the design method was described in the following website for instruction http:// crispor.tefor.net/crispor.py. The upstream primer (5 '-GATTGNNNNNNNNNNNNNNNNNNNNNNNNNNN-3') and the downstream primer (5 '-AAACNNNNNNNNNNNNNNNN-3') were synthesized separately. That is, sgRNA is a sequence of any 20 bases, the 5 '-end linker sequence of the upstream primer sequence is GATTG (SEQ ID No. 2), and the 5' -end linker sequence of the downstream primer is AAAC (SEQ ID No. 3).

(2) Annealing of

After the synthesized primers were diluted to a concentration of 100 ng/. Mu.L, 5. Mu.L of each of the upstream and downstream primers were mixed into a PCR tube, and the annealing procedure of the PCR instrument was: 5min at 95 ℃, 8s at-0.1 ℃ and annealing to 25 ℃. And adding water to the annealing product to dilute the annealing product by 10 times and storing the annealing product in a refrigerator at the temperature of 20 ℃ for later use.

pORE-Cas9/NtAN2 backbone enzyme cleavage

Enzyme digestion system (50 μ L):

the fragment was digested overnight at 37 ℃ and electrophoresed on a 1.5% agarose gel, and the backbone fragment was recovered using a gel recovery Kit (E.Z.N.A.gel Extraction Kit).

(3) Connection of

Products after annealing dilution are mixed one by one to form a mixed annealing product, and the mixed annealing product is connected with an enzyme-cut pORE-Cas9/NtAN2 skeleton vector, and a connecting system (10 mu L):

connecting for 2 hours at 16 ℃, purifying the connection product by using a gel recovery Kit (E.Z.N.A.gel Extraction Kit), transferring the connection product into escherichia coli through electrotransformation to form a gene knockout vector library, and shaking a large amount of bacteria and improving the quality of the particles for later use.

(4) Preparation of transformed Agrobacterium

The gene knockout vector library plasmid is transferred into agrobacterium LBA4404 through electric transformation, the transformed agrobacterium is evenly coated in YEB solid culture medium, and the transformed agrobacterium is obtained after overnight culture.

Example 4 transformation of plant explants with Gene knockout vector library and obtaining of resistant calli

The wild type safflower Dajinyuan tobacco is infected by the prepared agrobacterium tumefaciens, and the resistance callus is induced to form.

T-DNA tag selection of resistant calli: extracting the genome DNA of the resistant callus, and performing PCR amplification by using a specific primer of the NOS terminator to identify the existence of the T-DNA. The NOS primer sequences are as follows:

NOS-F：5’-GATTGAATCCTGTTGCCGGT-3’(SEQ ID No.4)；

NOS-R:5’-GTAACATAGATGACACCGCG-3’(SEQ ID No.5)。

the PCR product was subjected to agarose gel electrophoresis at a concentration of 1.5% to analyze whether it contained a specific 213bp band for the NOS terminator, and the results of the detection are shown in the following table:

and (3) displaying a detection result: after 3 batches of transformation, a certain proportion of green callus appeared in each batch, which may be false positive resistant callus formed along with red callus, and the green callus was negative after detection and the red callus was positive, indicating that the result detected by PCR is consistent with the result judged by color. The PCR detection method and the color determination method can both determine whether T-DNA is inserted into the callus, but the color determination method can be realized in actual experimental operation, is simpler and more convenient, and has almost no detection cost. In addition, false positive callus is eliminated in the callus stage, so that false positive regenerated plants are avoided, and the detection cost of target sites in the later stage is correspondingly reduced.

Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention.

Claims (5)

1. A recombinant vector capable of screening out false positive callus is characterized in that the recombinant vector comprises an NtAN2 gene element, an expression frame which is driven by an NtU4M promoter and terminated by a 35S terminator and used for expressing the NtAN2 gene is added into T-DNA, and the expression frame and other elements of gene editing form a linked recombinant vector; the whole expression frame sequence is SEQ ID No.1.

2. The recombinant vector capable of screening out false positive callus according to claim 1, wherein the NtAN2 gene is MYB transcription factor derived from tobacco for promoting synthesis of plant anthocyanin.

3. The recombinant vector capable of screening out false positive callus according to claim 2, wherein the expression cassette containing the NtAN2 gene is transferred into a plant cell and then can express and produce a protein for regulating anthocyanin synthesis, so that a large amount of red anthocyanin is accumulated in the plant callus, the positive callus and the false positive callus can be distinguished by naked eyes, and the elimination of the false positive callus can improve the efficiency of gene editing material creation.

4. Use of a recombinant vector according to any one of claims 1 to 3 for screening out false positive callus.

5. The use of the recombinant vector for screening out the false positive callus according to claim 4, which comprises the following steps:

(1) Preparation of NtAN2 gene elements and expression cassettes: the NtAN2 gene is driven by an NtU4M promoter, a 35S terminator is terminated, the sequence of the whole expression frame is SEQ ID No.1, primers with 15bp homologous sequences at two ends of a skeleton vector are used for PCR amplification at the upstream and the downstream, and a linearized DNA fragment of the expression frame is obtained by agarose gel electrophoresis and gel cutting purification;

(2) Construction of recombinant vectors: utilizing restriction enzyme KasI and HindIII to double-enzyme cut the skeleton vector pORE-Cas9, cutting gel by agarose gel electrophoresis, recovering purified linearized skeleton DNA, and carrying out recombination reaction on the linearized DNA of the NtAN2 expression frame and the purified linearized skeleton DNA; transforming the recombinant product into escherichia coli, and screening and sequencing to obtain a gene editing recombinant vector pORE-Cas9/NtAN2;

(3) Construction of gene knockout vector libraries: designing a 23nt specific sequence and upstream and downstream primers according to the sequence specificity of the target gene, carrying out PCR annealing connection and transforming agrobacterium;

(4) Transforming plant explants and resistant callus by using the gene knockout vector library to obtain: infecting wild tobacco by using the prepared agrobacterium, and inducing to form resistant callus;

(5) T-DNA tag selection of resistant calli: extracting the genome DNA of the resistant callus, and performing PCR amplification by using a specific primer of the NOS terminator to identify the existence of the T-DNA.

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